Proc. Nati. Acad. Sci. USA Vol. 78, No. 6, pp. 3639-3643, June 1981 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 , plasma 12,000 x g for 20 min in a Sorvall RC-2B refrigerated centri- membrane, , 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 , 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 : 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 (94,000), pyruvate kinase (59,000), per fraction) were collected from the bottom of the tube. In and an unidentified hepatic cytosolic phosphoprotein (46,000; see ref. addition to the above enzyme assays, the gradient fractions were 13). analyzed for several other subcellular marker enzymes by es- tablished techniques, namely NADH oxidase (marker for en- cipitated by anti-acetyl-CoA carboxylase antiserum, and the doplasmic reticulum and outer mitochondrial membrane) (17), 123,000-dalton microsomal phosphoprotein, by anti-ATP-ci- glucose-6-phosphatase (marker for endoplasmic reticulum) (18), trate lyase antiserum (not shown). monoamine oxidase (marker for outer mitochondrial mem- Distribution of Enzymatic Activities. Cytosolic and micro- brane) (19), 5'-nucleotidase (marker for plasma membrane) (17), somal fractions were prepared from livers of fasted/re-fed an- lactate dehydrogenase (marker for cytosol) (20), and galactosyl- imals and each was assayed for acetyl-CoA carboxylase, ATP- transferase (marker for Golgi apparatus) (21). The density of citrate lyase, and fatty acid synthetase activities (Table 1). These each fraction was determined by pycnometry. The protein con- data indicate that 43% of the total acetyl-CoA carboxylase ac- tent of each fraction was determined by the method of Lowry tivity present in the cytosolic plus microsomal fractions is pres- et al. (22). Each gradient fraction was subjected to sodium do- ent in the microsomes, while only approximately 13% of ATP- decyl sulfate/polyacrylamide gel electrophoresis employing citrate lyase and 7% of fatty acid synthetase total activities are slab gels (7.2% pH 9.81) as devised by Neville (23). present in the same fraction. Potential enzyme activities in other subcellular fractions (nuclei, mitochondria) were not de- RESULTS termined in the present study. Microsomal Phosphopeptides. Microsomal and cytosolic All three enzymes are well known to be induced in the tran- fractions were prepared from hepatocytes from fasted/re-fed sition between fasting states and states characterized by high rats after labeling with 32Pi for 60 min. The fractions were sub- rates oflipogenesis. The proportion of enzyme activity distrib- jected to polyacrylamide gel electrophoresis. Radioautography uted to the cytosolic and microsomal fractions (subsequently of the dried and stained gel indicated two major phosphopro- referred to as total activity) at two extremes oflipogenesis was, teins ofsubunit molecular weight 240,000 and 123,000 present therefore, explored. These data are also displayed in Table 1. in both the cytosolic and microsomal fractions (Fig. 1). We had The microsomal proportions oftotal ATP-citrate lyase and fatty previously identified the cytosolic proteins as being acetyl-CoA acid synthetase activity are essentially the same in fasting state carboxylase and ATP-citrate lyase, respectively (7, 10). The (low rates oflipogenesis) and the fasted/re-fed state (high rates identity ofthe microsomal phosphoproteins was established by of lipogenesis). In contrast, acetyl-CoA carboxylase activity in immunoprecipitation ofTriton-solubilized microsomal extracts the microsomal fraction rises from 6% to 43% of total with in- with specific antisera directed against each enzyme. The duction of lipogenesis by fasting/re-feeding. The absolute 240,000-dalton microsomal phosphoprotein is specifically pre- change in activities present in the microsomal and cytosolic frac- Downloaded by guest on September 25, 2021 Cell Biology: Witters et al. Proc. Natl. Acad. Sci. USA 78 (1981) 3641

Table 1. Distribution of lipogenic enzymes to cytosol and microsomes with nutritional induction Nutritional Acetyl-CoA carboxylase ATP-citrate lyase Fatty acid synthetase state C M M+C M/(M+C) C M M+C M/(M+C) C M M+C M/(M+C) Fasted 180 11 192 0.060 73 8 81 0.099 15 1 15 0.071 +5 ±5 ± 5 ±0.009 ±3 ±1 ±3 ±0.004 ±2 ±0.5 ±2 ±0.027 Fasted/re-fed 164 126 290 0.431 888 129 1009 0.128 221 16 237 0.066 + 5 ± 12 ± 15 ±0.022 ± 35 ± 18 ±44 ± 0.015 ± 13 ± 5 ± 15 ± 0.016 Cytosol (C) and microsome (M) fractions were isolated from livers ofrats either fasted for 18 hr or fasted for 72 hr and re-fed for 48 hr with a high- carbohydrate low-fat diet. Each number represents the mean (±SEM) of five paired nutritional experiments. Enzyme activities are expressed as milliunits per milliliter of cytosol or of microsomes resuspended to the same volume as that of the cytosol. The fractions contained 30-40 mg of cytosolic protein or 6-8 mg of microsomal protein per ml. M + C is the summation ofthe microsomal and cytosolic activities; the acetyl-CoA car- boxylase blank reaction (acetyl-CoA omitted) is too high to permit accurate assessment of total enzyme activity in the 12,000 x g supernatant.

tions (Table 1) indicates that during induction the cytosolic and g supernatant or incubation ofthis supernatantwith cyclic AMP, microsomal activities of ATP-citrate lyase and fatty acid syn- ATP, and Mg2" prior to microsome preparation decreases the thetase both increase to approximately the same extent. How- relative microsomal content; both of these conditions are as- ever, during induction, it is the microsomal activity of acetyl- sociated with enzyme protomerization (24, 25). Similar results CoA carboxylase that increases markedly (10-fold), with no were obtained when microsomes from fasted rats (stripped of change in the cytosolic fraction. any endogenous acetyl-CoA carboxylase by repetitive washing The association of acetyl-CoA carboxylase with the micro- with 0.2 M sodium acetate) were incubated with the cytosolic somal fraction appears to be dependent upon the polymeric fraction after treatment of this fraction with either citrate or state ofthe enzyme. Addition ofcitrate (10 mM) to the 12,000 x cyclic AMP/ATP/MG2+ (Table 2B). These data demonstrate g supernatant prior to high-speed centrifugation or initial ho- that the reassociation of the enzyme with microsomes is in- mogenization and microsomal preparation in a phosphate/ci- creased substantially by citrate and inhibited by preincubation trate buffer markedly increases the fraction oftotal cytosolic plus with cyclic AMP/ATP/Mg2e. Further support for the concept microsomal enzyme association with the microsomes (Table 2A). ofpolymeric association with the microsomes can be seen in the Conversely, the addition of malonyl-CoA to the 12,000 X determination of avidin-sensitive activities in each fraction; more than 90% of the cytosolic enzyme is inhibited by avidin, whereas less than 40% of the microsomal enzyme is inhibited Table 2. Distribution of acetyl-CoA carboxylase to cytosol and (not shown). Previous studies in other laboratories have indi- microsomes under differing preparation conditions cated that the protomeric form ofthe enzyme is avidin sensitive, Acetyl-CoA carboxylase whereas the polymer is relatively insensitive (24). Condition C M M + C M/(M + C) Because ofthe demonstration that enzyme polymer appeared A. Control (Tris) 79 71 150 0.472 to preferentially associate with the microsomes, it was impor- + citrate 29 166 194 0.852 tant to exclude the possibility that polymer was simply cosed- + malonyl-CoA 60 28 88 0.315 imenting with the microsomes during preparation. Sucrose + cyclic AMP/ATP/Mg2e 180 45 225 0.198 density gradient analysis indicates that the cytosolic enzyme, Pdcitrate 23 106 129 0.818 under polymerizing conditions in the presence of phosphate/ citrate, does not penetrate a 13.66-54.1% gradient (Fig. 2),* B. Control (Tris) 135 15 150 0.100 while a substantial fraction ofthe microsomal enzyme sediments + citrate 44 70 114 0.613 in a broad area with a peak at p = 1.14 g/ml (Fig. 3A). No fatty + cyclic AMP/ATP/Mg2e 159 8 167 0.049 acid synthetase and only traces of ATP-citrate lyase activities were associated with these fractions in the microsome gradient. In A, cytosol (C) and microsomes (M) were isolated from fasted/re- fed animals by high-speed centrifugation ofa 12,000 x g supernatant Polyacrylamide gel electrophoresis of each microsomal gradi- fraction prepared by liver homogenization in either 10 mM Tris-HCl, ent fraction revealed two peaks of a Coomassie blue-stained pH 7.2/2 mM dithiothreitol/0.25 M sucrose or 100 mM potassium phos- 240,000-dalton band (Fig. 3B); in the fractions above the gradi- phate, pH 7.2/10 mM sodium citrate pH 7.2/2 mM dithiothreitol/0.25 ent (22-24), this band corresponds to both acetyl-CoA carboxylase M sucrose. In the Tris isolation, either citrate (10 mM final concen- tration) or malonyl-CoA (100 uM final concentration) was added just and fatty acid synthetase activities. In the p range 1.120-1.145 prior to high-speed centrifugation or the 12,000 x g supernatant was g/ml, the 240,000-dalton band peak corresponds to the second incubated with 10 ,uM cyclic AMP/1 mM ATP/2 mM Mg2e for 20 min peak of acetyl-CoA carboxylase activity alone. at 37°C prior the second centrifugation. In each case, the microsomes Several subcellular marker enzymatic activities were also were resuspended into a buffer identical in composition to the corre- determined on the same gradient fractions in an effort to further sponding cytosolic fraction. In B, microsomes from 18-hr-fasted ani- mals were prepared and washed two times with Tris buffer containing define the potential subcellular locus ofacetyl-CoA carboxylase 0.2 M sodium acetate. These stripped microsomes (8.3 mg of micro- association. As shown in Fig. 3C, the region ofp between 1.10 somal protein) were incubated with a cytosolic fraction (28.3 mg cy- and 1.17 g/ml, which contains acetyl-CoA carboxylase activity, tosolic protein) from fasted/re-fed livers that had been previously in- also contains marker enzymes for plasma membrane, endo- cubated at 37°C for 20 min in the presence ofno additions (control), 10 plasmic reticulum, outer mitochondrial membrane, and Golgi mM citrate, or 10 ,uM cyclic AMP/1 mM ATP/2 mM Mg2e. The re- combined microsomal and cytosolic fractions were then recentrifuged apparatus; glucose-6-phosphatase activity (not shown, but as- at 105,000 x g for 60 min and the microsomal pellet was resuspended in a buffer equal in volume and composition to that of the superna- * Isolated rat liver acetyl-CoA carboxylase, ascertained to be a 42S spe- tant fraction. In both A and B, acetyl-CoA carboxylase is expressed cies on a separate 5-20% sucrose gradient in phosphate/citrate (24), as milliunits per total volume, as measured at 37°C in the preincu- also does not penetrate this gradient under these centrifugation bation assay at 5 mM citrate. conditions. Downloaded by guest on September 25, 2021 3642 Cell Biology: Witters et al. Proc. Natl. Acad. Sci. USA 78 (1981)

I- =, 80 Microsomal

- x <60 I-- 1- ^ ,SO { 40- a ¢ Ei G~) I ( d m ¢ 0 0.1 0.2 0.3 0.4 12 Antiserum, ,ul Fraction FIG. 4. Immunotitration ofrat liver microsomal and cytosolic ace- FIG. 2. Sucrose density gradient of a cytosolic fraction. Shown are tyl-CoA carboxylase. Curves from a representative experiment are the acetyl-CoA carboxylase (e) and lactate dehydrogenase (X) activi- shown. The cytosolic and microsomal fractions were prepared from the ties in a sucrose density gradient of a rat liver cytosol fraction. The liver of a fasted/re-fed rat, and the microsomal activity was resolu- broken line indicates the density of each fraction, as determined by bilized by washing with 0.2 M sodium acetate prior to assay. pycnometry at 4'C. Acetyl-CoA carboxylase activities were deter- mined with the preincubation assay at 5 mM citrate. U, unit. range. Therefore, the precise loci ofsubcellular acetyl-CoA car- sayed in other identical gradients in the absence of KPi) was boxylase association cannot be determined without further sub- recovered in the same density range as NADH oxidase. Mono- cellular fractionation. The bulk of the microsomal acetyl-CoA amine oxidase activity was also detectable through this density carboxylase activity, however, does not penetrate this gradient. Acetyl-CoA carboxylase in this region ofthe gradient might rep- resent protomer or polymer that is unassociated with mem- 1.3 120 -A branes, disrupted from loose association with a denser fraction, or in association with a subfraction ofmembrane(s) that does not 100- S-200 -1.0 Ocd penetrate the gradient. , 1.2 rt80 - .160s 0. Immunotitration of cytosolic and resolubilized microsomal 60 ~-120~, 0.6. enzyme indicated that the microsomal enzyme has a specific

-50 0. activity of 239 versus 48.5 for the cy- 1.1 uo g: 40 o x o milliunits/,ul-equivalent Q. tosolic enzyme (Fig. 4). It will be necessary to isolate both forms 4a 20- 40 o0.2E- to delineate any important kinetic and phosphorylation differ- 1.0I Pk, -0 ences. Preliminary data obtained by immunoprecipitation anal- B - 240,000 ysis of 32P-labeled cytosolic and microsomal enzyme from 32p- ^<123,000 labeled hepatocytes indicate that the 32p content of the micro- somal enzyme is about 40% higher than that of the cytosolic enzyme. ..~~~~~~: ,.. DISCUSSION The results of the present study suggest that liver microsomes 3.0 C may be a major locus ofintercellular fatty acid synthesis. Three

Fract-i1.0 5 lines ofevidence support this possibility. First, in the transition between states of low rates of fatty acid synthesis (fasting) and 2.0 -0.58 4 high rates (fasting/re-feeding), the proportion of total cytosolic -0.6 3 plus microsomal acetyl-CoA carboxylase in the microsomes in- 04.10 04 - creases from 6% to 43%. Second there appears to be preferential association of enzyme polymer with the microsomes, whereas ~~~~~~~~0 2 ) ~~0ZC.. 12 1~ conditions leading to enzyme protomerization lead to dissocia- tion. These data further suggest that, with nutritional induction 4 12 6 20 24 Fraction of lipogenesis in vivo, the position of the intracellular proto- mer-polymer equilibrium is shifted in the direction ofpolymer, FIG. 3. Sucrose density gradient of a microsomal fraction. The re- increasing its association with the microsomes. However, si- sults displayed are from a single representative sucrose density gra- multaneous induction of a microsomal isoenzyme cannot be dient of a rat liver microsomal fraction, run simultaneously with the excluded. Third, the specific activity ofthe microsomal enzyme gradient depicted in Fig. 2. (A) Distribution ofactivities ofacetyl-CoA is 5-fold higher than that of the cytosolic enzyme, as judged by carboxylase (e), fatty acid synthetase (X), and ATP-citrate lyase (A); the broken line represents the density at 40C of each fraction as de- immunotitration. termined by pycnometry. (B) Photograph of a Coomassie blue-stained ATP-citrate lyase and fatty acid synthetase activities are also polyacrylamide slab gel of gradient fractions 6-24. The gel was run as present in crude microsomal fractions and 32P-labeled ATP-ci- detailed in Materials and Methods, after application of 8.3 ,ul of the trate lyase was also identified in this fraction by immunopre- gradient fractions. The molecular weight standards indicate the mo- cipitation. However, neither enzyme activity sedimented sig- bilities ofpurified rat liver acetyl-CoA carboxylase (240,000) and ATP- nificantly with acetyl-CoA carboxylase into the sucrose gradients. citrate lyase (123,000). (C) Distribution ofactivities of 5'-nucleotidase presence of these two enzymes in the crude (z&), NADH oxidase (o), galactosyltransferase (A), and the protein con- Therefore, the centration (o), within the same gradient. U, unit, as described in the microsomes may represent either nonspecific trapping in the text. 105,000 x g pellet or loose associations that are easily disrupted Downloaded by guest on September 25, 2021 Cell Biology: Witters et al - Proc. Natl. Acad. Sci. USA 78 (1981) 3643

under gradient centrifugation conditions. It is tempting to spec- Ms. Martha Chambers for manuscript preparation. L.A.W. is the re- ulate, however, that the microsomes may be a locus ofassembly cipient of a Research Career Development Award (AM00520). This for the fatty acid-synthesizing enzymes. Lane and coworkers work was supported by Public Health Service Grant AM19720 and a (26) have previously suggested that acetyl-CoA carboxylase grant from the Juvenile Diabetes Foundation. polymer may represent a structural framework for such assem- 1. Abraham, S., Matthes, K. J. & Chaikoff, I. L. (1963) Biochim. bly. On the basis of the results of the present investigation, Biophys. Acta 70, 357-369. which indicate a high proportion ofenzyme activity on the mi- 2. Lorch, E., Abraham, S. & Chaikoff, I. L. (1963) Biochim. Bio- crosomes and preferential association ofpolymer with this frac- phys. Acta 70, 627-641. tion, the microsomes must be considered to be a possible ini- 3. Butterworth, P. H. W., Guchhait, R. B., Baum, H., Olson, E. B., Margolis, S. A. & Porter, J. W. (1966) Arch. Biochem. Bio- tiating locus for this assembly. phys. 116, 453-457. The precise locus or loci of subcellular localization ofacetyl- 4. Margolis, S. A. & Baum, H. (1966) Arch. Biochem. Biophys. 114, CoA carboxylase cannot be determined from the present data. 445-451. Previous work in our laboratory on adipose tissue identified a 5. Donaldson, W. E., Mueller, N. S. & Mason, J. V. (1971) 216,000-dalton [32P]phosphopeptide in cytosol and endo- Biochim. Biophys. Acta 248, 34-40. plasmic reticulum; it was absent from highly purified mito- 6. Janski, A. & Cornell, N. W. (1980) Biochem. Biophys. lRes. Com- mun. 92, 305-312. chondria and plasma membrane (27). Subsequent work dem- 7. Witters, L. A., Kowaloff, E. M. & Avruch, J. (1979) J. Biol. onstrated that this phosphopeptide was 32P-labeled acetyl-CoA Chem. 254, 245-249. carboxylase (7). These data then suggest that the endoplasmic 8. Lee, K.-H. & Kim, K.-H. (1979)J. Biol. Chem. 254, 1450-1453. reticulum may be the subcellular locus of acetyl-CoA carbox- 9. Brownsey, R. W., Hughes, W. A. & Denton, R. M. (1979) ylase association. Biochem. J. 184, 23-32. Comparison ofthe microsomal and cytosolic acetyl-CoA car- 10. Alexander, M. C., Kowaloff, E. M., Witters, L. A., Dennihy, D. T. & Avruch, J. (1979) J. Biol. Chem. 254, 8052-8056. boxylases indicates that the specific activity of the microsomal 11. Witters, L. A., Moriarity, D. & Martin, D. B. J. Biol. Chem. enzyme is 5-fold higher than that ofthe cytosolic enzyme. This (1979) 254, 6644-6649. difference may reflect various amounts of enzyme protomer or 12. Witters, L. A., Alberico, L. & Avruch, J. (1976) Biochem. Bio- polymer in these fractions or could be due to differences in the phys. Res. Commun. 69, 977-1003. phosphorylation state. Precise kinetic and structural definition 13. Avruch, J., Witters, L. A., Alexander, M. D. & Bush, M. A. of the microsomal and cytosolic enzymes will require isolation (1978) J. Biol. Chem. 253, 4754-4761. 14. Witters, L. A. & Vogt, B. (1981) J. Lipid Res. 22, 364-369. of each to homogeneity. 15. Katiyar, S. S. & Porter, J. W. (1974) Arch. Biochem. Biophys. We emphasize that the relative distribution of acetyl-CoA 163, 324-331. carboxylase activities between the cytosol and the microsomes 16. Majerus, P. W. & Kilburn, E. (1969) J. Biol. Chem. 244, in the present study may reflect the homogenization conditions 6254-6262. employed. We and others (4) have noted that the method of 17. Avruch, J. A. & Wallach, D. F. H. (1971) Biochim. Biophys. Acta homogenization, concentration of the homogenate, and buffer 233, 334-347. 18. Baginski, E. S., Foa, P. P. & Zak, B. (1974) in Methods of En- composition all influence the relative distribution of the en- zymatic Analysis, ed. Bergmeyer, H. U. (Academic, New York), zyme. Isolation procedures for the rat liver enzyme should take Vol. 2, pp. 876-880. into account possible significant loss of enzyme activity to the 19. K6chli, H. & von Wartburg, J. P. (1978) Anal. Biochem. 84, microsomal fraction during initial high-speed centrifugation. 127-135. In summary, our data demonstrate that a substantial fraction 20. Bergmeyer, H. V., ed. (1974) in Methods ofEnzymatic Analysis, of acetyl-CoA carboxylase is associated with the microsomes (Academic, New York), Vol. 1, pp. 481-482. 21. Podolsky, D. K. & Weiser, M. M. (1975) Biochem. Biophys. Res. during high rates of lipogenesis. Studies of the in vitro regu- Commun. 65, 545-551. lation of this enzyme must take into account possible effects of 22. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. this intracellular distribution on the modulation of enzyme ac- (1951)j. Biol. Chem. 193, 265-275. tivity and lipogenesis. 23. Neville, D. M. (1971) J. Biol. Chem. 246, 6328-6334. 24. Moss, J. & Lane, M. D. (1972)J. Biol. Chem. 247, 4944-4951. The authors acknowledge the assistance of Mr. Steven J. Rose for 25. Lee, K.-H. & Kim, K.-H. (1978)J. Biol. Chem. 253, 8157-8161. performing ATP-citrate Iyase assays, Dr. Daniel Podolsky of the Gas- 26. Gregolin, C., Ryder, E., Kleinschmidt, A. K., Warner, R. C. & trointestinal Unit ofthe Massachusetts General Hospital for performing Lane, M. D. (1966) Proc. Natl. Acad. Sci. USA 56, 148-155. the galactosyltransferase assays, and Ms. Barbara Vogt for technical 27. Avruch, J., Leone, G. R. & Martin, D. B. (1976) J. Biol. Chem. assistance. We also thank Dr. Joseph Avruch for helpful suggestions and 251, 1505-1510. Downloaded by guest on September 25, 2021