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THE SUBCELLULAR DISTRIBUTION OF RAT LIVER AND YEAST

GLUCOSE-6-PHOSPHATE DEHYDROGENASE

AND ITS ROLE IN REGULATION OF THE ENZYME

DISSERTATION

Presented in Partial Fulfillment of the Requirement for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Karen Elizabeth Reilly, B.S.

*****

The Ohio State University 1997

Dissertation Committee; Approved by John B. Allred, Advisor

Sylvia A. McCune Advisor Karla L. Roehrig Graduate Program in Food Science and Nutrition UMI Number: 9813339

UMI Microform 9813339 Copyright 1998, by UMI Company. All rights reserved.

This microform edition is protected against unauthorized copying under Title 17, United States Code.

UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT

GIucose-6-phosphate dehydrogenase (G6PD) belongs to the group of lipogenic

enzymes that all react similarly under lipogenic conditions. Specifically, when fasted

rats are refed a high carbohydrate diet, enzyme activity may increase up to ten times the

fasted level in 72 hours. Extensive research has been done to explain this phenomenon

for G6PD, and the general conclusion is that the overshoot is caused by increased

protein synthesis. However, the possibility that there is activation of the enzyme has

not been ruled out. Recent work has shown that other lipogenic enzymes are glycoproteins and may have GPI anchors that contribute to their regulation by shifting the enzymes from particulate to soluble fractions. G6PD was analyzed to see if it too shared these characteristics.

The sensitive digoxigenin-labeling method was used to confirm that G6PD is a glycoprotein. SDS-PAGE analysis showed matching migration patterns for protein, activity, antibody reactivity, and carbohydrate staining for both yeast and rat liver

G6PD. Treatment of yeast mitochondria with phosphatidyl inositol specific phospholipase c resulted in increased enzyme in the supernatant indicating that G6PD was bound to the mitochondria via a GPI anchor. Additionally, digitonin fractionation of rat liver mitochondria showed that G6PD is associated with the outer mitochondrial membrane. ii Male, Sprague Dawley rats were fed a high carbohydrate diet or fasted for 48

hours and refed a high carbohydrate diet and G6PD activity and quantity measured in

the cytosol and mitochondria to determine the subcellular distribution of the enzyme.

G6PD activity in the mitochondrial-free supernatant was 2.5 to 3.5 times greater in

refed rats compared to fed rats. In the mitochondria, the activity was up to twice as

high in refed rats. There was no difference in G6PD quantity between fed and refed rats

in either cellular compartment. Mitochondria had ten times as much enzyme as the mitochondrial-free supernatant, but the activity was only one tenth as much.

When expressed as actual enzyme specific activity (mU/amount enzyme), the mitochondrial-free supernatant G6PD was seven times higher in refed rats compared to fed rats. The refed rats had twice as much mitochondrial specific activity. Results showed that mitochondrial G6PD is very low in activity while the mitochondrial-free supernatant became much more active when fasted rats were refed a high carbohydrate diet. These results indicated that the overshoot in G6PD activity was due to increased activation of the enzyme which may be unrelated to the presence of carbohydrate attached to the protein or shifts in subcellular location.

Kinetic studies were performed with G6PD in fed and refed rats to explain better the increased activation of the enzyme. The Vmax for fed rats was 0.041mU/mg protein versus 0.096mU/mg protein for refed rats. These results were expected, however an unexpected difference in Km values was found. Fed rats had a Km of

28.8uM while refed rats had a Km of 55.9uM. These results show that there may be a different form of the enzyme present in refed rats that has distinct characteristics.

Ill Dedicated to my husband, John, and to the little one who provided a deadline for me to complete this project.

IV ACKNOWLEDGMENTS

There are many people to acknowledge who contributed to my graduate

experience. Foremost is my advisor. Dr. John Allred, who is a wonderful mentor and

has become a good friend. I realize that the advisor-student relationship is typically not

perfect, but I think in this case there was a good mesh of personalities which resulted in

a friendly, companionable work environment. Dr. Allred is a great role model for an

educator and researcher and some day I hope to be half as successful with my students as he is with his. I thank him for his guidance and patience, and seemingly photographic memory, but most of all his companionship.

I would like to thank Drs. Roehrig and McCune for taking the time to be on my committee and for their manuscript suggestions. I also appreciate having them as educators who fostered creative thinking and problem solving. And I thank them for their willingness to lend an ear or provide a piece of advice when the situation warranted. I thank Richard Jurin for the same ability.

Thank you to all my fellow students who became fnends and made my time at

Ohio State enjoyable—Jason Chou, Luther Chuang, Dave Smith, Heidi Senokozliefif,

Isabel Schuermeyer, and Kristin Pape. I truly don't think I could have survived without you. I'm grateful for the brainstorming sessions, one-on-one advice, and most of all the camaraderie. There's more to life than research and studies and we discovered that together. The best times were the times I could wander down the hail and find a

fiiendly ear to provide a little break for a while. Thank you all for that.

Finally, I would like to thank my husband, John. Your support over the past

four and a half years has been astounding. From the first stress-filled quarters when

going back to school was a foreign concept to the past few months when the final crush was on, you were always there to boost my spirits and pick up the slack. I know you say you're proud of me for what I've accomplished, but I'm just as proud of you for going through this with me.

VI VITA

November 22, 1968...... Bom - Akron, OH

1991...... B.S. Chemistry, John Carroll University

1991 - 1993...... Research Scientist New Projects Department The Lubrizol Corporation WicklifFe, OH

1993 - 1994...... Graduate Research Assistant The Ohio State University

1994 - Present...... USDA National Needs Fellow The Ohio State University

PUBLICATIONS

Research Publications

1. Reilly KE, Allred JB. (1995) Glucose-6-Phosphate Dehydrogenase from Saccharomyces cerevisae is a glycoprotein. Biochem. and Biophys. Res. Com. 216(3):993-8.

2. Allred JB, Reilly KE. Short-term regulation of acetyl CoA carboxylase in tissues of higher animals. (1996) Progress in Lipid Research, Elsevier Press, Oxford 35(4):371-85.

FIELDS OF STUDY

Major Field; Food Science and Nutrition

Vll TABLE OF CONTENTS

Abstract...... ii

Dedication ...... iv

Acknowledgments ...... v

Vita...... vii

List of Tables...... x

List of Figures...... xi

List of Abbreviations ...... xiii

Chapters:

Introduction ...... 1

1. Literature Review ...... 4 GIucose-6-Phosphate Dehydrogenase ...... 4 Lipogenic Enzymes ...... 5 Glycoproteins and GPI Anchors ...... 7 Digitonin Fractionation ...... 14 Particulate Forms of Lipogenic Enzymes ...... 16 Overshoot in G6PD Activity ...... 21

2. Methods and Materials ...... i5 Baker’s Yeast Studies ...... 35 Separation of Cytosol and Mitochondria ...... 35 Separation of Yeast Outer Mitochondrial Membrane and Treatment with PI-PLC ...... 36 Protein Determination ...... 37 Preparation of SDS-Boiled Samples ...... 37 Polyacrylamide Gel Electrophoresis ...... 38 Denaturing Gel ...... 38 Non-Denaturing G el ...... 38 viii Western Blot ...... 40 Antibody Detection of G6PD ...... 40 Quantitative Determination of G6PD ...... 41 G6PD Activity Stain of Non-Denaturing Gel ...... 43 Glycan Chain Detection of G6PD ...... 46 Enzyme Assays ...... 47 Animal Studies ...... 48 Animal Care ...... 48 Isolation of Livers ...... 50 Separation of Cytosol and Mitochondria ...... 50 Separation of Liver Mitochondrial Fractions (Digitonin Fractionation) ...... 51 Separation of Outer Mitochondrial Membrane in the Rat Liver... 52 Glycogen Determination ...... 54 Enzyme Kinetic Study of Rat Liver G6PD ...... 54 Materials...... 55 Statistics...... 55

3. Results and Discussion ...... 56 Yeast Studies ...... 56 Stability of Yeast G6PD ...... 59 Glycoprotein Analysis of Purified Yeast G6PD ...... 59 Treatment of Yeast Mitochondria with PI-PLC ...... 66 Animal Studies ...... 68 48-Hour Refeeding Study ...... 70 Short Term Refeeding Study ...... 79 Long Term Refeeding Study ...... 79 Enzyme Kinetic Studies for Rat Liver G6PD ...... 92 Electrophoretic Mobility Study for Rat Liver G6PD ...... 95

4. Conclusions ...... 100

References ...... 102

IX LIST OF TABLES

Table Page

2.1 Composition of Denaturing Polyacrylamide Gels ...... 39

2.2 Composition of Non-Denaturing Polyacrylamide Gels ...... 39

2.3 Composition of Diet ...... 49

2.4 Protease Inhibitors ...... 53

3.1 Specific Activity of G6PD in Yeast ...... 57

3.2 Quantity of G6PD in Yeast ...... 57

3.3 G6PD Specific Activity and Quantity in Rat Liver Mitochondrial-Free Supernatant ...... 71

3.4 G6PD Specific Activity and Quantity in Rat Liver Mitochondria ...... 73

3.5 G6PD Activity Per Quantity (mU G6PD/ug G6PD) in Rat Liver...... 75

3.6 Short Term Refeeding Study ...... 80

3.7 G6PD Quantity in Fed and Refed Rats ...... 86 LIST OF FIGURES

Figure Page

1.1 Structure of a Typical GPI-Anchored Protein ...... 12

2.1 Densitometer Readout from Rat Liver Quantitative Analysis ...... 42

2.2 Standard Curve for Yeast Studies ...... 44

2.3 Standard Curve for Rat Liver Studies ...... 45

3.1 Activity and Quantity of Yeast G6PD ...... 58

3.2 Stability of Yeast G6PD ...... 60

3.3 Molecular Weight Determination of Yeast G6PD ...... 62

3.4 Glycoprotein Detection of Yeast G6PD ...... 63

3.5 Glycoprotein Detection of Rat Liver G6PD ...... 65

3.6 Yeast G6PD Released by Phosphatidyl Inositol Specific Phospholipase C...... 67

3.7 Rat Liver Digitonin Fractionation ...... 69

3.8 G6PD Activity and Quantity in Rat Liver Mitochondrial-Free Super-^8-Hour Study ...... 72

3.9 G6PD Activity and Quantity in Rat Liver Mitochondria— 48-Hour Study ...... 74

3.10 G6PD Activity Analysis in Mitochondrial-Free Super— 48-Hour Study ...... 77

XI 3.11 G6PD Activity Analysis in Mitochondria—48-Hour Study ...... 78

3.12 G6PD Activity in the Mitochondrial-Free Supernatant— 36-Hour Study ...... 81

3.13 G6PD Activity in Mitochondria—3 6-Hour Study ...... 82

3.14 G6PD Activity in Fed Rats—36-Hour Study ...... 84

3.15 G6PD Activity in Refed Rats—36-Hour Study ...... 85

3.16 G6PD Activity Per Amount in Mitochondrial-Free Super— 36-Hour Study ...... 88

3.17 G6PD Activity Per Amount in the Mitochondria—3 6-Hour Study ...... 89

3.18 6PGD Activity in Mitochondrial-Free Supernatant— 36-Hour Study ...... 90

3.19 Glycogen Content in Rat Liver—3 6-Hour Study ...... 91

3.20 Rat Liver G6PD Kinetic Study—Velocity Plot ...... 93

3.21 Rat Liver G6PD Kinetic Study—Lineweaver-Burke Plot ...... 94

3.22 Densitometry Scan of G6PD Activity; Electrophoretic Mobility A—Fed Rats, B—Refed Rats ...... 97

3.23 Densitometry Scan of G6PD Antibody Reactivity: Electrophoretic Mobility A—Fed Rats, B—Refed R ats ...... 98

Xll LIST OF ABBREVIATIONS

ACC...... Acetyl CoA carboxylase Act...... Activity AdKi ...... Adenylate kinase ADP...... Adenosine diphosphate AMP...... Adenosine monophosphate ESA...... Bovine serum albumin OC ...... Degree Celsius Carbo...... Carbohydrate cAMP...... Cyclic adenosine monophosphate cDNA...... Complimentary deoxyribonucleic acid CoA...... Coenzyme A CytC ...... Cytochrome c oxidase Cyto...... Mitochondrial-free supernatant ECL...... Enhanced chemiluminescence reagent EDTA...... Ethylene diamine tetraacetic acid GC...... Gas chromatography G6P...... Glucose-6-phosphate G6PD...... Glucose-6-phosphate dehydrogenase GPI...... Glycosyl phosphatidyl inositol GSSG...... Oxidized glutathione GW ...... Greenawalt's H r...... Hour K ...... Thousand Kda ...... Kilodalton kg...... Kilogram L ...... Liter LPL...... Lipoprtoein lipase M ...... Molar MAO...... Monoamine oxidase MS...... Mass spectroscopy Max...... Maximum MDH...... Malate dehydrogenase Min ...... Minute Mito...... Mitochondria mg...... Milligrams xiii ml...... Milliliters mRNA...... Messenger ribonucleic acid mU...... Milliunits NAD...... Nicotinamide adenine dinucleotide NADP ...... Nicotinamide adenine dinucleotide phosphate NADPH...... Nicotinamide adenine dinucleotide phosphate (reduced form) ng...... nanograms nm ...... nanometers O.D...... Optical density PAGE...... Polyacrylamide gel electrophoresis PBS...... Phosphate-buffered saline PI-PLC...... Phosphatidyl inositol-specific phospholipase c PMSF...... Phenylmethyl sulfonylfluoride Prot...... Protein 6PGD...... 6-Phosphogluconate dehydrogenase Quant ...... Quantity S ...... Substrate SDS...... Sodium dodecyl sulfate SEM...... Standard error of the mean Soln ...... Solution Sp act...... Specific activity Super ...... Supernatant mM...... Millimolar TEMED...... N,N,N",N"-Tetramethylethylenediamine TBS...... Tris-buffered saline U...... Unit ug...... Micrograms uM ...... Micromolar V...... Velocity Vmax...... Maximum velocity VSG...... Variant surface glycoprotein w/v...... Weight/V olume

XIV INTRODUCTION

Glucose-6-phosphate dehydrogenase (G6PD) [E.G. 1.1.1.49] is the key regulatory enzyme for the pentose phosphate pathway. This enzyme catalyzes the reaction of glucose-6-phosphate to 6-phosphoglucono lactone which also produces reducing equivalents (NADPH) for a variety of biosynthetic processes, including fatty acid synthesis. For this reason it belongs to a class of enzymes known as lipogenic enzymes whose activities are known to increase under conditions of de novo lipid biosynthesis.

Animal studies have shown that refeeding a high carbohydrate diet to an animal that has previously been fasted results in increased lipogenesis and an overshoot in

G6PD activity compared to animals that have been fed a normal rat chow diet (1,2).

Many studies have been undertaken to determine why this great increase in activity occurs. Two main regulatory mechanisms that have been explored are increased protein synthesis and increased activation of the enzyme. Arguments for increased synthesis of

G6PD (3, 4, 5) have been strong, but the area of increased activation of the enzyme has not been fully explored (6, 7, 8). G6PD normally functions in the cytosol (9, 10), but particulate forms have also

been found in rat liver peroxisomes (11,12) and mitochondria (13, 14), and rabbit liver

microsomes (15). Particulate forms of other lipogenic enzymes have also been

characterized. ATP citrate lyase [E.C. 4.1.3.8] has been found in rat liver mitochondria

(16-18) and had more enzyme associated with the particulate fraction in fasted rats than

in fed rats (18). Acetyl CoA carboxylase [E.C. 6.4.1.2] has also been found in rat liver

mitochondria (19- 23) and showed a shift to soluble form upon fasting and refeeding

(20). It appears that these two lipogenic enzymes are ambiquitous in that they change

their cellular location based upon the physiological conditions of the cell.

Acetyl CoA carboxylase was proven to be a glycoprotein, and evidence

indicated that the enzyme was attached to a membrane via a glycosyl

phosphatidylinositol anchor (24). The enzyme was localized to the outer mitochondrial

membrane in this subcellular fraction (21). This particular linkage and association

would allow the enzyme to be cleaved from the membrane under lipogenic conditions

and could be a valuable means of regulation.

G6PD has many of the same characteristics as the other lipogenic enzymes,

specifically changes in enzyme activity concurrent with dietary changes. Since

particulate forms of some lipogenic enzymes have shown shifts in location upon

refeeding a high carbohydrate diet (18, 20), G6PD should also be analyzed to see

whether it exhibits this property. Additionally, acetyl CoA carboxylase is an inositol- containing glycoprotein and is attached to the outer mitochondrial membrane, and

G6PD should be investigated to determine if it shares these same characteristics. Specific Alms

The purpose of this research is to characterize G6PD as a glycoprotein,

determine if it has a glycosyl phosphatidylinositol anchor, and address the possibility

that the particulate form has a role in regulation of the enzyme under differing dietary

conditions. To accomplish these goals, yeast G6PD from Saccharomyces cerevisiae

was used in preliminary experiments due to its ready availability and unicellular

characteristics. The possible presence of carbohydrate was explored using the sensitive

digoxigenin labeling method and phosphatidyl inositol specific phospholipase c used to

examine the presence of a glycosyl phosphatidylinositol anchor. Male Sprague Dawley

rats were used to determine the distribution of liver G6PD between the cytosol and

mitochondria in fed, fasted, and fasted/ high carbohydrate refed animals. Both activity

and quantity of the enzyme were measured to assess possible shifts in subcellular

location of the enzyme. This research will add to the already great body o f knowledge concerning the regulation of G6PD by investigating the little-explored area of subcellular distribution of the enzyme. CHAPTER 1

LITERATURE REVIEW

Glucose-6-Phosphate Dehydrogenase

Glucose-6-phosphate dehydrogenase (G6PD) is a metabolic enzyme that is

widely distributed in nature and has been found in all organisms and cell types (9,25).

Its nucleotide sequence has been greatly conserved from Drosophila to humans (26, 27).

As the first enzyme in the pentose phosphate pathway, G6PD functions at a branch

point in metabolism in that it produces glycolytic intermediates, 5-carbon rings for

nucleotide synthesis, and NADPH for a number of reactions including fatty acid

biosynthesis. Thus, G6PD is involved in both carbohydrate and lipid metabolism.

Because of its presence in most cells and its pivotal role in metabolism, its

characteristics and regulation have been extensively studied.

G6PD is known to have three different active forms when analyzed by non­

denaturing polyacrylamide gel electrophoresis (4, 10, 28-31). These three forms are thought to be multiple subunits arranged in dimers, tetramers, and hexamers since the molecular weights are in a ratio of 1:2:3 (28). The inactive subumit has a reported molecular weight of 56-67,000 in yeast (27, 32) and several mammalian tissues (10, 28,

29, 33-35) while the main active forms are 121-130 lcDa(10,29, 34). Although multiple forms of G6PD exist, Watanabe and co-workers (36) found no difference in kinetic or

immunological properties among the different forms.

The reaction catalyzed by G6PD occurs primarily in the soluble fraction of the

cell (9, 10). It requires glucose-6-phosphate (G6P) almost exclusively as a substrate,

and the enzyme from most sources requires NADP to produce the reducing equivalents

(NADPH) for processes such as fat and cholesterol biosynthesis. Magnesium is also

required as an activator of the enzyme for most species. The products of the G6PD

reaction are 6-phosphoglucano lactone which is non-enzymatically converted to 6- phosphogluconate and NADPH. The oxidative phase of the pentose phosphate pathway concludes with the formation of ribulose-5-phosphate through the action of 6- phosphogluconate dehydrogenase (6PGD) via oxidative decarboxylation. NADPH is also produced at this step making these two reactions the major source of reducing equivalents in a cell (37).

Lipogenic Enzymes

G6PD is just one of a class of enzymes known as lipogenic enzymes. This group is responsible for fatty acid biosynthesis in organisms and includes ATP-citrate lyase, acetyl CoA carboxylase, fatty acid synthase, and malic enzyme. ATP-citrate lyase cleaves cytosolic citrate to form oxaloacetate and acetyl CoA. Acetyl CoA carboxylase adds a carboxyl group to acetyl CoA to form malonyl CoA as the first step in fatty acid biosynthesis while fatty acid synthase is necessary for fatty acid formation.

Malic enzyme also produces NADPH. These five enzymes share similar regulatory mechanisms in that the levels of all of them rise or fall together in response to nutritional changes (I). The enzyme activities of the lipogenic enzymes decrease with starvation and low levels of insulin and increase with carbohydrate refeeding (I). In isolated rat hepatocytes, glucose and insulin added to the media caused an increase in lipogenic enzyme mRNA concentration while fatty acids decreased the mRNA concentration (38). Increasing the amount of diet upon refeeding carbohydrate to previously starved rats caused an increase in lipogenic enzyme activity, transcriptional rate, and mRNA concentration (39).

Although not involved in fatty acid synthesis per se, there are other lipogenic enzymes involved in lipid metabolism that have similar activity patterns to the lipogenic enzymes. Their activities increase under lipogenic conditions. 3-Hydroxy-3- methylglutaryl-CoA reductase (HMG CoA reductase) is the rate-limiting enzyme in cholesterol biosynthesis. Lipoprotein lipase hydrolyzes fatty acids from circulating lipoproteins and deposits them in adipose cells. Research has shown that these two enzymes contain carbohydrate and can be classified as glycoproteins (40-43).

Particulate forms of other lipogenic enzymes have been found (16-23) and acetyl CoA carboxylase was shown to be a glycoprotein and possibly possesses a glycosyl phosphatidyl inositol anchor (24). Glycoproteins and GPI Anchors

Glycoproteins

A glycoprotein is a protein with a glycan chain (or oligosaccharide) covalently

attached. Increasing evidence has shown that most mammalian proteins contain sugar

chains (44). Glycoproteins have diverse functions such as maintenance of protein

conformation and solubility, proteolytic processing and stabilization of the polypeptide

against uncontrolled proteolysis, mediation of biological activity, intracellular sorting

and extemalization of glycoproteins, and embryonic development and differentiation

(45). The carbohydrate chains may contain any of seven sugars—galactose, glucose,

mannose, fucose, N-acetylneuraminic acid, N-acetylgalactosamine, and N-

acetylglucosamine. Sialic acid is usually present as well at chain termini (37). Glucose

is rarely found in a glycan chain. Typically a glycan chain has fifteen sugars with 1-30

glycan chains per glycoprotein. Therefore, the carbohydrate can be anywhere from I to

85% of the molecular weight of the glycoprotein (37). The different types of sugar residues and the almost unlimited number of linkages possible for a glycan chain allow for extensive branching and great diversity in glycoproteins.

Structurally glycoproteins can be classified into two groups. N-linked glycoproteins have an N-acetylglucosamine linked to an asparagine residue of the protein. The common sequence pattern is ASN-X-SER(THR) where X is any amino acid but proline or aspartate. 0-linked glycoproteins have an N-acetylglucosamine linked to a serine or threonine. This sequence pattern necessary for attachment is ASN-

Y-SER(THR) where Y is any amino acid but aspartate (37). Additionally, glycoproteins can be classified according to their sugar types (44). High mannose

chains contain only mannose and N-acetyl glucosamine residues. Complex chains

contain mixed sugars such as galactose, mannose, fucose, N-acetyl glucosamine, and

sialic acid but no mannose except in the anchoring core. Hybrid types are a mixture of

high mannose and complex types.

Many methods have been developed to help identify and characterize

glycoproteins. Gas chromatography/mass spectroscopy (GC/MS) can be used to detect

the presence of sugars. Lectins are available which are proteins from plants, which bind

sugars. Lectins can be immobilized on a column support and the potential glycoprotein

added to see if it becomes bound (37). Concanavalin A is a popular lectin that binds

sugars. It detects carbohydrate and can distinguish among the types of glycan chains

(44). Complex-type glycans which have two binding residues become bound to the Con

A column and are eluted with 5mM methyl a-glucopyranoside. High mannose-type

glycans become bound and are eluted only with 200mM methyl a-mannopyranoside.

Glycan chains with only one binding residue pass through the column.

Endoglycosidases such as endo F and endo H cleave certain N-acetylglucosamine

residues close to the polypeptide and are a useful tool in analyzing glycoproteins (44).

A suspected glycoprotein can be treated with an endoglycosidase and analyzed by gel

electrophoresis to determine whether a shift in molecular weight occurred indicating

loss of a glycan chain. If the glycan chain is too small to have an effect on molecular weight shifts, the glycan can be labeled and the absence of label after treatment

indicates the loss of carbohydrate. One popular method to label glycoproteins is with

8 tritiated borohydride (46). The protein is treated with galactose oxidase, which oxidizes

non-reducing terminal galactosyl and N-acetylgalactosamine residues to their

corresponding C-6 aldehydes. Next, the aldehydes are reduced back to their original

sugars with the addition of tritiated borohydride, which labels the sugar with a ^H.

Biochemical methods are then employed to detect the radiolabel.

A relatively new method for detecting carbohydrate attached to protein is simple

and involves no radioactivity. Adjacent hydroxyl groups on sugar residues can be

oxidized to aldehyde groups with periodate treatment. The potential glycoprotein is

then treated with the hapten digoxigenin, which becomes covalently attached to the

aldehyde by a hydrazide group (47). The digoxigenin-labeled glycoconjugate can then be detected using an anti-digoxigenin antibody conjugated with alkaline phosphatase or

horseradish peroxidase. Digoxigenin labeling can be performed on proteins in solution or immobilized on nitrocellulose. The antibody attachment is done after the protein has been immobilized on nitrocellulose. This method is highly specific and extremely sensitive for carbohydrate (47).

Glvcosvl Phosphatidvlinositol Anchors

Glycosyl phosphatidylinositol (GPI) anchors are a relatively newly discovered mechanism to attach proteins to membranes. Rather than having a hydrophobic region on the protein interact with the lipid bilayer of the membrane, the protein is bound with a phosphatidyl inositol linker attached to a glycan moiety of the protein (48). Proteins that utilize this kind of attachment are widely distributed between different organisms from yeast and mold to Drosophila and mammalian tissues (49). Although over 100

GPI-anchored proteins have been discovered, there is no evidence of their existence in prokaryotes or plants (50). Alkaline phosphatase was one of the first proteins shown to have a GPI anchor (51). It was shown to be released from mammalian tissue by treatment with phosphatidylinositol-specific phospholipase c (PI-PLC). This enzyme is believed to hydrolyze specifically phosphatidylinositol and phosphatidylinositol mannosides (48). Examples of other proteins characterized as having GPI anchors are placental alkaline phosphatase, decay accelerating factor, acetylcholinesterase,

5'nucleotidase, Thy-1 glycoprotein, and the variant surface glycoprotein (VSG) from

Trypanosoma bnicci (48, 52). Most of the GPI-anchored proteins that have been studied are anchored on the cellular surface, but this may be because it is easier to study that membrane than intracellular structures (51). Since this body of these proteins is so diverse, a protein would have to be specifically targeted and extensively studied to see if it also contains a GPI structure.

It was found that treatment of GPI-anchored proteins with PI-PLC released soluble, intact proteins and left the membrane moiety intact (48, 51). The molecular weight of released proteins was similar to that of native proteins. Since PI-PLC specifically cleaves between the phosphate and 1,2 diacylglycerol of phospholipids, it was likely that 1,2 diacylglycerol served as the membrane anchor (48). Some proteins believed to be GPI-anchored proteins may be resistant to release by PI-PLC (51). This is because PI-PLC from different sources may not have the same specificity for all proteins, and the individual proteins are not equally reactive to PI-PLC. Additionally,

10 some GPI-anchored proteins have a third fatty acid that attaches the inositol to the

membrane and these proteins are insensitive to PI-PLC treatment (52, 53). Recent work

using protein engineering of cell surfaces has shown that a clipped GPI-anchored

protein can be reattached to a membrane (53). This has introduced techniques for

genetically adding a GPI anchor to a non-GPI-linked protein and directing them to a

membrane.

A^o-inositol is not a normal constituent of proteins and its presence can easily

be confirmed by GC/MS analysis to indicate the possible presence of a GPI-anchored

protein (51). The diverse GPI-anchored proteins have other characteristics in common

as well. They all contain ethanolamine (1:1 molar ratio for VSG, 2:1 for Thy-1 and acetylcholinesterase), glucosamine (1 mol:l mol), and glycerol, phosphate, and fatty acid where analyzed (51). Other carbohydrates are also present although the same ones are not present in all GPI-anchored proteins. VSG contains mannose and galactose and

Thy-1 contains mannose and galactosamine (51). The fatty acid portion of the protein complex also differs. VSG contains only myristate while acetylcholinesterase contains a mixture including palmitate and Thy-1 contains stearate and two longer chain fatty acids (50, 51). The schematic for a typical GPI-anchored protein is shown in Figure

I.l. The 1,2 diacylglycerol is embedded in the membrane and the phosphatidyl inositol attached to it. The inositol is attached to a glycan chain of varied structure via a glycosidic linkage with glucosamine. The glycan is attached to an ethanolamine residue that is linked to the C-terminal end of the protein.

11 QLYCAN

tm

* Adapted from Reference 52.

Figure 1.1 Structure of a typical GPI-Anchored Protein*

As indicated in Figure 1.1, the anchoring domain of the GPI-anchored protein is attached at the C-terminal end. Studies with VSG showed that the cDNA sequence contained 17-23 amino acid residues at the C-terminus that were missing in the mature protein while Thy-1 had 31 residues removed during processing (51). They were probably removed and replaced with the glycan structure during processing. GPI- anchored proteins contain a similar hydrophobic N-terminus, which directs them to the endoplasmic reticulum for processing (49, 51). The C-terminal residues that are removed also have a hydrophobic region that includes several charged or hydrophilic residues (49, 51, 53). Otherwise there is no common amino acid sequence among the GPI proteins. The addition of the glycan moiety is rapid (within I minute for VSG)

which implies that the glycolipid structure is preconstructed before addition (49, 53).

Lipoeenic Enzymes Characterized as Glycoproteins

As mentioned previously, HMG-CoA reductase has been characterized as a glycoprotein. UT-1 cells are hamster cells, which have been genetically altered to express -500 times the normal amount of HMG CoA reductase and have been used to analyze this enzyme. Work by Liscum et a l(40) with UT-1 cells showed that HMG

CoA reductase is a transmembrane N-linked glycoprotein of the high mannose type.

The researchers showed that HMG CoA reductase bound to Con-A sepharose and exhibited a decrease in molecular weight when treated with endo H. Additionally, the enzyme incorporated [1,6^H]glucosamine into its structure. Volpe and Goldberg (41) using C -6 glial cells found that tunicamycin administration decreased HMG CoA reductase activity and cholesterol synthesis. Tunicamycin is an inhibitor of glycoprotein synthesis. The authors concluded that HMG CoA reductase is a glycoprotein, and the synthesis of the glycoprotein is necessary for activity.

Lipoprotein lipase (LPL) has not only been proven to be a glycoprotein (42), it has been shown to be attached to the cellular membrane by a GPI anchor (43). 3T3-L1 cells, which differentiate to adipocytes, are commonly used to investigate lipoprotein lipase. In the late 1970's Spooner et al (54) found that LPL activity was proportional to insulin levels. Insulin (10*^-10'^°M) increased LPL release from 3T3-L1 cells rapidly and increased its activity slowly. The authors concluded that the rapid release was

13 independent of energy metabolism and protein synthesis. In the late 1980's insulin was

linked to PI-PLC activity (43, 55). Saltiel and Cuatrecasas (55) used BC 3HI cells (from

a murine myocyte cell line) to show that insulin stimulated the activity of PI-PLC and

released a carbohydrate-containing substance from the cell membrane. Chan et al (43)

concluded that insulin was the predominant regulator of LPL in 3T3-L1 cells and the rapid release caused by insulin was due to the action of PI-PLC. The kinetics of release induced by insulin were the same as those of PI-PLC. Since HMG-CoA reductase and lipoprotein lipase are both glycoproteins and lipoprotein lipase has a GPI anchor, other lipogenic enzymes may have similar properties.

Digitonin Fractionation

In order to understand fully metabolic regulation, the location of all metabolites and enzymes must be fully established. A widely used technique to help elucidate the subcellular distribution of enzymes and pinpoint the location of particulate forms of enzymes is known as digitonin fractionation. Digitonin complexes with cholesterol and has been used for years to extract cholesterol in biological samples. In the fractionation procedure, digitonin is used to disrupt cellular membranes, which contain large amounts of cholesterol (28). If whole cells are used, the cholesterol content of the various membranes can be used to preferentially separate cell components by incubating with digitonin for various amounts of time (18). Plasma membrane cholesterol content is greater than outer mitochondrial and endoplasmic reticulum content, which is greater than inner mitochondrial membrane cholesterol content. This procedure has been used

14 to determine the distribution of some enzymes between the cytosol and mitochondria in

rat hepatocytes by comparing the rate of release of a given enzyme with that of known

marker enzymes (17).

If it has been established that an enzyme is mitochondrial, it is desirable to

determine where it is located in the mitochondria. The digitonin fractionation

procedure can be used on isolated mitochondria to disrupt the outer and inner

membranes (28, 56). Mitochondria are isolated by centrifugation and incubated with

digitonin at 0°C at a specified ratio of digitonin to protein. Although its exact

contribution is unknown (56), bovine serum albumin must be added to the reaction mix for complete results, perhaps to stabilize dilute enzyme protein. The resultant fractions are then centrifuged. The ratio of digitonin to mitochondria can be manipulated to obtain clean fractions of outer or inner mitochondrial membrane (56) or various concentrations used and the enzymes measured in the supernatant of the subsequent centrifugal spin to determine their release pattern (21). Some researchers have kept the digitonin concentration constant and varied the time of incubation to disrupt preferentially the mitochondrial membranes (16, 18).

A number of enzymes have been linked to specific fractions of the mitochondria and can be used as markers. For example, monoamine oxidase is a known outer mitochondrial enzyme and is released at low digitonin concentrations (28). Adenylate kinase is located between the inner and outer mitochondrial membranes and has a release pattern which starts at a lower digitonin concentration than monoamine oxidase

(28). This occurs because the enzyme is quick to 'leak' from its compartment once a

15 portion of the outer mitochondrial membrane has been disrupted. Cytochrome c

oxidase can be used as a marker for the mitochondrial matrix, and malate

dehydrogenase for the soluble matrix (21). Higher concentrations of digitonin or longer

incubation times are needed to release the latter two enzymes. The activities of the

enzyme in question and all marker enzymes are measured in each fraction and the

patterns of release compared to determine the location of the unknown enzyme (21).

This procedure has been used to localize ATP citrate lyase in the mitochondria (16-18)

and acetyl CoA carboxylase to the outer mitochondrial membrane (21).

Particulate Forms of Lipogenic Enzymes

O f the lipogenic enzymes, G6PD, ATP citrate lyase, and acetyl CoA

carboxylase are known to have particulate forms.

ATP Citrate Lvase

Janski and Cornell (19-18) have used the digitonin fractionation procedure to discover that ATP citrate lyase is found in the mitochondrial preparation of rat liver cells. Upon further investigation, when rats were fasted, 52% of the enzyme's activity was associated with the mitochondria compared to 21% for fed rats and 24% for fasted- refed rats (18). Thus, the change in the physiological state of the animal between fasted and fed or refed states caused ATP citrate lyase content to decrease in the mitochondria and increase in the cytosol. Presumably the enzyme was released from its particulate form. The addition of 20uM CoA to the digitonin fractionation medium also caused a

16 reduction in mitochondrial ATP citrate lyase while a reduction in cellular CoA

increased the particulate amount (18). Also, citrate and ATP added to the medium

caused the ATP-citrate lyase shift to the cytosol (16). The localization of the enzyme

with the mitochondrial membrane might allow it to easily bind citrate and efficiently

form acetyl CoA during times when the cell is not involved with fat synthesis. Citrate

could change the pH of the cell or the free magnesium concentration if it were to

accumulate. Due to these changes in ATP citrate lyase's location under differing

metabolic conditions it has been characterized as an ambiquitous enzyme.

Acetvl CoA Carboxvlase

Witters et a/ (19) were the first to report in recent years of an association of

acetyl CoA carboxylase with microsomes using ^^P labeled enzyme and

immunoprécipitation of rat liver hepatocytes. This was followed by work done by

Allred and co-workers (20-23) who discovered a mitochondrial form of acetyl CoA

carboxylase that was relatively inactive. The enzyme could be quantitated by

incubating SDS-denatured protein with [*'*C]methyl avidin, which binds to the biotin

moiety of acetyl CoA carboxylase, and subjected to polyacrylamide gel electrophoresis

(23). After exposure to X-ray film and fluorography, the protein could be quantitated by densitometry. Results showed that 50% of the acetyl CoA carboxylase in rat liver was associated with the mitochondria in fed rats, and fasting-refeeding shifted the amount toward the cytosol (20). In alloxan diabetic rats, the activity of acetyl CoA carboxylase is decreased along with the rate of lipogenesis. A theory for this

17 occurrence is that insulin needs to be present to synthesize the enzyme. Measurement

of acetyl CoA carboxylase in acutely diabetic rats, however, showed that there was less

enzyme in the cytosol but a greater amount in the mitochondria, which was mostly

inactive (22). It was proposed that insulin, in addition to a putative role in enzyme

synthesis, is also involved in the mobilization and activation of acetyl CoA carboxylase

from an inactive mitochondrial to an active cytosolic form.

Further studies of the quantitation of this enzyme revealed two antibody reactive

mitochondrial forms which, when added together, accounted for 75% of the total acetyl

CoA carboxylase quantity in the cell (21). 3.5% of the enzyme was microsomal and the remainder cytosolic. The specific activity of cytosolic acetyl CoA carboxylase was 16.4 mU/mg protein versus 1.8mU/mg protein for the mitochondrial form. Quantitatively, the cytosol had 3.1 nmol/g liver of the enzyme while the mitochondria had 5.7 and 4.3 nmol/g liver for the two forms. The actual specific activity of the enzyme could also be calculated. The cytosolic enzyme at 2.0 units/mg acetyl CoA carboxylase was 20 times more active than the mitochondrial enzyme at 0.09 units/mg acetyl CoA carboxylase showing that most of the acetyl CoA carboxylase was in an inactive mitochondrial form.

Digitonin fractionation was used to determine the location of the acetyl CoA carboxylase in the mitochondrial fraction (21). Both forms of mitochondrial acetyl

CoA carboxylase showed the same release pattern as monoamine oxidase indicating an association with the outer mitochondrial membrane. This location would allow the enzyme to be easily accessible for release or activation under lipogenic conditions.

18 Since acetyl CoA carboxylase was found to be associated with the outer

mitochondrial membrane, experiments were performed to determine whether it

contained a carbohydrate moiety, which could be used as an anchoring mechanism.

Bowers (24) determined that acetyl CoA carboxylase was a glycoprotein based on

several lines of evidence. GC/MS analysis confirmed the presence of mannose,

galactose, flicose, N-acetylglucosamine, and inositol in acetyl CoA carboxylase purified

from rat liver. The enzyme bound to a Con A sepharose column and was eluted by

methyl a-mannopyranoside. Additionally, gel electrophoresis followed by treatment

with the digoxigenin/anti-digoxigenin detection system showed a positive stain for

carbohydrate at the same mobility as the anti-acetyl CoA carboxylase antibody stain.

The presence of inositol associated with the purified enzyme introduced the possibility that acetyl CoA carboxylase has a GPI anchor.

Glucose-6-Phosphate Dehvdroeenase

Although G6PD is known to be a cytosolic enzyme and perform primarily in that part of the cell, particulate forms of the enzyme have been found. As early as 1965

Zaheer (13, 14, 57) discovered the presence of G6PD in rat liver mitochondria.

Extensive studies were performed on fresh and frozen liver in male and female rats.

Mitochondrial fractions were separated by centrifugation and the enzyme activity measured. It was discovered that the addition of Triton X-100 detergent to the reaction media helped maximize G6PD activity in mitochondrial fractions (57). Additionally, optimal G6PD activity was elicited by using a freeze-thaw cycle of rat liver

19 mitochondria (13). Since a combination of treatments was necessary to achieve full

activity, it was thought that G6PD must be firmly bound to the mitochondria. The

G6PD activity of the mitochondrial fraction was 20% that of the cytosolic fraction in

female rats and 40% in male rats. The female rats did not show a difference in G6PD

activity in the mitochondria compared to males although cytosolic G6PD activity was

higher in female rats. 6-PhosphogIuconate dehydrogenase was also found in the

mitochondria at 15% of the total cellular activity (13). Zaheer also purified G6PD from

both cytosol and mitochondria (13). Mitochondrial G6PD was found to be distinctly

different from cytosolic G6PD. It had a different optimal pH, Km value, and metal

requirement, but still required G6P and NADP as substrate. Reasons for the presence of

G6PD in the mitochondria and the difference in physical properties were unknown.

Particulate G6PD has also been found in rat liver peroxisomes (11, 12), rat renal cortical cells (58), and rabbit liver microsomes (15). G6PD in peroxisomes was thought to produce NADPH for chain elongation of saturated fatty acids (11). Stanton et a l(58) incubated rat renal cortical cells with epidermal growth factor (EOF) or platelet derived growth factor (PDGF) and noticed a 25% and 27% increase, respectively, in G6PD activity. It was thought that the increase was due to the release of G6PD from a structural element. Since the increase was rapid, protein synthesis was ruled out as the reason for the increase in activity. Ozols (15) isolated G6PD from rabbit liver microsomes and characterized its complete amino acid sequence. The sequence was not the same as that for cytosolic G6PD and the molecular mass was much larger (90 versus

56 IcDa). There were few homologous amino acid sequences. The microsomal G6PD

2 0 was blocked by a proglutamyl residue at the N-terminus and had carbohydrate attached at residue Asn-138 and Asn-263. Treatment of the microsomal enzyme with endo H, which preferentially cleaves high mannose glycoproteins, resulted in a shift in molecular weight when analyzed by SDS-P AGE, confirming the presence of an oligosaccharide and characterizing microsomal G6PD as a glycoprotein.

Overshoot in G6PD Activity

Factors Influencing G6PD Overshoot

As mentioned previously, one of the characteristics of lipogenic enzymes is an increase in activity with carbohydrate refeeding. When animals are fasted then refed a high carbohydrate diet, there is an overshoot in G6PD activity, which could be ten times the fasted level by 72 hours (59-63). Similar overshoots have been found in experiments with acetyl CoA carboxylase (64), fatty acid synthase (65), malic enzyme

(62) and ATP-citrate lyase (64, 66) which all showed an increase in enzyme activity when a high carbohydrate diet was fed. In most experiments, the animals were fasted for at least 48 hours and refed for various times. The peak in G6PD activity occurred between 48 and 72 hours (63).

Age and gender of the animals had differing effects on G6PD activity. Young rats of two months old had a greater response to starvation-refeeding than aged rats (22 months) while middle-aged rats (about 12-18 months) were similar in overshoot activity to young rats (67-69). Female rats had a higher G6PD activity than males when fed a normal chow diet (70) perhaps due to higher levels of estrogen in female rats (71).

21 When fasted and refed a high carbohydrate diet, male rats had a greater response to

refeeding than female rats (70-72) possibly due to the higher G6PD levels in female rats

under normal conditions.

Many theories have been proposed to explain the overshoot in G6PD activity.

Since the high increase in activity occurs when an animal has been fasted and not

merely transferred from a high fat or regular chow diet to the high carbohydrate diet,

Giminez and Johnson (73) pair fed rats to see if the overshoot could be due to increased

food intake after fasting. To mimic the effects of fasting, fed rats were maintained on a

high fat diet then switched to a high carbohydrate diet. Fasted-refed rats were fasted for

48 hours then refed the high carbohydrate diet in amounts equal to that ingested by the

fed group. The investigators found that transferring the rats from fat to sucrose resulted

in only a small increase in G6PD activity while the switch to a high sucrose diet after

fasting had a 10-20-fold increase in activity. Clearly, the overshoot was not due to

increased food intake since both groups of rats ingested the same amounts. There must

be something about the starved state that caused the induction.

The effect of fat in the diet on G6PD activity has also been investigated. Some

experimenters refed a high carbohydrate diet which had no fat (4, 25, 74-76,) while

others used a high carbohydrate diet containing fat (69, 77, 78). The increase in G6PD activity occurred in both groups of rats after a period of fasting. Dror et a l(77) found that when carbohydrate in the diet was replaced by fat, enzyme activity was as low as in fasted rats. Yagil and co-workers (79) performed experiments with mouse liver. They fed rats a 40% carbohydrate/ 25% fat diet and found that when fat was removed, there

2 2 was an overshoot in G6PD activity. They found that this induction would continue

through several fat, no-fat diet cycles (79).

The carbohydrate source seems to have an effect on the overshoot in G6PD

activity. Many experimenters have seen this overshoot with refeeding a high glucose or

fructose diet (4, 63, 64, 74, 80). Fitch et al (80) showed that fructose had a greater

effect on enzyme activity than glucose. G6PD activity has also been shown to increase

when animals were refed a diet high in glycerol (81). Sucrose, however, was shown to

elicit the greatest increase in G6PD activity (61, 75, 82).

When carbohydrate is consumed it triggers a release of insulin. Insulin then

causes a response in many other cells and activates many systems, including lipogenesis

and protein synthesis. For this reason, many investigators have examined insulin's role

in the carbohydrate-mediated increase in G6PD activity. The diabetic animal model is good for studying the effects of insulin administration. In diabetic rats, insulin increased G6PD activity to levels higher than in normal rats and this increase was found to be dose-dependent (83, 84). Fitch et al (85) found that diabetic rats had less of a change in G6PD activity than normal rats when a high glucose or fhictose diet was fed.

When normal rats were fed a chow diet (83) or one with high glucose or fructose (86) and injected with insulin, there was an increase in G6PD activity. Other experiments involved isolated hepatocytes from fed or fasted rats, which were then treated with insulin. Kurtz and Wells (87) discovered that glucocorticoids were needed as well as insulin to cause an increase in G6PD activity in hepatocytes isolated from fed rats.

Others have argued that insulin does not act as a direct inducer of G6PD. Rather it acts

23 by increasing appetite, which increases carbohydrate intake (88), or it has no effect on

induction by itself (63). Rudack et al (76) have argued that insulin does not directly

regulate the level of G6PD in vivo because the amounts required to produce an

overshoot in activity are not of a physiological nature.

G6PD and Protein Synthesis

By far the most popular theory explaining the increase in G6PD activity when

fasted rats are refed a high carbohydrate diet is that protein synthesis increases the

amount of G6PD present in cells. It seems logical that if there is a substantial increase

in activity there must be a large increase in the amount of enzyme to account for that

activity. Another theory is that there is an increase in active enzyme independent of

protein synthesis. The enzyme is present in adequate amounts, but there is some

activator mechanism which causes it to become more active. Although there is much

evidence pointing to increased enzyme synthesis, the activator theory is still plausible.

The first evidence that protein synthesis was required for the overshoot in G6PD activity caused by refeeding carbohydrate was the discovery that protein was needed in the diet to reach maximum inducible G6PD activity (3, 61, 89, 90). When a high carbohydrate/ no protein diet was compared to a high carbohydrate/ low protein diet, the overshoot in G6PD activity was more pronounced in the diet containing protein.

Adequate protein would be necessary for protein synthesis because dietary protein supplies essential amino acids required for building new proteins.

24 About the same time, investigators examined the activator theory. Rat liver

cytosol from induced rats was added to cytosol from non-induced rats to see if G6PD in

non-induced rats could be activated. The rationale was that if an activator was present

in the induced rat and the cytosol was mixed with the non-induced rat's cytosol, the non-

induced G6PD would also be activated. There was no indication that a change in

activity occurred when the two were mixed indicating that the overshoot was not due to the presence of a simple activator molecule (74, 79).

Blockers to protein synthesis were examined to see if they could reduce the induction of G6PD. Potter and Ono (3) showed that the increase in activity expected when rats were refed a high carbohydrate diet can be inhibited by ionizing radiation and puromycin administration in a dose-dependent manner. Another antibiotic, actinomycin

D, is known to block the transcription of DNA to RNA, and when administered blocked the increase in G6PD activity (75). Sassoon et a l(78) found that the activity overshoot was blocked when actinomycin D was administered at the start of refeeding but not when given four hours later. In an experiment with rats trained to eat their meal in a 2 hour time period. Mack et a/ (91) found that cyclohexamide could block the expected increase in G6PD activity completely if administered at the beginning of refeeding or two hours later while actinomycin D blocked it completely if given at zero hours and mostly if given at two hours. These experiments could point to a role of protein synthesis in the overshoot in G6PD activity if mRNA synthesis is triggered shortly after feeding begins.

25 8-Azaguanine can be incorporated into EINA instead of guanine during mRNA

synthesis, which also stops protein synthesis at the translational level. Szepesi and

Freedland (92) were the first to show that 8-azaguanine prevented the overshoot in

G6PD activity but did allow the activity to return to normal fed levels following fasting.

Mack et a l(93) starved rats for seven days, refed them a protein-ffee 90% sucrose diet

for two days, and then administered a 90% protein diet. There was an increase in G6PD

activity 2-3 times that of the fed rats during the high carbohydrate diet intake which was

increased further when the high protein diet was fed, even when 8-azaguanine was given at the start of the protein diet time period. They concluded that the increase in activity with the high sucrose diet paralleled the synthesis of mRNA and did not require protein while the increase in activity on the high protein diet was independent of mRNA synthesis.

Results fi-om experiments using protein synthesis blockers can be conflicting because of the effect these blockers have on food intake and overall health of the animals (74). Puromycin, actinomycin D, and cyclohexamide decrease food intake because they can destroy the intestinal lining (92). The decrease in G6PD induction could be correlated to a decrease in food intake or nutrient absorption. These same substances can be toxic to the animal, so the time of administration of the dose is important when investigating their effects. A better method to analyze the role of protein synthesis is to measure directly the amount of enzyme or the rate of synthesis.

A technique known as immunochemical titration has been used by many investigators (2, 7, 8, 36, 88, 94-96,) to measure the amount of G6PD present in cells.

26 This method is based on antibody binding to G6PD and the complex precipitating. An

antibody to G6PD would be prepared and combined with the appropriate amount of

liver supernatant. After incubation, the samples are centrifuged. A radioactive label could be used to quantitate the antibody/enzyme complex, or the supernatants measured for remaining activity. In most cases, there was a proportional increase in immunochemical precipitation and enzyme activity in induced rats indicating that more enzyme was present to cause the overshoot in activity. This strengthens the argument for increased synthesis of enzyme. Two groups, however, did not find a difference in enzyme quantity between fed and carbohydrate-refed rats. Hizi and Yagil (7) concluded that the change in activity upon refeeding was not due to synthesis of new enzyme in mouse liver. Kelley et al (8) did not find a difference between fed and refed animals in rat liver G6PD content and concluded that an activation-inactivation mechanism must be at work.

The discrepancies in results among the researchers may be attributable to non- specificity of antibody. When antibodies were prepared against impure G6PD, non­ specific antibody binding resulted (29). Dao et al (97) recognized that an antibody may not react with active and inactive forms of G6PD to the same extent. They prepared a monoclonal antibody which could react with palmitoyl CoA-inactivated G6PD. When radioimmunoassay was used to detect the quantity of G6PD, there were comparable amounts in rats that were fasted and refed a high carbohydrate diet and fasted and refed a high fat diet.

27 Winberry and Holten performed experiments to measure protein synthesis using radioactive labels (4). Hepatocytes were isolated from pellet-fed and fat-free, high carbohydrate-fed rats and pulse-labeled with [^H]leucine to measure synthesis directly.

A second method involved isolating polysomes and measuring the binding of '^^I-anti glucose-6-phosphate dehydrogenase to them (4). Both methods indicated there was increased enzyme synthesis in carbohydrate-refed rats.

Once it was widely accepted that the increase in G6PD activity caused by fasting and refeeding a high carbohydrate diet was due to protein synthesis, investigations began to determine what stage of protein synthesis was regulated. It could be at the level of mRNA transcription, mRNA stability, or some post- transcriptional event. Sun and Holten used a rabbit reticulocyte lysate system and isolated RNA to measure the amount of G6PD mRNA (98). They discovered a 2-3-fold increase in the amount of mRNA coding for the enzyme in carbohydrate-refed versus fed rats which they felt was insufficient to account for the 20-fold increase in enzyme synthesis. Therefore they concluded there was some post-transcriptional element working in addition to mRNA synthesis, possibly a change in the efficiency with which the mRNA is translated. In contrast, Miksicek and Towle (5) using a slightly different method measured G6PD mRNA activity and discovered a higher concentration of mRNA in fasted/refed rats indicating an elevated mRNA level leading to increased protein synthesis. At the same time Miksicek and Towle (5) measured mRNA template activity by cell-free translation of poly(A)-containing RNA in the rabbit reticulocyte

28 lysate system and discovered an increase in template activity, which paralleled the

increase in G6PD activity.

Both of these groups used an in vitro measurement of mRNA as an indirect

measurement of mRNA levels. More recent studies to assess the transcriptional role in

protein synthesis involved hybridizing isolated mRNA to cDNA. Kletzien's group (99)

developed a cDNA probe for G6PD and discovered that there is an increase in

hybridizable mRNA in fasted-refed rats compared to normal rats. Ultimately, the

transcription rate of G6PD mRNA was measured and it was found to increase in tandem with mRNA levels and enzyme activity when rats were fed a high carbohydrate diet

(25, 39, 100). It was discovered that transcription rates reached a maximum of three­ fold in six hours while mRNA concentrations reached a maximum of 10 fold in 16 hours. It was felt that the combined increase in transcription rate and mRNA concentration was enough to account for the increased G6PD activity in starved-refed rats.

The next stage in the exploration of the reasons behind the overshoot in G6PD activity involved determining what was the primary inducer of the protein synthesis.

Once again the role of insulin was investigated. Since it is difficult to assess the exact role of hormones in intact animal systems, cell systems of isolated hepatocytes from rats that underwent dietary treatment were used. Insulin or dexamethasone (a synthetic glucocorticoid), and a carbohydrate source of glucose, fructose, or glycerol were added to the culture medium. Insulin alone and dexamethasone alone caused an increase in

G6PD mRNA (38, 101, 102). When both were administered, the results were additive.

29 Interestingly, dexamethasone increased the level of mRNA without causing an increase

in the rate of synthesis of G6PD. This indicated that a combination of hormones may

be necessary for full induction. Other investigators have looked at the role of glucagon

in G6PD induction. Garcia and Holten (94) discovered that glucagon prohibits the

induction of G6PD by a decrease in protein synthesis and cAMP also decreases G6PD

synthesis. Rudack et a lalso discovered that glucagon and cAMP decrease the activity

of G6PD (76) and that this effect could involve inactivation of enzyme or a decrease in

protein synthesis (103).

Other Regulatory Mechanisms for G6PD Induction

Protein synthesis is just one of the many forms of regulation that keeps an organism in homeostasis. Many enzymatic reactions are controlled by the amount of substrate or product available in a system. The amount of substrate limits the operating capacity of an enzyme. Obviously there must be substrate available for the enzyme to act. As the availability of substrate increases, the reaction rate increases until the enzyme becomes saturated and is acting at its maximum velocity (Vmax). The substrate necessary for G6PD is glucose-6-phosphate (G6P) with NADP as a cofactor.

G6P is readily available in a cell under lipogenic conditions as a metabolite of glucose, and it is possible that the increased substrate upon feeding a high carbohydrate diet could account for an increased G6PD activity. This simple hypothesis is not supported

(104). When thyroxine and estrogen were given to animals on a carbohydrate free diet, there was still an elevation in G6PD activity (105). Also, the induction was much

30 greater in rats that were fasted prior to a carbohydrate diet compared to those switched

from a high fat diet or regular chow diet. If the availability of glucose, and

consequently G6P, was the main regulator, there would be an overshoot in enzyme

activity under all conditions where a high carbohydrate diet is fed.

The immediate products of the G6PD reaction are 6-phosphoglucono lactone

and NADPH. 6-Phosphoglucono lactone is immediately changed by non-enzymatic

hydrolysis to 6-phosphogluconate. There is rarely a build-up of either 6-

phosphoglucono lactone or 6-phosphogluconate that could directly inhibit G6PD. The

ratio of NADPH/NADP, however, could have an immediate effect on G6PD activity.

Krebs and Eggleston (104, 106) have found that NADPH inhibits G6PD and believed the enzyme was actually regulated by overcoming this inhibition. Out of over a hundred substances tested in in vitro conditions, only GSSG was found to overcome the inhibition. GSSG can change the NADPH/NADP ratio through the action of glutathione reductase. NADPH is consumed in the reaction and reduced glutathione formed. Ayala et al (6, 107) have also performed experiments with NADPH/NADP ratios and discovered that an increase in consumption of NADPH caused by increased fatty acid synthesis or detoxification reactions was accompanied by an increase in

G6PD activity, but when NADPH was not consumed the enzyme activity levels did not increase. Thus, there is a regulatory role for this product.

Dietary fatty acids have been examined to determine if they play a role in the regulation of G6PD. Dietary fat has been shown to cause an 8-fold decrease in G6PD activity when rats were switched from a no-fat diet to one containing 1 5% fatty acids

31 (88). This decrease was found to coincide with a decreased food intake, however, and these researchers decided that fatty acids do not have a direct effect on G6PD activity or

synthesis (88). Other researchers have found that fatty acid consumption does decrease

G6PD activity (65, 83). Muto (65) gave fatty acids to rats adapted to a high carbohydrate/no fat diet and saw a decrease in G6PD activity. Sassoon et a l(63) found no evidence that fatty acids act as a feedback inhibitor. A direct role of fatty acids as an inhibitor of G6PD in the liver has not been established.

A common regulatory mechanism for enzyme regulation is that of covalent modification. An enzyme could be altered in such a way that it becomes more or less active. For example, acetyl CoA carboxylase undergoes phosphorylation/ dephosphorylation cycles, which alter its activity. Since acetyl CoA carboxylase and

G6PD are both lipogenic enzymes, G6PD has been investigated to see if undergoes some form of covalent modification. Thus far only one group has found that G6PD can be phosphorylated but only on tyrosine residues (108) and not serine/threonine residues as are other lipogenic enzymes (109). The researchers stated that this reaction was carried out in vitro and there is no evidence that this phosphorylation occurs physiologically. As this is the only mention of phosphorylation of G6PD, it seems unlikely that the increased activity observed upon refeeding a high carbohydrate diet is caused by this type of covalent modification.

Compartmentation of enzymes is another form of regulation. Some enzymes are active only in certain cells or cellular compartments. Since they are separated from another compartment by a membrane, their action is limited to the environment in

32 which they are present. For example, the reactions to carry out fatty acid biosynthesis take place in the cytosol while those for fatty acid oxidation occur in the mitochondria.

Although G6PD is present in all cell types, it has typically been found only in the cytosol of cells. Since the reaction catalyzed by G6PD occurs in the cytosol, it is logical that is where it is predominantly found. However, G6PD has also been found in the particulate fraction of some cells. The reason for its presence there has not been elucidated, but perhaps there is some form of regulatory mechanism that requires its presence in a particulate form.

Another type of enzyme regulation related to compartmentation is that of ambiguity. The subcellular distribution of an ambiquitous enzyme depends upon the physiological state of the organism. The enzyme can exist in more than one distinct intracellular location depending on the metabolic state of the tissue. The most characterized ambiquitous enzyme is hexokinase. Normally hexokinase is found in the cytosol where it metabolizes glucose, but many researchers have found that hexokinase is also membrane-bound on the mitochondria or microsomes (110-115). It has been shown by digitonin fractionation that hexokinase is associated with the outer mitochondrial membrane in rat kidney (111) and rat brain (112). In rat liver hepatocytes, it was shown that glucokinase (hexokinase IV) could be rapidly released from its bound form by incubation with glucose in the presence of insulin (113).

Hexokinase's mitochondrial location gives it preferred access to ATP generated by oxidative phosphorylation. When the cell needs to metabolize large amounts of glucose.

33 hexokinase is released to move closer to the cell membrane to catch glucose as it enters

the cell.

Although many of the regulatory mechanisms of G6PD have been investigated,

the subcellular distribution and possible involvement of a giycan moiety have not been

thoroughly examined. G6PD possesses many of the same characteristics as other

lipogenic enzymes in that activity is dependent on the metabolic state of the organism

and can change with dietary manipulations. Acetyl CoA carboxylase, a rate limiting

enzyme for fatty acid biosynthesis has been shown to be a glycoprotein with an

association with the outer mitochondrial membrane. This association has been shown to change in response to changes in diet and the presence of insulin. This release of

enzyme and conversion to an active form is a form of short-term regulation compared to long term regulation such as protein synthesis. Although many investigators have reported that the overshoot in G6PD activity upon refeeding a high carbohydrate diet to previously fasted animals is due to protein synthesis, other studies suggest that there are additional short-term regulatory mechanisms of this enzyme.

34 CHAPTER 2

METHODS AND MATERIALS

Bakers Yeast Studies

Separation of Cvtosol and Mitochondria

Type n Baker's yeast {Saccharomyces cerevisiae) was used to detect the presence of G6PD in mitochondria (116). Six grams of yeast were placed in 60 ml of 3- mercaptoethanol buffer (2GmM Tris-Cl, 25mM EDTA, 0.2M 3-mercaptoethanol, pH

8.0) and incubated at 30°C for 30 minutes. The yeast mixture was poured into centrifuge tubes and centrifuged at 3000 g for 10 minutes. The supernatant was discarded and the pellet washed with 60 ml 0.6M mannitol/20mM Tris-Cl, pH 7.5. The yeast was centrifuged again at 3000 g and the supernatant discarded. The pellet was suspended in 60 ml of the 0.6M mannitol buffer and the mixture equilibrated at 30°C.

Lyticase (10,OOOU) and 0.2mM PMSF were added and the yeast incubated at 30°C for

90 minutes to help degrade the outer cell membrane. The yeast cells were centrifuged at 3000 g for 10 minutes, the supernatant discarded, and the pellet washed in 60 ml of

0.3M mannitol. The centrifuge and wash steps were repeated. The supernatant was again discarded and the pellet homogenized with a Sorvall Omnimixer at high speed for

15 seconds in 60 ml of 0.3M mannitol. The homogenate was centrifuged at 700g for 20

35 minutes. The supernatant was preserved and centrifuged at 27K g for 20 minutes. The

supernatant was carefully decanted and saved (mitochondrial-free supernatant). The pellet was resuspended in 30 ml of 0.3M mannitol, centrifuged as above, and the supernatant discarded. The pellet (mitochondria) was resuspended in 5 ml of 0.3M mannitol. The cytosol and mitochondria were assayed for protein and G6PD activity and boiled in SDS-mix for polyacrylamide gel electrophoresis (PAGE).

Separation of Yeast Outer Mitochondrial Membrane and Treatment with

Phosphatidvlinositol Specific Phospholipase C

The mitochondria were separated from yeast cells as described and the final pellet resuspended in 4 ml of 0.3M mannitol. The mitochondria were assayed for protein. Recrystallized digitonin was used to separate the outer mitochondrial membrane from the mitochondria (21, 55). Digitonin was weighed at a concentration of

0.6mg digitonin/mg protein and placed in 0.3M mannitol buffer. It was dissolved by heating then cooled. The mitochondria and digitonin were mixed 1:1 to achieve the above concentration and incubated on ice for 20 minutes. The mitochondria were evenly distributed in hard-sided centrifuge tubes and centrifuged in an ultracentrifuge at

4°C at 40K g for 30 minutes. The supernatants were poured into clean tubes and the pellets discarded. The tubes were centrifuged at 4°C at 160K g for 60 minutes. The supernatant was saved and the pellets suspended in 2 ml total volume 0.3M mannitol.

The pellet contained the outer mitochondrial membrane. Both fractions were assayed for G6PD activity. The outer mitochondrial membrane had 1 mg/ml BSA added to

36 preserve enzyme activity. For phosphatidylinositol specific phospholipase c treatment,

500|ii of pellet, SOjiI of 20% non-idet P-450 detergent, and lU of phosphatidylinositol

specific phospholipase c were incubated at 37°C for 60 minutes (117). A control was

also incubated that had water substituted for the phospholipase c. After incubation, the

samples were centrifuged at 15Kgat 4°C for 10 minutes. The supernatant was saved

and the pellet suspended in 570pi of 0.3M mannitol with 0.2% non-idet. The fractions

were assayed for G6PD activity and boiled in SDS-mix for quantitative analysis.

Protein Determination

Protein was determined by the procedure of Bradford (118), which utilizes the

concept of protein-dye binding. Bovine serum albumin (BSA) was used to produce a

standard curve. The samples were read on a Coleman Junior n, model 6120

spectrophotometer at 595nm.

Preparation of SDS-Boiled Samples

Samples to be used for denaturing electrophoresis and dot-blot analysis were

denatured by boiling in a sodium dodecyl sulfate (SDS) solution composed of 3.3%

SDS and 11% sucrose with bromophenol blue as indicator (21). SDS mix was heated in

a boiling water bath and 1-2 equal volumes of sample rapidly injected into boiling SDS mix. The solution was heated for 4-6 minutes. Samples were either used immediately for electrophoresis or frozen for later analysis.

37 Polyacrylamide Gel Electrophoresis

Denaturing Gel

The denatured enzyme was subjected to SDS-PAGE analysis using a 3% (w/v) acrylamide stacking gel and a 12.5% (w/v) acrylamide separating gel. The gel compositions are shown in Table 2.1. Protein standards and purified G6PD were loaded onto the same gel. The gel was run at a current of 7.5 milliamps in buffer containing

0.4M glycine, 50mM Tris, and 1% (w/v) SDS until the gel front reached one inch from the bottom of the glass plates (119). Gels were removed and placed in a solution of

Towbin’s Buffer (25mM Tris-Cl, 192mM glycine, pH 8.8, diluted 4:1 with methanol)

(120). For protein staining, individual lanes were cut from the gel and stained in a solution of 0.6% Coomassie blue R-250, 50% methanol, and 10% acetic acid.

Non-Denaturing Gel

The non-denaturing gel was 8% (w/v) polyacrylamide with no SDS and no stacking gel (Table 2.2). Samples were prepared by suspending the enzyme in a sucrose solution with bromophenol blue as a tracking dye. The cathode buffer had 10 mM

NADP added. The gel was run at a current of 3 milliamps overnight (119). A portion of the gel was stained for protein and another portion was stained for activity. Proteins in the remainder of the gel were transferred by Western Blot onto nitrocellulose (120).

38 12.5% Separating Gel 3% Stacking Gel 30% acrylamide/0.8% BIS 6.7 ml 2.0 ml 30% acrylamide (no BIS) 10 ml — 1.5M Tris-Cl, pH 8.7 10 ml —— 1.5M Tris-Cl, pH 86.8 — 2.0 ml distilled water 13 ml 15.5 ml 20% SDS 200 til 100 til 13% ammonium persulfate 100 nl 100 til TEMED 20 til 10 til

Table 2.1 Composition of Denaturing Polyacrylamide Gels

8% Separating Gel 30% acrylamide/0.8% BIS 6.7 ml 30% acrylamide (no BIS) 3.9 ml 1.5M Tris-Cl, pH 8.7 10 ml distilled water 19.1 ml 13% ammonium persulfate 100 til TEMED 20 til

Table 2.2 Composition of Non-denaturing Polyacrylamide Gels

39 Western Blot

After PAGE, samples were transferred electrophoretically onto nitrocellolose membrane (120). The gel was placed on top of the nitrocellulose and sandwiched between two pieces of filter paper. The transfer was completed in cold Towbin’s Buffer at 0.5 amps in 3 hours. The nitrocellulose was removed and completely dried before further treatment was performed.

Antibody Detection of G6PD

The nitrocellulose membrane containing G6PD was blocked with a 5% solution of non-fat dried milk solids dissolved in IMB (Immunoblot buffer: lOmM Tris Cl, 0.15

M NaCl, pH 7.6, 0.2% Triton X-100) for one hour. The nitrocellulose was washed three times, 10 minutes each with IMB and then incubated with anti-G6PD antibody in

IMB for one hour (121). After washing, the nitrocellulose was incubated with Protein

G-peroxidase in IMB for one hour. The nitrocellulose was washed thoroughly with

IMB and the peroxidase detected with ECL reagents and exposure to X-ray film.

Manufacturer's (Amersham) instructions were followed for the use of ECL detection reagents. Briefly, Solution 1 and Solution 2 were mixed in equal amounts to give 0.125 ml ECL reagents per cm^ of membrane. The membrane was immersed in the detection reagents for one minute, wrapped in plastic wrap, and exposed to X-ray film for various short periods of time. The film was then developed.

40 Quantitative Determination of G6PD

Yeast samples were analyzed for G6PD quantity by separating the cytosol from

the mitochondria and boiling the samples in SDS-mix. Four different amounts of each

fraction were added to wells of a 12.5% SDS-PAGE gel with a 3% stacking gel along

with a G6PD standard at different concentrations. The proteins were then transferred to

nitrocellulose via Western Blotting. Rat liver samples were transferred to nitrocellulose

using a BIO-RAD Bio-Dot® Microfiltration apparatus. The mitochondrial-free

supernatant and mitochondria were separated and boiled in SDS-mix. Nitrocellulose

was wetted and placed in the apparatus which was then prepared according to the

manufacturer's instructions. Samples and standards of varying concentrations were

added to individual wells and allowed to adhere to the nitrocellulose by gravity

filtration. After rinsing with IMB and drying by vacuum filtration, the nitrocellulose

with proteins was removed from the apparatus and allowed to dry completely. G6PD

from both yeast and rat liver samples was detected using the anti-G6PD antibody

procedure.

Each gel lane or dot blot was scanned using an LKB 2222-020 Ultrascan XL

Laser Densitometer and the peak area recorded. A sample readout from the densitometer for rat liver is shown in Figure 2.1. Four distinct bands can be seen corresponding to four different wells with increasing known amounts of sample. The

G6PD standard readouts for rat liver were similar. The yeast samples and G6PD standards were added to individual wells of a gel, and their readouts would have one peak for each gel lane. For the G6PD standards, a graph was made of the peak area in

41 i.n

I.M

i.st

I.JIT I.Z T t

T'Xiltie# (n) ♦ T-JUrt : iS : 74 f-iU» : 1/2 ( : 29 lierou 1

Figure 2.1 Densitometer Readout from Rat Liver Quantitative Analysis

42 O.D. units V . the amount of G6PD in the well or blot and the linear slope of the curve calculated. A sample of a yeast standard curve is shown in Figure 2.2, and a rat liver standard curve in Figure 2.3. For the samples, the peak area in O.D units was plotted v. the amount of protein in the sample and the slope calculated. The relative amount of

G6PD/amount protein was determined by dividing the slopes:

O.D units divided by O.D. units = ng G6PD/mg protein mg protein ng G6PD

A similar analysis can be performed for activity by graphing the activity of G6PD v.

O.D. units for each sample and looking at the slopes as a comparison of activity and relative quantity.

G6PD Activity Stain of Non-denaturing Gel

After removing the gel from the electrophoresis unit, lanes were cut out and placed in 50mM Tris-Cl, pH 7.4 before being stained for G6PD activity. The gel was placed in an activity stain consisting of the Tris buffer with O.SmM glucose-6- phosphate, 0.2mMNADP, 0.1 mM magnesium acetate, 0.2mg/ml nitroblue tétrazolium, and 0. Img/ml phenazinemethylsulfate (122). The gel was stained for less than 5 minutes and destained in distilled water.

43 6.0

5.0

4.0 3 Q d 3.0

2.0

1.0 10 20 30 40 50 60 70

G6PD (ng)

Figure 2.2 Standard Curve for Yeast Studies

44 3.5

3.0

2.5 c 3 Q d 2.0

5

1.0 0.60 0.96 1.32 1.68 2.04 2.40

G6PD (ng)

Figure 2.3 Standard Curve for Rat Liver Studies

45 Giycan Chain Detection of G6PD

After electrophoresis and Western blot transfer, gels containing G6PD were

probed for carbohydrate using a periodate-digoxigenin anti-digoxigenin kit from

Boehringer Mannheim (47). The manufacturer's directions for Method B were followed

with minor modifications. Specifically, after proteins were transferred to the

nitrocellulose, the blots were washed in PBS (phosphate buffered saline—50mM

BCH2PO4, 150mM NaCl, pH 6.5) for 15 minutes. All subsequent steps in the giycan

chain analysis up to the detection step were performed in buffer which contained 0.2%

Triton X-100. Carbohydrate on the nitrocellulose was oxidized with lOmM sodium

meta-periodate in lOOmM sodium acetate buffer pH 5.5 for 20 minutes. After the

nitrocellulose was washed three times for 10 minutes each with PBS, it was incubated

with digoxigenin succinyl-e-amidocaproic acid hydrazide in lOOmM sodium acetate

buffer pH 5.5 (1:5000) for one hour. After this and all the following incubation steps, the nitrocellulose was washed in TBS (Tris buffered saline—0.05M Tris Cl, 0.15M

NaCl, pH 7.5) three times for 10 minutes each. The nitrocellulose was blocked with giycan chain blocking reagent for one hour. It was incubated with anti-digoxigenin peroxidase FAB fragments in TBS (1:2000) for one hour. The nitrocellulose was incubated with peroxidase labeled anti-peroxidase in TBS (1:2000) for one hour to amplify the signal. The nitrocellulose was incubated with Protein G-peroxidase in TBS

(1:4000) for one hour to further amplify. Finally, the nitrocellulose was washed thoroughly with TBS before the peroxidase was detected with ECL detection reagents and exposed to X-ray film as described previously.

46 Enzyme Assays

G6PD activity was measured according to Rudack el a l(76). The reaction

mixture consisted of 0.12M Tris-CI, pH 8.0 with 2mM giucose-6-phosphate, 0.9mM

NADP, and l0.4mM magnesium acetate. The cuvette had a volume of 0.7 ml and the

reaction was started by the addition of enzyme solution. The activity was measured by

following NADPH production at 340nm in a Perkin Elmer Lambda 4B UV/VIS

spectrophotometer. Activity was calculated from the slope and expressed as mU/ml enzyme where one unit (U) of enzyme produces Ipmol ofNADPH/min. All mitochondrial preparations had 0.2% non-idet P-450 detergent added.

6-Phosphogluconate dehydrogenase (6PGD) activity followed the procedure of

Dror et a l(123). The reaction mix contained 0.12M Tris-Cl, pH 8.0, 1.67mM 6- phosphogluconate, 1.7mM NADP, and 3.4mM magnesium acetate. The assay proceeded as the G6PD reaction outlined above.

Monoamine oxidase (MAO) activity was assayed by the procedure of Tabor et al. (124). The enzyme solution was added to a cuvet containing 0.9 ml of 200mM

KH2PO4, pH 7.2 and lOpM benzylamine. The reaction was monitored at 250nm in the

Perkin Elmer spectrophotometer.

Malate dehydrogenase (MDH) activity was measured by the procedure of Ochoa

(125). A pre-mix consisting of 0.25M Tris-Cl, pH 7.4, 1.5mM NADH, 7.6mM oxaloacetate, and water was prepared. Enzyme solution was added to 0.7 ml of pre-mix and the production of NAD followed at 340nm. Activity was calculated from the slope of the line.

47 Adenylate kinase (ADK) activity was measured according to Schnaitman and

Greenawalt (28). The reaction mix contained 70mM Tris-Cl, pH 8.0, 0.75 mM NADP,

15mM glucose, 10 lU hexokinase, 0.4 lU G6PD, 0.45mM KCN, 3mM ADP, and SmM

magnesium acetate. The reaction mix was allowed to sit for 5 minutes to digest all

AMP. The enzyme solution was added to 0.9 ml of the mix and the production of

NADPH followed at 340nm.

Cytochrome c oxidase was measured according to Poyton (126). The buffer

system was 40mM KH2PO4 with 0.5% TWEEN, pH 6.7. In 25 ml of buffer, 20mg of

cytochrome c and 2mg dithionite were dissolved. The enzyme was added to 1 ml of

buffer solution and the reaction monitored at 550nm.

Animal Studies

Animal Care

Male, Sprague Dawley rats weighing between 150 and 200 grams were housed in pairs in galvanized, wire mesh bottomed cages. The procedures were approved by

ILACUC. All rats had ad libitum access to a high carbohydrate diet and water for at least one week prior to the beginning of an experiment (22). The composition of the diet is described in Table 2.3. The animals were maintained on a twelve hour light/dark cycle with the light cycle being between 7pm and 7am. Food was removed from fasted rats at 7am for a period of 48 hours. For refed rats, food was removed for 48 hours and rats were refed the high carbohydrate diet beginning at 7am and continuing for the

48 Ingredient ______kg______% dry weight Dextrose 580 58 Casein 210 21 Cellufil 130 13 Vitamins' 20 2 Minerals** 40 4 Com Oil 20 2 a. Composition of the vitamin mix supplement titurated in dextrose (g/kg) : a - tocopherol (lOOIU/g) 5.0, L-ascorbic acid 45.0, choline chloride 75.0, d-calcium pantothenate 3.0, inositol 5.0, menadione 2.25, niacin 4.5, PABA (para- aminobenzoic acid) 5.0, pyridoxine HCl 1.0, riboflavin 1.0, thiamine HCl 1.0, vitamin A acetate 900.000 U/kg, calciferol (D2) 100.000 U/kg, biotin 20 mg/kg, folic acid 90 mg/kg, vitamin B12 1.35 mg/kg. b. Mineral mix US? XTV contains (g/kg); ammonium 0.57, cupric sulfate 0.48, ferric ammonium citrate 94.33, manganese sulfate 1.24, potassium iodide 0.25, sodium fluoride 3.13, calcium carbonate 68.6, calcium citrate 308.3, calcium biphosphate 112.8, magnesium carbonate 35.2, magnesium sulfate 38.3, potassium chloride 124.7, dibasic potassium phosphate 218.8, sodium chloride 77.1.

Table 2.3 Composition of Diet

49 period of time specified in each experiment. Fasted rats were housed singly with free

access to water for the duration of their fast.

Isolation of Livers

Rats were weighed prior to killing. In all experiments, fed rats were taken at the

same time as the experimental fasted or refed rats. Rats were killed by cervical

dislocation following a blow to the head. Livers were quickly removed and placed on

ice (22). Livers were weighed and 0.3M mannitol added at two times the liver weight.

Homogenization was performed with four up and down strokes of a Potter-Elvehjem

homogenizer at full speed.

Separation of Cvtosol and Mitochondria

For the separation of mitochondria, 6 ml of homogenate from each liver was

placed in a chilled centrifuge tube along with 18 ml of 0.3M mannitol (20). The tubes

were centrifuged at 700 g for 20 minutes and the supernatants poured into clean tubes.

The tubes were centrifuged at 7800 g for 20 minutes. The supernatant was discarded

and the pellet resuspended in 16 the original volume of 0.3M mannitol. The suspension

was centrifuged at 7800 g for 20 minutes. The supernatant was discarded and the pellet

resuspended in 5 ml of 0.3M mannitol. The centrifugation was repeated and the pellet

resuspended in I ml of 0.3M mannitol. The mitochondria were then assayed for protein and various enzyme activities.

50 The separation of mitochondrial-free supernatant which contained cytosol and

microsomes, was performed from the original homogenate (20). Volumes were

equalized in chilled centrifuge tubes and centrifiiged at 27,000 g for 20 minutes.

Cheesecloth was rinsed with distilled, deionized water, chilled, and placed in a filter

funnel. The supernatants from the centrifugation were poured through the cheesecloth

into individual containers kept on ice. The mitochondrial-free supernatant was analyzed

for protein and various enzyme activities.

Separation of Rat Liver Mitochondrial Fractions (Digitonin Fractionation)

The separation of outer and inner mitochondrial membranes from rat liver was performed according to Greenawalt (56) as modified by Allred and Roman-Lopez (21).

Livers were extracted from fed rats and homogenized in two times their volume of

Greenawalt's buffer (70mM sucrose, 220mM mannitol, 2mM HEPES, pH 7.4, with

O.Smg/ml BSA added just prior to use). Three more volumes of Greenawalt's buffer

(GW) were added and the homogenate poured into chilled centrifuge tubes and centrifuged at 700 g for 10 minutes. The pellet was discarded and the supernatant spun again. The pellet was discarded and the supernatant poured into clean, chilled tubes.

The cell contents were centrifuged at 7800 g for 20 minutes to separate the mitochondria. The supernatant was discarded and the pellets resuspended in 16 the original volume of GW buffer. The tubes were centrifuged at 7800 g for 20 minutes and the supernatant discarded. The pellet was resuspended in Vi the original volume of

GW buffer. The process was repeated and the pellet was resuspended in a final volume

51 of 4 ml GW buffer. The protein content of the mitochondria was determined. Digitonin was added to centrifuge tubes containing mitochondria at the following concentrations:

0, 0.05, 0.1, 0.2, 0.4, and 0.6 mg digitonin/mg mitochondrial protein. The tubes were incubated on ice for 10 minutes and centrifuged at 15Kgfbr 10 minutes. The supernatants from this spin were assayed for G6PD, adenylate kinase, malate dehydrogenase, cytochrome c oxidase, and monoamine oxidase activities to determine the location of G6PD in the mitochondria.

Separation of Outer Mitochondrial Membrane in Rat Liver

Solutions of protease inhibitors were prepared as in Table 2.4. 0.5 ml of each solution was added to 0.5 L Greenawalt's buffer (127). Livers were extracted from fed rats and homogenized in two times the volume of Greenawalt's buffer with the protease inhibitors. Two more volumes of the buffer were added and the solution poured into centrifuge tubes. The procedure was then followed as in the digitonin fractionation procedure with the final 7800 g pellet suspended in 3 ml of GW buffer with protease inhibitors. The protein content was determined and the digitonin treatment performed with 0.2mg digitonin/mg mitochondrial protein. After incubation, the mixture was centrifuged at 27K g for 20 minutes. The pellet was discarded and the supernatant poured into small, hard-sided centrifuge tubes which were centrifuged at 160Kg for one hour. The supernatant was saved and the pellet washed by rinsing four times with GW buffer with no BSA. The pellet was suspended in 2 ml of GW buffer and assayed for

G6PD activity along with the supernatant from the final spin.

52 mg volume

Soin A 1 ml ethanol PMSF 17.33 TLCK 3.6 TPCK 3.6

Soin B benzamidine 156.7 1 ml water

Soin C NaNs 100 1 ml water

Soin D 1 ml water pepstatin I.O leupeptin 1.0 trypsin inhibitor 1.0

Table 2.4 Protease Inhibitors

53 Glycogen Determination

Glycogen content was measured in frozen samples of rat liver mitochondrial-

free supernatant by the method of Roehrig and Allred (128). The mitochondrial-free

supernatant was treated with aminoglucosidase to degrade the glycogen to glucose.

Aminoglucosidase was dissolved in 50mM acetate buffer, pH 4.5 to a concentration of

~4U aminoglucosidase per assay. The reaction mixture consisted of IOO|il

aminoglucosidase solution, 400 |il water, and 10 |il of the mitochondrial-free

supernatant. The mixture was incubated at 55°C for 10 minutes. A glucose

oxidase/peroxidase capsule was dissolved in 100 ml water and 0.8 ml of o-dianisidine

solution was added. lOOjjJ of the incubated samples were added to 2 ml of glucose oxidase reagent. Samples were incubated at 37°C for 30 minutes and the absorbance read at 500nm on a Coleman spectrophotometer. A standard curve was generated using purified glucose and the amount of glucose in the samples calculated from the curve.

Enzyme Kinetic Study of Rat Liver G6PD

Mitochondrial-free supernatant was isolated from livers of fed rats (20). An enzyme activity pre-mix was made consisting of 0.12M TRIS-Cl, pH 8.0, 0.9 M NADP, and 10.4mM magnesium acetate. A 2mM G6P in TRIS-Cl solution was made and various volumes added to the pre-mix in a cuvet. TRIS was used to make the total G6P volume equal 0.1 ml. The actual G6P concentrations varied from 0 to 200^M. A 20 (il aliquot of the mitochondrial-free supernatant was added to the cuvet, mixed, and assayed for G6PD activity. The activities and G6P concentrations were used to 54 construct velocity curves and Lineweaver-Burk plots to calculate the Vmax and Km of

G6PD in rat liver.

Materials

Enzymes, substrates, and reagents were purchased from Sigma Chemical

Company, St. Louis, MO with the exception of the following: sodium dodecyl sulfate,

bromophenol blue, pre-stained protein standards, biotinylated standards, glycine, acrylamide, ammonium persulfate, TEMED, nitrocellulose membranes, and Coomassie blue were obtained from BIO-RAD, Hercules, CA ECL detection reagents were purchased from Amersham, Arlington Heights, IL. The components of the giycan detection kit, sodium meta periodate, digoxigenin succinyl-e-amidocaproic acid hydrazide, and anti-digoxigenin POD FAB fragments were obtained from Boehringer

Mannhein Biochemicals, Indianapolis, IN. Kodak XAR-2 X-ray film and developing reagents were purchased from E.G. Baldwin and Associates, Hilliard, OH. The components of the rat diet were obtained from U.S. Biomedical Corp., Cleveland, OH.

Statistics

A one-way analysis of variance (ANOVA) was performed to determine overall differences in some experiments. Differences between individual means were determined using Tukey's pair-wise comparisons. Paired t-tests were performed to determine differences in some cases. In all types of comparisons, a p-value of 0.05 was used to define differences. Statistics were done using the Minitab statistical package.

55 CHAPTERS

RESULTS AND DISCUSSION

Yeast Studies

The first of these experiments used Type II Baker's yeast, Saccaromyces cerevisiae, to determine the distribution of glucose-6-phosphate dehydrogenase between the cytosol and mitochondria. Yeast cells were homogenized as described in the methods section and the cytosol and mitochondrial fi’actions separated. Both subcellular fractions were assayed for G6PD activity and subjected to SDS-PAGE to quantitate the amount of G6PD. Table 3.1 depicts the specific activity of G6PD in yeast cells while Table 3.2 shows the quantity of G6PD in cytosol and mitochondria. The data show that a considerable amount of the G6PD activity is localized in the mitochondria although the cytosol has more than twice the specific activity as the mitochondria. The cytosol also has 2.7 times the quantity/mg protein than the mitochondria. These comparisons can easily be seen in Figure 3.1. Since the specific activity and the quantity of G6PD in yeast fractions have been measured, the activity of

G6PD per quantity could be determined for each fraction. The cytosol had 5.37 ± 0.45 mU G6PD/ug G6PD while the mitochondria had 1.20 ± 0.11 mU G6PD/ug G6PD.

Thus, the cytosolic G6PD appears to be about 4.5 times more active than the mitochondrial form. 56 cyto mito cyto/mito experiment mU/mg prot mU/mg prot 1 39.33 17.98 2.19 2 59.15 22.42 2.64 3 81.25 33.15 2.45 4 * 29.11 * 5 46.36 18.36 2.53 6 47.93 21.37 2.24

mean 54.80 23.73 2.41 SEM 7.3 2.5 0.1

" denotes missing data SEM is the standard error o f the mean

Table 3.1 Specific Activity of G6PD in Yeast

cyto mito cyto/mito experiment ug/mg prot ug/mg prot 1 2.821 1.005 2.808 2 3.422 1.213 2.821 3 0.573 0.215 2.671 4 0.526 0.316 1.664 5 1.139 0.306 3.721

mean 1.696 0.611 2.737 SEM 0.60 0.21 0.33

SEM is the standard error o f the mean

Table 3.2 Quantity of G6PD in Yeast

57 70 5.0

ok_ Q. 60 2 - 4.0 a . 15 o 50 « o O) - 3.0 E 40 CO 3 E 03 E 30 3 - 2.0 5k > 20 C Ü - 1.0 cd < 10 3 a o CO 0 0.0 Cyto Mito

Activity %%%) Quantity

Figure 3.1 Activity and Quantity of Yeast G6PD

58 Stability of Yeast G6PD

It is known that G6PD loses its in vitro activity over time. Since the quantitation procedure required that the G6PD be handled over a period of several hours or days and previous researchers have shown that some antibodies do not react with inactive enzyme (97) it was necessary to determine whether the enzyme loses its antibody reactivity as well. To assess the effect of time on G6PD activity and antibody reactivity, a time course study was performed. Purified yeast G6PD was prepared in

0.3M mannitol and incubated at 37 °C. At zero time and every ten minutes an aliquot was removed. Part was assayed for G6PD activity and part spotted onto nitrocellulose using the dot blot apparatus. The quantitation procedure was then performed to assess antibody reactivity. The results are shown in Figure 3.2. G6PD rapidly lost its activity upon heating and by 60 minutes was unable to form product. The antibody reactivity, however, remained relatively stable over time. Measuring G6PD activity must be performed quickly after isolation of cell fractions, but storage of the enzyme should not be detrimental to quantitative analysis using the yeast anti-G6PD antibody.

Glvcoprotein Analvsis of Purified Yeast G6PD

Since it was shown that G6PD has a particulate form, it was examined to determine whether it was also a glycoprotein. The first step was to measure the molecular weight of yeast G6PD. This was done by performing SDS-PAGE as described with purified yeast G6PD and biotinylated protein standards of known molecular weight. After transferring the proteins to nitrocellulose, the standards were

59 0.12 10.0

0.10 8.0

0.08 3 Quantity, a E c 0.06 3 > d o < 4.0 0.04

2.0 0.02 Activity

0.00 0.0 0 10 20 30 40 50 60 70 80

Time (mln)

Figure 3.2 Stability of Yeast G6PD

60 detected by incubation with streptavidin-peroxidase followed by ECL treatment. The

subunit molecular mass of G6PD was calculated to be 56kDa (Figure 3.3). This is the

reported molecular weight of the enzyme from a variety of microbial and mammalian

sources (15, 36, 79) and is close to the molecular weight of 57,229 calculated from the

amino acid sequence of Baker's yeast G6PD (26, 27).

The results of the glycoprotein detection for purified yeast G6PD are in Figure

3.4. Panel A shows lanes that were cut from a non-denaturing gel or from lanes

transferred to nitrocellulose from the same gel. Lane I shows one major protein band

detected by Coomassie Blue stain. Lane 2 is a G6PD activity stain that corresponds

with the anti-G6PD antibody reaction in Lane 3. Lanes 2 and 3 do show an additional

band with a much slower mobility that could be due to aggregation of molecules. Lane

4 contains proof that G6PD is a glycoprotein as shown by probing the nitrocellulose for

carbohydrate using the highly specific periodate-digoxigenin glycan detection method that labels carbohydrate. The glycoprotein stain matches the antibody reactivity and activity stain migrations. Panel B shows results from a denaturing gel followed by

Western Blot. Lane I was stained with Ponceau S which detects proteins. Lane 2 is the antibody stain and had one major band with another band of slightly faster mobility.

Lane 3 is the result of the carbohydrate detection and matches the mobility of the antibody stain. These results show that the same protein that has G6PD antibody reactivity is a glycoprotein.

61 100000 -

ta I T ry . irfL»

10000 0-00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

Relative Distance

Figure 3.3 Molecular Weight Determination of Yeast G6PD

62 B 12 3

Lane 1—Coomassie blue protein stain Lane I—Ponceau S protein stain Lane 2—G6PD activity stain Lane 2—G6PD antibody stain Lane 3—G6PD antibody stain Lane 3—Carbohydrate stain Lane 4—Carbohydrate stain

Figure 3 4 Glycoprotein Detection of Yeast G6PD

63 Figure 3.5 shows the results of a denaturing gel and Western Blot of partially

purified rat liver G6PD. Lane I was stained with Ponceau S and shows a double band

of protein. Lane 2 was reacted with anti-G6PD antibody and contains one big band.

Lane 3 is the glycoprotein detection which also exhibited double banding. Since the

antibody lane only showed one band, it is presumed that the preparation was

contaminated with another glycoprotein. As with the yeast results, this experiment

shows that rat liver G6PD is a glycoprotein. Thus, cytosolic G6PD as well as

microsomal G6PD fi-om rat liver (15) has carbohydrate attached.

Preliminary studies were performed to characterize the glycan nature of yeast

G6PD. Purified yeast G6PD was added to a Con A sepharose column, but did not bind to the column. This indicated that the glycan chain has a different carbohydrate structure which must not bind to the Con A column and perhaps is not N-linked. (44).

Treatment of G6PD with N-glycosidase F [B.C. 3.2.2.18] under the same conditions which removed the glycan chain from transferrin, failed to remove it from G6PD. Since

N-glycosidase F catalyzes the hydrolysis of asparagine-1 inked carbohydrates with no regard to glycan specificity (129), it appears that the carbohydrate is not linked to an asparagine residue on G6PD.

There is one particular class of glycoproteins that does not link the glycan chain to the protein at an asparagine residue. Proteins that have GPI anchors have part of their carboxyl terminal sequence cleaved and the pre-formed glycan chain is attached to the newly exposed a-carboxyl group (50). There is no specific amino acid requirement known for this addition. One characteristic of GPI-anchored proteins is a hydrophobic

64 F f

Lane I—Ponceau S protein stain Lane 2—G6PD antibody stain Lane 3—Carbohydrate stain

Figure 3.5 Glycoprotein Detection of Rat Liver G6PD 65 C-terminal region with hydrophilic residues interspersed in the sequence. Sequencing has shown that rat liver microsomal (15) and yeast cytosolic (27) G6PD both have a hydrophobic C-terminal region, but the sequencing was performed on isolated protein which would already have been processed and the C-terminal residues removed.

Nonetheless, there is enough evidence to investigate whether G6PD is a GPI-anchored protein.

Treatment of Yeast Mitochondria with Phosphatidyl Inositol Specific Phospholipase C

Once it was ascertained that yeast G6PD is present in the mitochondria and contained carbohydrate, it was speculated that it might be associated with the mitochondrial membrane. Isolated yeast outer mitochondrial membrane was treated with phosphatidyl inositol specific phospholipase c (PI-PLC) as described in the methods section to determine if it could be released from the membrane. After treatment, if the G6PD were cleaved, it should appear in the supernatant since it would no longer be associated with a particle. The results are shown in Figure 3.6. Both the activity and quantity of G6PD in the supernatant of PI-PLC treated mitochondrial membrane was much higher than in the control which was not treated with the cleavage enzyme. The activity was eleven times higher and the quantity five times higher in the treated samples compared to the control. Thus, G6PD was released from the mitochondria by PI-PLC which is highly specific for cleaving GPI-anchored proteins

(48). This evidence along with the detection of a glycan chain attached to cytosolic

G6PD indicates that G6PD is attached to the mitochondria via a GPI anchor in yeast.

6 6 c

o E < 0) _> A O CC

Quantity Activity

Control '^/ / À Treated

* Experiment was performed once.

Figure 3.6 Yeast G6PD Released by Phosphatidyl Inositol Specific Phospholipase C

67 Animal Studies

Preliminary studies using male Sprague Dawley rats showed that G6PD was

present in both the cytosol and mitochondria. At this point it seemed logical to

ascertain in which portion of the mitochondria the G6PD existed. The digitonin

fractionation method was used with isolated rat liver mitochondria to peel away

mitochondrial layers. The activity of G6PD in the supernatant was measured as well as

that of marker enzymes to pinpoint G6PD's location. The digitonin concentration with

the highest activity for each enzyme was designated 100% and all other concentrations

expressed as a percentage of that. The results of the digitonin fractionation are shown

in Figure 3.7. The patterns of release of the marker enzymes was typical of the

digitonin fractionation procedure (21). Adenylate kinase was released first at the lowest digitonin concentration. Monoamine oxidase was released next. Malate dehydrogenase, the mitochondrial matrix enzyme marker, was released at a slightly lower digitonin concentration than cytochrome c oxidase as the inner membrane had holes poked in it. G6PD was clearly different from malate dehydrogenase and cytochrome c oxidase indicating that it was not a matrix or inner mitochondrial membrane enzyme. G6PD did show a pattern of release that was similar to either adenylate kinase or monoamine oxidase. Therefore, according to these results

G6PD could be associated with the outer mitochondrial membrane or present in the inner mitochondrial membrane space.

It was assumed that G6PD was associated with the outer mitochondrial membrane and the possibility of this relationship was further explored in rat liver by

6 8 120

100 o 3a co o 80 B c "5 60 < X es 2 40 o 20

0.00 0.10 0.20 0.30 0.40 0.50 0.60

Digitonin (mgjmg protein)

G6P MAO A AdKi ▼ MDH ♦ CytC

*Experiment was performed twice.

Figure 3.7 Rat Liver Digitonin Fractionation

69 isolation of the mitochondrial membrane with the digitonin fractionation method with

protease inhibitors added to the isolation buffer. Digitonin was used at a concentration

of 0.2mg digitonin/mg protein, then the outer mitochondrial membrane isolated by

centrifuging at 160K g. The pellet and supernatant were assayed for G6PD activity.

The pellet, containing the outer mitochondrial membrane, had a total activity of 0.60mU

compared to the supernatant's activity of 1.26 mU. This indicated that G6PD was

associated with the outer mitochondrial membrane but dissociated during high

centrifugal spins.

G6PD in yeast and rat liver was shown to contain carbohydrate, the yeast form

was shown to have a GPI anchor linkage, and the rat liver form was associated with the

outer mitochondrial membrane. Since acetyl CoA carboxylase was also proven to be a glycoprotein (24) and exhibited shifts from particulate to cytosolic forms upon fasting and refeeding (20), G6PD was analyzed to see if a link could be made between the overshoot in activity and shifts in location upon dietary treatment.

48 Hour Refeeding Studv

The first rat refeeding study involved three groups of rats. The fed group was allowed to eat the high carbohydrate diet a d libitum. The fasted group was fasted for 48 hours. The refed group was fasted for 48 hours, then refed the high carbohydrate diet for 48 hours. Each group had four rats. The procedure for the preparation of the mitochondrial-free supernatant and the mitochondria was performed and G6PD activity and quantity measured.

70 G6PD sp act G6PD Quantity (mU/mg prot) (ug G6PD/mg prot)

Fed 0.0658 ±0.013“ 0.509 ±0.11“ Fasted 0.0316 ±0.002“ 0.381 ±0.08“ Refed 0.2329 ±0.016" 0.349 ± 0.08“

Values are expressed as mean ± standard error of the mean n=4 rats per group a—Values with different superscripts in a column are significantly different (p < 0.05)

Table 3.3 G6PD Specific Activity and Quantity in Rat Liver Mitochondrial-Free Supernatant

The mitochondrial-free supernatant G6PD specific activity exhibited expected results upon fasting and refeeding (Table 3.3, Figure 3.8). Fasted rats had half the G6PD activity of fed rats. Refed rats showed a significant overshoot in G6PD activity that was

3.5 times that found in fed rats. Unexpected results occurred when measuring the quantity of G6PD in the mitochondrial-free supernatant. There were no significant differences (p < 0.05) in G6PD quantity in the fed, fasted, or refed groups. This is in direct opposition to reports by many investigators that an overshoot in activity is due to increased enzyme synthesis (2, 4, 88, 94-96). Since investigators have also found no appreciable change in the degradation rate of the enzyme, rapid turnover cannot account for increased synthesis without a change in quantity. Evidence presented here indicates that the antibody reactive quantity of G6PD in the cytosol does not increase when fasted rats are refed a high carbohydrate diet. This is in agreement with Hizi and Yagil (7) and Kelly et al (8) who also found no change in quantity upon refeeding.

71 0.25

So 0 .2 0 - a. "5 o o» O) 0.15 - E I Q. Z3 ■ 2 (O & O 0.10 - > < a. oC co 0.05 - 3 a

0.00 Fed Fasted Refed

Activity Quantity

Figure 3.8 G6PD Activity and Quantity in Rat Liver Mitochondrial-Free Super— 48-Hour Study 72 G6PD sp act G6PD Quantity (mU/mg prot) fug G6PD/mg prot)

Fed 0.015510.004=^ 2.73 +0.37“ Fasted 0.0063 ±0.00 r 3.25 ±0.60“ Refed 0.0249 ± 0.005 ’’ 2.62 ±0.27“

Values are expressed as mean ± standard error o f the mean n=4 rats per group a—Values with different superscripts in a column are significantly different (p < 0.05)

Table 3.4 G6PD Specific Activity and Quantity in Rat Liver Mitochondria

G6PD specific activity in the mitochondria was an order of magnitude smaller than in the mitochondrial-free supernatant. As shown in Table 3.4 and Figure 3.9, fasted rats again had half the activity of fed rats, but refed rats had 1.6 times the activity of refed rats. A significant (p < 0.05) overshoot in G6PD activity occurred in the mitochondria compared to fasted rats, but it was not as pronounced as in the the mitochondrial-free supernatant in all groups when expressed per milligram of protein. Although the fasted rats appeared to have more G6PD, there was no significant difference (p < 0.05) in mitochondrial G6PD quantity among the three groups.

Results presented here for G6PD differ from those found by Allred et al for acetyl CoA carboxylase (20). These investigators found that acetyl CoA carboxylase quantity increased in the cytosol when rats were refed a high carbohydrate diet, and quantity in the mitochondria decreased under the same conditions. When rats were fasted, mitochondrial acetyl CoA carboxylase quantity increased while cytosolic

73 0.25

S o 0.20 a a. 5 "5 2 a O) a> 0.15 E E S' 3 CL (D & (3 0.10 3a» ü < C Q. es CO 0.05 3 a

0.00 Fed Fasted Refed

Activity Quantity

Figure 3.9 G6PD Activity and Quantity in Rat Liver Mitochondria—48-Hour Study

74 decreased. It was proposed that a shift from an inactive mitochondrial storage form to

an active cytosolic form occurred when excess carbohydrate was present upon

refeeding. G6PD does not appear to have this same shift in enzyme. The amounts in

each fraction stay relatively constant under the different dietary conditions.

The actual specific activity of G6PD could be calculated and expressed as activity of G6PD per amount of G6PD (Table 3.5). In refed rats, the mitochondrial-

27K g Super Mitochondria

Fed 0.135 ± 0.02* 0.005 ± 0.01 ^ Fasted 0.105 ± 0.04* 0.002 ± 0.00* Refed 0.953 ± 0.33 0.009 ± 0.01 "

Values are expressed as mean ± standard error o f the mean n=4 rats per group a—Values with difterent superscripts in a column are significantly different (p < 0.05)

Table 3.5 G6PD Activity Per Quantity (mU G6PD/ug G6PD) in Rat Liver

free supernatant G6PD was seven times as active per ug G6PD as in the fed rats while fasted rats had slightly less activity. Although the quantity of G6PD remained nearly the same in all groups, the refed rats had significantly (p<0.05) more activity for the same amount of G6PD. This points to an increased activation of G6PD to account for the great overshoot in enzyme activity with a high carbohydrate diet. G6PD appears to become more active in the refed state.

In the mitochondria, the G6PD activity per amount was almost twice as high in refed rats compared to fed rats when expressed as enzyme quantity but this difference

75 was not significant. There was a significant difference between the fasted and refed rats

in the mitochondria (p<0.05) when activity per amount was compared. The data show

that the mitochondrial G6PD is much lower in activity per amount than the

mitochondrial-fi'ee supernatant G6PD although much more G6PD appeared to be

present. When the mitochondrial-free supernatant is compared to the mitochondria, the

G6PD specific activity is 27, 52, and 100 times more active in fed, fasted, and refed

rats, respectively. In comparison, acetyl CoA carboxylase specific activity was 20

times more active in the mitochondrial-fi'ee supernatant than in the mitochondria in

refed rats (21). Both of these enzymes appear to have a large quantity of inactive

enzyme in the mitochondria.

The procedure to quantitate G6PD can also be used to look at the specific activity of G6PD by graphing the activity (in mU) versus quantity (O.D. units) read fi-om the densitometer. Thus the slope of the graph gives a measure of the relative activity of G6PD per quantity of G6PD. Figure 3.10 shows the results of the activity analysis in the mitochondrial-fi’ee supernatant while Figure 3.11 has the mitochondria results. The average slope was found for each group—fed, fasted, or refed rats. Since the fasted rats had the smallest slope (and change in activity), the slopes for the other groups were determined relative to the fasted rats’ slope. In the mitochondrial-ffee supernatant, refed rats had a slope four times that of fed rats. This indicated that a large change in G6PD activity was accompanied by a small change in G6PD quantity when rats were refed. In the mitochondria, refed rats had a larger change in activity per quantity than fed rats, but it was not as pronounced as in the mitochondrial-free

76 0.30 Refed

0.24

3 E 0.18

> o < 0.12 Fed 0.06

Fasted 0.00 0.0 1.0 2.0 3.0 4.0 5.0

O.D. Units

Figure 3.10 G6PD Activity Analysis in Mitochondrial-Free Super—48-Hour Study

77 2.58-03 Refed

2.08-03

Fed ^ 1.5e-03

> I 1 .Oe-03

5.08-04 Fasted

0 . 08+00 0.0 1.0 2.0 3.0 4.0 5.0

0 .0 . Units

Figure 3.11 G6PD Activity Analysis in Mitochondria—48-Hour Study

78 supernatant. These two methods of looking at the change in activity per quantity of

G6PD, actual enzyme specific activity and the graphical method, are consistent with

one another. Both indicate that G6PD becomes more active when fasted rats are refed a

high carbohydrate diet. This is consistent with an activator theory to explain the

overshoot in activity in contrast to previous investigations which ruled out this theory

(74, 79).

Short Term Refeeding Studv

To define better the subcellular distribution of G6PD with changes in dietary state, it was decided to examine G6PD activity in rats refed for shorter amounts of time.

Rats were refed a high carbohydrate diet after a 48-hour fast and livers extracted at 0, 4, and 8 hours into the refeeding cycle. Fed rats were examined at each of these same time points. There were three rats per group at each time point. Table 3.6 shows that in this short period of refeeding, the G6PD activity of the refed rats reached the activity of the fed rats at 8 hours but there was no overshoot in activity in such a short period of time.

This indicated that a timed refeeding study of longer duration should be performed.

Long Term Refeeding Studv

Male Sprague Dawley rats were fasted for 48 hours then refed the high carbohydrate diet. Livers were extracted at 0, 6, 12, 18, 24, and 36 hours after refeeding. Fed rats were examined at the same time points as a control. There were 3 rats per group. Activity and quantity of G6PD were studied in this experiment.

79 Fed Refed Time sp act quant sp act quant hours mU/mg prot ug G6PD/ mU/mg prot ug G6PD/ mg prot mg prot

Cyto 0 0.231 ±0.04 0.097 ± 0.02 0.100 ±0.01 0.083 ±0.01 4 0.193 ±0.02 0.072 ± 0.02 0.089 ±0.01 0.060 ±0.01 8 0.127 ±0.02 0.064 ±0.01 0.130 ±0.02 0.077 ±0.01

Mito 0 0.055 ±0.02 0.215 ±0.04 0.048 ±0.03 0.295 ± 0.02 4 0.072 ± 0.02 0.361 ±0.08 0.046 ± 0.03 0.465 ± 0.05 8 0.055 ±0.01 0.422 ± 0.08 0.085 ±0.01 1.037 ±0.18 data expressed as mean ± SEM

Table 3.6 Short Term Refeeding Study

Additionally, the activity of 6PGD and the glycogen content of the liver were determined.

The specific activity of G6PD in fed rats remained relatively stable over time

(Figure 3.12 and 3.13). In the mitochondrial-ffee supernatant, G6PD activity in the refed rats equaled the fed rats at 12 hours refeeding and was doubled at 36 hours. In the mitochondria, the refed rats' G6PD activity equaled the fed rats at 12 hours and the activity was only slightly higher at 36 hours. An overshoot in activity occurred in the mitochondria but not to the same extent as in the mitochondria-ffee supernatant. These activity values show good agreement with the values from the short-term refeeding study at the same time points although the activity at 36 hours was higher than the refeeding activity in the 48 hour study.

80 0.30 o Refed k. a .

o 0.20 0 3 E 3 Fed E 0.10 > u < Q. Dark Light Dark w 0.00 0 6 12 18 24 30 36

Hours Refed a High Carbo Diet

Figure 3.12 G6PD Activity in the Mitochondrial-Free Supernatant—36-Hour Study

81 0.10 2 a 0.08 13 R efed O O) 0.06 E D E F ed 0.04

> o 0.02 it < a Light Dark CO 0.00 0 6 12 18 24 30 36

Hours Refed a High Carbo Diet

Figure 3.13 G6PD Activity in Mitochondria—36-Hour Study

82 The specific activity of G6PD was also expressed as tnU/g liver based on the amount of protein present in the liver cytosol and mitochondria determined previously in this laboratory (data not shown). As the total amount of protein is similar in fed and refed rats, the same relationships occurred as when the activities were expressed as specific activity.

Figure 3.14 shows the slight diurnal variation that occurs in G6PD activity in fed rats in both the mitochondrial-ffee supernatant and mitochondria. As rats fed during the dark cycle (0 to 12 hours), the G6PD activity increased in the mitochondrial-ffee supernatant and decreased in the mitochondria. The opposite occurred during the light cycle in which activity decreased slightly in the mitochondrial-ffee supernatant and increased slightly in the mitochondria. This diurnal pattern suggests that there may be some shift in G6PD activity from the mitochondria to the cytosol while rats are feeding and then a shift back to the mitochondria when rats are less actively feeding. The

G6PD activity in refed rats did not show the same patterns (Figure 3.15). The activity increased steadily in both the mitochondrial-free supernatant and mitochondria. There does appear to be a plateau in activity in the mitochondria after 24 hours.

The relative quantity of G6PD was again determined for all time points in the study and expressed as ng G6PD/mg protein in Table 3.7. In the mitochondrial-ffee supernatant there was no significant difference (p > 0.05) in G6PD quantity between fed and refed rats at any of the time points over the course of the study. There was also no difference in quantity in fed rats when compared at different time points and the same can be said for the refed rats. The mitochondria did not show a significant difference

83 12.0

0.8 o 9.0

Cyto 0.6 3 6.0 ir 3 E E >» 3.0 o 0.2 o < < Dark Light Dark 0.0 0.0 0 6 12 18 24 30 36

Hours Refed

Figure 3.14 G6PD Activity in Fed Rats—36-Hour Study

84 0.3 0.12 o Cyto

o o L— O. 0.09 o . 0.2 " r t "êô o o O) 0.06 Oî E t 3 Z) E 0.1 E >. 0.03 >s ’> o L ight Dark o < < o . 0.0 0.00 a . ûO co 6 12 18 24 30 36

Heurs Refed

Figure 3.15 G6PD Activity in Refed Rats—36-Hour Study

85 ng G6PD/mg prot Time (hours) Fed Refed

Cyto 0 2.87 ±0.51 1.41 ±0.17 6 2.58 ±0.19 2.29 ±0.15 12 2.79 ±0.35 2.26 ±0.13 18 2.09 ± 0.06 1.73 ±0.11 24 2.03 ±0.19 2.12 ±0.18 36 2.64 ± 0.28 1.87 ±0.27

Mito 0 8.74 ± 0.86 7.52 ±2.95 6 7.50 ± 1.18 4.67 ± 0.96 12 11.82 ± 1.24 14.00 ± 1.57 18 6.94 ± 1.61 9.19 ±1.90 24 5.49 ± 0.86 9.01 ± 1.83 36 2.32 ±0.35 8.56 ±0.15 n=j rats per group data expressed as mean ± SEM

Table 3.7 G6PD Quantity in Fed and Refed Rats

between the groups over time with the exception of the 36 hour time point. The fed rats had a much lower quantity at 36 hours in the mitochondria which was unexpected and may be explained by experimental error during the quantitation procedure. The quantity was expected to be similar to that of the 12-hour time point. Both the fed and refed rats had a peak in mitochondrial G6PD quantity at 12 hours. The mitochondrial

G6PD quantity was higher than in the mitochondrial-free supernatant and showed a build-up with feeding during the light cycle.

When the data were expressed as mU G6PD/ng G6PD, the same pattern emerged that was seen in the 48-hr study. In the mitochondrial-free supernatant (Figure

8 6 3.16), there was very little variation in activity per quantity over time in the fed rats. In the refed rats, however, there was a greater change in activity versus quantity as the rats were refed longer, indicating a large change in activity with a small change in quantity.

With the exception of the 36-hour time point (which could be in error), the fed rats showed slight diurnal variation in activity per quantity in the mitochondria (Figure

3.17). In refed rats, the activity per quantity increased over time in the mitochondria but not to the same extent as in the mitochondrial-ffee supernatant. These results again show that there is an increase in G6PD activity without a large change in G6PD quantity consistent with an activator theory.

The activity of 6-phosphogluconate dehydrogenase in the mitochondrial-free supernatant and mitochondria was studied in this experiment. Figure 3.18 shows the specific activity in the mitochondrial-free supernatant. There was no change in activity over time in the fed rats. The fasted rats (zero time point for the refed rats) had very low activity values. The refed rat values equaled the fed rat values at 12 hours and was slightly higher in 6PGD activity by 36 hours. This overshoot in 6PGD activity has been reported by others (4, 62, 91). 6GPD activity was below detectable levels in the mitochondria for both the fed and refed rats. However, Zaheer et al{\2) did find 6PGD activity in the mitochondria using different isolation techniques.

Liver glycogen was measured in frozen samples of mitochondrial-free supernatant from both fed and refed groups at each time point to see if there was a relationship to the overshoot in G6PD activity (Figure 3.19). For all time points, the fed rats ranged from 3 to 4% liver glycogen. There was very little change in glycogen even

87 0.17 R e fe d a> 3 & Q a . CD 0 oE Q Q. CO 0

o <

0.00 '

Hours Refed a High Carbo Diet

Figure 3.16 G6PD Activity Per Amount in Mitochondrial-Free Super—36-Hour Study

88 0.028

Fed 3 0.021 Q 0. CD a £ 0.014 ta Q Q. CO 0 Refed 0.007 Ü < :!■ T Î Light Dark 0 000 0 6 12 18 24 30 36

Hours Refed a High Carbo Diet

Figure 3.17 G6PD Activity Per Amount in the Mitochondria—36-Hour Study

89 0.16 R e fe d

% 0.14 -r wo CL 5 o O) 0.12 - E 3 _E ^ 0.09 - > o < COQ. 0.07 -

0.05 12 18 24 30 36

Hours Refed a High Carbo Diet

Figure 3.18 6PGD Activity in Mitochondrial-Free Supernatant—36-Hour Study

90 1

1

a»o 8 Refed o

4 " Fed

D ark Light Dark

0 6 12 18 3024 36

Hours Refed a High Carbo Diet

Figure 3.19 Glycogen Content in Rat Liver—36-Hour Study

91 when going from an eating to a non-eating cycle. The refed rats went from 1.5%

glycogen in the fasted state to 10.5% at 18 hours to 6% at 36 hours, showing an increase

in liver glycogen upon refeeding a high carbohydrate diet. Unlike the G6PD activity

response that continuously increased upon refeeding, the glycogen content in the refed

rats peaked and then decreased.

Other parameters were explored to try to gain insight into why there was a change in activity upon carbohydrate refeeding without a change in enzyme quantity.

Kinetic and electrophoretic mobility experiments were performed.

Enzyme Kinetic Studies for Rat Liver G6PD

A study was performed to examine the differences, if any, in the Vmax and Km for G6P between the two groups. Three rats were fasted for 48 hours then refed the high carbohydrate diet for 36 hours. Livers were extracted and the mitochondrial-free supernatant separated. Three fed rats were examined at the same time. The mitochondrial-free supernatant from the three rats in each group were then pooled and the activities measured as described in the methods section. The experiment was performed twice and the results averaged. Figure 3.20 is the velocity plot for rat liver for the fed and refed rats while Figure 3.21 is the Lineweaver-Burk plot for each. In the analysis, the Vmax for fed rats was 0.041 mU/mg protein versus 0.096mU/mg protein for refed rats. This was expected since there is a great difference in specific activity between fed and refed rats. The activity of the enzyme under both dietary conditions was also measured using the standard assay with an excess of substrates. Although

92 5.0 Refed 4.0

0ü 3.0 1 Fed

2.0 o <

1.0

0.0 0 40 80 120 160 200

[G6P]

Figure 3.20 Rat Liver G6PD Kinetic Study—Velocity Plot

93 2.0 Fed

1.5 Refed

t 1.0 > I

0.5

0.0 -0.05 0.00 0.05 0.10 0.15

1/[G6P] (1/S)

Figure 3.21 Rat Liver G6PD Kinetic Study—Lineweaver-Burk Plot

94 those values were lower (fed = 0.021 mU/mg prot and refed = 0.052 mU/mg prot), the

ratio of the Vmax values, fedrrefed were about 0.45 for each part of the experiment.

There was also a difference in Km values that was unexpected. Fed rats had a Km of

28.8uM while refed rats had a Km of 55.9uM, twice as high. A difference in Km values

has been shown before. Yagil and Hizi got a Km of 12uM for non-induced mice and

18uM for induced mice (79), but that difference is minor by comparison. In this

experiment it appears that there are two distinct enzymatic forms of G6PD with their

own separate kinetic properties. One is present in fed rats, and the other is dominant in

refed rats.

Electrophoretic Mobilitv Studv for Rat Liver G6PD

The electrophoretic mobility for G6PD was analyzed in non-denaturing gels for

fed and refed rats. Three rats were fasted for 48 hours then refed the high carbohydrate

diet for 36 hours. Livers were extracted and the mitochondrial-ffee supernatant

separated. Three fed rats were examined at the same time. The mitochondrial-ffee

supernatants from the three rats in each group were then pooled. The samples were

placed in wells, in duplicate, on a non-denaturing gel and electrophoresed. One set of

lanes was analyzed for G6PD activity and the other transferred to nitrocellulose and analyzed for antibody reactivity. The activity-stained gel and antibody-reacted film were read on the densitometer with all lanes lined up so they were read from the same starting point. Lower numbers on the densitometiy scale correspond with a greater mobility distance and the Y-axis is in O.D. units.

95 Figure 3.22 shows that both the fed and refed rats had similar mobility patterns

when analyzed for activity. There was one broad band between 109 and 123 mm on the

densitometer scale with a minor one after 135. The mobility pattern from the antibody reactivity analysis was also similar for both the fed and refed rats (Figure 3.23). There were three major bands at 110, 123, and 135mm, with the band at 123mm being most prominent. The presence of three major forms is consistent with work by many other researchers (33, 36, 122). It is assumed that the three bands are dimers, tetramers, and hexamers of G6PD which has an inactive subunit (122). The most active form is the one with the greatest mobility, the dimer (36). This is consistent with the results presented here as the greatest activity is with the fastest moving band. However, in this experiment there appears to be a smear of activity that blends the two fastest moving bands when the peaks from the activity stain are compared to those from the antibody stain. Most of the quantity of G6PD is associated with the middle band, presumably the tetrameric form in contrast to work by Watanabe and Taketa who found 90% of the quantity associated with the dimer (36).

The results of this experiment show that there could be a shift in mobility of antibody-reactive quantity from the second band to the first when going from the fed to refed states. The ratio of the area under band 1 to the area under band 2 is 0.84 for fed rats and 1.03 for refed rats. There could be a shift to the fastest moving band (and the one considered most active) upon refeeding.

The results from these experiments show that G6PD is a glycoprotein and is attached to the outer mitochondrial membrane via a GPI anchor much like acetyl CoA

96 L2 — ).a-

i.ii

u;:

i.u.

I.U.

I.H

I.W til KS

Figure 3.22 Densitometry Scan of G6PD Activity; Electrophoretic Mobility A—Fed Rats, B—Refed Rats 97 k s- .V

I

: w-

J.TI-

i.n.

I.N

IH UI us 111 m ui t

i.n

151

1.(1

m til UI Its 131 135 USUI

Figure 3.23 Densitometry Scan of G6PD Antibody Reactivity: Electrophoretic Mobility A—Fed Rats, B—Refed Flats 98 carboxylase. Unlike the rate-limiting enzyme for fat biosynthesis, however, G6PD does not appear to show a shift from the mitochondria to the cytosol upon refeeding a high carbohydrate diet after a fast. There was no change in overall G6PD quantity in the mitochondria or mitochondrial-ffee supernatant under differing dietary conditions. The enzyme is much more active in refed rats compared to fed rats. Mitochondrial-ffee supernatant G6PD is seven times more active per amount of G6PD in refed rats compared to fed rats. In the mitochondria, refed activity is twice as active. There must be more than one regulatory mechanism at work. G6PD could be clipped from the mitochondria under conditions when insulin is present, but there is also an activation mechanism at work that is independent of carbohydrate attachment.

99 CHAPTER 4

CONCLUSIONS

Results from both yeast and rat experiments indicate that glucose-6-phosphate

dehydrogenase is a glycoprotein. The digoxigenin carbohydrate detection method

showed that the same protein that had G6PD antibody reactivity and activity contained

carbohydrate. Treatment of yeast with phosphatidylinositoi specific phospholipase c

released G6PD from mitochondria indicating a GPI linkage. Studies with rat liver

showed that G6PD is associated with the outer mitochondrial membrane since it had a

pattern of release similar to monoamine oxidase with digitonin fractionation and was

present in the outer mitochondrial fraction upon centrifugation. These properties by themselves, however, do not explain the increase in enzyme activity upon refeeding a fasted rat a high carbohydrate diet.

When rats were refed a high carbohydrate diet, there was a characteristic overshoot in G6PD activity in both the mitochondrial-free supernatant and the mitochondria. There was no change in quantity in either fraction and no indication that the enzyme shifted from one compartment to another in response to dietary manipulations. When analyzing the actual specific activity of the enzyme in the mitochondrial-free supernatant, G6PD in refed rats was seven times more active per

1 0 0 amount of G6PD than in fed rats. In the mitochondria it was twice as active. Kinetic

studies showed that refed rats had a higher Vmax and Km than fed rats indicating more

than one form of the enzyme.

These results show that G6PD becomes more activated without a change in

enzyme quantity when rats are refed a high carbohydrate diet after fasting. This

activation appears to have nothing to do with protein synthesis or a shift in enzyme

location from mitochondria to cytosol. At this time the mechanism for the activation of

the enzyme remains in question.

The results of these experiments lead to studies that could be performed in the

future. The glycoprotein moiety of G6PD should be further analyzed. The point of carbohydrate attachment should be elucidated and the presence of sugars and inositol confirmed by GC/MS. Experiments should also be performed to determine what activator mechanism of G6PD is present in refed rats. Unfortunately this would involve a shotgun method of research as currently there is no known activator mechanism for

G6PD.

1 0 1 REFERENCES

1. Gibson DM, Lyons RT, Scott DP, Muto Y. (1971) Synthesis and degradation of the lipogenic enzymes of rat liver. \xiAdv. Enz. Reg. 10:187-204.

2. Iritani N. (1992) Nutritional and hormonal regulation of lipogenic-enzyme gene expression in rat liver. Eur. J Biochem. 205:433-42.

3. Potter VR, Ono T. (1961) Enzyme pattern in rat liver and Morris hepatoma 5123 during metabolic transitions. Cold Springs Harbor Symposia on Quantitative Biology. 26: 355-62.

4. Winberry L, Holten D. (1977) Rat liver G6PD. J. Biol. Chem. 252(1):7796-801.

5. Miksicek RJ, Towle HC. (1982) Changes in the rate of synthesis and messenger RNA levels of hepatic G6PD and 6PGD following induction by diet or thyroid hormone. J. Biol. Chem. 257:11829-35.

6. Ayala A, Fabregat I, Machado A (1991) The role ofNADPH in the regulation of G6PD and 6PGD in rat adipose tissue. Mol. Cell. Biochem. 105:1-5.

7. Hizi A, Yagil G. (1974) On the mechanism of G6PD regulation in mouse liver:3 the rate of enzyme synthesis and degradation. Eur. J. Biochem. 45:211-21.

8. Kelley D, Watson J, Mack D, Johnson BC. (1975) Mutr. Rep. Int. 12:121-35.

9. Anstall HB, Trujillo JM. (1967) G6PD: its purification and properties. Am. J. Clin. Path. 47(3) :296-302.

10. Kanji MI, Towes ML, Carper WR. (1976) G6PD: Purification and partial characterization. J. Biol. Chem. 251(8):2255-7.

11. Panchenko LF, Antonenkov VD. (1984) G6PD of rat liver peroxisomes. Experentia 40:467-8.

12. Antonenkov VD. (1989) Dehydrogenases of the pentose phosphate pathway in rat liver peroxisomes. Eur. J. Biochem. 183:75-82.

1 0 2 13. Zaheer N, Tewari KK, Krishnan PS. (1967) Mitochondrial forms of G6PD and 6PGD in rat liver. Arch Biochem. Biophys. 120:22-34.

14. Zaheer N, McClean P. (1972) Evidence for the existence and functional activity of the pentose phosphatepathway in the large particle fraction isolated from rat tissues. Biochem. Biophys. Res. Comm. 46:167-74.

15. Ozols J. (1993) Isolation and the complete amino acid sequence of luminal endoplasmic reticulum G6PD. Proc. Natl. Acad. Sci. USA. 90:5302-6.

16. Janski AM, Cornell NW. (1980) Association of ATP citrate lyase with mitochondria. Biochem. Biophys. Res. Comm. 92:305-12.

17. Janski AM, Cornell NW. (1980) Subcellular distribution by rapid digitonin fractionation of isolated hepatocytes. Biochem. J. 186:423-9.

18. Cornell NW, Janski AM, Rednon A. (1885) Compartmentation of enzymes: ATP citrate lyase in hepatocytes from fed or fasted rats. F ed Proc. 44:2448-52.

19. Witters LA, Friedman SA, Bacon GW. (1981) Microsomal acetyl-CoA carboxylase: evidence for association of enzyme polymer with liver microsomes. Proc Natl Acad Sci U SA 78(6):3639-43.

20. Allred JB, Roman-Lopez CR, Pope TS, Goodson J. (1985) Dietary dependent distribution of acetyl CoA carboxylase between cytoplasm and mitochondria of rat liver. Biochem Biophys Res Commun 129(2):453-60.

21. Allred JB, Roman-Lopez CR. (1988) Enzymatically inactive forms of acetyl CoA carboxylase in rat liver mitochondria. Biochem. J. 251:881-5.

22. Roman-Lopez CR, Allred JB. (1987) Acute alloxan diabetes alters the activity but not the total quantity of acetyl CoA carboxylase in rat liver. JN utr 117(11):1976-81.

23. Roman-Lopez CR, Goodson J, Allred JB. (1987) Determination of the quantity of acetyl CoA carboxylase by [14C]methyl avidin binding. J. Lipid Res. 28(5):599-604.

24. Bowers DF. (1991) Identification and characterization of cetyl CoA carboxylase as a glycoprotein. Ph D. dissertation. The Ohio State University, Columbus, OH..

25. Prostko CR, Fritz RS, Kletzien RF. (1989) Nutritional regulation of hepatic G6PD. Biochem J. 258:295-9.

103 26. Nogae I, Johnston M. (1990) Isolation and characterization of the ZWFl gene of Saccharomyces cerevisiae, encoding G6PD Gene 96:161-9.

27. Persson B, Jomvall H, Wood I, Jeffery J. (1991) Functionally important regions of G6PD defined by the Saccaromyces cerevisiae enzyme and its differences from the mammalian and insect forms. Eur. J. Biochem. 198:485-91.

28. Schnaitmen C, Greenawalt JW. (1968) Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J. Cell Biol. 38:158-75.

29. Holten D. (1972) Relationships between the multiple molecular forms of rat liver G6PD. Biochem. Biophys. Acta. 268:4-12.

30. Grigor M. (1984) Multiple molecular forms of rat mammary G6PD: proposed role in turnover of errzyvaeArch. Biochem. Biophys. 229:612-22.

31. Martins RN, Stokes GB, Masters CL. (1985) regulation of the multiple molecular forms of rat liver G6PD by insulin and dietary restriction. Biochem. Biophys. Res. Comm. 127:136-42.

32. Jeffery J, Persson B, Wood 1, Bergman T, Jeffery R, Jomvall H. (1993) G6PD: Stmcture-flinction relationships and the Pichia jadinii enzyme structure. Eur. J. Biochem. 212:41-9.

33. Taketa K, Watanabe A. (1971) Interconvertible microheterogeneity of G6PD in rat liver. Biochim. Biophys. Acta 235:19-26.

34. Hizi A, Yagil G. (1974) On the mechanism of G6PD regulation in mouse liver:3 purification and properties of the mouse-liver enzyme. Eur. J. Biochem. 45:201- 9.

35. Dao ML, Watson WR, Delaney R, Johnson BC. (1979) Purification of a new high activity form of G6PD from rat liver and the effect of enzyme inactivation on its immunochemical reactivity. J. Biol. Chem. 254(19):9441-7.

36. Watanabe A, Taketa K. (1973) Immunological studies on G6PD of rat liver. Arch. Biochem. Biophys. 158:43-52.

37. Murray RK. (1988) Glycoproteins and proteoglycans, in Harpers Biochemistry (Murray RJC, Granner DK, Mayes PA Rodwell VW., eds.) Appleton and Lange, Norwalk/San Mateo, pp 589-606.

104 38. Fukada H, Katsurda A, Iritani N,. (1991) Nutritional and hormonal regulation of mRNA levels of lipogenic enzymes in primary cultures of rat hepatocytes. J Biochem. 111:25-30.

39. Iritani N, Nishimoto N, Katsurda A, Fukada H. (1992) Regulation of hepatic lipogenic enzyme gene expression by diet quantity in rats fed a fat-free, high carbohydrate diet. J. Nutr. 122:28-36.

40. Liscum L, Cummings RD, Anderson RGW, DeMartino GN, Goldstein JL, Brown MS. (1983) 3-Hydroxy-3-methylgIutaryi-CoA reductase: a transmambrane glycoprotein of the endoplasmic reticulum with N-linked ‘high mannose’ oligosaccharide. Proc. Natl. Acad. Sci. USA 80:7165-9.

41. Volpe JJ, Goldberg RI. (1983) Effect of tunicamycin on 3-hydroxy-3- methylglutaryl coenzyme A reductase in C-6 glial cells. J. Biol. Chem. 258(15):9220-6.

42. Amri EZ, Varmier C, Etienne J, Ailhaud G. (1986) Maturation and secretion of lipoprotein lipase to cultured adipocyte cells. II. Effects of tunicamycin on activation and secretion of the enzyme. Biochem Biophys. Acta. 875:334-43.

43. Chan BL, Lisanti MP, Rodriguez-Boulan E, Saltiel AR. (1988) Insulin-stimulated release of lipoprotein lipase by metabolism of its phosphatidyl inositol anchor. Science 241:1670-2.

44. Kobata A. (1992) Structures and functions of the sugar chains of glycoproteins. Eur. J. Biochem. 209:483-501.

45. Olden K, Bernard BA, Humphries MJ, Yeo T, Yeo K, White SL, Newton SA, Bauer HC, Parent JB. (1985) Function of glycoprotein glycans. TIBS. 10:78-82.

46. Gahmberg CG, Tolvanen M. (1994) Nonmetabolic radiolabeling and tagging of glycoconjugates. Meth Enz 230:32-7.

47. Haselbeck A, Hosel W. (1993) immunological detection of glycoproteinson blots based on labeling with digoxigenin. Meth. In Mol. Biol. 14:161-73.

48. Low MG, Ferguson MA, Futerman AH, Silman I. (1986) Covalently attached phosphatidylinositoi as a hydrophobic anchor for membrane proteins. TIBS May 212-5.

49. Doering TL, Masterson WJ, Hart GW, Englund PT. (1990) Biosynthesis of glycosyl phosphatidyl inositol membrane anchors. J. Biol. Chem. 265(2):611-4.

105 50. Menon AK. (1994) Structural analysis of glycosyl phosphatidylinositoi anchors. Meth. in Enz. 230:418-42.

51. Low MG. (1987) Biochemistry of glycosyl-phosphatidylinositol membrane protein anchors. Biochem. J. 244:1-13.

52. Udenfriend S. Kodukula K. (1995) How glycosylphosphatidylinositol-anchored membrane proteins are made. Anna. Rev. Biochem. 64:563-91.

53. Medof EM, Nagarajan S, Tykocinski ML. (1996) Cell-surface engineering with GPI-anchored proteins. FASEB J. 10:574-586.

54. Spooner DM, Chemick SS, Garrison MM, Scow RO. (1979) Insulin regulation of lipoprotein lipase activity and release in 3T3-L1 adipocytes. J. Biol. Chem. 254(20): 10021-9.

55. Saltiel AR, Cuatrecasas P. (1986) Insulin stimulates the generation from hepatic plasma membranes of modulators derived from an inositol glycolipid. Biochemistry. 83:5793-7.

56. Greenawalt JW. (1974) The isolation of outer and inner mitochondrial membranes. Meth. in Enz. 31:310-23.

57. Zaheer N, Tewari KK, Krishman PS. (1965) Exposure and solubilization of hepatic mitochondrial shunt dehydrogenases. Arch. Biochem. Biophys. 109:646.

58. Stanton RC, seifter JL, Boxer DC, Zimmerman E, Cantley LC. (1991) Rapid release of bound G6PD by growth factors. J. Biol. Chem. 266(19): 12442-8.

59. Tepperman HM, Tepperman J. (1958) Effects of antecedent food intake pattern on hepatic lipogenesis. Am. J. Phys. 193:55-64.

60. Glock GE, McClean P. (1955) A preliminary investigation of the hormonal control of the hexose monophosphate oxidative pathway. Biochem. J. 61:390-7.

61. McDonald BE, Johnson BC. (1965) Metabolic response to realimentation following chronic starvation in the adult male rat. J. Nutr. 87: 161-7.

62. Fitch WM, Chaikoff JL. (1960) Extent and patterns of adaptation of enzyme activities in livers of normal rats fed diets high in glucose and fructose. J. Biol. Chem. 235(3):554-7.

63. Sasoon HF, Watson JJ, Johnson BC. (1968) Diet-dependence of rat liver G6PD levels. JN utr. 94:52-6.

106 64. Abraham S. Kopelovich L, Chaikoff EL. (1964) Dietary and hormonal regulation of the hepatic citrate cleavage enzyme. Biochim. Biophys. Acta. 93:185-7.

65. Muto Y, Gibson DM. (1970) Selective damping of lipogenic enzymes of liver by exogenous polyunsaturated fatty acids. Biochem. Biophys. Res. Comm. 38:9-15.

66. Komacker MS, Lowenstein JM. (1965) Citrate and conversion of carbohydrate into fat. Biochem. J. 94:209-15.

67. Fukada H, Katsurada A, Iritani N. (1990) Effects of aging on transcriptional and post-transcriptional regulation of malic enzyme and G6PD in rat liver. Eur. J. Biochem. 188:517-22.

68. Bois-Joyeux B, Chanez M, Aranda-Haro P. Peret J. (1990) Age dependent hepatic lipogenic enzyme activities in starved-refed rats. DiabetesMetab. 16: 290-5.

69. Mack DO, Watson JJ, Johnson BC. (1975) Effect of dietary fat and sucrose on the activities of several rat hepatic enzymes and their diurnal response to a meal. J. Nutr. 105:701-13.

70. Holten D, Carlos JR, Reichert LK, Nakayama R. (1993 Regulation of liver glucose- 6-P dehydrogenase levels in female rats. Comp Biochem Physiol 104(1): 115-8.

71. Hansen RJ, Jungermann K. (1987) Sex differences in the control of G6PD and 6PGD. Biol. Chem. Hoppe-Seyler. 368:955-62.

72. Berdanier CD. (1981) Effects of estrogen on the responses of male and female rats to starvation refeeding. J. Nutr. Ill: 1425-29.

73. Giminez MS, Johnson BC. (1981) Pair feeding in the dietary control of G6PD. J Nutr. 111:260-5.

74. Tepperman HM, Tepperman J. (1963) On the response of hepatic G6PD activity to changes in diet composition and food intake pattern. mAdv. Enz. Reg. 1:121-36.

75. Johnson BC, Sassoon HF. (1967) Studies on the induction of liver G6PD in the rat. in Adv. in Enz. Reg. 5:93-106.

76. Rudack D, Chisolm EM, Holten D. (1971) Rat liver G6PD, regulation by carbohydrate, diet, and insulin. J. Biol. Chem. 246:1249-54.

77. Dror Y, Sassoon HF, Warson JJ, Mack DO, Johnson BC. (1973) Fat versus sucrose as the nonprotein caloric portion of the diet of rats. J. Nutr. 103:342-6.

107 78. Sasoon HF, Dror Y, Watson JJ, Johnson BC. (1973) Dietary regulation of liver G6PD in the rat: starvation and dietary carbohydrate induction. J. Nutr. 103:321- 35.

79. Yagil G. Shimron F, Hizi A. (1974) On the mechanism of G6PD regulation in mouse liver: 1. Characterization of the system. Eur. J. Biochem. 45:189-200.

80. Fitch WM, Hill R., Chaikoff JL.(1959) Effect of fructose feeding on glycolytic enzyme activity of normal rat liver. J. Biol. Chem. 234(5): 1048-51.

81. Takeda Y, Inoue H, Honjo BC, Tanioka H, Daikuhara Y. (1967) Dietary response of various key enzymes related to glucose metabolism in normal and diabetic rat liver. Biochem. et Biophys. Acta. 136:214-22.

82. Potter VR, Gebert A, Pitot HC. (1966) Enzyme levels in rats adapted to 36-hour fasting. In: Advances in Enzyme Regulation, vol. 4 ed. by G. Weber. Pergamon Press, New York, p. 247.

83. Weber G. Convery JJ. (1966) Insulin; inducer of G6PD. Life Sci. 5:1139.

84. Berdanier CD, Schubeck D. (1979) Interaction of glucocorticoids and insulin in the responses of rats to starvation-refeeding. JNutr. 109:1766-71.

85. Fitch WM, Hill R., Chaikoff JL.(1959) Hepatic glycolytic enzyme activity in the Alloxan-diabetic rat: response to glucose and fructose feeding. J. Biol. Chem. 234(11):2811-3.

86. Freedland RA, Cunliffe TL, Zinke JG. (1966) Effect of insulin on enzyme adaptation to diet and hormones. J. Biol. Chem. 24(22)1:5448-51.

87. Kurtz JW, Wells WW. (1981) Induction of G6PD in primary cultures of adult rat hepatocytes. J. Biol. Chem. 256(21): 10870-5.

88. Gozukara EM, Frolich M, Holten D. (1972) The effect of unsaturated fatty acids on the rate of synthesis of rat liver G6PD. Biochem. Biophys. Acta. 286:155-63.

89. Katsurada A, Iritani N, Fukada H, Noguchi T, Tanaka T. (1986) Effects of dietary nutrients on lipogenic enzyme and mRNA activities in rat liver during induction. Biochim. Biophys. Acta. 877:350-8.

90. Katsurada A, Iritani N, Fukada H, Matsumura Y, Noguchi T, Tanaka T. (1989) Effects of nutrients and insulin on transcriptional and posttranscriptional regulation ofG6PD synthesis in rat liver. Biochem et Biophys Acta 1006:104-10.

108 91. Mack DO, Watson JJ, Johnson BC. (1975) Regulation of G6PD and 6PGD in the meal fed rat J. Nutr. 105:714-7.

92. Szepesi B, Freedland RA. (1969) Differential requirement for de novo RNA synthesis in the starved-refed rat: inhibition of the overshoot by 8-azaguanine after refeeding. J. Nutr. 99:449-58.

93. Mack DO, Watson JJ, Chandler AM, Johnson BC. (1974) Induction of G6PD by a 90% carbohydrate diet and 8-Azaguanine insensitive induction of G6PD following a transfer from the 90% carbohydrate diet to a 90% protein diet. J. Nutr. 104:12-7.

94. Garcia DR, Holten D. (1975) Inhibition of rat liver G6PD synthesis by glucagon. J. Biol. Chem. 250(10):3960-5.

95. Peavy DE, Hansen RJ. (1975) Immunological titration of rat liver G6PD from animals fed high and low carbohydrate diets. Biochem. Biophys. Res. Comm. 66:1106-11.

96. Peavy DE, Hansen RJ. (1976) Increased synthesis of G6PD with glucose diet. Fed. Proc. 35:282.

97. Dao ML, Johnson BC, Hartman PE. (1982) Preparation of a monoclonal antibody to rat liver G6PD and the study of its immunoreactivity with native and inactivated enzyme. Proc. Natl. Acad Sci. USA. 79:2860-4.

98. Sun JD, Holten D. (1978) Levels of rat liver G6PD messenger RNA. J. Biol. Chem. 2530:6832-6.

99. Kletzien RF, Prostko CR, Stumpo DJ, McClung JK, Dreher KL. (1985) Molecular cloning of DNA sequences complementary to rat liver G6PD mRNA. J. Biol. Chem. 260(9):5621-4.

100. Katsurada A Iritani N, Fukada H, Matsumura Y, Nishimoto N, Noguchi T, Tanaka T. (1990) Effects of nutrients and hormones on transcriptional and posttranscriptional regulation of fatty acid synthase in rat liver. Eur. J. Biochem. 190:435-41.

101. Fritz RS, Stumpo DJ, Kletzien RF. (1986) G6PD mRNA sequence abundance in primary cultures of rat hepatocytes. Biochem. J. 237:617-9.

102. Stumpo DJ, Kletzien. RF. (1984) Regulation of G6PD mRNA by insulin and the glucocorticoids in primary cultures of rat hepatocytes. Eur. J. Biochem. 144:497-502.

109 103. Rudack D, Davis B. Holten D. (I97I) Regulation of rat liver G6PD levels by adenosine 3',5' monophosphate. J. Biol. Chem. 246(24):7823-4.

104. Krebs HA, Eggleston LV. (1974) The regulation of the pentose phosphate cycle in rat liver. Adv. in Enz. Reg. 12:421-34.

105. Tepperman HM, Tepperman J. (1964) Pattern of dietary and hormonal in induction of certain NADP-Iinked liver enzymes. Am. J. Phys. 206:357-61.

106. Eggleston LV, Krebs HA. (1974) Regulation of the pentose phosphate cycle. Biochem. J. 138:425-35.

107. Ayala A, Fabregat I, Machado A. (1990) Possible involvement of NADPH requirement in regulation of G6PD levels in rat liver. Mol. Cell. Biochem. 95:107-15.

108. Napier MA, Lipari MT, Courter RG, Cheng CHK. (1987) Epidermal growth factor receptor tyrosine kinase phosphorylation of G6PD in vitro. Arch. Biochem. Biophys. 259(2):296-304.

109. Munday MR, Campbell DO, Carling D, Hardie DG. (1988) Identification by amino acid sequencing of three major regulatory phosphorylation sites on rat acetyl CoA carboxylase. Eur. J. Biochem. 175:331-8.

110. Purich DL, Fromm HJ, Rudolph FB. (1973) The hexokinases: kinetic, physical, and regulatory properties, in Adv. in Enzymology and Related Areas of Molecular Biology, vol 329, pp. 249-326.

111. Parry DM, Pederson PL. (1984) Intracellular localization of rat kidney hexokinase. J. Biol. Chem. 259(14):8917-23.

112. Parry DM, Pederson PL. (1990) Glucose catabolism in brain. J. Biol. Chem. 265(2): 1059-66.

113. Agius A, Peak M. (1993) Intracellular binding of glucokinase in hepatocytes and translocation by glucose, fructose,and insulin. Biochem. J. 296:785-96.

114. Bessman SP, Geiger PJ. (1980) Compartmentation of hexokinase and creatinine phosphokinase, cellular regulation and insulin action. Curr. Top. Cell. Reg. 16:55-86.

115. Uyeda K. (1992) Interaction of glycolytic enzymes with cellular membranes. Curr. Top. Cell. Reg. 33:31-46.

1 1 0 116. Wiley WR. (1974) Isolation of spheroplast and membrane vesicles from yeast and filamentous fungi. Meth. in Enz. 31:609-26.

117. Ikezawa H. Taguchi R. (1981) Me/A. in Enz. 71:731-41.

118. Bradford MM. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-54.

119. Laemmli UK. (1970) Cleavage of structural proteins during assembly of the head of Bacteriophage T4.Nature 227:680-5.

120. Burnette WN. (1981) “Western blotting”: Electrophoretic transfer of proteins from SDS-PAGE gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated Protein A. Anal. Biochem. 112:195-203.

121. Reilly KE, Allred JB. (1995) Glucose-6-Phosphate Dehydrogenase from Saccharomyces cerevisae is a glycoprotein. Biochem. and Biophys. Res. Com. 216(3):993-8.

122. Schmukler M. (1970) The heterogeneity and molecular transformations of G6PD of the rat. Biochem. et Biophys. Acta 214:309-17.

123. Dror Y, Sassoon HF, Warson JJ, Johnson BC. (1970) G6PD assay in liver and blood. Clin. Chim. Acta. 28:291-8.

124. Tabor J. Biol. Chem. 208:645.

125. Ochoa S. (1954) Malic dehydrogenase from pig heart. Meth. in Enz. 1:735-8.

126. Poyton RG, Goehring B, Drosle M, Sevarino KA, Allen La, Zhao XJ. (1985) Cyrochrome c oxidase from Saccharomyces cerevisiae. Meth. Enz. 260:97-102.

127. Allred JB, Harris GJ, Goodson J. (1983) Regulation of purified rat liver acetyl CoA carboxylase by phosphorylation. J. Lipid Res. 24:449-55.

128. Roehrig KL, Allred JB. (1974) Direct enzymatic procedure for the determination of liver glycogen.Anal. Biochem. 58:414-21.

129. Takahashi N. (1992) in Handbook of Endoglycosidases and Glycoamidases (Takashi, N. and Muramatsu, T., eds), CRC Press, Boca Raton, FL/London, PP. 183-98.

Ill