Fatty acid synthetase of mammalian brain, liver and adipose tissue regulation by prosthetic group turnover

Biochimica et Biophysics Acta, 326 (1973) 293-304 8 Elsevier Scientific Publishing Company, Amsterdam

- Printed in The Netherlands

BRA 56355

FATTY ACID SYNTHETASE OF MAMMALIAN BRAIN, LIVER AND ADIPOSE TISSUE REGULATION BY PROSTHETIC GROUP TURNOVER*

JOSEPH J. VOLPE and P. ROY VAGELOS Departments of Biological Chemistry, Pediatrics and Neurology Washington University School of Medicine, St. Louis, MO. 63110 (U.S.A.) (Received August

Iph,

1973)

SUMMARY

The role of prosthetic group turnover in the regulation of the fatty acid synthetase of mammalian brain, adipose tissue and liver has been studied. The fatty acid synthetase was labeled in the 4’-phosphopantetheine prosthetic group of the acyl carrier protein by administration to rats of calcium [3H]pantothenate. By determining the time course of incorporation of radioactivity into both CoA, the precursor for the 4’-phosphopantetheine moiety of acyl carrier protein, and the fatty acid synthetase, it was possible to estimate the rate of prosthetic group turnover. The turnover rate was found to be extremely rapid in all three tissues. In brain, the prosthetic group is replaced completely in IO-I I h and this is an order of magnitude faster than the turnover of the enzyme complex, previously shown to occur with a tt of 6.4 days. In liver and adipose tissue, the prosthetic group turnover is slightly faster than in brain. Thus, the rapid turnover of the prosthetic group, first shown in Escherichiu coli, also occurs in three different mammalian tissues and may represent a general biological phenomenon. Since turnover of the protein of fatty acid synthetase of brain does not change at all in various nutritional states when turnover of liver synthetase changes dramatically, we considered the question of whether the striking turnover of the prosthetic group in these tissues also responded differently to those states. Distinct changes in the efficiency of the exchange of the 4’-phosphopantetheine between CoA and fatty acid synthetase were observed only in the liver but not in the brain. Increased exchange in liver during fat-free feeding was discovered and the earlier observation of diminished exchange during starvation was confirmed. We also evaluated by immunochemical methods the possibility that rapid regulation of fatty acid synthetase is mediated by changes in catalytic efficiency. Quantitative precipitin analyses of liver extracts from animals during the first hours after onset of starvation or upon refeeding after starvation demonstrated that changes in enzyme activity were related entirely to changes in enzyme content. Thus, the data did not support the possibility that fatty acid synthetase activity is decreased by changes

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294

in catalytic efficiency due to removal of the 4’-phosphopantetheine or to inhibition by metabolites.

prosthetic group

INTRODUCTION

Fatty acid synthetase is the multienzyme complex responsible for the synthesis of palmitic acid from acetyl-CoA and malonyl-CoA. The long-term regulation of this complex in brain and liver during development and in various nutritional states has been shown to be related to changes in the amount of enzyme, and the roles of synthesis and degradation of enzyme in causing these changes in content have been elucidated’. The mechanisms of rapid regulation of the synthetase are unknown. Alteration of synthetase activity by certain metabolites (e.g. stimulation by hexose diphosphates’, inhibition by malonyl-CoA3 and palmityl-CoA4*5) has been demonstrated in vitro. However, no data have established any physiological role for these factors (see for review6). A unique mechanism for rapid regulation of the synthetase may involve the 4’-phosphopa~tetheine of the acyl carrier protein. In Esc~erjc~ia coli the turnover of this prosthetic group was shown to occur at a much faster rate than the turnover of acyl carrier protein”‘. Similarly, rap’rd p rosthetic group turnover was demonstrated in rat liverg, and this turnover is markedly diminished during starvation”. The enzymes involved in the turnover of the 4’-phosphopantetheine of acyl carrier protein have been isolated from E. coli and characterized. Holo-acyl carrier protein synthetaser’*t2 catalyzes reaction I, and acyl carrier protein hydrolaser3 catalyzes Reaction 2. CoA i- apo-acyl carrier protein Mg2+\ holo-acyl carrier protein-t- 3’,5’-ADP (I) Holo-acyl carrier protein Mn2

apo-acyl carrier protein + 4’-phosphopantetheine

(2)

The object of the present work was to determine whether rapid prosthetic group turnover could play a role in the regulation of fatty acid synthetase in mammalian brain. To determine whether rapid prosthetic group turnover might represent a general phenomenon in mammals, we also studied adipose tissue as well as liver. We utilized immunochemical techniques to study the rapidity of this turnover, to determine whether factors known to regulate the fatty acid synthetase alter the turnover, and to determine whether such alterations might provide insight into the possible mechanisms underlying the turnover. In addition, we evaluated the possibility that rapid regulation of hepatic fatty acid synthetase is mediated by changes in catalytic efficiency. METHODS

Materials Malonyl-CoA (P-L ~ochemicals~, NADPH (Sigma), di~othreitol (Sigma), 2-mer~ptoe~anol (Eastman), fatty acid-poor albumin (Per&x), Freund’s complete adjuvant (DiFco), DEAEcellulose (DE+) (Whatman), Sepharose 48 (Pharmacia),

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295

acetyl phosphate (Sigma), phosphotransacetylase (Sigma), ATP (Sigma), cyclic AMP (Sigma), transfe~n (Sigma) and CoA (P-L Bi~he~~ls), were obtained from the designated sources. Acetyl-CoA was synthesized by the method of Simon and Shemin14. ~3H]Pantothenate was prepared by New England Nuclear by catalytic exchange utilizing calcium pantothenate purchased from Sigma. The calcium [3H]pantothenate was purified by thin-layer chromatography on silica gel (Analtech) with distilled water as solvent15. [14C]Palmi~l-CoA was purchased from New England Nuclear. Sodium dodecylsulfate was purchased from Sigma and recrystallized in ethanol before use. Animals and diets Animals employed wererats of the Sprague-Dawley strain (National Laboratory Animals, St. Louis, MO.). Rats were fed either Purina Laboratory Chow or a fat-free diet (Nutritional Biochemical@ as indicated. Lighting conditions were controlled and consisted of 12 h of light from 08.00 to 20.00 h and 12 h of darkness from 20.00 to 08.00 h. Ail experiments were begun at the same time of day; thus, zero time was always 09.00 h. PuriJication of radioactive fatty acid synthetase and assays For the preparation of radioactive fatty acid synthetase, ten 200 g rats were starved for 24-48 h and refed a fat-free diet for 72 h. On the morning of each day of refeeding each animal received I mCi of r3H]pantothenate by intraperitoneal injection. The fatty acid synthetase was purified from liver by a modi~cation16 of the procedure of Burton et al.“. To this modified scheme was added passage of the solution prepared from the final (NH4),S04 step over a column (0.9 cm x60 cm) of Sepharose 4B equilibrated and eluted with 0.5 M potassium phosphate buffer (pH 7.0) and 0.003 M dithiothreitoll. Purity was deduced from measurements of specific activity @o-moo nmoles of NADPH oxidized per min per mg of protein), cochromato~phy of radioactivity, enzyme activity and protein on DEAE-cellulose and Sepharose 4B, and coelectrophoresis of radioactivity and protein on discontinuous polyacrylamide gel electrophoresis in the standard alkaline system”. Fatty acid synthetase activity was determined by the spectrophotometric assay as previously described’. Protein concentrations were determined by the microbiuret method”. ~mmuno~agica~ procedures Specific antibodies were prepared against pure rat liver fatty acid synthetase, which is immunologically identical to the brain enzyme as previously reported’. Immunolo~~l identity of the adipose tissue synthetase with the liver and brain enzymes was shown in a similar manner. The preparation of antibodies against liver synthetase was described previouslyl. Immunochemical titrations and quantitative precipitin reactions were carried out as described by Kabat and Meyerzo. isolation of radioactive fatty acid sy~thet~e and CoA for study of prosthetic group turraover Turnover experiments were carried out by injecting i3H]pantothenate, IO pLei per g of body weight and sacrificing the rats at various time intervals. Fatty acid synthetase was partially purified from crude extracts by a 22-38x (NH4)2S04

296

J. J. VOLPE, P. R. VAGELOS

fractionation and the enzyme was isolated from this fraction by immunopr~ipi~tion* . The supematant solution from the (NH&SO.+ fractionation was used for isolation of COA”~~~. CoA specific radioactivity was determined as previously described10,22. RESULTS

Nature of the radioactivityin the immwoprecipitates Although we had established previously that the specific antibody quantitatively precipitates the fatty acid synthetase of both brain and liver’, it was critical to demonstrate that bound radioactive CoA or acyl-CoA derivatives were not also contained in the i~unopr~ipi~t~. Thus, a series of experiments was performed with imm~opr~ipi~t~ prepared from 22 to 38 % (NH&SO, fractions of brain, adipose tissue and liver. Significant amounts of bound radioactive CoA in the immunoprecipitates were ruled out by the following: (I) dissociation of the precipitate in I M acetic acid, followed by incubation at 25 “C for 30 min and gel filtration over Sephadex G-50 (fine) in I M acetic acid resulted in the appearance of all the radioactivity with the protein in the void volume; (2) suspension of the precipitate in I M HCl, followed by incubation at IOO “C for IO min and precipitation of protein with IO% trichloroacetic acid resulted in no release of acid-soluble radioactivity. Bound CoA would be dissociated from protein by the first procedure and cleaved at the pyrophosphate bond by the second procedurez3. The 4’-phospho~ntetheine of acyl carrier protein is cleaved from the protein upon incubation at pH 12 and 70 “C (ref. 23). To obtain pres~ptive evidence that the radioactivity in the immunopr~ipi~t~ was in the 4’-phosphopantetheine of the acyl carrier protein component of the fatty acid synthetase, the precipitates were dissolved in NaOH (pH 12) incubated at 70 “C for 60 min, and protein was precipitated with ro % trichloroacetic acid. At least 95 % of the total radioactivity was found to be acid-soluble after this treatment, indicating it had been cleaved from the protein. The experiments described above do not rule out the possibility that bound radioactive long chain acyl-CoA derivatives were present in the immunoprecipitates. The immunoprecipitates from brain, adipose tissue and livers of chow-fed animals were dissolved in I % sodium dodecylsulfate, incubated at room temperature for 24 h, heated to 75 “C for 5 min and chromatographed on Sepharose 4B. The elution profile of the radioactivity of the immunoprecipi~te prepared from brain is shown in Fig. I. A single major peak of tritium radioactivity, eluting slightly before transferrin (mol. wt 76ooo), was consistently observed. Essentially no tritium radioactivity eluted with [r4C]palmityl-CoA which was added as a marker for long chain acyl-CoA derivatives. In a separate experiment where a great excess of [14C]palmityl-CoA was added to nonradioactive fatty acid synthetase before immunoprecipitation, the palmityl-CoA was similarly separated from the protein. Immunoprecipitates of adipose tissue and liver, similarly dissociated in I % sodium dodecyl sulfate and chromatographed on Sepharose 4B, gave results similar to those obtained with brain. The fact that between 80 and 95% of the radioactivity in the immunoprecipitates from brain, as well as adipose tissue and liver, was eluted with the protein rather than the palmityl-CoA indicated that immunopr~ipi~tion was an effective procedure for determining specific radioactivity of fatty acid synthetases of these three tissues. Immunopr~ipi~t~ of liver from starved animals, dissociated in I % sodium

PROSTHETIC

GROUP TURNOVER

OF FATTY ACID SYNTHETASE

297

240 -

FfWTION

NWBER

Fig. I. Elution protlle of immunoprecipitated brain fatty acid synthetase chromatographed on Sepharose 4B in I % sodium dodecyl sulfate. The immunoprecipitate was dissolved in I % sodium dodecyl sulfate, 0.1 M sodium phosphate (PH 7.0), 0.05 M z-mercaptoethanol and o.oo25 M NalEDTA and incubated as described in the text. Blue dextran and [“QpalmitylCoA were added as markers and the sample was eluted with the same solution used in dissolution of the precipitate. o-o,sH; o---o, W.

dodecyl sulfate and chromatographed on Sepharose 4B, gave elution profiles with 50 % of the radioactivity in the protein peak and 50 % in the pahnityl-CoA peak. The specific radioactivities of hepatic fatty acid synthetase obtained by purification by the standard method was compared with those obtained by immunoprecipitation for animals in various nutritional states (Table I). The data show that similar fatty acid synthetase specific radioactivities were obtained by enzyme purification and immunoprecipitation in liver preparations from chow-fed and fat-free-fed animals, whereas the specific radioactivity obtained by immunoprecipitation of the starved animal preparation was twice as high as that of the purified fatty acid synthetase. Thus, these dam confirm those obtained with sodium dodecyl sulfate chromatography of the immunoprecipitates and suggest coprecipitation of acyl-CoA derivatives. This is not surprising, since in the liver of the starved animal there is a marked increase in total TABLE I SPECIFIC RADIOACTIVITY OF LIVER FAT-I-Y ACID SYNTHETASE OR IMMUNOPRECIPITATION

BY PURIFICATION

Animals were chow-fed, fat-free-fed or starved, and specific radioactivity of liver fatty acid synthetase was determined by purification or immunoprecipitation as described in the text. Livers were pooled from five animals for each nutritional state. Single purifications were carried out. Duplicate determinations of two levels of enzyme were utilized for the immunoprecipitation data. Values did not vary more than 5 ‘A and numbers shown are means. Nutritional state

Purification

Immunoprecipitation

Chow fed Fat-free fed Starved

4820 7500 2325

5080 7ooo 4600

Fatty acid synthetase

specijc

radioactivity

(dpmlnmole)

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298

concentration of long chain fatty acyl-CoA derivatives’ Y24and at least a 2-fold increase in specific radioactivity of CoA (see below). No significant change in specific radioactivity of CoA in brain was observed under any of the experimental conditions, and the specific radioactivity of CoA in brain was only 5-15 % that of CoA in liver. In view of the over-estimate by immunoprecipitation of specific radioactivity of hepatic synthetase during starvation, we used the standard purification method for the experiments with starved animals. Prosthetic group turnover in liver, brain and adipose tissue

We considered first the question of whether the turnover of the 4’-phosphopantetheine prosthetic group of brain and liver fatty acid synthetase is different, since the turnover of the enzyme complex from these tissues differs markedly’.Thus 150 g chow-fed rats were given [3H]pantothenate, and pairs were sacrificed at various time intervals thereafter until 48 h after the injection. The pantothenate is incorporated into CoA and the acyl carrier protein of fatty acid synthetase. The time course of incorporation for both tissues is shown in Figs 2 and 3. Values for only the first 24 h after injection are shown because maximum incorporation was observed by 24 h in all experiments.

$j 4

8 TIME

12

16

(hours)

20

24

:

I 4

6 TIME

I

I

I

12

16

20

24

(hours)

Fig. 2. The time course of specific radioactivity of fatty acid synthetase and CoA in brain after a single intraperitoneal injection of [3H]pantothenate. The enzyme and CoA were isolated as described in the text. Brains were pooled from two animals for each point. Duplicate determinations of two levels of enzyme were utilized for each point. Values did not vary more than 5 % and numbers plotted are means. Percent maximum specific radioactivities are plotted; 100% values were 1290 and 4030 dpm/nmole for CoA and fatty acid synthetase, respectively. q --- q, fatty acid synthetase (FAS); o-o, CoA. Fig. 3. The time course of specific radioactivity of fatty acid synthetase and CoA in liver after a single intraperitoneal injection of fwjpantothenate. See legend to Fig. 2 and text. 100 ‘A values were 2730 and 5500 dpm/nmole for CoA and fatty acid synthetase, respectively. [II--- U, fatty acid synthetase (FAS); o-o, CoA.

Several points are evident from the data for brain (Fig. 2). First, label was rapidly incorporated into both CoA and synthetase after the intraperitoneal injection. Second, the incorporation of label into the synthetase lagged behind the incorporation into CoA. This suggests a precursor-product relationship, as demonstrated earlier in E. coli’ and in rat liverg. Third, at the point of maximum incorporation the specific radioactivities of both CoA and fatty acid synthetase, rzgo and 4030 dpm/nmole,

PROSTHETIC GROUP TURNOVER OF FATN

ACID SYNTHETASE

299

respectively, were of approximately similar magnitude. This also supports the concept of a precursor-product relationship. The discrepancy in the values for specific radioac~~ty was reproducible (data from two additional experiments are not shown). Although it may be related to experimental error, particularly the isolation and assay of nanomolar quantities of CoA, the possibility also exists that separate pools of CoA or other pantothenate-containing compounds occur within the cell and that the specific radioactivity of CoA, which is specifically a precursor to acyl carrier protein, is higher than that in the 38 % satd (NH&SO4 supe~a~nt solution. Fourth, and most critical, the close, parallel course of the curves for both CoA and the synthetase indicate that the turnover of the prosthetic group must be very rapid. In fact, the curve for the fatty acid synthetase never lags more than IO-I I h behind that for CoA, and thus, the whole pool of the synthetase must reach equilibrium with that of CoA in approximately that time. This time period should be compared to the t+ of 154 h obtained for the enzyme complex with E3H]leucine which was used to label the protein’. The data for liver (Fig. 3) are noteworthy for several reasons. The incorporation of label into CoA and enzyme was more rapid than in brain. The rate of appearance of radioactivity in CoA of brain lagged 1-3 h behind the rate in liver. The slower passage of a variety of compounds into adult brain relative to adult liver is well known. The precursor-product relationship of CoA and fatty acid synthetase in liver is suggested by the relations~p of the curves, as noted for the brain. At the point of maximum incorporation the specific radioactivities of both CoA and enzyme, 2730 and 5500 dpm/nmole, were of similar magnitude. This relationship of CoA and synthetase radioactivity was noted in two additional experiments (data not shown). Compartmentalization of CoA pools may occur in liver as in brain, but the possibility of experimental error in the absolute measurements of CoA cannot be ruled out. Of greatest significance, the turnover of the prosthetic group of liver fatty acid synthetase was very rapid, and the whole pool of enzyme must reach equilibrium with CoA in no more than 4-5 h. The t* of the protein of the enzyme complex in adult liver is 67 h (ref. I). Since prosthetic group turnover was shown to be rapid in brain as well as liver, we considered whether this rapid turnover might be a general phenomenon in mammals. A third tissue in which fatty acid synthesis is an impo~nt process is adipose tissue. Study of prosthetic group turnover in this tissue (Fig. 4) indicated that in adipose tissue, as in brain and liver, there is evidence for CoA as precursor of the label in fatty acid synthetase and that the rate of turnover of prosthetic group is of the same order of rapidity as that noted in the other tissues. Prosthetic group ~rnover in various ~#tritio~al states Turnover of the fatty acid synthetase of brain does not change under nutritional conditions in which dramatic changes of turnover of the synthetase occur in liverl. We considered whether the striking turnover of the prosthetic group in these tissues also responded differently to these states. Thus, animals were either fed Purina chow or the fat-free diet for 7 days or starved for 48 h. After intraperitoneal ad~nistm~on of [3H]~to~enate, animals were sacrificed after 7,r3,24 and 48 h. Peak levels of specific radioactivity of both CoA and fatty acid synthetase were reached by 24 h (13 h in liver) in each tissue. The ratio of fatty acid synthetase to CoA specific radioactivities at this point is a measure of the

J. J. VOLPE, P. R. VAGELOS

4

6

12 TIME

16

20

24

I

Ihours)

Fig. 4. The time course of specific radioactivity of fatty acid synthetase and CoA in adipose tissue after a single intraperitoneal injection of [3H]pantothenate. See legend to Fig. 2 and text. 100 % values were 4480 and 8820 dpm/nmole for CoA and fatty acid synthetase respectively. q --- q, fatty acid synthetase (FAS); o-o, CoA.

efficiency of the exchange of 4’-phosphopantetheine between CoA and fatty acid synthetase (Table II). In brain, the efficiency of prosthetic group exchange was not altered in any of the nutritional states. In contrast to brain, upon feeding a fat-free diet there was an apparent increase in the efficiency of the exchange in liver. In addition, during starvation there was a marked decrease in the efficiency of the exchange, confirming an earlier report lo . Essentially identical data for both brain and liver were obtained in a second experiment. TABLE II EFFICIENCY

OF PROSTHETIC

GROUP

EXCHANGE

IN BRAIN AND LIVER

The animals were fed either Purina chow or the fat-free- diet for 7 days or were starved for 48 h. Specific radioactivities of fatty acid synthetase and CoA were determined as described in the text. For each point organs from two animals were pooled, and duplicate immunoprecipitations of two levels of enzyme were carried out. Values did not vary more than 5 % and numbers shown are means. Tissue

Nutritional state

Fatty acidsynthetase specific radioactivity/ CoA specific radioactivity ratio

Brain Brain Brain

Chow fed Starved Fat-free fed

3.21 3.48 3.14

Liver Liver Liver

Chow fed Starved Fat-free fed

2.08 o.57* 3.28

* The value for specific radioactivity of fatty acid synthetase was obtained by purification of the enzyme by the standard method (Methods).

Prosthetic group turnover in vitro The rapid turnover of fatty acid synthetase prosthetic group apparent in brain, liver and adipose tissue is analogous to that occurring in E. coZi7s8,and in this microorganism the enzymes responsible for this turnover, holo-acyl carrier protein synthetase and acyl carrier protein hydrolase, have been studied in detail’1-‘3. To pursue

PROSTHETIC GROUP TURNOVER OF FATTY ACID SYNTmTASE

301

the analogy further, we attempted to demonstrate hydrolase activity in mammalian liver. Incubation of substrate quantities of fatty acid synthetase, labeled in its 4’phosphopantetheine moiety, under conditions optimal for the E. coliacyl carrier protein hydrolase, resulted in no fatty acid synthetase inactivation and no loss from the complex of the labeled prosthetic group. Incubation of the fatty acid synthetase in the presence of crude liver extract supplemented with ATP, cyclic AMP or CoA also failed to demonstrate prosthetic group hydrolysis. Because the in vivu studies of prosthetic group turnover in liver suggested that the efficiency of the exchange is directly correlated with the rate of fatty acid synthesis, we attempted to demonstrate turnover in vitro under conditions optimal for fatty acid synthesis. Thus, experiments similar to those described above were carried out in the presence of acetyl-CoA, malonyl-CoA and NADPH, the substrates required for fatty acid synthesis. Again, there was no release of the radioactive 4’-phosphopantetheine. Thus, although the in vivo studies strongly suggest the functioning of acyl carrier protein hydrolase in rat tissues, this enzyme has not yet been demonstrated in vitro. Absence of a~ro~~~te~ during starvation and refeeding In previous studies of the synthetase complex’ we had shown that there is no evidence for an immunolo~c~y reactive, enzymatically inactive species in the liver of starved vs chow-fed or fat-free-fed animals. However, these analyses were carried out after relatively long periods when steady states had been reached. In the present studies, an effect of altered prosthetic group turnover was sought in the first hours after food deprivation. Fatty acid synthetase apoprotein, though enzymatically inactive, should be immunologically reactive with antibody directed against the holoenzyme. Thus, quantitative precipitin analysis could be used to detect the apoprotein. Pairs of rjo-g animals were sacrificed at o, 4, IO, 17, 24 and 28 h after the onset of fasting. The hepatic specific activities of fatty acid synthetase in the 105ooo xg su~rna~t solutions were: oh, 4.60; 4 h, 3.44; IO h, 3.50; 17 h, 2.75; 24 h, 2.22; and 28 h, 2.03. Quantitative precipitin analyses revealed equal equivalence points for all extracts (Fig. 5). Thus, there is a constant and equal amount of immunoprecipitable enzyme present per unit of enzyme in all extracts and, therefore, there is no evidence for immunologically reactive, enzymatically inactive fatty acid synthetase apoprotein or any inhibited form of the enzyme. 2d

Haurr

starved

5

IO ENZYME

IS

20

25

30

35

ADDEO, unit8

Fig. 5. Quantitative precipitin rcwtions of fatty acid synthetase in liver during starvation. Twelve 150-g rats were starved for the intervals shown in the upper left hand comer of the figure. At each time point a pair was sacrificed, ropoo x g supematant solutions of liver were prepared and pooled, and ~u~t~~tive precipitin analyses were performed. See text for details.

J. J. VOLPE, P. R. VAGELOS

302

Another possible time for the occurrence of fatty acid synthetase without its prosthetic group would be during the first hours of refeeding a fat-free diet. Thus, 150 g animals were starved for 48 h and then refed a fat-free diet. Pairs of animals sacrificed at o, 4,8, 13.5 and 24 h had hepatic fatty acid synthetase specific activities in the ro5oooxg supernatant solutions of 2.2, 1.5, 2.2, 3.6 and 14.8, respectively. Quantitative precipitin analyses of these extracts resulted in identical equivalence points (Fig. 6). Thus, again there was an equal amount of immunoprecipitabie enzyme per unit in ail extracts, and no evidence was obtained for the occurrence of fatty acid synthetase without its prosthetic group.

5

10 ENZYME

15 ADDED,

20

25

35

units

Fig. 6. Quantitative precipitin reactions of fatty acid synthetase in liver during refeeding a fat-free diet after 48 h of starvation. Ten 150-g animals were studied. See Iegend to Fig. 5 and text.

DISCUSSION

The present investigation dealt with the role of prosthetic group turnover in the regulation of the fatty acid synthetase of mammalian brain, liver and adipose tissue. Prosthetic group turnover was found to be extremely rapid in ail three tissues that were studied. In brain the turnover of the enzyme complex, represented by a t+ of 6.4 days’, is very much slower. The prosthetic group of the brain enzyme was replaced completely in IO-I I h. Thus, the prosthetic group turns over completely in a period of time equivalent to 5-10 % of that required to turn over one-half of the protein of the complex. In liver, the turnover of the complex is significantly faster than in brain and is represented by a tt of 2.8 days’. The prosthetic group of the liver enzyme was replaced in 4-5 h, confirming the basic observations of Tweto et al.‘. Thus, as in brain, the prosthetic group turns over completely in a period of time equivalent to ~-IO% of that required to turn over one-half the protein of the whole complex. In adipose tissue the prosthetic group was also replaced in a matter of several hours. Thus, it is apparent that the phenomenon of prosthetic group rapid turnover pertains in three different mammalian tissues. The enzymes responsible for fatty acid synthetase prosthetic group turnover, hoio-acyl carrier protein synthetase and acyi carrier protein hydrolase, demonstrated in E. coli11-13,have not yet been demonstrated in any mammalian tissue. The inability to demonstrate the activity of the hydroiase in mammalian liver synthetase in vitro,raised the possibi~ty that its activity was dependent on ATP and/or cyclic AMP. The recent observation that activity of acetyi-CoA carboxyiase can be altered in vitro

PROSTHETIC GROUP TURNOVER OF FATTY ACID SYNTHETASE

303

suggests the possibility that this initial by phosphorylation-dephosphorylation2s enzyme in fatty acid biosynthesis is, in fact, regulated in this manner. We were unable to demonstrate any such mechanism operative in the regulation of the fatty acid synthetase. The additional possibility that the activity of the hydrolase varies with the rates of fatty acid synthesis by the synthetase was suggested by the alterations of prosthetic group turnover observed in viva during fat-free feeding and starvation. However, the function of the hydrolase could not be demonstrated in vitro even when fatty acid synthetase was actively synthesizing fatty acids. The failure of brain prosthetic group turnover to respond to dramatic nutritional influences such as starvation or fat-free feeding is consistent with the failure of brain fatty acid synthetase activity, as well as fatty acid synthetase synthesis and degradation, to respond to such factors ‘. The difference in response of the prosthetic group turnover in brain and liver is another major difference between the regulation of the synthetase in these two tissues. The finding of rapid turnover of the 4’-phosphopantetheine moiety in brain suggests a means for rapid regulation of the fatty acid synthetase, already shown to be an important enzyme in brain’*26. That prosthetic group turnover plays an important role in regulation of hepatic fatty acid synthetase is suggested by the observations that during the feeding of a fat-free diet and starvation, states accompanied by “marked changes in synthetase activity, there are distinct changes in the efficiency of the exchange of q’-phosphopantetheine between CoA and fatty acid synthetase. The original observation of markedly diminished exchange during starvation” was confirmed, and the increased exchange during fat-free feeding was discovered. The enzymatic mechanisms of these effects remain unclear, however, and the possibility that the effect is at the level of CoA metabolism has not been ruled out. Prosthetic group turnover may be involved in very rapid regulation of fatty acid synthetase in brain, adipose tissue and liver. Quantitative precipitin analyses provided some insight into mechanisms that might operate to mediate such regulation in liver. If the change in turnover in liver during starvation is caused by decrease in activity of holo-acyl carrier protein synthetase while the hydrolase continues to function, then fatty acid synthetase without its prosthetic group should be present during the early hours of fasting. In addition, since during starvation the complex is degraded at a greatly accelerated rate, apo-acyl carrier protein, unassociated with the other components of the fatty acid synthetase, might be present and, on refeeding a fat-free diet, might be incorporated into the synthetase. By quantitative precipitin analyses, we obtained no evidence for such an enzymatically inactive, immunologically reactive species. All equivalence points were equal and thus all differences in activity were related to differences in content of intact holo-enzyme. These data also do not support a regulatory role for palmitylCoA or other metabolites as inhibitors or activators of fatty acid synthetase, since such inhibited or activated species should be immunologically reactive. ACKNOWLEDGEMENTS

This investigation was supported in part by Grant GB-38676X from the National Science Foundation, Grant I-ROI-HGIo~o~ from the National Institutes of Health, and the Allen P. and Josephine B. Green Foundation.

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