69-10599 GOMES, Benedict, 1933

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This dissertation has been 69-10,599 microfilmed exactly as received GOMES, Benedict, 1933- BEEF LIVER MITOCHONDRIAL AMINE OXIDASE; PURIFICATION AND STUDIES ON SOME PHYSICAL AND CHEMICAL PROPERTIES.

University of Hawaii, Ph.D., 1968 Biochemistry

University Microfilms, Inc., Ann Arbor, Michigan BEEF LIVER MITOCHONDRIAL AMINE OXIDASE;

PURIFICATION AND STUDIES ON SOME

PHYSICAL AND CHEMICAL PROPERTIES

A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE

UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN BIOCHEMISTRY SEPTEMBER 1968

BENEDICT GOMES

Dissertation Committee:

Kerry T. Yasunobu, Chairman Morton Mandel Lawrence H. Piette Robert H. McKay John B. Hall DEDICATION

TO MY MOTHER Acknowledgements

To the East-West Center of the University of

Hawaii; the National Institute of Health; and the

Hawaii Heart Association for fellowships.

To Drs. I. Igaue and H. J. Kloepfer for their assistance in the enzyme purification.

To Mrs. Tomi Haehnlen and Kazi Sirazul Islam

for drawing figures. TABLE OF CONTENTS

LIST OF TABLES ••••••••••••••••••••••.•••••••••• vi

LIST OF FIGURES •••••.•.••••••••••.••••••••••••. viii

ABBREVIATIONS .••••••.•.•.••••••••••.••••••••••• xi

ABSTRACT •..•.•.•.••.•••••••••••••••.••••••••••. xii

I. INTRODUCTION. •••.•.•••.••••••••..•••••••••. 1

A. Historical Background of Amine 2 Oxidase Studies •••..••••••••••.••••

B. Physiological Significance •.••••••• 5

C. Statement of the Problem...... 6

II. MATERIALS AND METHODS .•••••••.••••••••••.•• 8

A. Ma t e ria 1 s •••••••••••••••••.••••••.. 8

1. Materials and Reagents Obtained Comme r cia11y ••••••••••••••••••• 8

2. Materials Obtained as Gifts ••.• 10

B. Methods...... 12

1. Preparation of Adsorbents and Ionexchange Materials •••••••••• 12

(a) Alumina C/'...... 12

(b) Calcium phosphate gel..... 12

(d) Hydroxy1ap a t i te ••••••••••• 12

(e) Starch (for Electrophoresis) 12

(f) Sephadex G-200 ••••••••••••• 13

(g) Agarose (Bio-Gel A-1.5) gel. 13 ii

2. Electrophoresis .••.•.••••.. 13

(a) Starch Block Electrophoresis 12

(b) Polyacrylamide Gel Electrophoresis .•.•••• 14

3. Ultracentrifuge Studies •••• lS

(a) Sedimentation Velocity. lS

(b) Sucrose Density Gradient •...... •.••.• 16

4. Preparation of Mitochondria •. 17

S. Measurement of Enzymatic Activity ..•.•...•....••••.•. 17

6. Determination of Hydrogen Peroxide •••••••.•.•.•••••••• 18

7. Determination of the Partial Specific Volume, V •..•••••••• 20

8. Determination of Molecular We igh t •....•....•...••••..•. 20

(a) Mol. Wt. by gel filtration method 20

(b) Mol. Wt. from sedimen tation coefficient, Stoke'~ radius, and the partial specific vo 1 ume ...••.....••••••. 21

9. Metal Analyses ..•..•••••••.•• 23

(a) Copper ...... 23 (b) Cobalt ...... 23 (c) Iron ...... 23 (d) Manganese ...... 23

( e) Molybdenum •••• 0 •••••••• 23 iii

10. Determination of Riboflavin ••••• 23

11. Determination of Purine •••••••• 24

12. Determination of Adenine ••••••• 25

13. Determination of Ribose ••••.••• 26

14. Determination of Phosphorus •••• 26

15. Analysis of Phospholipid .•••••• 27

16. Determination of the Sulfhydryl Groups...... 27

III. RESULTS 29

A. Purification and Purity Studies ••.•• 29

1. Purification of the Mitochon drial Amine Oxidase •.••••••••••• 29

Calcium phosphate gel t rea tmen t. ••.•.••••••••.....••.•• 29

DEAE-cellulose column chromatography 31

Hydroxylapatite column chromatography...... 31

2. Studies on the Purity of the En z ym e .•••••.••••••••••.•••••••• 40

(a) Rechromatography on DEAE- cellulose ••.••••••••.•••••• 40

(b) Rechromatography on hydroxylapatite column 43

(d) Analytical starch block electrophoresis •••••••••••• 43

(e) Free boundary electrophoresis 52

(f) Polyacrylamide gel electrophoresis •.•.•••••••. 52

(g) Ultracentrifuge studies •••• 52 iv

B. Kinetic Properties ••••••••.•••••.••. 52

1. Activity of the Enzyme •.•••••••• 52

2. Effect of Temperature on the Enzyme Activity...... 59

3. Effect of pH on the Enzyme Activity...... 64

4. Substrate Specificity 64

5 • Inhibitor Specificity 64

(a) Product inhibition ..••.•.•• 64

(b) Inhibition by sulfhydryl reagents ••••••••••••.•.•••• 68

(d) Inhibition by aldehyde reagents •..•••••••••••••••. 76

C. Physical Properties .••..•.•••.•••••. 85

1. Spectral Properties ••••••.••.••. 85

2. Sedimentation Coefficients •••••• 85

3. Partial Specific Volumes •.•••••• 92

4. Molecular Weights .•••••••••••••. 92

(a) Molecular weights determined by Agarose gel filtration.. 92

(b) Molecular weights determined from Stoke's radii, sedimen tation coefficients, and par t i a 1 s p e c ifi c volum e s ••. 9 6

(c) Molecular weights determined from sedimentation-diffusion coefficients and Stoke~s radii •••..••••.•••..•.•••.. 102 v

5. Frictional Ratios .••.••••••.•••• 102 D. Chemical Properties ••.••••••••.•••.. 106

1. Metal Content ••••••••••••.•••.•. 106

2. Phosphorus Content ••••.••••••••. 106

(a) To ta 1 pho sphorus ••••.•••••• 106

(b) Phospholipid Phosphorus 112

( c) Flavin dinucleotide phosphorus •••••••••.•.••••• 112

3 • Organic Prosthetic Group •••••••• 114

4. Sulfhydryl Groups .••.••••••••••• 118

IV. DISCUSSIONS.AND.CONCLUSION •.••••.••••.••••• 131

V. SUMMARy ••••••••••.•.•••••..••••••••••••• o. 150

VI. BIBLIOGRAPHy...... 153 LIST OF TABLES

I. Purification of Beef Liver Mitochondrial Amine Oxidase .•.•••••••.••••••••••.••.••• 37

Modified Procedure for the Preparation of Amine Oxidase (FLOW SHEET) ••••••••••.• 38- 39

II. Substrate Specificities of the two Amine Oxidase Components ••••••.•••••••••.•••••• 67

III. A. Inhibition of Amine Oxidase by Sul- fhydryl Reagents ••••••••••••••••.•••• 73

III. B. Inhibition of Amine Oxidase by Sul- fhydryl Reagents ...•.•.••.••••...•••• 74

III. C. Inhibition of Amine Oxidase by Sul- fhydryl Reagents •••••••••••••..•.•••• 75

IV. Inhibition of Amine Oxidase by Metal Che1ating Agents •••••.•.•••••••••..•••••• 81 V. The Effect of Aldehyde Reagents on the Enzyme Activity ••••••..••••.•••••.••••••• 84

VI. A. Sedimentation Coefficients at Different Protein Concentrations of the Mito- c h 0 n dria1 Am ine 0 x ida s e ••••....•••••• 93

VI. B. Sedimentation Coefficients by Sucrose Density Gradient ••••••••••••••.•.•••• 94

VII. Agarose Gel Filtration Data of Standard Proteins, Blue Dextran 2000, and Amine Oxidase Components ••.•..••••••••...•.•••• 95

VIII. Molecular Parameters Obtained from Gel Filtration Data •.•••••••••.••••••..•.•••• 101

IX. Physical Parameters of the Mitochondrial Amine Oxidase •••.••••••••••.•.••••.••.••• 103

X. Molecular Weights of the Amine Oxidase Components by three Methods •••••••.•••••• 104

XI. Frictional Ratios of the Amine Oxidase Comp 0 n e n t s •..••••••••.•••..•••••...•••••• 105 vii

XII. Metal Content of Amine Oxidase ••••••••••• 111

XIII. Phosphorus content of Mitochondrial Amine Oxidase 113

XIV. A. Riboflavin, Adenine, Ribose, and Nucleotide Phosphorus Content of Mitochondrial Amine Oxidase...... 119

XIV. B. Riboflavin, Adenine, Ribose, and Nucleotide Phosphorus Content of Mitochondrial Amine Oxidase •••.•••.•••.•.••••.••.•• 120

XIV. C. Pyridoxal Content of Phosphorylase a and of the Mitochondrial Amine Oxidase Components •••••.•••..•••••.••••.•.••• 121

XV. Number of Titratab1e Sulfhydryl Groups in the Mitochondrial Amine Oxidase Components 128

XVI. A. Properties: 1a. Kinetic Parameters of Mitochondrial Amine Oxidase •••.••• 145

XVI. B. Properties: lb. Kinetic Parameters of Mitochondrial Amine Oxidase •.••••• 146

XVI. C. Properties: 2. Molecular Parameters of Mitocnondria1 Amine Oxidase ••••••• 147

XVI. D. Properties: 3. Chemical Parameters of Mitochondrial Amine Oxidase ••••••• 148 LIST OF FIGURES

1. Chromatography of the partially purified amine oxidase on the DEAE-cellulose column. 33

2. Hydroxylapatite column chromatography of the partially purified mitochondrial amine oxidase 0...... 36

3. Rechromatography of the purified enzyme component. 2 on the DEAE-cellulose

col urn n ...... •. 0•••••••••••••••••••••••••••• 42

4. Rechromatography of purified component 2 on hydroxylapatite •.•••.•.••••••••••••.•••• 45

5a. Chromatography of amine oxidase component 1 on Sephadex G-200 ••••••.••••••••••••••••••• 47

5b. Chromatography of amine oxidase component 2 on Sephadex G-200 •.•••••••.•••••..••.•••..• 49

6. Migration of the amine oxidase .components on starch block electrophoresis •••.••••••.•••• 51

7. Electrophoretic pattern of component 2 54

8. Polyacrylamide gel electrophoresis of . amine oxidase components 1 and 2 •••.••••••• 56

9. Sedimentation pattern of the amine oxidase component 1 •••.•••••••••••.•••••••••••••••• 58 lOa. Effect of temperature on the enzymatic activity 61 lOb. Effect of temperature on the activity of the amine oxidase •.••••.•••.••••••••••••••• 63

11. Effect of pH variation on the activity of the enzyme components 1 and 2 ••••••.••.•••••••. 66

12. Product inhibition studies •••••••••.••••••• 70 l3a. Inhibition of amine oxidase by sulfhydryl reagents ....•.•...... •...... 72 l3b. Lineweaver-Burk plot of benzylamine oxidation in the absence and presence of p-CMB •••••.• 78 ix

13c. Lineweaver-Burk plot of the benzy1amine oxidation in the presence and absence of p - CMB ••••••••.•.••••••.••••••••••••••••••• 80

14. Lineweaver-Burk plot of benzy1amine oxidation in the presence and absence of

cupr izone ...•...... 0••••••••••••• 83

15a. Absorption spectrum of the purified enzyme

component2 ....•....•. o ••••••••••••••••••• 87

15b. Reduction of the enzyme component 2 by substrate and sodium dithionite .•••••••••. 89

16. Sedimentation coefficients of amine oxidase component 1 at varying protein concentrations 91

17. Agarose gel fYl:tration data of various standard proteins and of the Dextran 2000, and amine oxidase components •••••••••••••• 98

18. Correlation of Kd with Stoke's radius ••.•• 100 19a. Copper content of the enzyme •••••••••••••• 108

19b. Iron content of the enzyme .••"...... 110

20. Flavin content of the enzyme •••••••••••••• 116

21a. p-Ch1oromercuribenzoate titration of component 1 •.••.•••••••••••••••.•••••••••• 123

21b. p-Ch1oromercuribenzoate titration of the component 1 in the presence of urea •••••• 125

21c. p-Ch1oromercuribenzoate titration of the component 2 ••••••••••••••••.••••••••.••.• 127

22. Activity of amine oxidase component 1 during p-CMB titration ••••••••••••••••.•• 130 x·

LIST OF ABBREVIATIONS o A Angstrom

DEAE-cellulose Diethylaminoethyl-cellulose

M Molar concentration mg mill igram ml milliliter o Degree(s) Centigrade mu mill imicron (s) s Sedimentation coefficient s(obs) Observed sedimentation coefficient Sedimentation coefficient corrected s20, w to water as solvent at 20 0 • S Svedberg Constant (1 S =s20,Wx 10- 13 sec) • TCA Trichloroacetic acid cm Cent ime t e r (s) ug microgram (s) mole gram molecule

a tom (s) microa tom (s)

mole (s) micromole (s) mp.mo le (s) millimicromole(s)

Michaelies Constant

Ki Inhibition Constant -SH group(s) Sulfhydryl group(s)

D Diffusion coefficient

N Normal concentration

% Percent p-CMB parachloromercuribenzoate ABSTRACT

Beef (steer) liver mitochondrial amine oxidase was prepared according to the method reported earlier (Adv.

Pharmacol., ~, Part A, 43, 1968). In addition to the usual preparation with high activity, (component 2, specific activity of 8,000) another component of the enzyme (component 1) with lower activity (specific activity 3,000) was isolated (Biochem. Biophys. Res.

Commun., submitted). Studies were made on some physical and chemical properties of these two components.

The amine oxidase components were bright yellow in color; they were thermolabile, and unstable at room temperature. The rate of inactivation of component 2 was faster than that of component 1. The optimum pH for activity was found to be 9.2. Both the components were non-competitively inhibited by p-chloromercuribenzoate.

Metal chelators like cuprizone, 8-hydroxyquinoloine,

~-phenanthroline inhibited the enzyme components. Ammonia or aldehyde reagents did not have significant effects on

the activity. Both the components had almost the same substrate specificity.

The molecular weights of the enzyme component 1 was found to be 400,000 by the gel filtration technique,

396,000 ~ 10,000 on the basis of Stoke's radius,

sedimentation coefficient, and partial specific volume, xii

and 425,000~ 10,000 on the basis of sedimentation

diffusion method. These values for component 2 were

1,300,000, 1,195,000, and 1,355,000, respectively.

The sedimentation coefficients of component 1 and component 2 were 14.4 + 0.3 and 20.6, respectively.

Metal analyses of the enzyme yielded 1 gram atom of copper per 400,000 grams or 3 gram atoms of the metal per mole of component 2. Other metals, such as cobalt,

iron, manganese, and molybdenum were examined and found

to be either absent or insignificant (J. Biol. Chem.,

241, 2774, 1966).

Both the components of the mitochondrial enzyme were found to be flavoproteins. This was amply proved

(1) from their riboflavin content as determined micro biologically, and spectrophotometrically (Biochem.

Biophys. Res. Commun., 23, 324, 1966), (2) from a steady

increase of riboflavin during purification processes,

and (3) from the spectrum of flavo-peptide obtained from pronase digest of the enzyme. Besides, the prosthetic

group was found to contain ribose (Biochem. Biophys. Res.

Commun., 29, 562, 1967), adenine and phosphorus in

integral values suggesting that the "flavin prosthetic"

group was a flavin adenine dinucleotide of unknown

structure. Accordingly, component 1 contained 4 and

component 2 contained 12 FAD or FAD-like substance per mole, respectively. xiii

Examination of the sulfhydryl groups revealed that components 1 and 2 of the enzyme contained 28 and 86 titratable sulfhydryl residues, respectively in their molecules, and that they were not directly involved in enzyme catalysis. In addition, the enzyme was found to contain 24 and 106 moles of phospholipid in components

1 and 2, respectively.

Finally, it appeared that the high molecular weight component was the native form from which the small molecular component arose during the purification of the enzyme, although no interconversions were observed with the purified enzyme preparations. I. INTRODUCTION

Enzymology has gained enormous popularity in a very short time as compared to other disciplines in biochemistry. It has also become of great importance in other health related fields such as microbiology, pharmacology, toxicology, pathology, medicine, etc.

However, to a biochemist, the enzyme has a very special significance since life itself depends on a network of complex biochemical reactions which are catalyzed by enzymes.

One may ask questions such as how do enzymes act?

What makes them so unique as to be able to mediate such complex biological reactions? What are their sizes and shapes? What are they made up of? Biochemists and physical chemists have attacked these questions with vigour in an effort to answer these questions and con siderable progress has occurred. (Vide the work of

Phillips group on lysozyme, the work of numerous labora tories with ribonuclease, the results of Lipscombs laboratory on carboxypeptidase, etc.). However, the studies have all been made with enzymes which can be readily isolated from cells. On the other hand, mito chondrial enzymes are in a different class because of the fact that they are difficult, in general, to free 2 from the mitochondria or from the mitochondrial fragments. Nevertheless, experience has shown that it is essential to obtain the homogeneous enzyme in order to obtain meaningful physicochemical values. Thus, a considerable amount of time and effort must be spent in order to work out precise isolation procedures in the case of mitochondrial enzymes. Once the purifica~ion procedure is developed, various properties of the pure enzyme from the mitochondria can be investigated like those of the more readily isolatable enzymes. The informations derived from these investigations can thus provide reasonable answers to those questions mentioned above.

A. Historical Background of Amine Oxidase Studies

Amine oxidase is the common name for a group of enzymes which catalyze the following general reaction:

R-CH2NH2 + H20 + 02 = R-CHO + NH3 + H202. The enzyme was first described by Hare in 1928 (1) as catalyzing the oxidative deamination of tyramine. She termed this enzyme, tyraminase.

It was soon realized that this enzyme was widely distributed not only in animals, but in plants, and in bacteria. The early history and distribution of amine oxidase have been described in numerous review articles

(2- 4) . 3

Although the general reaction shown is catalyzed by a typical amine oxidase, it was soon observed that there occurred certain differences among these enzymes depend ing on the sources they were obtained from. It was found that those enzymes, which were mainly bound to the mitochondria of animals had a substrate specificity distinct from those of the animal plasma, plant, or bacterial enzymes. Thus, the mitochondrial enzymes were found to attack tyramine, tryptamine, catechol amines and other "biogenic" (5) monoamines, and benzylamine. Unlike the diamine oxidase of hog kidney

(6-8) or that of pea seedlings (9-14), the histaminase of pig plasma (15-17), or the amine oxidase of beef plasma (18), the mitochondrial amine oxidase did not attack cadaverine, histamine, or putrescine; nor did it catalyze the breakdown of spermine and spermidine.

Moreover, the aldehyde reagents which are known-to inhibit histaminase (19,20) and related oxidases (21-23), did not inhibit the mitochondrial amine oxidase. These observations indicated, that the mitochondrial enzymes were a class of enzymes distinct from those of the kidney diamine oxidase, pig plasma histaminase, or beef plasma enzyme which are known to be copper-pyridoxal enzymes (24). Studies have been made on the purifica tion and properties of a number of mammalian plasma and kidney enzymes. Thus, Blaschko and Bufoni purified 4 and crystallized pig plasma histaminase (25) and studied various physical and chemical properties (26) of it.

Yamada and Yasunobu purified, crystallized, (18) and

investigated the properties of beef plasma enzyme

(27,28). McEwen reported on the purification of and kinetic studies on human (29,30) and rabbit (31) plasma enzymes. Purification has also been reported for amine oxidase in insects (32) and in microorganisms (33).

On the other hand, little progress in the purifi cation of the mitochondrial amine oxidase has occurred due to the particulate nature (34) and the relative

insolubility of this enzyme (24). Although the isola

tion of partially purified mitochondrial amine oxidase has been reported (35), a highly-purified preparation

that could be employed for studying the properties of

this enzyme was not available. It is only recently that the purification problem has largely been overcome by using special techniques. Thus, Barbato and Abood (36)

liberated the enzyme from the insoluble mitochondrial

structures by using a non-ionic detergent, Triton X-IOO.

Some workers used sonication (37), and sonication in the presence of substrate (38), to release the enzyme from particulate structures.

Recently, Erwin and Hellerman (39) purified the

amine oxidase from the bovine kidney mitochondria by 5 using digitonin as the solubilizing agent. Tipton (40) used repeated sonication and thawing to liberate the

amine oxidase from pig brain mitochondria. These authors

also made some investigations on the properties of their preparations.

B. Physiological Significance

Earlier literature suggested that amine oxidase was involved in the detoxication (2) or in the oxidative deamination of biologically active amines in animal

systems (41-45). Since certain members of the biogenic

amines are associated with hypertension (46) and hyper sensitivity (47), the amine oxidases were considered to be involved in the enzymatic removal of these amines.

In other words, the amine oxidases are involved in the

"detoxication" of the biologically active amines (48).

The rich supply of this enzyme in the intestinal mucosa

indicates a protective function. The enzyme, it is reported, thus prevents many amines formed in the gut by bacterial decarboxylases, from entering the general circulation. This protective role has been supported by recent findings on the effects of monoamines when the oxidative deamination activity was blocked by amine oxidase inhibitors (49,50). Some workers, at the same

time reported that the products of enzymatic deamination of monoamines alter significantly the pattern of 6

car~ohydrate metabolism in some tissues. Barondes (51) reported that a number of aldehydes stimulate the glucose oxidation in beef anterior pituitary slices and suggested that the aldehydes originating from biogenic amines by enzymatic deamination are responsible for this. Moreover, pep pills or psychic energizers such as tranylcypromime, phenelzine (49), or pargyline (50) are potent inhibitors of mitochondrial amine oxidase.

This finding suggests that mitochondrial amine oxidase may possibly be important in maintaining the normal' mental state of human individuals by regulating the levels of catecholamines and other biogenic amines in

their systems. c. Statement of the Problem

The objective of this work was to purify the beef

liver mitochondrial amine oxidase and to study some physical and chemical properties of this enzyme. The

enzyme is a very special one since it is tightly bound

to the insoluble membrance of the mitochondrion (34,52).

Many laboratories attempted its purification without

apparent success. In this laboratory a 50-fold

purfication of the enzyme was achieved (53) for the

first time and a reasonably pure preparation was 7 available for the preliminary study of some of its properties. Recently, our laboratory improved the purification method to a great extent and highly purified preparations with very high activity were obtained (55).

More recently, better yields and multiple enzyme com ponents with amine oxidase activity have been isolated.

The present work will describe investigations of the highly purified enzyme components and will include the following major aspects: (i) Purification and demon stration of purity (ii) effects of various physical factors such as pH, temperature, etc., on the enzymatic activity; (iii) effects of various inhibitors; (iv) sedimentation behavior; (v) molecular weight determina tion; (vi) determination of metal components; (vii) studies on cofactors; (viii) determination of the number of sulfhydryl groups; and (ix) other properties of the enzyme. II. MATERIALS AND METHODS

A. Materials

1. Materials and Reagents Obtained Commercially

(a) J. T. Baker Chemical Co., New Jersey

2,6-Dimethy1 Pyridine (Lutidine)

(b) Bio-Rad Laboratories, California

Agarose (Bio-Ge1 A-1.S m) Beads, 100-200 mesh

Agmatine Sulfate

Cadaverine Dihydroch1oride

Ferritin

G1ucose-6-Phosphate, Disodium Salt

n-Hepty1amine

Trimethylene diamine Dihydroch1oride

(d) Carl Schleicher & Schue1 Co., New Hampshire

Diethy1aminoethy1 (DEAE)-ce11u1ose

(e) Cyc10 Chemical Corporation, California

Di,thioerythrito1

(f) Difco Laboratories, Michigan

Yeast Extract

(g) Eastman Organic Chemicals, New York

Acry1amide

Amido Schwarz

1-Amino-2-Naphthol-4-Su1fonic Acid Benzy1amine 9

Butane Diamine Dihydroch1oride

N,N-Methy1ene-Bis-Acry1amide

N-(1-Naphthy1)-Ethy1ene Diamine Dihydro-

chloride

N,N,N',N'-Tetramethy1ethy1ene Diamine

Tyramine Hydrochloride

(h) Fisher Scientific Company, New Jersey

Nessler's Reagent

(i) The G. Frederick Smith Chemical Company, Ohio

Bis-Cyc1ohexanone Oxa1dihydrazone (Cuprizone)

4,7-Dipheny1-1,10-Phenanthro1ine

(Bathophenanthro1ine)

Hydroxy1ammonium Chloride, 10% Solution,

Iron-Free

Sodium Acetate, 10% Solution, Iron-Free

Standard Iron Solution

(j) Hawaii Meat Co., Honolulu, Hawaii

Steer (Beef) Liver

(k) Mann Research Laboratories, Inc., New York

o-Dianisidine

Kynuramine Dihydrobromide

(1) Matheson Coleman & Bell, Ohio

Potato Starch

(m) Nutritional Biochemicals Corporation, Ohio

Bovine Serum Albumin (2 x recrystallized) 10

(n) Pharmacia Fine Chemicals, Inc., New Jersey

Blue Dextran 2000

Sephadex G-25, Coarse Grade

Sephadex G-200

(0) Pierce Chemical Company, Illinois

Cholic Acid

(p) Sigma Chemical Company, Missouri

DL-Arterenol (Norepinephrine) Hydrochloride

Catalase (6 x recrystallized)

p-Chloromercuribenzoic Acid, Sodium Salt

Cytochrome c, Type V, From Beef Heart

Flavin-5-Phosphate (FMN) Sodium Salt

Mescaline Sulfate

Spermidine Trihydrochloride

Spermine Tetrahydrochloride

Tryptamine Hydrochloride

(q) Worthington Biochemical Corporation, New Jersey

Peroxidase (from Horse Raddish)

Phosphorylase a

(r) Upjohn Research Laboratories, Michigan

5-Hydroxytryptamine (Serotonine) Sulfate

(s) Van Waters & Rogers, Inc., California

Phenol (Folin-Ciocalteau) Reagent

2. Materials Obtained as Gifts

~. coli K 12, from Dr. Morton Mandel 11

Dept. of Biochem. & Biophys. UH

E. coli C 406, from Dr. John B. Hall

Dept. of Biochem. & Biophys. UH

Triton x-lOO, from Rohm & Hass, Pennsylvania 12

B. METHODS

1. Preparation of Adsorbents and Ion Exchange

Materials

(a) Alumina Cy. was prepared according to

Willstatter and Kraut (55).

(b) Calcium phosphate gel was prepared by

the method of Keilin and Hartree (56).

was treated according to the procedure of Peterson

and Sober (57). The dry material was allowed to

sink freely in lN NaOH and the suspension was

filtered on a sintered glass filter. Washing with

lN NaOH was repeatedly done until no more yellow

color was removed. The material was now treated with

sufficient lN HCl to make a strongly acid suspension,

which was immediately filtered and washed free of acid

with water. The filtered substance was again sus-

pended in lN NaOH, washed free of alkali with water,

and finally suspended in the selected starting buffer.

(d) Hydroxylapatite was prepared by the

method of Tiselius et al (58).

(e) Starch (for starch electrophoresis) was

treated by the procedure described by Fine and

Costello (59). 13

Commercially obtained potato starch was suspended in approximately 3 volumes of distilled water and allowed to settle. The supernatant was decanted, removing suspended impurities and fine starch particles. After it was washed 3 times with water, the starch was washed 3 times with the buffer in which electrophoresis was conducted. The starch, thus treated, was kept under the same buffer in the cold room (at 0-4 0 ) for routine use.

(f) Sephadex G- 200, obtained as a dry powder, was added to excess water and was allowed to stand for 3 days with occasional stirring and decantation. The swollen gel was washed 3 times with the starting buffer at 0_4 0 before packing the column.

(g) Agarose (Bio-Gel A-l.5 m) beads, 100

200 mesh, was obtained in 0.001 M tris-EDTA buffer medium containing 0.02% sodium azide as a preserva tive. The agarose column was exclusively washed free of azide and tris-buffer by running large volume of starting buffer through the column.

2. Electrophoresis

(a) Starch block electrophoresis

Starch block electrophoresis was done according to the method of Fine and Costello (59). 14

Blocks were prepared with starch in plastic trays

(44 x 3 x 1.5 cm). For the electrophoresis run, potassium phosphate buffer, pH 7.4, with an ionic strength of 0.1, was employed. Samples were dialyzed against the same buffer for 3 hours with

2 changes and were applied in amounts of 10 to 20 mg enzyme in 2 m1 portions. Separation was effected in the cold room (at 0 - 4 0 ) with a voltage of about

400 volts and between 10 and 15 mA per block for 18 -

24 hours.

(b) Polyacrylamide Gel Electrophoresis

Polyacrylamide gel electrophoresis was done according to the method described by Taber and

Sherman (60) with an alteration in solution (a). In the present experiment, it consisted of the following composition per 100 m1 of solution: 8 m1 1N KOH,

1.9 gm glycine, and 0.077 m1 N,N,N',N'-tetramethy1- ethylene diamine, pH 10.3. The gel system used contained 3.75% acry1amide. Gel columns (65 x 6 mm) were prepared in 95 x 6 mm i.d. pyrex tubes. They

\ . were soaked in solution (a) wh~ch was diluted to the same concentration that occurred in the gel, for 2 days to diffuse out any unreacted materials. Samples containing 50 to 100 ug in 10 to 20 u1 quantities 15 were applied to the gel columns, layered on with diluted lutidine-glycine buffer, pH 8.3, and run in the same buffer at a potential of 410 volts and 3 mA per tube for 2 hours. At the completion of electrophoresis, gel columns were removed from the tubes and stained by immersing them in a solution of Amido

Schwarz for 45 minutes. The gel columns were destained by washing with 7.5% acetic acid and stored in the same acid solution.

3. Ultracentrifuge Studies

(a) Sedimentation velocity measurements were made in a Spinco Model E Analytical Ultracen- trifuge equipped with a RTIC unit for controlling the rotor temperature within + 0.1°. The conventional 12 mm aluminum cell with a 4° sector shaped centerpiece was used for all runs. The speed employed was 35,600 rpm (73,684 x g) using a rotor type An-D and the rotor temperature was 22.5 0 • The sedimentation coefficient was calculated by using the following equation:

1 dx (i) s= dt w"2 x where x is the distance of the boundary from the axis of rotation in centimeters, t is the time in seconds, and w is the angular velocity in radians 16

per second (211 rpm) . The observed sedimentation 60 coefficients (sobs) were corrected to the standard conditions (S20,w) in terms of the density and viscosity of water as the solvent at 20 0 according to

Svedberg and Peterson (61).

(b) Sucrose density gradient centrifugations were carried out according to the procedure described by Ames and Martin (62). The present method, however, differed only in that a Beckman Model L 2-65 Ultra- centrifuge, with a swinging bucket rotor, SW-4l in which 14 x 89 mm cellulose nitrate tubes, were used.

A linear sucrose gradient, made from a 20% and 5% sucrose in 0.1 M potassium phosphate buffer, pH 7.4" containing 1 x 10- 4 M dithioerythritol, was used in all experiments. Gradients of 11.5 ml in each tube, prepared by using a Buchler Polystaltic Pump were equilibrated for 4 to 8 hours at 0-4 0 in the cold room and centrifuged at 25,000 rpm (75,000 x g) for

16 hours at 0 0 after applying samples.

4. Preparation of mitochondria

Beef liver mitochondria were prepared by the method of Schneider and Hodgeboom (63). Select steer livers, obtained immediately after slaughtering, were 17

brought from the Hawaii Meat Company. Membranes,

large blood vessels, and bile ducts were removed.

Weighed liver slices were homogenized in 9 volumes

of cold 0.25 M sucrose with a Waring blendor for

2 minutes at 0-4 0 • The homogenate was centrifuged in

a Model PR-2 International Refrigerated Centrifuge

at 700 x g for 10 minutes. The supernatant was

carefully decanted and re-centrifuged at 5000 x g in

a Sorval Refrigerated Centrifuge, Model RC 2-B for 10

to 15 minutes. The opalescent supernatant, together

with a pink partially sedimented layer of particles

above the firmly packed pellet of mitochondria, was

discarded. The mitochondrial pellet was washed two

times with one-third the original homogenizing volume

of 0.25 M sucrose and then centrifuged at 24,000 x g

for 10 minutes. The washing procedure waS repeated

once with 1.15% KCl solution and the mitochondria,

thus prepared, were stored frozen in 0.01 M potassium

phosphate buffer at pH 7.4.

5. Measurement of Enzymatic Activity

The enzymatic activity was determined by the

spectrophotometric method of Tabor, Tabor and

Rosenthal (64) using benzylamine as the substrate. In

this work, 2.85 ml cif 0.2 M potassium phosphate buffer,

pH, 7.4, were added to a I-cm cell containing 0.1 ml 18

of enzyme solution and the sample was mixed. To this cell, 0.05 ml of 0.1 M benzylamine solution was added to make a total volume of 3 ml and a final subtrate concentration of 1.67 mM. The assay solution was mixed by inversion. A blank was prepared likewise except that the substrate was omitted. Readings were made at 250 m¥ initially and then subsequently every minute for 5 minutes.

One unit of enzymatic activity was defined as the amount of enzyme that produced a change in absorbance of 0.001 per minute at 250 mp at 25 0 •

Specific activity was expressed as the number of units of activity per milligram of enzyme. The enzyme protein was measured by the method of Lowry ~ al (65) using bovine serum albumin as the standard. In activity measurements, the amounts of enzyme used showed activity in the range of 10 to 50 spectrophoto metric units.

6. Determination of Hydrogen Peroxide

Substrate specificity of the amine oxidase was determined for various amines by a method developed by McEwen (29) by coupling the normal reaction with peroxidase in the presence of o-dianisidine (66).

In this reaction, the peroxide formed as a product of the oxidative deamination of amines by amine oxidase 19 converts peroxidase to peroxide-peroxidase complex

(complex 2) which then converts o-dianisidine to a reddish brown compound as shown in the following reactions:

R-CH -NH2+ H20 + 02 Amine> R-CHO + NH3 + H202 Oxidase

Peroxidase + H202--~>~p-p-Complex (Complex 2)

H3CO OC H3 -----.:::>~Peroxidase p-p-Complex + H2 N -0---0-- NH2 o-Dianisidine H3CO OC H3 + HN ==0==0= NH Reddish Brown Color For this experiment, 5 mg of horse radish peroxidase and 8000 units of amine oxidase were dissolved in

99 ml of 0.1 M potassium phosphate buffer, pH 7.4. To this enzyme mixture was added 1 ml of o-dianisidine solution made by dissolving 10 mg o-dianisidine (2 x rec~y- stallized) in 1 ml 95% ethanol. The resulting enzyme- chromogen (approximately 4 x 10-5M) solution was filtered. To 2.9 ml aliquots of this solution were added 0.1 ml of the amine solutions being assayed, so that the final concentrations of these amines were the same (3.3 x

10-3M) in all tubes. A reagent blank was prepared in the same way except that the amines were omitted.

After 15 minutes, the reddish brown color was measured at 450 m)l against the blank. 20

7. Determination of the Partial Specific Volume, V

Since the term (l-Vp) is contained in the

equation for molecular weight determination by the

sedimentation-diffusion method, Stoke's law, and by

the sedimentation equilibrium method, the partial

specific volume, V, has to be determined. The measurement of this parameter was done by the method of Schachman (67). Accordingly, the

densities of solvent and solution were measured pycnometrica11y, and the amount of protein in

solution was determined. The apparent partial

specific volume was calculated by using the following

equation:

Vapp = 1/do-1/x(d-do )/do .... (ii) where x is the concentration of protein in grams per mi1i1iter of solution, and do and d are the densities

of solvent and solution, respectively.

8. Determination of Molecular Weight

(a) Molecular weight by the gel filtration

method

Gel filtration techniques published by

Whitaker (68) and Andrews (69) were employed for the molecular weight determination. Agarose (Bio-Ge1

A-l.S m) gel was packed in a 120 x 1.9 (i.d.) cm column, and the column was equilibrated with 0.05 M potassium phosphate buffer, pH 7.4, containing 0.01 M 21 mercaptoethanol. The same buffer was used for elution of protein standards and markers for the determination of

'elution vOlume' and 'void volume.' The void volume was determined by passing~. coli (44) through the column and measuring the turbidity due to these organisms. The column was then calibrated with standard proteins of known molecular weights before running the enzyme sample in the column.

The elution volume is defined as the volume of buffer eluted corresponding to the peak concentration of the solute. Fractions of 3 ml were collected and a standard curve was constructed by plotting the ratios of the elution volumes to the void volume against the logarithms of the molecular weights according to the method of Whitaker (42).

(b) Molecular weight from sedimentation coeffici ent, Stoke's radius, and the partial specific volume

Elution volume is a function of the molecular radius (or the Stoke's radius) of a protein molecule upon chromatography on a gel column (45). A calibrated gel filtration column can be used for the estimation of

Stoke's radius of a macromolecule present even in the impure form, provided a method for the assay of the macromolecule is available. The molecular or Stoke's radius is determined from the gel filtration data 22 presented in terms of a distribution coefficient, Kd, which is a function of the molecular size. This parameter is defined as follows:

(iii) when Ve = elution volume, Vo = void volume, and Vi= 1 volume inside the gel grain. When the Kd~ values of the standard proteins are plotted against their molecular (Stoke's) radii, a linear curve is obtained

(72). The molecular or Stoke's radius of a macromolecule can easily be determined from the constructed standard curve.

The sedimentation coefficient of a macromolecule can be determined by the sucrose density gradient technique or by the conventional sedimentation velocity method (if the material is pure). The molecular weight and the frictional ratio of the macromolecule can be accurately determined, if the partial specific volume is reasonably known. The molecular weight and the frictional ratio, therefore, can be determined from the relationship defined by the following classical equations:

6:ff n M = Nas ( iv) (l-Vp ) 1 (3V M)3 fifo = al 4.1T N .. (v) when M is the molecular weight, n is the viscosity of the medium, a is the Stoke's radius, s is the sedimentation 23

coefficient, V is the partial specific volume, p is the

density of the medium, f/fo is the frictional ratio and N is the Avogadro's number.

9. Metal Analyses

Purified amine oxidase components were analyzed

for their metal contents. In these experiments, 5 mg of

the purified enzyme were used for each analysis.

Copper was determined in the purified components

according to the method of Peterson and Bollier (73).

The assay solution was prepared both by extraction of

copper with 10% trichloroacetic acid and by wet ashing

(74) . Cobalt estimation was done by the ~tomic absorp-

tion spectrophotometric method of Fuwa et a1. (75) •

Iron was analyzed by the procedure of Peterson (76) on

dry or wet ashed samples of the enzyme. Manganese was

determined by atomic absorption spectrophotometry as

described by Fuwa et a1. (75) . Molybdenum was measured

by the method described by Sandell (77).

10. Determination of Riboflavin

Riboflavin was determined both microbiologically

and spectrophotometrica11y.

In the microbiological assay, the growth response

of Lactobacillus casei was measured as a function of

riboflavin concentration according to the method of Snell

and Strong (51). As these micro-organisms depend on 24 riboflavin as a growth factor, a medium containing all the necessary growth factors except for riboflavin was prepared.

The growth of the micro-organisms in the media with varying concentration of riboflavin was measured by titrating the acid produced in each tube. A standard curve was constructed by plotting the volume of acid produced and the known concentrations of riboflavin added to each tube. The riboflavin content in the unknown sample was determined from the standard curve.

The spectrophotometric determination of the flavin component of the enzyme was based on the reduction of the 450 m~ absorptkn upon addition of sodium hydrosu1fite

(Na2S204) to the enzyme. The difference between the absorbances at 450 m~ before and after the addition of hydro sulfite was a measure of the flavin nucleotide content of the enzyme. The flavin concentrations were calculated from the molar absorbancy index of FAD at m~ 4 2 1 450mr (E450 = 1.13 x 10 cm mo1e- ). 11. Determination of Purine

Microbiological assay of the purine content of the enzyme was made by measuring the growth response of a special mutant of E. coli (E. coli C 406) which requires purines in addition to other nutrients for their growth.

The medium was prepared according to the procedure of

Sedat and Sinsheimer (79), and the organisms were grown in 100 m1 aerated cultures. After 12 hours, the organisms 25 were harvested and suspended in a sterile 0.9% NaCl solution. A set of tubes each containing 9 ml of medium with increasing concentrations of adenine were inoculated with 1 ml of E. coli suspension. A blank was similarly prepared with the exception that it did not contain any adenine. The standard as well as the blank tubes were incubated at 37 0 for 18 hours after which the turbidity was measured at 650 m~. A standard curve was drawn by plotting turbidity (O.D. at 650 m~) against adenine concentrations.

Enzyme samples were hydrolyzed for purine deter mination according to the method of Vischer and Chargaff

(80). For the experiment, 4 mg of purified enzyme were hydrolyzed in 5 ml 1 N H2S04 at 1000 for 1 hour. The precipitated protein was filtered and washed 3 times with 0.5 ml portions of 0.1 N H2S04. After adjusting the pH to 6.8 with 2 N KOH and the volume to 10 ml with water, the filtrate was employed for purine determination

~n the same way as standard.

12. Determination of Adenine

For this assay, 12 mg of pure enzyme were hydrolyzed in 1 N H2S04 exactly in the way stated above for the purine determination. However, the pH of the sample was adjusted, instead, to a pH of 1 with 10 N KOH. Adenine was determined by the colorimetric method of Koritz et ale (81). The determination is based on a color 26 reaction of adenine with N-(1-naphthy1) ethylenediamine hydrochloride after its reduction with zinc dust and diazotization with NaN0 2 . The absorbance of the red color developed is measured spectrophotometrica11y at

505 mp.

13. Determination of Ribose

The ribose content of the enzyme was measured by the orcinol test first proposed by Bia1 (82) and later modified by many others (83,84). In this case, 4 mg of pure enzyme were first hydrolyzed with 0.5N KOH for

48 hours at 25 0 to liberate all the ribose quantitatively

(85) as purine nuc1eotides. The hydrolysate was then adjusted to pH 1-2 by dropwise addition of 20% HC104.

The modified method of Dische (86) was employed for the quantitative dete~mination of ribose.

14. Determination of Phosphorus

Phosphorus was determined by the u1tramicro chemical method described by Bartlett (87). In this experiment, 5 mg enzyme were precipitated with ice cold trich10ro-acetic acid (TCA) such that the final TCA concentration was 7%. The precipitated enzyme was centrifuged and the supernatant was discarded. The enzyme precipitate was washed 3 times with 5 m1 portions of 1% ice cold TCA. The enzyme precipitate was then wet ashed and analyzed for phosphorus according to the method referred to above (87). 27

15. Analysis for Phospholipid

The procedure for the extraction of lipids from the enzyme is described by Folch ~ al. (88) . S amp 1es containing 5 mg of enzyme in a volume of 1.5 ml were extracted with 5 m1 of a 2:1 ~hloroform-methanol mixture in 50 m1 glass-stoppered conical centrifuge tubes.

The chloroform extract was transferred to a fat-free filter paper, and filtered into a 25 ml volumetric flask.

The extraction was repeated 3 more times. Occasionally, the aqueous and ch~oroform layers were separated by centrifugation when they did not separate clearly. The lipid content was determined on 20 ml aliquots by the method mentioned above (88). Phospholipid was measured by determining the phosphorus present in the lipid extract according to the method of Bartlett (87).

16. Determination of the Sulfhydryl Groups

The sulfhydryl groups were determined by the spectrophotometric method of Boyer (89). In order to pre~ent air oxidation of the sulfhydryl groups, the

0.05 M potassium phosphate buffer, pH 7.0, was equi1i- brated with nitrogen. Since the enzyme readily precipitated at the neighborhood of pH 5, determinations were made at pH 7.0 (a) in the absence of urea, and (b) in the presence of 8 M urea. Twice recrystallized p-ch1oromer- curibenzoic acid (p-CMB, absorbance index, E232 mJl =

1.69 x 104 cm2 mo1e- 1 in 0.05 M potassium phosphate 28 buffer, pH 7.0) was used for the titration of the -SH groups. Increasing amoun~of p-CMB (3 x 10- 4 M) solution were added to a constant amount (0.4 mg) of enzyme in an initial volume of 1 ml. Titration in the presence of urea was done by adding sufficient 10 M urea (in buffer) to the enzyme solution so that the final urea concen tration was 8 M. The p-chloromercuribenzoate

(3 x 10- 4 M) solution used in this titration also contained 8 M urea. In all experiments, appropriate blanks were used to correct absorbances due to protein and p-CMB. Spectrophotometric readings were taken at

250 m~l hour after each addition of p-CMB. III. RESULTS

A. Purification and Purity Studies

1. Purification of the Mitochondrial Amine Oxidase

The amine oxidase was prepared from steer (beef)

liver mitochondria according to the method described

by Yasunobu, Igaue, and Gomes (54) except for some

alterations. The method referred to above comprised

(a) homogenization of the purified mitochondria by

using Potter-Elvehjem homogenizer, (b) extraction of

the enzyme with Triton X-lOa (a non-ionic detergent),

phosphate gel treatment, (e) DEAE-cellulose column

chromatography, followed by (f) hydroxylapatite

column chromatography, and finally (g) electrophore-

sis in the starch block. In the present purification

procedure, however, alterations were made in the

calcium phosphate gel and in the hydroxylapatite

column chromatography steps which are described in

some detail as follows:

(d) Calcium phosphate gel treatment

The reddish brown enzyme solution from the third

step of purification was dialyzed against 4 liters

of 0.01 M potassium phosphate buffer for 2 hours at

0-4 0 , after which the dialyzing buffer was changed

and the dialysis was continued for an additional 30

2 hour period against the same buffer. To this dialyzed enzyme, was added enough calcium phosphate gel to make an overall p~otein to gel ratio of 1.4.

The mixture was gently stirred while adding the gel and the stirring was continued for 15 minutes. The gel-enzyme mixture was centrifuged in a Model PR-2

International Refrigerated Centrifuge for 20 minutes at 850 x g using rotor No. 276 a. The supernatant

(S) was decanted into a 2 liter beaker and was saved

for the_total absorption later.

The gel (Gl.l) was eluted with 200 ml of 0.1 M potassium phosphate buffer, pH 7.6, and then twice successively with 200 ml portions of 0.2 M buffer, pH 7.6. The eluates (termed Sl.2,Sl.3, and Sl.4 in

the Flow Sheet) were combined and desalted in a

Sephadex G-25 (coarse grade) gel column (4.5 x 45 cm)

and was again treated with calcium phosphate gel such

that the protein to gel ratio was 1.1 on the basis of

the initi~l protein concentration. The mixture was centrifuged. The gel (G c ) was discarded and the yellow supernatant (S3) was saved.

The supernatant (S) saved above for the total

absorption was treated with calcium phosphate gel on

the initial protein to gel ratio of 1.5 and the mixture was stirred slowly for 1 hour. The gel obtained by centrifugation of this mixture absorbed ------~--~. ---.... _-_.------

almost all the enzyme activity from the supernatant

(8). The gel containing the enzyme was washed with

about 1 liter of 0.01 M potassium phosphate buffer,

pH 7.4, and was centrifuged. The supernatant (8i.l)

was discarded and the gel (G2.l) was eluted in the

same manner as described for gel (Gl.1) above. All

the eluted fractions (82.2,82.3, and 82.4) were

combined with the supernatant (83) kept aside above.

The combined supernatant (combined supernatant) had

a final volume of 1.2 liters to 1.5 liters. The

enzyme contained in this solution was concentrated,

desalted, and passed through a DEAE-cellulose column.

(e) DEAE-cellulose column chromatography

The DEAE-cellulose column chromatography was

carried out in the same way as described earlier (54).

The present work, however, differed from the earlier

(54) one in that, the enzyme fractions having

specific activities of 1,500 to 4,000 were collected

(Figure 1). The procedure for concentrating and

desalting the enzyme before applying it to the

hydroxylapatite column was similar to that reported

previously (54).

(f) Hydroxylapatite column chromatography

The hydroxylapatite column (2.9 x 18 cm) was

equilibrated with 0.01 M potassium phosphate buffer, V> r->

Figure 1. Chromatography of the partially purified mitochondrial

amine oxidase on the DEA~-c~llulose column. Protein (about 340 mg),

containing 7 x 105 units of activity was applied to a column (45 x 2.2 cm)

which was equilibrated with 0.01 M potassium phosphate buffer, pH 7.4.

Gradient elution (900 ml of 0.1 M potassium phosphate buffer in mixing

flask and 900 ml of 0.2% Triton X-lOO in the same buffer in the

reservoir) was used to elute the enzyme. Fractions of 12 ml were col-

1ected at a flow rate of 0.75 m1 per minute. The symbols used are:

-0-0-, enzyme activity (units per milliliter); -e-e-, protein concen-

tration (milligram per milliliter); -~-~-, specific activity (units per

milligram of protein), and -x-x-, Triton X-lOO concentration (%). 33

£_01 X A11J\ 1.1::>'1 ::>1.~1::>3dS 0 10 00 to V (\J (IWI OW) N13.l0~d q 0 0 0 0

001 -x NO.lIH.l 0 10 \ 0 \ \ .&; \ 0 -u x 0 0 \ Q) \ \ E 10. 0 en -a:: w 0 en en m .-I c QJ 0 :i!: :::> l-< :l -(,) Z eo 0 ... 10 -.-I u.. CD Z J::.< -,::, 0 ~ 0 I- 0 0 U 0- CD « a:: LL

10 r-

o r-

\ L----L --L ----'I 0 o o N (IWI s~!un) A.1IJ\I.1~'1 34 pH 7.4 and the desalted enzyme concentrate (80 ml to 100 ml) from the DEAE-cellulose step was applied to it. The column containing the absorbed enzyme was washed with about 200 ml of equilibrating buffer, and the enzyme fractions were collected by the stepwise elution with 200 ml each of 0.1 M, 0.2 M and 0.2 M buffer containing 0.15% potassium cholate. The buffer used for elution was of pH 7.6 (Figure 2).

Two bright yellow fractions of enzyme were obtained--one eluted with 0.1 M or 0.2 M buffer

(component 1), and the other with 0.2 M buffer containing cholate (component 2). The two fractions, components 1 and 2 had specific activities in the order of 2,000 to 4,000, and 6,000 to 7,500, respectively.

The final step of purification of these fractions was achieved by the starch zone electrophoresis under the same conditions published earlier (54). The enzyme components 1 and 2 after starch zone electrophoresis attained specific activities of

3,000 to 4,000 and 7,000 to 9,000, respectively.

Table I summarizes all the steps involved in this purification as developed by Yasunobu ~ ale (54) and the attached FLOW SHEET briefly describes the newly modified procedure. w VI

Figure 2. Hydroxylapatite column chromatography of the partially purified mitochondrial amine oxidase. One hundred and twenty milligrams of protein containing 3.6 x 105 units of enzyme activity was applied to a column (2.9 x 15 cm) which had been equilibrated with 0.01 M potassium phosphate buffer, pH 7.4. Fractionation of the enzyme was made by stepwise elution with, A, 0.01 M potassium phosphate buffer, pH 7.4;

B, 0.1 M fo1~owed by 0.2 M potassium phosphate buffer, pH 7.4; and C,

0.2 M potassium phosphate buffer, pH 7.4 plus 0.15% potassium cho1ate.

Curve -6-6- indicates enzyme activity, and curve -0-0- indicates protein concentration. ·I~ A B ~ C , -- .....E If) 0' I E 0- Component 2 - X 4 Z -lJJ --E l- ..... 0 ." 0:: 0-... 0- c -=' I I T .., 1.0 >- 2 t: >- l- I L~ II~ IT \~ .., 0.5 «0

Pooled - J 60 I~ Pooled ~l I 00 Fractions Fractions FRACTION NUMBER (6 ml each)

Figure 2 W 0\ TABLE I

Purification of Beef Liver Mitochondrial Amine Oxidase*

Purification Step Volume Total Total Specific Yield Purifi Solubility protein units** activity % cation (ml) (mg) xlO- 3 (units/mg)

1. Mitochondrial 1,800 37,080 4,860 131 100 1 Insoluble homogenate

2. Triton X-100 plus 0.15 720 14,976 4,392 293 90 2 Insoluble saturated (NH4)2S04

3. 0.25-0.40 saturated 515 7,151 2,987 417 62 3 Insoluble ( NH 4)2S04 4. Calcium phosphate 80 432 1,088 2,519 22 19 Insoluble gel eluate

5. DEAE-cellulose eluate 54 133 631 4,750 13 37 Insoluble

6. Hydroxylapatite eluate 5 45 356 7,900 7.3 60 Soluble

7. Starch block 16 31 249 8,050 5.1 61 Soluble electrophoresis

* From 39 gm (dry weight) of purified mitochondria. **A unit of enzyme activity is the amount of enzyme required to change the absorbance 0.001 per minute at 250 mp using the spectrophotometric assay of Tabor ~~. in which benzyl amine is used as the substrate. Insoluble means that detergent is required for solubility. Soluble means that no detergent is needed and that the enzyme precipitates instead of floats when ammonium sulfate is added to precipitate the enzyme. VJ ...... 38

FLOW SHEET

Modified Procedure for the Preparation of Amine Oxidase

STEP IV. Calcium Phosphate Gel Treatment (54)

Reddish Brown Enzyme SOlutionl from STEP III C54) • I (1) Dialyzed for 4 hr.

(2) Ca-ph gelI (1:4, prote~n:ge1). added I (3) Centrifuged

(1) Eluted with 0.1 M (1) Ca- ph gel (1: 5 , protein: gel) added I cenLifUged (2) Centrifuged

Supernatant S2.1 Discarded

(1) Eluted 2 x with 0.2 (1) Washed with 0.01 M K-ph buffer, pH 7.6 K-ih buffer, pH 7.4 I (2) Centrifuged (2) Centrifuged.

Supernatant Gel, G2.2 Supernaiant S1. 3 and S1. 4 S2.2 Discarded Discarded

(1) Combined S2.2, and S2.3, and S2.4 (2) Ca-ph gel (1: 1, protein:ge1) ~ added Combined (3) ce+trifuged Supernatants -t. ,j, !' Gel, Gc Supernatant S3 Discarded 39

!combined Supernatants I I (1) Concentrated by Amm-S04 (0.4 satd ) (2) Desalted in SrPhadex G-25 Column

Eluate (100 to 150 ml)

STEP V. DEAE-cellulosl Column Chromatography (54) I STEP VI. Hydroxylapatite Column Chromatography (54) .j, ~ ~ Component 1 Component 21

STEP VII. !Starch Zone STEP VII. Starch Zone E lectrrPhOreSiS Elect ophoresis

\ Purified Purified Component 1 Component 2 40

2. Studies on the Purity of the Enzyme

It is a necessary condition to ascertain that

the enzyme is reasonably pure, before studying its

properties. Numerous tests, therefore, should be

employed to prove that the enzyme consists of only

one protein. Since each test establishes a certain

degree of purity, all the tests together will confirm

whether the enzyme is very pure or not.

Tests employed to study the homogeneity of a

protein or an enzyme are based on the measurement of

certain physical properties of the macromolecule.

In order to determine the degree of purity of the

mitochondrial amine oxidase, the following experiments

were done:

(a) Rechromatography on DEAE-ce11u1ose

After the final step of purification (starch

block electrophoresis step), component 2 was rechro

matographed on the DEAE-ce11ulose column (25 x 1.6

cm), which was equilibrated with 0.01 M potassium

phosphate buffer, pH 7.4. Gradient elution,

using 250 m1 of 0.01 M buffer containing 0.5 M

sodium chloride in the reservoir and 250 m1 of 0.01

M potassium phosphate, pH 7.4, in the mixing chamber,

was made. As shown in Figure 3, a single component

was observed. .p I-'

Figure 3. Rechromatography of the purified enzyme component 2 on the DEAE-ce11u10se column. Protein, 15.1 mg, containing 1.18 x 105 units of activity, was applied to a column (25 x 1.6 cm) which had been equi1i- brated with 0.01 M potassium phosphate buffer pH 7.4. Gradient elution was used to elute the enzyme (250 ml of 0.01 M buffer in the mixing chamber and 250 m1 of 0.5 M sodium chloride in 0.01 M buffer in the reservoir).

Fractions of 5.4 m1 were collected at a flow rate of 0.3 m1 per minute.

The symbols indicate: -0-0-0-, enzyme activity; -0-0-, protein concentration; and -6-6-, sodium chloride concentration. 42

(lW/OW) NI3J.0~d d I

v (W) I:>DN 0 l\. I \ • \ • 0-- U) E \ V \ !J It) \ ,/ / - ., ~..o 0 0: ....0.... / IJJ ~_o...... /0 m ~ C""') __-0----0- , /0 ::::;)

\ • \ •

~--1.-_--_---J._--__...L- --IO\ eN £_01 X(lW / SI!Un) A..1IAIJ.:>\1 43

(b) Rechromatography ~ hydroxylapatite column

After the starch block electrophoresis step,

component 2 was subjected to hydroxylapatite column

chromatography. It was applied on a hydroxylapatite column

(10 x 1.6 cm), pre-equilibrated with 0.01 M potassium

phosphate buffer, pH 7.4. Fraction 2 was eluted as

one peak with 0.2 M buffer, when stepwise elution with 0.1 M, and 0.2 M buffer, was used. The result is

shown in Figure 4.

A Sephadex G-200 gel column (120 x 1.9 cm)

chromatography of the fractions 1 and 2 are shown in

Figures 5a and 5b. A single peak was observed for

each component.

(d) Analytical starch block ~lectrophoresis

Starch block electrophoresis was performed as

described by Fine and Costello (59) in the section,

Materials and Methods. At the close of the experiment,

1/2 cm transverse sections were cut and eluted separately with 2 ml of 0.1 M potassium phosphate buffer, pH 7.4,

containing 1 x 10-4 M dithioerythritol. Protein and

activity determinations were made on the eluates. Both

components moved as single bands as shown in Figure 6.

The first component moved a distance of 4.8 cm and the

second, a distance of 6.8 cm from the point of origin

towards the anode. .p .p-

Figure 4. Rechromatography of purified component 2 on hydroxylapatite.

Protein, 8.75 mg, containing 6.9 x 104 units of activity, was applied to

a column (10.5 x 1.6 cm) which was equilibrated with 0.01 M potassium

phosphate buffer, pH 7.4. Stepwise elution was used. Fractions of 2.2 m1 were collected at a flow rate of 0.2 ml per minute. The symbols represent:

A, 0.1 M potassium phosphate buffer, pH 7.4; B, 0.2 M potassium phosphate bufferi pH 7.4; -~-~- is enzyme activity, and -o-o-is protein concentration.

\, 45

It) (lWI f»W) N131.0~d d ~ .--.....------~--__r--__,~

o v

...... <1 - .."., E m _<1-- C\I --- l-l z ::l 00 0 "..I N Z r=.. 0 i-- «0 0:: LL

I--..L..-.....!------_L...-. ---JO en v £_01 X (IWI Sl!un) Al.IAll.:l'1 46

Figure Sa. Chromatography of amine oxidase

components 1 on Sephadex G-200. Three milligrams of

enzyme component 1 in 1.5 ml of 0.05 M potassium

phosphate buffer, pH 7.4 containing 0.01 M mercapto

ethanol were applied to the column and eluted with

the same buffer. Fractions of 3 ml were collected

Curve -~-~- represents protein, and curve

-0-0- represents activity. 47

(\I 7 1 0 -E - 6 1\ ...... X 0 C' -E 5 E ...... - II) Z ·c 4 0.20 W -~ 3 0.15 b0:: -~ .- a.. - 2 .--> 0.10 (,)« I 0.05

o 4 20 24 28 32 36 40 FRACTION NUMBER (3 ml each)

Figure Sa 48

Figure 5b. Chromatography of amine oxidase

component 2 on Sephadex G-200. Two milligrams of

enzyme component 2 in 1.5,ml of 0.05 M potassium

phosphate buffer, pH 7.4, containing 0.01 M

mercaptoethanol were applied to the column and eluted

with the same buffer. Fractions of 3 ml were

collected at 0_4 0 • Curve -0-0- indicates activity

and curve -~-~- indicates protein concentration. 49

rt) I 8.0 0- X -E 6.0 ""en 2.0 -= E -,c :::J ""C' - E >- 4.0 - -~ Z > 1.0 IJJ- ~ ~- 0 0 2.0 0:: « Q.

o FRACTION NUMBER (3 ml

Figure Sb VI a

Figure 6. Migration of the amine oxidase components in starch block electrophoresis. About 10 mg of each component in 2 rol buffer, pH 7.4 with ionic strength of 0.1, were used. Electrophoresis continued for l7~ hours at 410 volts and 15 mA per block. Charging of protein in the block and electrophoresis run was carried out in the cold room (0-4 0 ). Curve-o-o- indicates activity and curve -t:.-b.- indicates protein concentration. 10. i j

If) 8 I Component 2 0 ~ - E X " --E 6 1.2 e 6 ,~ - "en 6 z .- '\ I, -C Component 1 If ILl- 7 ::J 4 ~ ,~/ \.l d\ .8 I I, o - a:: I, 0. >- I' t: 2 , ' .4 > I I-- U « + 024 6 8 10 STARTING ZONE ( centimeter) t Vt.... Figure 6 52

(e) Free boundary electrophoresis

Purity check of component 2 was made in the Perkin-

Elmer electrophoresis apparatus. The Fraction 2 was dissolved and dialyzed against potassium phosphate buffer of 0.1 ionic strength, pH 7.4, containing 0.2 M mercapto ethanol for 4 hours with 2 changes of buffer. Electro

0 phoresis was carried out in the same buffer at 4 • A single peak was observed (Figure 7).

(f) Polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis was carried out as

described in the Methods section (39). Component 1 moved a distance of 2.2 cm and component 2 moved 1.2 cm from the

starting zone as shown in Figure 8.

(g) Ultracentrifuge studies

The sedimentation behavior of the enzyme component 1 was studied in phosphate buffer, pH 7.4. The sedimentation pattern shown in Figure 9, indicates homogeneity of the

component.

B. Kinetic Properties

1. Activity of the Enzyme

The mitochondrial amine oxidase components 1 and 2 have specific activities of 3000 to 4000 and 7000 to 9000,

respectively. Although both the components are unstable,

component 1 shows relatively high stability as compared to

component 2. In 0.1 M potassium phosphate buffer, pH 7.4

containing 1 x 10- 4 M dithioerythritol at 0 - 4 0 , component ---_.._-_ _-_.. __ .

'" ;

Figure 7. Electrophoretic pattern of component 2.

A 0.5% solution of the enzyme (specific activity, 8000) dissolved in 0.1 ionic strength of potassium phosphate buffer, pH 7.4, containing 0.02 M mercaptoethanol, was used. The electrophoretic pattern of the ascending limb was photographed after 75 minutes. The run was made at 4 0 and the field strength was 9.28 volts per cm2 . The mobility was calculated to be -5.21 x 10-5 cm2 volt- l sec-I. 54

Figure 7 VI VI

Figure 8. Polyacrylamide gel electrophoresis of amine oxidase components 1 and 2. (a) separation of component 1 from component 2 when a mixture of both was applied; (b) a single band of component 1; and (c) single bands of component 2. Electrophoresis runs were carried out under the conditions described in the Materials and

Methods Section. 56

00 I.n "

Figure 9. Sedimentation pattern of amine oxidase component 1.

Sedimentation studies of a 3.6 mg enzyme dissolved in 0.1 M potassium phosphate buffer, pH 7.4, were made at 22.5°, and at 35,600 rpm.

Photogralphs we re taken at 8 minu te interva1 s a f terattaining top speed. 58 59

was found to be stable at least for a week, after which the

activity declined gradually. Component 2 deteriorated

faster than component 1 under the same conditions. Stability, moreover, was found to be a function of the enzyme concen-

tration. The more concentrated the enzyme components in

solution were, the more unstable they were. Freezing

destroyed component 2 in a day and component 1, in a few

days.

2. Effect of Temperature ~ the Enzyme Activity

The effects of temperature on the partially purified

enzyme after the DEAE-cellulose step, as well as on the

purified enzyme components 1 and 2 were studied and the

results are shown in Figures 10aand lab. The partially

purified enzyme from the DEAE-ce11ulose column incubated

for 15 minutes at various temperatures, and was found to be

unstable at any temperature above 30 0 • The purified enzyme

component 2, under the same conditions, was found to be

more stable than the partially purified enzyme. Component 2

retained the initial activity up to 40 0 whereafter the

activity dropped progressively with increasing temperatures.

The partially purified enzyme, on the other hand, retained

0 only 65% of the initial activity at 40 • Furthermore, the

purified enzyme component 2 was more heat stable in the

presence of 1 x 10-4 M dithioerythrito1. Similar findings

were obtained with the enzyme component 1. 60

Figure lOa. Effect of temperature on the

enzymatic activity. For the experiments, 0.5 ml

samples of the enzyme (1.94 mg per ml, specific

activity, 3920) after the DEAE-cellulose step were

diluted in 4.5 ml of 0.1 M potassium phosphate

buffer, pH 7.4, and preinculated for 15 minutes at

temperatures from 25-60°. The solutions were cooled

to 25°, and the activity of 0.05 ml aliquots was

determined at 250. The standard assay method was

used. 61

0.4.------.,

-.-c E "'~ 0.3 E o an N C; 0.2 o o <1 ->- 0.1 ~ -> ~- o« 20 30 40 50 60 TEMPERATURE (OC)

Figure lOa 62

Figure lOb. Effect of temperature on the

activity of the amine oxidase. For this experiment,

720 units of enzyme component 2 (specific activity,

7,650) in 2 m1 of 0.1 M potassium phosphate buffer,

pH 7.4, were incubated for 15 minutes at various

temperatures ranging from 25 to 60°. The solutions

were cooled to 25°, and the activity of 0.1 m1

a1iquots was determined under the usual assay

conditions a t2 5 ° . " 63

150 ....>- >- ....- 100 0

~ 0 50

25 30 35 40 45 50 55 60 65 TEMPERATURE (OC)

Figure lOb 64

3. Effect of ~ ~ ~ Enzyme Activity

The effect of variations of pH on the activity of the enzyme components 1 and 2 were studied. As found with the partially purified enzyme (53) the pH optima of the enzyme components 1 and 2 were pH 9.1-9.2 which are in close agreement with the value reported earlier by Hare (1).

Both these values, however, are different from those reported by others (39, 40). The buffer systems used were

0.2 M potassium phosphate-pyrophosphate buffers. Figure 11 illustrates the eff~ of pH variation on the activity of the enzyme components.

4. Substrate Specificity

The substrate specificities of the two components were determined with various amines by a method developed by

McEwen (12) in which the usual amine oxidase assay was coupled with the peroxidase-o-dianisidine color reaction (41)

Table II summarizes the results on various amines including an amino acid.

5. Inhibitor Specificity

Inhibition studies provide good tools to track down dertain specific groups or moieties which might be involved in the cataytic function of an enzyme. Accordingly, the following inhibition studies were made.

(a) Product inhibition

As many enzymes are inhibited by a product of the reaction they catalyze (feed back inhibicion), the effect CJ\ VI

Figure 11. Effect of pH variation on the activity of the enzyme

components 1 and 2. Potassium pyrophosphate buffer (0.2 M) was used.

Each reaction mixture contained 0.05 ml of the enzyme component 1 (0.12 mg per milliliter, specific activity 3,200), 1.67 mM of benzylamine in

a total volume of 3 mI. For component 2, the reaction mixture contained

0.05 ml of the enzyme solution (0.07 mg per milliliter, specific activity,

7,100). Other conditions were like those of component 1. Activity at various pH values was measured by the usual method. 66

C\I c -CD c c -CD o c 0- o E 0- E o o u u

o o o rr> C\I ( U!W/ OW OSZ aov ) A.LIAll.~'f 67

TABLE II

Substrate Specificities of the Two Amine Oxidase Components*

Sub s trate Relative Specificity of Components

Component 1 Component 2

Monoamine Benzylamine 100 100 Heptylamine 82 79 Tryptamine 18 32 Tyramine 13 30 Mescaline 0 0 Serotonin 3 5

Diamine Kynuramine 43 52 Agmatine 0 0 Butanediamine 0 0 Cadaverine 0 0 Histamine 0 0 Trimethy1enediamine 0 0

Catecholamine Norepinephrine 41 46 Epinephrine 21 25

Polyamine Spermidine 0 0 Spermine 0 0

Basic amino acid Lysine 0 0

*Each reaction mixture contained 45 units of enzyme (specific activity 5,600, and 7,800 for first and second components respectively), and 3-3 mM of substrate in 0-06 M potassium phosphate buffer, pH 7-0_ The table shows relative substrate specificity with different amines at 25 0 _ 68 of NH on the re~ion of the enzymeoomponents was investigated. 3 The activity was examined in the presence of various concen trations of (NH4)ZS04' A 50% inhibition of the enzyme

(after the DEAE-cellulose step) was observed at an (NH4)ZS04 concentration of 0'4 M. The results are shown in Figure lZ.

(b) Inhibition ~ sulfhydryl ±eagents

Since there are many reports on the inhibition of mitochondrial amine oxidase by sulfhydryl reagents, and reports that the enzyme is a sulfhydryl enzyme (90-93), effects of various sulfhydryl reagents on the enzymatic activity of the partially purified enzyme and that of the two purified components were investigated.

Mercuric chloride, silver nitrate, p-chlormercuribenzoate

(p-CMB), cadmium sulfate, and sodium arsenite were studied from amongvarious mercaptide forming reagents. All but arsenite had inhibitory effects. Cadmium showed 60% inhibition in the activity of the partially purified enzyme only when its concentration was prohibitively high (Figure l3a,

Tables III A, III B, and III C). However, these results did not show conclusively whether the inhibition was

indicative of the participation of sulfhydryl group(s) in

the enzyme activity or was due to a general effect of heavy metals on enzyme.

As p-CMB is a most widely used thiol reagent, it was

employed to examine the type of inhibition produced by a

thiol reagent. Accordingly, activities of the two components 0\ \0

Figure 12. Product inhibition studies. The reaction mixture contained 35 units of amine oxidase component 1 (specific activity,

3,250). The ordinate shows the percentage of inhibition and the abscissa shows the molar concentrations of ammonium-sulfate. 70

10 o

IJJ ~ t- O (/) ro I 0 ~ ~ .-I :::> ,..Q) - ::l 2 be -r-! ~ 0 r:.. 0 ~ ~ <[

LLJ ...J d 0 ~

o o o U) v C\I NOIJ.18IHNI J.N30~3d -..J....

Figure l3a. Inhibition of amine oxidase by sufhydryl reagents.

Each reaction mixture contained 46 units of amine oxidase (specific activity, 4,200), plus 1.7 mM benzylamine as substrate and various concentrations of inhibitors shown in the plot. The ordinate(s) indicates percent of activity and the abscissa gives the molar concentrations of the inhibitors used. 100

90 AgN03 eOr >- \ \ \f t-- 70 >- t- 60 tHgCI2 O

~. I v -7 -6 -5 -4 -3 -2 LOG MOLE INHIBITOR ...... N Figure 13a 73

TABLE III A

Inhibition of Amine Oxidase by Sulfhydryl Reagents*

Inhibitor Concentration Inhibition pM %

p-Chloromercuribenzoate 5'00 37'5

2'50 22'7

Silver nitrate 5'00 37·5

2'50 10' 9

Mercuric chloride 1'00 100'0

0"25 0"0

Cadmium sulfate 1000'00 60·0

Sodium arsenite 1000'00 0'0

* Standard assay conditions were maintained, and each reaction mixture contained 46 units of enzyme with a specific activity of 4,200. 74

TABLE III B

Inhibition of Amine Oxidase by Sulfhydryl Reagents*

Inhibitor Concentration for 50% Inhibition pM

Mercuric chloride 0-56

Silver nitrate 3-31 p-Chloromercuribenzoate 4-27

Cadmium sulfate 513-00

*The table shows the inhibitor concentrations at which 50% inhibition occurs in the enzyme_ Standard conditions were used in each determination and each reaction mixture contained 46 units of enzyme having a specific activity of 4,200_ 75

TABLE III C

Inhibition of Amine Oxidasea by Sulfhydryl Reagents

Inhibitor Inhibitor % Inhibition Conc. pM Component 1 Component 2 p-Chloromercuribenzoate 2.5 26 24

Silver nitrate 5.0 91 96

Mercuric chloride 1.0 96 91

0.5 72 80

Sodium arsenite 1000 0 0

Cadmium chloride 330 45

Iodoacetic acid 50 0 0

Iodoacetamide 1000 Ob_1Oc

N-ethylmaleimide 1000 Ob_ 34c aThirty-two units of compon~ 1 (specific activity 3290) and 35 units of component 2 (specific activity 7,050) were used for these experiments. Mole ratios of the inhibitors to enzyme components varied from 200 (for HgC1 2 ) to 4,000,000 (for alkylating reagents) considering the molecular weights of 400,000 and 1,280,000 for components 1 and 2, respective ly. Activity was measured by the usual benzylamine assay method. blnhibitions after incubating for 2 hours. clnhibitions after incubating for 24 hours. 76 at various substrate concentrations were determined at a fixed concentration of p-CMB (5 x 10-6 M). A Lineweaver-Burk plot, as shown in Figure l3b demonstrates that p-CMB is a non-competitne inhibitor of the component 1. The same type of inhibition was also observed with component 2 (Figure l3c).

Substances like cyanide, azide, phenanthroline, and some other metal chelating reagents inactivate the enzymes which contain heavy metal(s) (e.g. iron, copper, molybdenum, manganese, etc.) as prosthetic group(s). In order to determine whether a metal was involved in the activity of the mitochondrial enzyme, the effect of some metal chelators on the activities of the enzyme was investigated. Table IV summarizes the results of these studies. In addition, the type of inhibition produced by cuprizone is illustrated in

Figure 14.

(d) Inhibition Ex. Aldehyde Reagents

Aldehyde or carbonyl reagents may in some cases act like inhibitors by combining with a carbonyl group in the enzyme itself, or with a cofactor or prosthetic group

(e.g. pyridoxal phosphate) as in the case of the plasma amine oxidase (28). Studies were made with both enzyme components to determine whether or not they are inhibited by carbonyl reagents. The results show the effect of some of the well known carbonyl reagents (Table V). "

Figure l3b. Lineweaver-Burk plot of benzylamine oxidation in

the absence and presence of p-CMB. Each reaction mixture contained

45.5 units of enzyme component 1, specific activity 3,550. The

ordinate gives the reciprocal of the activity in terms of the change in absorbance at 250 mpper minute and the abscissa gives the reciprocal , of the molarity of the benzylamine. I is the concentration of p-CMB used. 78

N 0..

0 0 \ " en CO

to- X 10

-I

N I ~ V I '------_--J 10 I ..... \0

Figure l3c. Lineweaver"Burk plot of benzylamine oxidation in the presence and absence of p"CMB. Each reaction mixture contained

52 units of enzyme component 2, specific activity 7,600. The ordinate gives the reciprocal of the activity in terms of the change in absorbance at 250 mll per minute and the abscissa gives the reciprocal of the molarity of benzylamine. I is the concentration of p"CMB used.

, 80

- 0 0 0 '" 0 en II 0)

X ~ &0 .. (D ..... &0 ", q- 0 '0 l:'"l...... QJ rt> - ~ 0'\\ ;:l X bO -.-I \;\ (\J -len ~ 00 0

-I ~Ol X T (\J " I rt> I q- I

&0 I 81

TABLE IV

Inhibition of Amine Oxidase by Metal Che1ating Agents*

Chelators Final Inhibition Concentration % mM

None 0 0

Bis-cyc1ohexanone oxa1 0.3 76 dihydrazone (Cuprizone)

Neo cupro ine 0.3 33

8-Hydroxyquiono1ine 3.0 90

Sodium diethy1dithio 3.0 24 carbamate o-Phenanthro1ine 0.3 19 a"a -Dipyridine 3.0 10 Ethy1enediaminetetraacetate 3.0 0

Sodium azide 30.0 0

NaCN 30.0 0

*The enzyme after the DEAE-ce11u1ose column chromatography was assayed llsing the kynuramine assay of Weissbach et a1. (94). For the experiments, 0.1 m1 of partially purified enzyme, specific activity 3360, was preincubated with the che1ating agent mentioned in the table for 15 minutes at 26 0 and" then assayed. (Xl N

Figure 14. Lineweaver-Burk plot of benzy1amine oxidation in the presence and absence of cuprizone. Each reaction mixture contained

22 units of enzyme, specific activity 3,500. The ordinate gives the reciprocal of the activity in terms of the change in absorbance at

250 mp per minute and the abscissa gives the reciprocal of the molarity of benzylamine. I is the concentration of the cuprizone. 83

~ LO I 0 rt> I CD X 0 LO.. X v -1(1)

-::t ~

OJ C\I l-l ==' be .~ r:.. 84

TABLE V

The Effect of Aldehyde Reagents on the Enzyme

Activity*

Inhib itor Final Inhibition Concentration % }lM Component 1 Component 2

Hydroxylamine 330 0 0

Phenylhydrazine 3.3 31 30 p-Nitropheny1hydrazine 3.3 53 44

Semicarbazide 33 1 0

Hydrazine 33 2 2

Potassium benzoate 3.3 10 2

*The reaction mixture contained 35 units of enzyme. The mole ratio of inhibitor to enzyme components 1 (M.W. 407,000) and 2 (M. W. 1,280,000) varied between 1200(for phenylhydrazine, etc.) and 120,000 (for hydroxylamine), and 2440 (for p-nitropheny1, etc.) and 240,400 (for hydroxylamine), respectively. The results did not change on preincubation of the enzyme with the inhibitors for 10 minutes at 25 0 • 85 C. Physical Properties

1. Spectral Properties

The enzyme components are bright yellow in color at the final step of purification in contrast to the plasma enzyme which is pink in color. The absorption spectrum of component 2 of the mitochondrial amine oxidase is mown in

Figure 15a. The spectrum of the enzyme in 0.1 M potassium phosphate buffer, pH 7.4, was taken in Beckman DK-2 Ratio

Recording SpectrophotomSEr. The absorption spectrum differs from that of a typical f1avoenzyme. However, the 450 mp peak is indicative of the presence of flavin. There are, in addition, an absorption maximum at 410 m~ and a shoulder at 480 mp. When the enzyme was treated with substrate

(benzy1amine) or sodium hydrosu1fite (Na2S204), the 450 m~ shoulder disappeared (Figure 15b). The peak at 450 mp and the 480 m~ shoulder were partially restored when air was carefully admitted. Component 1 showed similar spectral properties.

2. Sedimentation Coefficients

A sedimentation pattern of component 1 has been shown in Figure 9. Runs were made in 0.1 M potassium phosphate buffer, pH 7.4, in the Spinco analytical ultracentrifuge.

Also, sedimentation coefficients were determined in the preparative ultracentrifuge. Figure 16 summarizes the sedimentation coefficients determined at various protein concentra t ions. The values obtained for component 1 was 00 (J\

Figure 15a. Absorption spectrum of the purified enzyme component 2. The enzyme with specific activity of 7800, was used. The concentration of the enzyme was 0.56 mg/m1 and the enzyme was dissolved in 0.1 M potassium phosphate buffer, pH 7.4. 89

1.0

UJ o 0.4 z « en o0:: CJ) ~ 0.5 480

0.1

240 280 320 360 400 450 500 550

ex> WAVE LENGTH m)J ..... Figure lSa 00 00

Figure l5b. Reduction of the spectrum of enzyme component 2

by substrate and sodium dithionite. Spectra of the purified enzyme

(2.15 mg/ml; specific activity, 7950), ; the benzylamine (150 mole per mo~ of enzyme) reduced enzyme, -.-.-; and the sodium

dithionite (5 mole per mole enzyme) reduced enzyme, ----. Spectra were taken in 0.1 M potassium phosphate buffer, pH 7.4 aerobically. 289

1.0

LLI () 0.4 Z

0.1

240 280 320 360 400 450 500 550 WAVE LENGTH m)J 00 Figure 15b \0 1.0 o

Figure 16. Sedimentation coefficients of amine oxidase component 1 at varying protein concentrations, Sedimentain behavior was studied in

0.1 M potassium phosphate buffer, pH 7.4 at 22.5°, Runs were made at 35,600 rpm (73,684 x g) using the rotor type An-D. 91

- 10 0 Z I 0 00 l-- v

3 . Par t i a 1 SP e c i f i c Vo 1 um e s

Table VII shows the results of the determination of partial specific volumes of components 1 and 2. For these experiments, the enzyme components were dialyzed for 4 hours in 0.05 M potassium phosphate buffer, pH 7.4 with

three changes. The buffer from the last dialyzate was used as the solvent. The relative densities of the enzyme

solution and solvent were determined pycnometrically at

The partial specific volumes of the enzyme

components 1 and 2 were calculated as described by

Schachman (67) and were found to be 0.782 cm 3 jg and

0.805 c m3Jg respectively.

4. Molecular Weights

(a) Molecular weights detemined by Agarose (Bio Gel

A-~ m) gel filtration

Molecular weights for the two components of the

mitochondrial enzyme were estimated by the gel filtration

technique using agarose gel columns. Table VII lists the

elution volumes of the standard (or marker) proteins, and

Blue Dextran 2000. The void volume was found to be 93 ml

by using E. coli which is excluded by the agarose gel used (7Q). 93

TABLE VI A

Sedimentation Coefficients at Different Protein

Concentrations of the Mitochondrial Amine Oxidase*

No ° Protein Average 13 conceno s20 , w x 10 sec s20 w x 1013 sec % ,

1. 0°45 14°3

2. 0°27 14°6

0~20 3 ° 14·3 14°4 + 0°3

4o 0°14 14°5

5 ° 0°44 14°1

*Runs were made in 0°1 M potassium phosphate buffer,

pH 7°4, at 35,600 rpm, using analytical rotor, type

An-D a t 22 ° 5 0 ° 94

TABLE VI B

Sedimentation Coefficients by Sucrose Density Gradient*

Species s20 , w x 1013 sec

Component 1 14·5 + 0·2

Component 2 20"6 + 0"4

'>'(Runs were made in 20% - 5% sucrose gradients in 0"1 M

potassium phosphate buffer containing 1 x 10- 4 M

0 dithioerythrito1 at 75,000 x g for 16 hours at 0 • The

values are averages from 2 runs. 95

TABLE VII

Agarose Gel Filtration Data of Standard Proteins, Blue Dextran 2000, and Amine Oxidase Componentsa

Species Mol. Wt. V b V· Iv c log Mol. Wt. e e 0 x 10- 3 (ml)

Standard

Cyt. c 12.4 207 2.23 4.09

BSA (monomer) 65- 70 174 1. 87 4.845

Catalase 250 149 1. 61 5.398

Ferritin 747 130 1. 39 5.874

Blue Dextran 2,000 105 1.13 6.310

Amine Oxidase

Component 1 141 1. 52 5.61

Component 2 114 1. 23 6.114 aEquilibration and elution were done with 0.05 M potassium phosphate buffer, pH 7.4 containing 0.01 M mercaptoethano1. b Ve = Elution volume.

cVe/vo = Ratio of elution volume to void volume (Vo )' Vo volume was determined by using E. coli K 12 which is ex c 1 u de d by a 11 kind s 0 f s e p h a dex and a gar 0 s e gel s ( 70) . 96

Figure 17 shows the linear relationship of the logarithm of molecular weights to the ratios of elution volumes of

the proteins to void volume (Ve/Vo). The ratios Ve/Vo were found to be 1.52 and 1.23 for component 1 and component 2

corresponding to logarithms of molecular weights of 5.61 and

6.114 respectively. These values correspond to molecular weights of 400,000 for component 1 and 1,300,000 for

component 2.

(b) Molecular weights determined from Stoke's Radii,

Sedimentation coefficients, and Partial Specific Volumes

Stoke's radii for components 1 and 2 were estimated

from the known linear relationship of the distribution

coefficient (Kd) to the molecular (or Stoke~s) radius

(Figure 18) (from agarose gel filtration data). The

Stoke's radii of various proteins and the two amine oxidase

components are illustrated in Table VIII. Stoke's radii

for components 1 and 2 were found to be 60 ~ and 106 ~, respectively. Sedimentation coefficients as mentioned in

section 7 above were 14.4 + 0.3 and 20.6 for the two

components. Partial specific volumes were determined by

the method of Schachman (67) and were found to be

0.782 cm3 /g for component 1 and 0.805 cm 3 /g for component 2.

Placing these values in equation 3 (page 15), molecular weights were calculated as 396,000 + 10,000 and 1,195,000

for component 1 and component 2 respective1y~ 97

Figure 17. Agarose gel filtration data of various standard proteins and Blue Dextran 2000, and amine oxidase components. Agarose gel was equilibrated in a 1.9 x 120 cm column with 0.05 M potassium phosphate buffer, pH 7.4, containing 0.01 M mercaptoethano1. Fractions of 3 m1 were

0 collected. The work was conducted at 0_4 • 98

2.0 O~~:~:::ase

o MAO~", > ...... o~ Q) Ferritin > MA02~ Blue De xtran"-0,

1.0

4 5 6 6.5 LOG MOLECULAR WEIGHT

Figure 17 99

Figure 18. Correlation of Kd with Stoke's

radius. Agarose gel filtration data were plotted

as described by Porath (72). 100

0.9 .------. "o~

0.8 - O~ (monomerl

O~atalase 0.7 I K 3 ~~ d

0.6 - o Ferritin

0.5 -

0 .4 '--__.J...-11__-'--__...&.-I __...L--I __--'--I __---' 20 40 60 80 100 120 o STOKE'S RADIUS, A

Figure 18 101

TABLE VIII

Molecular Parameters Obtained from Gel Filtration Data

Species Mol. Wt. Stoke's Ve Kd x 10- 3 radius (ml) 0 A

Standard

Cyt. c 12"4 10 207 0·675

BSA (monomer) 65- 70 35 174 0·480

Catalase 250 52 149 0·335

Ferritin 747 79 130 0"220

Amine Oxidase Components

Component 1 60 141 0·32

Component 2 106 114 0·124 • 102

If Stoke's radius of a macromolecule is known, the diffusion coefficient can be calculated from the equation

k T (vi) D 6 na where T absolute temperature, n = viscosity of the medium, and k = Boltzmann's constant. Diffusion coefficients for components 1 and 2 were calculated to be 3.8 x 10- 7 cm 2 sec- l and 2.03 x 10- 7 cm 2 sec- l , respectively. Placing these values in Svedberg's well known equation

R T.s (vii) M D(l-Vp) molecular weights obtained were 423,000 and 1,355,000 respectively for components 1 and 2. Various molecular constants and the molecular weights of the two components determined by three different methods, are summarized in

Tables IX and X, respectively.

5. Frictional Ratios

The frictional ratios of the amine oxidase components are shown in Table XI • These values were calculated from the molecular weights of the two components by using the equation (v). Values obtained for components 1 and 2 were

1.17 and 1.46, respectively, indicating that the component 1 is more ~herical than the component 2. 103

TABLE IX

Physical Parameters of the Mitochondrial Amine Oxidase

Species Stoke'sa b d Radius s20, W V o A (X 1013 sec) cm 3 Jg

Component 1 60 14.4 + 0.3 3.8 0.78

Component 2 106 20.6 + 0.4 2.0 0.80

lJ3 aStoke's (molecular) radii were determined from the Kd vs Stoke's radius standard plot (Figure 18). b The sedimentation coefficient of component 1 is the average of five values (Table VI A), and that of component 2 is the average of 2 values determined by sucrose density gradient technique.

cDiffusion coefficients of the two components were determined from the equation, D = k T 6 na dV is the partial specific volume determined by the method of Schachman (67). 104

TABLE X

Molecular Weights of the Amine Oxidase Components by three Methods

Method Component 1 Component 2

Gel Fi1trationa 408,000 + 9,000 1,300,000 + 70,500

6 nNasb 396,000 17,000 1,195,000 90,500 M = + + (l-"Vp)

M = RTs c 423,000 + 10,000 1,355,000 + 27,500 D(l-Vp)

Average 406,000 + 14,700 1,280,000 + 91,500 aThe molecular weight determinations are based on three gel filtration runs. bMo1ecu1ar weights are estimated from five sedimentation coefficient values for component 1 and two from two values for component 2. cSame as that for molecular weight determinations from Stoke's 1awb . 105

TABLE XI

Frictional Ratios of the Amine Oxidase Components

Species Stoke's Frictional Radius Ratio o A f/f o *

Component 1 60 1. 17

Component 2 106 1. 46

*Frictiona1 ratios for the two enzyme components

were calculated by using the following equation:

f/ f = a/(3VM)1/3 o (4 N) 106

D. Chemical Properties

1. Metal Content

Metal analyses were made according to the methods mentioned earlier in Section 8 under Materials and Methods.

The copper content was determined in a number of purified preparations of component 2 which yielded values ranging from 0.15 to 0.17 pg/mg protein. In addition, copper content was measured in each step of purification (Figure 19a).

Determinations were also made for other metals such as cobalt, iron, manganese, and molybdenum. Iron was present in insignificant amount (0.02 pm/mg protein) and was considered to be a contaminant as determined by measuring its content in each purification step (Figure 19b). Other metals examined were found to be absent. The results of these determinations are summarized in Table XII.

2. Phosphorus Content

(a) Total phosphorus

Total phosphorus was determined in purified components 1 and 2 by the method referred to earlier (87) . Total phosphorus content determined in a number of preparations yielded average values of 2.56 + 0.05 pg and 3.37 + 0.03 pg per milligram of component 1 and component 2, respectively.

These values correspond to total phosphorus contents of

0.0815 + 0.0015 pmole and 0.1095 + 0.0008 p mole per milligram of protein, respectively, for component 1 and component 2. t-' o "

Figure 19a. Copper content of the enzyme. The copper content and specific activity of the enzyme were determined at each step of the purification procedure. The copper contents were determined by the method of Peterson and Bollier (73).

.) 108

0 _0 0 0 \ ex> \ , 0 - 0 , .....0 \ >- \ 0 \ 0 I- - 0 - \ (0 -> \ I- \ 0 - 0 u , 0 <{ 10 0 \ 0 ell , 0 U 0- - 0 \ v LL .-l CI.l \ U l-< , 0 ::s 0 W bO , - 0 ..-l CL ~ \ rt> C/) 0 0 \ 0 \ - 0 W \ C\J ~ \ I 0 >- J _ 0 N I 0 Z / _/ W o- _----0 I 0------;--- I I <0 10. V rt> C\J -. 0 0 0 0 0 0 'NI3J.OHd OWl H3ddO~ orl I-' o \0

Figure 19b. Iron content of the enzyme. The iron content and the specific activity were determined at each step of the purification procedure. Iron was determined according to the method of peterson (76). 110

0.6

0.5

«0 ::E 0.4

0- E ...... 0.3 0- ::J..

0.2

0.1

o 1000 2000 3000 7000 8000 SPECIFIC ACTIVITY

Figure 19b 111

TABLE XII

Metal Content of the Amine Oxidase

Metals rg/mg Protein Method

Coppera 0'15- O' 17 Microchemical (46)

Ironb 0'02 Micro chemica 1 (49)

Cobaltb 0'00 Atomic Absorption (48)

Manganeseb 0'00 Atomic Absorption (48)

Molybdenumb 0'00 Micro chemica 1 (50)

aCopper was determined in purified component 2 only. bThese determinations were made on the enzyme after the DEAE-cellulose step. As these metals were either absent or in insignificant amounts, they were not investigated in purified enzyme. 112

(b) Phospholipid phosphorus

Lipid was extracted from a number of purified preparations of component 1 and component 2 by the method of

Fo1ch et al. (88). Phosphorus determinations were made on the extracted lipid according to the method of Bartlett (87).

From a number of determinations. average values obtained were 1.84 + 0.01 pg and 2.71 + 0.05 pg per milligram of protein for component 1 and 2. respectively. These values correspond to phospholipid content of 0.059 + 0.0005 ~M and

0.086 + 0.02 ~M per milligram of protein (considering

1 g-atom phosphorus per mole of phospholipid).

These values were calculated by subtracting phospholipid phosphorus from total phosphorus which yielded values of

0.67 pg and 0.679 pg per milligram of protein. respectively. for components 1 and 2. All these values are summarized in

Tab 1 e XIII. 113

TABLE XIII

Phosphorus Content of Mitochondrial Amine Oxidase

Species Totala Phospho1ipidb Nucleotide c Phosphorus Phosphorus Phosphorus

p.atom/mg p.atom/mg }latom/mg Protein Protein Protein

Component 1 0-0814 0-059 0-0216

Component 2 0-1094 0-086 0-0219

aTotal phosp~orus was determined in three different preparations and figures shown for the two components are the average values of these determinations_ The phosphorus contents were determined by the method of Bartlett (87). bphospholipid phosphorus was determined on the total lipid extracted according to the method described by Fo1ch etal_ (88).

cNucleotide phosphorus was estimated by subtracting the phospholipid phosphorus from the total phosphorus. 114

3~ Organic Prosthetic Group

Riboflavin determinations were made on the enzyme during the various steps of purification of component 2. The results are shown in Figure 20. In the first step, the riboflavin content was high due to the presence of other flavo-enzymes and free-riboflavin. But in the two subsequent steps, there was marked decrease in riboflavin resulting from the removal of contaminating flavo-proteins. Thereafter, the riboflavin content increased proportionately with the specific activity of the enzyme. In addition, the riboflavin content was determined microbiologically in a number of purified preparations and a value of 1.2 ~g or 3.3 mrmo~ riboflavin per milligram of protein was obtained corresponding to a value of 0.33 moles riboflavin per 100,000 grams of protein.

The most accurate result for the determination of riboflavin was obtained spectrophotometrically. By this method, an average value of 10.3 mpmoles and 10.1 mpmoles flavin per milligram of protein were obtained for the purified components 1 and 2, respectively. These values correspond to 1.03 moles and 1.01 moles of riboflavin per

100,000 grams of protein for the two components.

Adenine determination was made both microbiologically and micro chemically. Microbiological assay yielded a value of 0.513 ~g or 3.8 m?moles adenine per milligram of protein indicating an adenine content of 0.38 moles per 100,000 grams of component 2. Precise values, however, were obtained from t-' t-' V1

Figure 20. Flavin content of the enzyme. The flavin content and specific activity of the enzyme were determined at each step of the purification procedure according to the method described by Snell and

Strong (78).

.,,, 116

0 0 0 CD 0 0 0 to- o 0 0 >- U) I-- 0 > 0 I-- 0 U 10 0 « N Cll 0 l-I 0 ;:l 0 co 0 -LL -,-I V - ~ 0w 0 a.. 0 CJ) 0 rt)

0 0 0 C\I

0 0 0

o o CD C\I Nf3~O~d Ow / NIA\fl.:1081~ On 117 microchemical (spectrophotometric) determination which yielded values of 1.42 ~g and 1.38 ~g or 10.5 m~ moles and 10.2 m~ moles adenine per milligram of enzyme components 1 and 2, respectively. These values correspond

to 1.05 moles and 1.02 moles respectively of adenine per

100,000 grams of component 1 and 2.

The microchemical determination of ribose gave a value of 1.55 fg or 10.3 m~ moles of ribose per milligram of protein indicating a ribose content of 1.04 moles per

100,000 grams of enzyme component 2.

The nucleotide phosphorus contents in the enzyme components were determined by subtracting the phospholipid phosphorus from total phosphorus contents in the enzyme components 1 and 2 as shown in Table XIIlin the preceeding section. The values calculated were 0.67 pg and 0.679 pg corresponding to 21.6 m~atoms and 21.9 m~atoms of phosphorus per mi11~am of protein of the enzyme components 1 and 2, respectively. These values suggest that there are 2.16 gram atoms and 2.19 gram atoms of phosphorus per 100,000 grams of enzyme components 1 and 2, respectively.

In addition to the investigation of the "flavin prosthetic" group in the amine oxidase components, examinations were made of the pyridoxal content of the enzyme by the microbiological procedure described by Miyazawa (95). In

these experiments, phosphorylase a was used as a standard.

In MAO, there was 0.03 ~g pyridoxal per milligram of 118

purified enzyme component 2 as compared to 1.4 pg per miligram of phosphorylase a. These results ~ield values

of 0.07 moles per mole of enzyme component 2 (MW 1,280,000)

as compared to 4.3 moles of pyridoxal per mole of phosphory

lase a (MW 500,000). All these results are summaried in

Tables XIV A, XIV B, and XIV C.

4. Sulfhydryl Groups

Sulfhydryl groups were determined for enzyme component 1

as well as component 2. The results are shown in Figures 2la,

b, and c. Enzyme component 1 was titrated with increasing

amounts of p-CMB solution. The break point indicated that

there were 6.95 titrable sulfhydryl groups per 100,000 grams

of component 1 (Figure 2la). This value did not change

when the p-CMB titration was done in the presence of 8 M

urea. The value obtained in the latter case was found to

be 7 sulfhydryl groups per 100,000 grams of protein

(Figure 2lb). When the p-CMB titration experiment was

performed on enzyme component 2, a value of 7.15 sulfhydryl

residues per 100,000 gram of protein were obtained

(Figure 2lc). These results are summarized in Table XV.

In a separate experiment, the activity of the enzyme

component 1, during the p-CMB titration experiments, was

simultaneously measured. About 86% of the activity was

retained when all the titrable sulfhydryl groups in enzyme

component 1 had reacted with p-CMB as shown in Figure 22.

A similar result was obtained with component 2. TABLE XIV A

Riboflavin, Adenine, Ribose, and Nucleotide Phosphorus Content of Mitochondrial Amine Oxidase

Material mumole/mg Protein Method Reference

Component 1 Component 2

Riboflavin - - 3.3 Microb iolog ica1 (57) 10.3 10.1 Spectrophotometric (69) Adenine -- 3.8 Microb iolog ica 1 (52) 10.5 10.2 Microchemical (54) Ribose -- 10.3 Microchemical (59) Nucleotide Phosphorus 21. 6 21. 8 Ultramicrochemical (68)

...... \0 120

TABLE XIV B

Riboflavin, Adenine, Ribose, and Nucleotide Phosphorus Content. of Mitochondrial Amine Oxidase

Material Mole/100,000g Enzyme

Component 1 Component 2

Riboflavin 1"03 1"01

Adenine 1"05 1"02

Ribose 1"03

Nucleotide Phosphorus 2"16 2"18 121

TABLE XIV C

Pyridoxal Content of Phosphorylase aa and of the Mito

chondrial Amine Oxidase Componentsb

Species Pyridoxal Content C Mole/mole of Enzyme

Component 1

Component 2 0'07

Phosphorylase a

aHighly purified rabbit muscle phosphorylase was used for these analyses. bPurified enzyme component ~ with specific activity of 8000 was used for these analyses. cLactobacillus casei ATCC NO 7469 was used for the determination of pyridoxal as described by Miyazawa (95) • .... l'-l l'-l

Figure 2la. p-Chloromercuribenzoate titration of component 1.

The preparation had a specific activity of 3560. The initial solution contained 0.4 mg of enzyme in 1 ml of 0.05 M potassium phosphate buffer at pH 7.0, which had been flushed with nitrogen. To this solution was added increasing amount of p-CMB (3 x 10-4 M) solution in the same buffer. The mixture of enzyme and p-CMB was incubated for 1 hour at room temperature (25 ) after each addition of p-CMB and optical density measured subsequently in a Beckman DU spectrophotometer for a total period of 12 hours. 0.20 /0-0 0 0- ::s- 0.16 E /0 0 It) 0.12 N 0 0 0

0.04

o 2 4 6 8 10 12

No. OF - SH GROUPS (moles p CMB)/I050 PROTEIN .... N W Figure 21a t-' N ~

Figure 2lb. p-Chloromeruribenzoate titration of the component 1

in the presence of urea. The reaction mixture contained 0.399 mg of component 1 (specific activity of 3,560) in 1 ml of 8 M urea prepared

in 0.05 M potassium phosphate buffer pH 7.0, which had been saturated with nitrogen. The enzyme was allowed to stand in the above mixture for 150 minutes before titration with p-CMB. Other conditions were identical to that described in Figure 2la.

:-J t-~ 125

z -LaJ I- 0 I a: 0 Q. N 0- U) 0 0 "-- 0 -OJ :E 0 u C- .0 eo f/) ..-l N Q) I Q) 0 - l-< 0 ;:l bO E or! U) ~ "'-0 -en Q. ~o ::> v a:0 ~ (.!) 0, N :x: 0 en I u. 0 0 10 V rt) C\l . 0 0 q 0 0 0 0 0 0 0 0 z rfw OgZ aov t-' N 0\

Figure 21c. p-Ch10romercuribenzoate titration of the component 2.

The initial reaction mixture contained 0.4 mg enzyme protein (specific activity 7850) in 0.05 M potassium phosphate buffer, pH 7.0. The buffer was flushed with nitrogen before using in this experiment.

The titration was made by adding increasing amounts of p-CMB (of initial concentration of 3 x 10- 4 M) to the enzyme solution. Absorbances were measured under identical conditions described in Figure 21a. 0.3

0 0-0 0-0- 0.2 l-

:l. I E 0 10 C\J / /0 0 0.1 0 0 /

TABLE XV

Number of Titrab1e Sufhydry1 Groups in the Mitochondrial Amine Oxidase Components*

Species Number of SH/100,OOO g Enzyme No Urea 8 M Urea

Component 1 6 .95 7: 0

Component 2 7 ".10

*The number of sulfhydryl groups was determined by p-CMB titration according to the method of Boyer (89). 129

Figure 22. Activity of amine oxidase component 1 during p-CMB titration. For this experiment 0.4 mg enzyme

(specific activity of 4,020) in 1 ml of 0.05 M potassium phosphate buffer, pH 7.0, flushed with nitrogen was used.

To this enzyme solution were added increasing quantities of p-CMB and 0.01 ml samples were withdrawn one hour after each addition of p-CMB, and the activity measured at 25°. 130

>- 110 t: > 100 .... 90 «0 .... 80 Z 70 l.LJ a:0 60 l.LJ a.. 50 40 10 20 30 40 50 60 MOLE RATIO (pCMB :ENZ) Figure 22 IV. DISCUSSION AND CONCLUSIONS

Since Cotzias and Dole (96) reported that rat liver amine oxidase is predominantly associated with the mitochondrial fraction, many attempts have been made to localize this enzyme in the mitochondria. The problem, however, of subcellular localization of amine oxidase is complicated by the fact that the so called mitochondrial fraction is biochemically and morphologically heterogeneous

(97-100). Extensive studies of the subcellular fractions from tissue homogenates have shown that many enzymes or enzyme systems concerned with respiration and intermediary metabolism are associated with the mitochondria.

Investigations have been carried out in recent years to separate and characterize the mitochondrial membranes and localize the enzymes and the chemical components, whose location has been somewhat uncertain for a long time. Thus,

Parsons et al. (101, 102) have reported the separation of the "inner" and the "outer" membranes of the rat liver mitochondria. Levy et al. (103) have used digitonin to remove the outer membranes of rat liver mitochondria to investigate the structure of the "inner" membrane. Advantage has been taken of the use of digitonin to remove the "outer" mitochondrial membrane by Schnaitman, Erwin, and Greenwalt

(104), who reported that the mitochondrial amine oxidase is localized in the "outer" membrane of the rat liver mitochondria. 132

Recently, DeRobertis et a1. (105) subfractionated the mitochondrial fraction of the rat brain into five sub-

fractions which consisted of (i) myelin, (ii) membranes

and fragmented cholinergic endings, (iii) cholinergic

nerve endings, (iv) non-cholinergic nerve endings, and

(v) the free mitochondria. Subfractions (iv) and (v)

accounted for about 37% and 61%, respectively, (comprising

together, 98%) of the total amine oxidase activity. The

localization of the amine oxidase in the non-cholinergic

synapses led to suggestions that this enzyme plays a role

similar to that of cholinesterase in the cholinergic ones

(106). In fact, amine oxidase has been shown to control

the levels of neural hormones (e.g., epinephrine, nor-

epinephrine) by catalytically removing them when they are

present in excess (2, 107).

These possibilities give considerable interest to the

investigation of the various integral properties of the

mitochondrial amine oxidase, the physiological role of

which is not completely known. However, the complex

structure of the mitochondrion itself, (104), the presence

of multiple enzyme complexes of the electron transport

system and the citric acid cycle (108-110) the similar

distribution of the amine oxidase and the succinate

dehydrogenase (111), and above all, the firm attachment of

these enzymes or enzyme systems to the mitochondrial 133 structural protein or to the lipid, or both, made it difficult to purify the mitochondrial amine oxidase, and many attempts, as mentioned earlier, have led only to the partial purification of the enzyme (35, 36).

Initial investigations in this laboratory to purify

the amine oxidase from beef liver mitochondria, however, resulted in a fifty-fold purification of the enzyme (53).

In these preparations, the enzyme was eluted from the DEAE cellulose column as a final step of purification and the specific activity was 4,000 to 4,500. Later, the highly purified preparations with very high activity were obtained by extending the purification proc_edure to include hydroxylapatite column chromatography and starch block

electrophoresis steps. The outcome was the separation of two major fractions with specific activities of 3,000

to 4,000 (112), and 7,000 to 9,000 (112, 113). They are

termed component 1 and component 2, respectively, throughout

this presentation. Preliminary studies done on a few properties of the earlier preparations of mitochondrial

enzyme agreed well with later findings and a brief

discussion of these results will be made.

The substrate specificity, behavior towards various

inhibitors, and pH optima, indicated that the beef liver mitochondrial enzyme was the well known, classical mitochondrial amine oxidase. The results of the earlier

investigations on the substrate specificity agreed well with 134 the findings on purified enzyme components. In contrast to the observation made by Gorkin (35) a few years ago, that there were two amine oxidases present in rat liver mitochon dria with different substrate specificities, the beef liver mitochondrial enzyme components were found to possess the same substrate specificity (11 2 ). The degree of deamination in case of tryptamine and tyramine differed a little, but both the components showed similar activities on all the amines investigated (Table II). This finding also differs from that recently reported by Ragland (114).

However, it should be realized that different methods of purification were used by the various investigators as well as different analytical methods to detect the multiple forms of the enzyme.

Both the components were unaffected by aldehyde reagents indicating that a pyridoxal prosthetic group was not present in them (Table V). The slight inhibition shown by p-nitrophenylhydrazine or phenylhydrazine was possibly due to the benzene rings rather than the hydrazine groups that these compounds contain. This was evident from the fact that sodium benzoate inhibited the enzyme components whereas hydrazine, semicarbazide, or hydroxylamine were without effect. Metal chelators, such as cuprizone, neocuproine, 8-hydroxyquinoline, o-phenanthroline, diethyldithiocarbamate, etc. (Table IV) produced significant inhibition. These findings suggested the presence of a 135 metal in the mitochondrial enzyme components. Ethylenediaminetetraacetate (EDTA), sodium azide, or sodium cyanide, on the other hand, did not show any inhibition.

These differential effects, however, need not be considered as contradictory since EDTA and a few other chelating agents which are known to form highly stable complexes with metal ions in aqueous solutions were found to be relatively weak chelators of protein bound metals (115).

Preliminary results suggested that the beef liver mitochondrial amine oxidase was sensitive to sulfhydryl reagents. The various sulfhydryl reagents investigated required concentrations of 1 x 10- 3 M to 1 x 10- 6 M to produce a 50% inhibition of the enzyme. In the presence of

4.3 x 10- 6 M p-CMB, the oxidation of benzylamine was inhibited by 50%. Similar observations were made by Lagnado and

Sourkes (92) with the rat liver mitochondrial enzyme.

When p-CMB inhibition was examined with purified enzyme components 1 and 2, very interesting results were obtained. With a 370-fold molar excess of p-CMB component 1 showed a 26% inhibition in the enzyme activity (Table IIIC).

When a 800-fold molar excess of p-CMB was used, enzyme component 2 was inhibited only by 24% (Table III C). Line- weaver-Burk plots (Figures 13 b anc c) showed that p-CMB was a non-competitive inhibitor of both the enzyme components.

The Michaelis-Menten constants, Km, for the purified enzyme components 1 and 2 were 3.1 x 10- 4 M and 2.9 x 10-4 M at

0 25 , and the inhibition constants, Ki , under the same 136 conditions were 1.9 x 10- 5 M and 1.6 x 10- 5 M, respectively, with p-CMB.

The sedimentation coefficient was determined for a number of purified preparations of component 1. The values obtained ranged from 14 S to 14.7S. The average of 5 such determinations yielded a sedimentation coefficient of 14.4 + 0.3 when corrected to standard conditions at 20 0 in water and extrapolated to zero protein concentration.

When the sedimentation coefficient was determined by sucrose density method, a value of 14.7 + 0.3 S was estimated for component 1, and 20.6 + 0.4 S for component 2.

The Stoke's (molecular) radii estimated from gel filtration o 0 data were 60 A and 106 A for components 1 and 2, respectively.

Partial specific volumes determined for components 1 and 2 were 0.78 cm3 jg and 0.80 cm3 /g, respectively. These values of the partial specific volumes determined are markedly higher than those usually obtained for most proteins which yield values in the range of 0.7 to 0.75 cm3 jg. These differences in partial specific volumes of the two components can be explained by the fact that these enzyme components contain significant amounts of phospholipid, and proteins containing lipid materials in their molecules yield higher partial specific volumes in the hydrated form (71).

Diffusion coefficients calculated from Stoke's radii of components 1 and 2 were found to be 3.8 x--10- 7 cm2 sec- 1 and

sec~l, respectively. 137

When the molecular weight was determined from the gel filtration data by the method of Whitaker (68), a value of

400,000 was obtained for component 1. This value was found to be 396,000 when calculated from Stoke's radius, sedimentation coefficient, and partial specific volume according to the equation (iv) of the Materials and Methods

Section. The molecular weight calculated for component 1 from the sedimentation and diffusion coefficients were

423,000. The molecular weights determined by these three methods were 1,300,000 1,195,000, and 1,355,000,respective1y, for component 2. These molecular weights for the two beef liver mitochondrial amine oxidase components differ from those reported by Erwin and Hellerman (39) for bovine kidney mitochondrial monoamine oxidase. These authors reported a molecular weight of 290,000 calculated from a sedimentation coefficient of 10.6 S, an assumed partial specific volume of 0.75, and an apparent diffusion constant

(D20) of 3.5 x 10- 7 cm2 sec- l calculated from Sephadex G-200 gel filtration data by the method of Ackers (116). Same molecular weight (290,000) was reported by Youdim and

Sourkes (38) for rat liver monoamine oxidase. These authors, however, showed a sedimentation coefficient of 6.3 S for their enzyme in contrast to 10.6 S for that of Erwin and

Hellerman (39). On the other hand, the molecular weight reported by Tipton (40) for pig brain mitochondrial enzyme was 102,000 as determined by Sephadex G-200 gel filtration. 138

In addition to his major fraction with the above molecular weight, he eluted another small fraction which corresponded to a molecular weight of 435,000, which, he considered might be a tetrameric form of the lower molecular weight fraction. In these regards, the low molecular weight component or component 1 of beef liver enzyme corresponds to the tetrameric form of Tipton's enzyme, and the high molecular weight fraction or component 2 represents a trimer of component 1. There are many reasons to support this contention which will be discussed later.

Of all the metals analyzed (Table XII) only copper was found to be present in significant amounts. A number of highly purified fractions of the enzyme component 2 yielded values ranging from 0.15 pg to 0.17 ~g of copper per milligram of protein. On the basis of molecular weight of

1,300,000, the enzyme contains 3 gram atoms of copper per mole of component 2. The preliminary assumption that a metal participated in the catalytic activity of the enzyme could not be conclusively proved. However, bis-cyc1ohexanone oxa1dihydrazone (cuprizone), 'a specific che1ating agent for copper, produced mixed type of inhibition of the DEAE cellulose eluted enzyme (Figure 14). In addition, sodium diethy1dithiocarbamate, a-hydroxyquinoline, a,a -bipyridy1, etc, inhibited the enzyme, indicating the presence of a metal in it. The determination of other metals demonstrated that they are either absent or present in negligible amounts 139

(Table XII). The copper content, nevertheless, showed an initial drop in the first two steps of purification, and indicated slight but steady increase in the subsequent steps of purification (Figure 19a). Erwin and Hellerman, in this respect, reported similar findings with beef kidney mitochondrial monoamine oxidase. They estimated a copper content of 0.15 pg to 0.19 pg per milligram of their enzyme. Youdim and Sourkes (38) on the other hand, found that the rat:1iver mitochondrial enzyme contains

0.12% iron and 0.034% copper, corresponding to 1.2 pg iron and 0.34 pg copper per milligram of their enzyme protein.

In beef liver mitochondrial enzyme, however, iron occurred only in the crude enzyme and the largest amount found was

0.07 pg/mg of protein in the second step of purification.

The iron content dropped sharply in the subsequent steps as shown in Figure 19b, and is considered to be an impurity.

The result obtained in this work suggests that copper is the only metal present in significant amount. The reason it did not respond to cyanide, or EDTA, is that many chelating agents which chelate metals in aqueous solutions fail to do so when the metal is bound to a protein (115). Copper ions, in addition, react with amino acids or proteins more strongly than do any other metal ions (117). When copper is a prosthetic group of an enzyme, the copper ion cannot be separated by any amount of dialysis. A drastic treatment is necessary to remove it 140 from the protein (117).

rhe beef liver mitochondrial amine oxidase contains a flavin which is covalently linked to the enzyme protein.

Due to this tenacious attachment to the enzyme molecule, it was not easy to remove it from the enzyme and characterize it. Despite this difficulty, however, the enzyme has conclusively been shown to be a flavoenzyme by physical, chemical, and microbiological methods. The spectrum, however, does not resemble that of a typical flavoenzyme (118), but has an absorption peak at 4S0 mp and a shoulder at 480 mp(Figure lSa) which are reducible by the substrate"benzylamine and by sodium hydrosulfite

(Figure lSb). This property of the enzyme has been used for the spectrophotometric determination of the flavin dinucleotide cofactor in both the components of the enzyme.

That the enzyme was a flavoprotein was inferred from the following observations: (i) The yellow color of the enzyme was intensified with each step of purifica~ion. (ii) The purified yellow-peptide of the pronase digest of the enzyme exhibited spectral properties characteristic of riboflavin, or flavin nucleotides. ~iii) The yellow colored material promoted the growth of L. casei which cannot thrive without riboflavin. (iv) The riboflavin content increased steadily in the subsequent steps of purification and was proportional to the specific activity of the enzyme

(Figure 20). About 1.3 moles of riboflavin per 400,000 g of component 2 were determined by the microbiological method. 141

The most accurate results were obtained by the spectrophoto metric method which yielded a value of 4.2 moles of flavin nucleotide per 400,000 g of component 1 and 4.05 moles per

400,000 g of component 2.

Once it was confirmed that the beef liver enzyme contained riboflavin, it was necessary to determine if it were a flavin di- or mono-nucleotide. Flavin mononucleotide does not contain ribose or adenine. It was, therefore, decided to investigate the adenine and ribose content of

the enzyme. Microbiological assay of the enzyme hydrolyzate yielded a value of 1.48 moles of purine (probably adenine) for 400,000 g of enzyme component 2. A microchemical method (81), however, demonstrated conclusively that the enzyme contained 4.3 moles of adenine per 400,000 g of component 1 and 4.02 moles per 400,000 g of component 2.

The ribose content of the enzyme was then determined to be about 4.1 moles of ribose per 400,000 g of enzyme component 2. Next the phosphorus content of the purified enzyme was then measured. Since the mitochondria are rich in phospholipids, the determination of the nucleotide phosphorus was not easy. Total phosphorus and the phospholipid phosphorus were estimated for both the enzyme components and the difference between total phospherous and phospholipid phosphorus yielded the values for the nucleotide phosphorus per 400,000 grams of enzyme component,

1 and 2, respectively. These results suggest that the flavin nucleotide is flavin adenine dinucleotide or FAD. 142

Since 1 mole of FAD contains 1 mole each of riboflavin, ribose, and adenine, and 2 gram atoms of phosphorus, these results further suggest that the beef liver mitochondrial amine oxidase contains 4 moles of FAD per mole of component 1, and 12 moles of FAD per mole of component 2. Microbiological determination of riboflavin yielded low values which cannot be explained satisfactorily. The value obtained by this method accounts for only 33% of that determined spectro photometrically. The low value estimated by microbiological method was also obser~ed in the case of succinate dehydro genase (119) where a value, 29% of the exact flavin content of that enzyme, was estimated.

That the FAD was catalytically involved in the enzyme was suggested by the observation that the 450 mp peak and the 480 mp shoulder were bleached by the substrate benzylamine and by sodium hydrosulfite. Specific inhibitors of the enzyme were found to prevent the reduction of the visible maximum at 450 mp. Those which were not substrates, did not bleach the 450 mp absorption band (113).

Determination of total phosphorus yielded a value of

132 gram atoms of phosphorus per mole of enzyme component 2.

When phosphorus determinations were made on the lipid extracts of the enzyme, 24 moles ~nd 106 moles of phospho lipids per mole of enzyme were found in components 1 and 2, respectively. These values correspond to 0.059 mp mole and

0.086 mp mole of phospholipid, respectively, per milligram 143

of component 1 and component 2. These values are in

agreement with the result of Erwin and Hellerman (39) who

estimated a value of 0.06 mp mole of phospholipid for their

enzyme. As already mentioned, this high phospholipid

content in components 1 and 2 is responsible for the high partial specific volumes of these proteins. The partial

specific volumes determined for component 1 and component 2,

did not agree, therefore, with the assumed partial specific volume of 0.75 reported by Erwin and Hellerman (39).

Determination of the sulfhydryl residues revealed that

100,000 grams each of component 1 and component 2 contained

7 and 7.1 titratable -SH groups, respectively. These values

correspond to 28 -SH groups per mole of component 1 and

86 per mole of component 2. Erwin and Hellerman (39)

found 8 titrable thiol residues per 100,000 grams of

their enzyme. They have reported further, that the activity

of their enzyme declined with increasing p-CMB concentrations

and was completely inhibited when 7.3 moles of p-CMB per

100,000 grams of protein were added. This indicated that

the inhibition was complete when all the detectable thiol groups were titrated.

In the present work, however, contrary results were obtained. The residual activity was as much as 86% in component 1 and around 30% in component 2 when all the -SH groups of these components were titrated with p-CMB. Both the components of the enzyme exhibited the non-competitive 144 type of inhibition (Figures l3b and c). Alkylating agents such as iodoacetic acid, iodoacetamide, N-ethylmaleimide, etc., which are potent inhibitors of certain sulfhydryl enzymes (120-122) were found to be apparently ineffictive in inhibiting the enzyme (Table III P). Further, the inhibition due to p-CMB was observed to be reversed on dialysis. From these findings, it appears that the sulfhydryl groups are not involved in the catalytic function of the enzyme. They are possibly associated with the conformational requirement of the enzyme.

Finally, the various properties of the beef liver mitochondrial amine oxidase are summarized in Tables XVI A,

XVI B, XVI C, and XVI D. It seems that there are at least

2 components of the enzyme, one possibly being the polymeric form of the other. The similarities in their substrate specificities (Table II), inhibition by various chelating agents (Table IV), non-competitive inhibition with p-CMB etc. (Figures l3b and c), support this contention.

Evidence derived from chemical characterization of the two components demonstrates that they have the same FAD and sulfhydryl group contents, relative to their molecular weights. The most convincing evidence is the fact that the two components have approximately the same amino acid composition (123). It is also a reasonable assumption that the higher molecular weight fraction or component 2 is the parent enzyme and that the lower molecular weight fraction TABLE XVI A

Properties: 1 a. Kinetic Parameters of Mitochondrial Amine Oxidase

a c Species Substrate Specific Stability Optimal R'om

Activity pH (X 104 M) eub / mg Protein

Component 1 Benzylamine 3000-4000 Thermolabile 9.2 3.1

Component 2 Benzylamine 7000- 9000 Thermolabile 9.2 2.9

aAmong 17 mono-, di-, and poly-amines examined (Table II), benzylamine proved to be the best substrate and was used in all enzyme determinations. beu = enzyme unit; one unit is defined as the amount of enzyme which produces a change in absorbance of 0.001 per minute at 250 mu at 25 0 • cOptimal pH was determined on the enzyme after DEAE-cellulose step and was not done after final purification of the two components. dMichaelis-Menten constant, Km, was determined at pH 7.4, using 0.2 M potassium phosphate buffer, and benzylamine substrate at 250.

t-' .po VI TABLE XVI B

Properties: 1 b.' Kinetic Parameters of Mitochondrial Amine Oxidase

Species Inhibition Ki e

Product Sulfhydrylb Metal C Aldehyded (X 105 M) Inhibitiona reagents Chela tors reagents (NH 4+)

Component 1 + + 1.9 Component 2 + + 1.6 a(NH4)2S04 was used as for product inhibition. bThiol reagents used inhibited in the order Hg+-S Ag+· = p-CMB > Cd++. cCuprizone, Neocuproine, and 8-hydroxyquiloline (Table IV) inhibited, whereas cyanide, azide, and EDTA, did not. dAmong aldehyde reagents, only phenylhydrazine, and p-nitrophenylhydrazine showed some inhibition (Table V). eInhibition constant, Ki' was determined by using p-CMB which inhibited non-competitively...... I: 0\ TABLE XVI C

Properties: 2. Molecular Parameters of Mitochondrial Amine Oxidase

c d Species Stoke's V s20,W n Mol. Wt. e f/ f o Radiusa Partial (X 1013 sec) (X 107cm2sec- l ) Specific A Volume

cm 3/gb

Component 1 60 0.78 14.4 3.8 407,000 1. 17

Component 2 106 0.80 20.6 2.0 1,280,000 1. 46 aStoke's radii for components 1 and 2 were determined from gel filtration data according to Siegel and Monty (45). bpartial specific volumes were determined pycnometrically (67). cSedimentation coefficient for component 1 was determined by sedimentation velocity method (61) and that of component 2 by sucrose density gradient centrifugation (37) . dniffusion coeffkients were calculated from Stoke's radii and the use of the following equation, n = kT/6J1Na...... po. eMolecular weights of the two amine oxidase components are the average values of " those determined by three methods--gel filtration, the method based on Stoke's law, and the sedimentation-diffusion method. TABLE XVI D

Properties: 3. Chemical Parameters of Mitochondrial Amine Oxidase

Species Coppera Phospholipidb FAD c Pyridoxal d -SH residuee

( g- a tom ) ( mole ) ( mole ) ( mo 1e ) ( number ) ( per ) ( per ) ( per ) ( per ) ( per ) (mo Ie Enzymf!) (mole Enzyme) (mole Enzyme) (mole Enzyme) (mo Ie Enzyme)

Component 1 24 4 28

Component 2 3.1 106 12 0.07 86

aCopper was determined by the microchemical method of Peterson and Bollier (76). bphospholipid was extracted by the method of Folch et al. (88) and quantitated by phosphorus determination (87). -- --

cRiboflavin, ribose, adenine, and nucleotide phosphorus were in the proportion of 1:1:1:2 as found in FAD.

dPyridoxal was determined by the method of Miyazawa (94).

eSu~fhydryl groups were determined by the method of Boyer (89).

I-' -I:' 00 149

or component 1 is derived from component 2. Results of the phospholipid determinations show that component 2 has a higher phospholipid content than that of component 1, suggesting a release of component 1 from the lipid-enzyme complex of the parent molecule or component 2, since it is unlikely that component 1 with a proportionately lower phospholipid content will aggregate to yield a trimer with a proportionately higher phospholipid content. The frictional ratios of component 1 and component 2 were calculated to be

1.17 and 1.46, respectively, suggesting that component I is more spherical than component 2.

The question whether two or more than two components are present in the beef liver mitochondrial enzyme as separate components or whether they are artifacts of the purification procedure merits further investigation.

Moreover, since Tipton's (92) investigations with the pig brain mitochondrial enzyme demonstrated a major component and a minor component with molecular weights of 102,000 and

435,000, respectively, another form with a molecular weight in the order of 100,000 may be isolated by the sonication procedure. Sonication procedure was not used in the present study. However, it is hoped that the properties of the two enzyme components outlined in this dissertation will provide some insight to the other workers who intend to study this aspect as well as other properties of this enzyme. V. SUMMARY

Beef liver mitochondrial amine oxidase was purified

in this laboratory by extraction with a nonionic detergent

Triton X-lOO, ammonium sulfate fractionation, column chromatography, and electrophoresis. Two fractions, which

are described as component 1 and component 2 in the text, were isolated. Various purity studies were made on these purified enzyme components.

The enzyme components 1 and 2 were thermolabile and lost 25% and 40%, respectively of their activity on standing at room temperature for 7-8 hours. Freezing resulted in prompt loss in activity due to denaturation of both the components.

When the substrate specificity of the components were examined, they showed similar specificities towards

the amines tested. Lysine and diamines except kynuramine, and all polyamines, were not deaminated by either enzyme component.

Certain metal chelators like cuprizone, 8-hydroxy quinoline,a ,a -bipyridine, and neocuproine inhibited the enzyme, whereas other chelating agents like EDTA, cyanide, or azide did not. Aldehyde reagents did not show signifi cant inhibition of either component. Certain thiol reagents like p-CMB, HgC12, AgN03, etc., which form metal 151

mercaptides with the enzyme sulfhydryl groups moderately inhibited the enzyme at high concentrations. p-Chloro- mercuribenzoate was a noncompetitive inhibitor of both

components. The Km for components 1 and 2 were 3.1 x 10- 4 M and 2.9 x 10- 4 M, respectively. The corresponding

Ki values in the presence of p-CMB were 1.9 x 10- 5 M and

The product, NH3 (in the ionized form,

NH4+ ), did not have an inhibitory effect.

Sedimentation studies showed that the sedimentation

coefficients of components 1 and 2 were 14.4 + 0.3 Sand

20.6 + 0.4 S, respectively. Frictional ratios of 1.17 and

1.46, respectively, for components 1 and 2 were calculated,

indicating that component 1 is more spherical than

component 2. Molecular (or Stoke's) radii calculated from o 0 gel filtration data were 60 A for component 1 and 106 A

for component 2. Diffusion coefficients for components 1

and 2 were calculated to be 3.8 x 10- 7 cm2 sec-land 2.0 x 10- 7 cm2 sec- 1 , respectively. The partial specific

volumes were estimated pycnometrica1ly and were found to

be 0.78 cm3 jg for component 1 and 0.80 cm3 }g for com-

ponent 2. Molecular weights as determined by the gel

filtration method were 400,000 and 1,300,000 for components

1 and 2, respectively. Stoke's Law yielded molecular weights of 396,000 + 10,000 and 1,195,000 and Svedberg's 152

equation (S and D), values 425,000 ~ 10,000 and 1,355,000,

respectively. Averages of the molecular weights determined

by these three methods were 406,000 ~ 14,700 and 1,280,000

+ 91,500, respectively, for components 1 and 2.

Metal analyses of the enzyme yielded values of 0.15

to 0.17 pg copper per milligram of the enzyme protein.

This value corresponded to 1 gram atom of copper per mole

of component 1 or 3 gram atoms of copper per mole of

enzyme component 2. The presence of iron was insignificant

and was considered to be an impurity. Cobalt, manganese,

and molybdenum were found to be absent.

The prosthetic group , FAD, is covalently linked to

the enzyme. There is 1 FAD per 100,000 grams of either

component suggesting the presence of 4 moles of the

dinucleotide in component 1 and 12 moles in component 2.

Phospholipid is present in markedly large amounts in

the enzyme to the extent of 24 moles per mole of component

1 and 106 moles per mole of component 2.

There are 28 -SH groups in a molecule of the compo nent 1 and as many as 86 such residues in component 2.

The substrate specificity, the inhibitor specificity,

amino acid composition, and other properties of component

1 and component 2 are remarkably similar suggesting that one is the polymeric form of the other. The ratio of the molecular weights, FAD contents, and numbers of -SH groups

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