Evidence for Association of Enzyme Polymer with Liver Microsomes (Lipogenesis/Enzyme Regulation) LEE A
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Proc. Nati. Acad. Sci. USA Vol. 78, No. 6, pp. 3639-3643, June 1981 Cell Biology Microsomal acetyl-CoA carboxylase: Evidence for association of enzyme polymer with liver microsomes (lipogenesis/enzyme regulation) LEE A. WITTERS, STEVEN A. FRIEDMAN, AND GEOFFREY W. BACON Diabetes Unit and Medical Services, Massachusetts General Hospital and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114 Communicated by P. Roy Vagelos, February 25, 1981 ABSTRACT Fatty acid synthesis is traditionally viewed as cellular distribution of the enzymes of lipogenesis. The results being confined to the cytosolic cellular fraction, although a sub- ofthe present study suggest that the microsomes may be a major stantial body of data indicates that both microsomes and mito- locus of fatty. acid synthesis in the liver. chondria are capable ofinitiating fatty acid synthesis and may con- tain acetyl-CoA carboxylase [acetyl-CoA:carbon-doxide ligase MATERIALS AND METHODS (ADP-forming), EC 6.4;1.2], fatty acid synthetase, and ATP-ci- Materials. Male C-D rats (130-150 g) were obtained from trate Iyase [ATP citrate (pro-3S)-lyase; ATP:citrate oxaloacetate- Charles River Breeding Laboratories. Sodium [32Plphosphate lyase (pro-3S-CH2COO- - acetyl-CoA; ATP-dephosphorylat- ing), EC 4.1.3.8] activities. We have identified 32P-labeled acetyl- and NaH14CO3 and [3H]AMP were obtained from New England CoA carboxylase and 32P-labeled ATP-citrate Iyase by immuno- Nuclear. Adenosine monophosphate, tyramine, malonyl-CoA, precipitation of a rat hepatocyte microsomal preparation. In the glucose 6-phosphate, NADH, and NADPH were obtained from transition between the fasting state (low rates of lipogenesis) and Sigma. Leuco-2',7'-dichlorofluorescein diacetate was obtained fasting/re-feeding (high rates), the fraction of total cytosolic plus from Eastman. Horseradish peroxidase was purchased from microsomal acetyl-CoA carboxylase in the microsomes increases Worthington. The sources of reagents used in other enzyme from 6% to 43%, whereas the microsomal proportion oftotal fatty assays are as previously published (11). acid synthetase and ATP-citrate Iyase remains approximately Methods. Microsomes and cytosolic fractions of liver were 10%. Microsome isolation conditions favoring carboxylase poly- prepared in the following manner. Rats were killed by cervical merization (presence of citrate) promote microsomal association, dislocation and the liver was rapidly removed and rinsed in 10 whereas conditions favoring enzyme protomerization (malonyl- mM Tris HCl, pH 7.2/2 mM dithiothreitol/0.25 M sucrose at CoA, preincubation with cyclic AMP/ATP/Mg2+) diminish this 40C. The liver was then homogenized in the same buffer with association. The microsomal enzyme has a 5-fold higher specific a Dounce homogenizer (20 strokes with a loose pestle for whole activity than the cytosolic enzyme as determined by immunotitra- liver; 40 strokes with a tight pestle for hepatocyte pellets) in the tion. Sucrose density gradient analysis of the microsomal fraction above buffer [1 g/2 ml ofwhole liver or 2.0 x 10 cells (= 1 g) indicates that a substantial portion of carboxylase activity sedi- per 2 ml for hepatocytes]. The homogenate was centrifuged at ments with marker enzymes for endoplasmic reticulum, plasma 12,000 x g for 20 min in a Sorvall RC-2B refrigerated centri- membrane, Golgi apparatus, and outer mitochondrial membrane, fuge. The supernatant obtained was then centrifuged at 105,000 while cytosolic enzyme or isolated enzyme incubated under poly- x g for 60 min in a Beckman L2 centrifuge. This supernatant, merizing conditions does not penetrate the gradient. These data termed cytosol, was removed, and the volume was measured. suggest that the microsomes may be a significant locus offatty acid The pellet, termed microsomes, was resuspended in an iden- synthesis initiated with association ofacetyl-CoA carboxylase poly- tical volume ofbuffer by homogenization, employing a glass rod mer with this fraction. with Vortex mixing. In experiments designed to resolubilize Fatty acid synthesis and the activities of three important lipo- microsomal acetyl-CoA carboxylase, the microsomes were re- genic enzymes, ATP-citrate lyase [ATP citrate (pro-3S)-lyase; suspended in the above buffer containing sodium acetate (200 ATP:citrate oxaloacetate-lyase (pro-3S-CH2 COO- acetyl- mM) and recentrifuged at 105,000 X g for 60 min. This super- CoA; ATP-dephosphorylating), EC 4.1.3.8], acetyl-CoA car- natant was designated resolubilized microsomal acetyl-CoA car- boxylase [acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC boxylase. In immunoprecipitation experiments, the micro- 6.4.1.2], and fatty acid synthetase, have generally been re- somes were resuspended in the homogenization buffer containing garded as being confined principally to the cytosolic cellular 1% Triton X-100 and the solubilized microsomal proteins were fraction. However, there are several studies to indicate that isolated after recentrifugation. both microsomes and mitochondria are capable ofinitiatingfatty Hepatocytes were prepared by collagenase digestion of the acid synthesis and contain all three enzymatic activities (1-6). isolated perfused rat liver (12). 32p labeling and cell incubations We have been interested in the role ofcovalent enzyme phos- were as described (13). 32P-Labeled fractions were subjected phorylation in the regulation of lipogenesis. Previous studies to polyacrylamide gel electrophoresis with subsequent radioau- from our laboratory and others (7-10) have indicated that both tography as described (13). Immunoprecipitation was carried acetyl-CoA carboxylase and ATP-citrate lyase are subject to out by a published method (7). hormonally induced changes in enzyme phosphorylation. Dur- Rats were prepared for experiments after an 18-hr fast ing the course of these studies, it was recognized that these (fasted), or were fasted for 72 hr and re-fed for 48 hr with a low- phosphoproteins were not confined to the cytosolic fraction. fat high-carbohydrate diet (fat-free test diet, ICN). These findings have prompted the reinvestigation of the sub- Enzyme Assays. Acetyl-CoA carboxylase was assayed with a preincubation assay under conditions previously reported The publication costs ofthis article were defrayed in part by page charge (11). One unit of acetyl-CoA carboxylase activity is 1 ,umol of payment. This article must therefore be hereby marked "advertise- H4C03- fixed into malonyl-CoA per minute at 37°C. ATP-ci- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. trate Iyase activity was measured by a published method (10). 3639 Downloaded by guest on September 25, 2021 3640 Cell Biology: Witters et al. Proc. Natl. Acad. Sci. USA 78 (1981) In blank reactions of each extract, the ATP was omitted. One Cyto- Micro- unit of ATP-citrate lyase activity is 1 ,umol of NADH oxidized plasmic somal per minute at 250C. Fatty acid synthetase was assayed by the method of Katiyar and Porter (15). In blank reactions of each extract, the malonyl-CoA was omitted. One unit of fatty acid _ 240,000 synthetase activity is 1 umol of NADPH oxidized per min at w_._ 250C. £_ I 4 123,000 Immunotitration. Resolubilized microsomal enzyme and cy- _- 94,000 tosolic enzyme (adjusted to the same acetate concentration) were assayed in the presence ofvarious amounts ofacetyl-CoA carboxylase antiserum raised in sheep against homogeneous rat liver enzyme (14). A preincubation assay was employed with a 30-min incubation of enzyme in the presence of5 mM sodium -59,000 citrate, 5 mM MgCl2, and antiserum prior to initiating the re- action. Under these conditions, we routinely see only an 80% - inhibition of enzyme activity at maximal antiserum concentra- 46,000 tions, presumably due to antibody-bound enzyme that remains catalytically active; this has been noted by other investigators (16). Under immunoprecipitation conditions (not shown), there is 100% precipitation of activity with this antiserum. Immu- notitration curves were constructed and the immunotiter was determined by extrapolation ofthe initial slope ofthe inhibition curve to zero activity. The specific activity ofthe enzyme is then determined as a ratio ofactivity in the absence of antiserum to the immunotiter and is expressed as milliunits of activity per ,.l-equivalent of antiserum. Gradient Analyses. The microsomal and cytosolic fractions _ Dye front were analyzed by sucrose density gradient centrifugation. Cy- ~ tosolic and microsomal fractions were prepared as above, except FIG. 1. Cytoplasmic and microsomal (32P]phosphopeptides. Shown that the homogenization buffer was 100 mM potassium phos- is a radioautograph of a sodium dodecyl sulfate/polyacrylamide gel, phate, pH 7.20/10 mM sodium citrate/2 mM dithiothreitol/ previously electrophoresed, stained and destained, and dried. The left- 2% (wt/wt) sucrose. The cytosolic and microsomal fractions (1.0 hand gel was derived from electrophoresis of 20 ug of 32P-labeled cy- ml) were then layered over a sucrose density gradient tosol from 32P-labeled hepatocytes offasted/re-fed rats; the right-hand sucrose in the same buffer (12.2 ml)]. gel is 20 ,ug oflabeled microsomal protein. Both fractions were isolated [13.66-54.1% (wt/wt) as detailed in the text. The molecular weight standards are purified Centrifugation was in an SW-41 rotor in a Beckman L265 ul- rat liver acetyl-CoA carboxylase (240,000), rat liver ATP-citrate lyase tracentrifuge at 4°C for 45 min. Twenty-four fractions (0.55 ml (123,000), glycogen phosphorylase