ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 197, No. 2, October 15, pp. 379-387, 1979

Subunit Structure of the Mammalian Fatty Acid Synthetase: Further Evidence for a Homodimerl

STUART SMITH2 AND ALAN STERN

Bruce Lyon Memorial Research Laboratory, Children’s Hospital Medical Center, 51st and Grove Streets, Oakland, Calijornia 94609

Received April 4, 1979; revised June 18, 1979.

Immunochemical procedures and limited proteolysis have been used to investigate the subunit structure of fatty acid synthetase from rat mammary gland. Specific antibodies were raised against the two thioesterase I domains obtained from the fatty acid synthetase by treatment with trypsin. The antibodies precipitated both subunits of the dissociated fatty acid synthetase, indicating that both subunits contained a single thioesterase I domain. An analysis of the time course of limited trypsinization of the fatty acid synthetase, labeled in its two thioesterase I domains with [1,3-14C] diisopropylphosphofluoridate, indicated that each subunit was susceptible to tryptic attack at identical locations and that the thioesterase I domains occupied a terminal locus at one end of each polyfunctional polypeptide chain. The most plausible explanation for these results is that the mammalian fatty acid synthetase is a homodimer.

Fatty acid synthetase multienzyme com- plexes exist in nature in various forms of polyfunctional polypeptide enzyme. In Saccaromyces cerevisiae and Aspergillus fumigatus, six copies of two nonidentical subunits (a&) are assembled into a com- plex of molecular weight approximately 2.4 x lo6 (l-3). Each subunit contains several of the partial activities of the complex, but none of the activities are found on both sub- units. On the other hand, the multienzyme Mycobacterium smegmatis appears to be an oligomer of identical polyfunctional poly- peptide chains (4). In animals, the fatty acid synthetases are found as dimers of approxi- mate M, 0.5 x lo6 (see Ref. (5) for re- view). Whether the subunits are identical or nonidentical has been a matter of some controversy (see Ref. (6) for review). The classical methods of distinguishing between homo- and heterodimer, such as tryptic

’ Dedicated to the late Feodor Lynen. Supported in part by Grants AM16073 and RR05467 from the National Institutes of Health, DHEW, and Grant BMS 7412723 from the National Science Foundation.

’ Most of this work was carried out while the author was the recipient of an Established Investigatorship of the American Heart Association.

peptide mapping, are not feasible because of the large size of the subunits. In this study we have used a novel approach to address this issue. We used (i) an immuno- globulin probe to determine whether the two thioesterase I domains of a mammalian fatty acid synthetase are present in a single subunit or one on each subunit and (ii) limited proteolysis to determine whether the thioesterase I domains are in the same location of each of the subunits.

EXPERIMENTAL PROCEDURES

Preparation of enzymes. Fatty acid synthetase was isolated from lactating rat mammary gland (7). The two thioesterase I domains were removed by limited trypsinization and purified by ammonium sulfate precipitation and gel filtration on Sephadex G75 (8, 9). The core of the fatty acid synthetase remaining after removal of the thioesterase I domains was purified by gel f&ration on Sepharose 6B (10). This preparation, referred to as trypsinized fatty acid synthetase, contains no demonstrable fatty acid synthetase activity and retains only about 0.2% of the palmityl-CoA-hydrolyzing activity of the native enzyme (10); however, it retains all of the other partial activities of the native enzyme (10,ll).

Preparation of antibodies. Rabbit antibodies were prepared against fatty acid synthetase (12) and thio-

379 0003-9861/79/12037309$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

380 SMITH AND STERN

e&erase I (7) and purified by ammonium sulfate precipitation and ion-exchange chromatography (13).

Immunodifision studies. The procedures were based on methods established by Ouchterlony (14) and Mancini et al. (15). Agarose gels (1%) were pre- pared in 0.1 M NaCl/O.Ol M sodium phosphate buffer (pH 7.2)/5 mM EDTAlO.01 M sodium azide for Ouch- terlony double diffusion analyses. For analyses of the Mancini type, anti-thioesterase I immunoglobulins were mixed with the agarose, at 5o”C, prior to pouring of the gels. Samples were applied to the gels at 0-4°C and the subsequent diffusion was carried out at 0-4°C for 48 h. Precautions were taken to ensure that the samples remained cold at all times, since the subunits reassociate rapidly on warming to room temperature (16). Gels were washed and then stained with Coomas- sie brilliant blue (17).

Quantitative immunoprecipitin reactions. Reac- tions, carried out as described previously (12), were allowed to take place at 0-4°C for 2 days. Every pre- caution was taken to ensure that the samples remained cold at all times, in order to avoid reassociation of subunits. Agglutinated proteins were collected (12) and assayed as described by Lowry et al. (18).

Rocket immunoelectrophoresis. The procedure described by Weeke (17) was adapted for use with a Pharmacia flat-bed electrophoresis apparatus. Aga- rose gels (1%) containing anti-fatty acid synthetase immunoglobulins in sodium barbital/Tris/glycine buf- fer, pH 8.8, p = 0.02 (17) were poured to a depth of 1 mm. The same buffer at a higher ionic strength, p = 0.04, was used as electrode buffer. Wells held 8 ~1 of sample. Electrophoresis took place for 16 h at 4 v/cm. Gels were washed and stained with Coomassie brilliant blue (17).

Sodium dodecyl sulfate polyacrylamide gel electro- phoresis. The procedure was described in an earlier publication from this laboratory (8).

Labeling of fatty acid synthetase with radioactive diisopropylphosphojfuoridate. The active sites of the two thioesterase I domains were labeled with either [l-3H(N)]diisopropylphosphofluoridate (18.5 Ci/mol) or [1,314C]-diisopropylphosphofluoridate (10 Ci/mol) as described previously (9).

Limited trypsinization of [1,3W]diisopropylphos- phojluoridate-treated fatty acid synthetase. Fatty acid synthetase (4.3 mg/ml) labeled with [1,3Y+ diisopropylphosphofluoridate (2.1 mol/21 Cilmol of enzyme) was treated with trypsin (2 pg/ml) in 0.05 M sodium phosphate buffer (pH 7)/l mM EDTAB mM dithiothreitol at 30°C. Portions of the incubation mixture were removed at intervals and treated with trypsin inhibitor to stop the reaction. Proteins were denatured (7) and subjected to sodium dodecyl sulfate- polyacrylamide electrophoresis. The following molecu- lar weight standards were used: myosin (200,000), Escherichia coli RNA polymerase subunits (a = 39,000, p = 155,000, p’ = 165,000), glycogen de-

branching enzyme (lSO,OOO), phosphorylase (100,000) and bovine serum albumin (67,000).

Dissociation offatty acid synthetase 13 S dimer into 9 S subunits. Fatty acid synthetase was dissociated by storage at 0-4°C for 5 days in 0.25 M potassium phosphate buffer (pH 7)/l mM EDTA/l mM dithiothre- itol (19). That dissociation was complete, was con- firmed by sucrose density gradient centrifugation (20), with beef liver catalase (11.3s) as a reference standard (21).

Protein determinations. The concentrations of fatty acid synthetase, trypsinized fatty acid synthetase, thioesterase I, and purified immunoglobulins were determined from the A,,, using the published absorption coefficients (8, 10).

Materials. The sources of materials have been described in recent publications (7- 11).

RESULTS AND DISCUSSION

Immunochemical Approach

The objective of the immunochemical experiments was to determine whether the two thioesterase I domains (M, = 35,000) are present, one on each of the two fatty acid synthetase subunits (M, = 240,000). Antibodies were raised against the thio- esterase I component of the fatty acid synthetase. These antibodies react speci- fically with the thioesterase I domains and do not cross-react with the core of the multienzyme remaining after trypsinization (9). Fatty acid synthetase (13 S dimer) was dissociated into 9 S subunits and diffused against anti-fatty acid synthetase immuno- globulins in agarose containing various amounts of anti-thioesterase I immuno- globulins (Fig. 1). Undissociated fatty acid synthetase, trypsinized fatty acid synthe- tase, and thioesterase I yere included in other antigen wells. In the absence of anti- thioesterase I immunoglobulins, fatty acid synthetase subunits gave a sharp immuno- precipitin line. As expected, lines of partial identity (spurs) were formed when either trypsinized fatty acid synthetase or thio- esterase I was present in an adjacent well. When anti-thioesterase I immunoglobulins were present in the agarose, precipitin rings formed around the wells containing thioesterase I and the wells containing fatty acid synthetase subunits. The diam- eter of the rings decreased as the con- centration of anti-thioesterase I immuno-

SUBUNIT STRUCTURE OF FATTY ACID SYNTHETASE 381

FIG. 1. Double diffusion of fatty acid synthetase, trypsinized fatty acid synthetase, and thioesterase I against anti-fatty acid synthetase antibodies in the presence and absence of anti-thioesterase I antibodies in the agarose. The three diffusion plates shown in this experiment are representative of several, covering a wide range of antibody concentrations with several different batches of fatty acid synthetase. (A) No anti-thioesterase I immunoglobulins in the agarose. (B) Anti-thioesterase I immunoglobulins (0.7 mgiml) included in the agarose. (C) Anti-thioesterase I immunoglobulins (3.6 mg/ml) included in the agarose. On each plate, center wells contained 7 ~1 of anti-fatty acid synthetase immunoglobulins (15 mg/ml). Outer wells contained the following: 1, 0.15 M NaC110.01 M sodium phosphate buffer, pH 7.2; 2 and 5, fatty acid synthetase 9 S subunits (0.7 mg/ml); 3, fatty acid synthetase 13 S dimer (0.7 mgiml); 4, trypsinized fatty acid synthetase (0.7 mgiml); 6, thioesterase I(O.1 mgiml).

globulins was increased. As expected, no precipitin ring was found around the well containing trypsinized fatty acid synthe- tase. In the agarose gel containing the highest concentration of anti-thioesterase I immunoglobulins, no immunoprecipitin line was formed between thioesterase I and anti-fatty acid synthetase immunoglob- ulins, indicating that all of the enzyme had been precipitated within the immunopre- cipitin ring, by the anti-thioesterase I immunoglobulins. It should be noted that the concentration of thioesterase I domains were the same in the wells containing free thioesterase I and fatty acid synthetase; vis. 2.9 PM. In the case of the fatty acid syn- thetase subunits, a distinct immunoprecipi- tin line, formed by the anti-fatty acid synthetase immunoglobulins, persisted out- side the immunoprecipitin ring. Clearly the fatty acid synthetase contained some polypeptide which was not recognized by anti-thioesterase I antibodies. This pre- cipitin line, unlike that formed in the absence of anti-thioesterase I, did not spur with the line formed by the trypsinized fatty acid synthetase. In this experiment, identical results were obtained when fatty acid synthetase was introduced to the antigen well as either the 13 S dimer or the 9 S subunits.

The results of this experiment were confirmed and extended with quantitative immunoprecipitin reactions (Fig. 2). Fatty

acid synthetase 9 S subunits were titrated with anti-thioesterase I immunoglobulins, the antibody-antigen precipitate was re- moved by centrifugation, and the super-

75

.

verus mti-TE 1

wsw anti-FAS

I I , 2.5 5.0 7.5

Antibody (pg)

FIG. 2. Quantitative immunoprecipitin reactions between anti-fatty acid synthetase, anti-thioesterase I, and fatty acid synthetase subunits. Dissociated fatty acid synthetase subunits (8.4 pg) were titrated with anti-fatty acid synthetase immunoglobulins (M) or anti- thioesterase I immunoglobulins (A) in a volume of 0.38 ml and the precipitates were assayed for protein. The supernatants from the titration of fatty acid synthetase subunits versus anti-thioesterase I immuno- globulins were mixed with 5.5 mg of anti-fatty acid synthetase immunoglobulins in a volume of 0.68 ml and the precipitates were assayed for protein (0).

382 SMITH AND STERN

natant was mixed with anti-fatty acid synthetase immunoglobulins. At equiv- alence, anti-thioesterase I immunoglobulins precipitated a total of 22 pg of complex from 8.4 pg fatty acid synthetase. This corresponds to approximately 3 antibody molecules per fatty acid synthetase subunit. In comparison, anti-fatty acid synthetase immunoglobulins precipitated 72 pg of complex from 8.4 pg fatty acid synthetase 9 S subunits, equivalent to 12 antibody molecules per fatty acid synthetase subunit. The supernatants remaining after titration of fatty acid synthetase to equivalence, with anti-thioesterase I immunoglobulins, gave 16 pg antibody-antigen precipitate when mixed with anti-fatty acid synthetase immunoglobulins.

Since the number of anti-thioesterase I antibody molecules associated with the fatty acid synthetase 9 S subunits at equivalence was close to the minimum required for lattice formation, it was conceivable that some soluble anti-thio- esterase I-fatty acid synthetase subunit complexes might have been formed which subsequently were precipitated by anti- fatty acid synthetase immunoglobulins. To test this possibility, fatty acid synthetase labeled on its two thioesterase I domains with [3H]diisopropylphosphofluoridate (1.8 mol inhibitor/m01 fatty acid synthetase dimer) was dissociated into 9 S subunits and treated with anti-thioesterase I immuno- globulins. The immunoprecipitate contained 99.3% of the radioactivity. This indicated clearly that both thioesterase I domains were precipitated with anti-thioesterase I antibodies and ruled out the possibility that soluble antibody-antigen complexes remained after the reaction. However, since not all of the protein in the fatty acid synthetase 9 S subunit preparation was precipitated by anti-thioesterase I anti- bodies, the experiments did not rule out the possibility that both the thioesterase I domains resided on a single subunit. Such an arrangement would allow only one sub- unit to react with anti-thioesterase I immunoglobulins while both would be recognized by anti-fatty acid synthetase immunoglobulins. If this were the case, then one would predict that half of the fatty

acid synthetase protein would remain in the supernatant after treatment with anti- thioesterase I immunoglobulins. Alterna- tively, if the thioesterase I domains were present, one on each subunit, both subunits would be precipitated by the anti-thio- esterase I immunoglobulins and the amount of non-antibody protein remaining in the supernatant would necessarily be very small, since the fatty acid synthetase preparation consisted of more than 95% 240,000 M, polypeptides, as determined by sodium dodecyl sulfate-polyacrylamide electrophoreis (8). Thus, quantitation of the amount of antigen remaining in the super- natant, that was recognized by anti-fatty acid synthetase, would be expected to distinguish between these two possibilities. To this end, we used the technique of rocket immunoelectrophoresis.

Fatty acid synthetase 9 S subunits were titrated to equivalence with anti-thio- esterase I immunoglobulins and the pre- cipitate was removed by centrifugation. Portions of the supernatant were analyzed by rocket immunoelectrophoresis, using both fatty acid synthetase and trypsinized fatty acid synthetase as standards (Fig. 3). Trypsinized fatty acid synthetase was included, since the immunoprecipitin line formed by the component of the fatty acid synthetase not recognized by anti-thio- esterase I immunoglobulins gave an ap- parent reaction of identity with that formed by trypsinized fatty acid synthetase, when these antigens were diffused against anti- fatty acid synthetase immunoglobulins (Fig. 1). In practice, only a slight difference was detected in rocket heights when equal amounts of fatty acid synthetase and trypsinized fatty acid synthetase were compared. The results showed that when 9 S subunits were treated with anti-thio- esterase I antibodies, the amount of protein remaining in the supernatant which was recognized by anti-fatty acid synthetase antibodies, corresponded to only 3.5% of the initial fatty acid synthetase protein. This result demonstrated clearly that the anti- gen in the fatty acid synthetase preparation which was recognized by anti-fatty acid synthetase, but not by anti-thioesterase I immunoglobulins, was not a subunit lacking

SUBUNIT STRUCTURE OF FATTY ACID SYNTHETASE 383

a thioesterase I domain, but was a minor component of the preparation.

Conceivably the component could be either a nicked fragment of the fatty acid synthetase formed during purification and/ or storage, or a non-fatty acid synthetase- derived impurity. Present evidence indi- cates the former possibility to be the more likely: (i) The purified fatty acid synthetase contains as its major “impurity” a compo- nent of molecular weight approximately

123456789lOlll2l3

FIG. 3. Rocket immunoelectrophoresis of fatty acid synthetase-derived antigen not recognized by anti- thioesterase I antibodies. Fatty acid synthetase, 9 S subunits (20 pg), was reacted with anti-thioesterase I immunoglobulins (4.2 mg) in a volume of 100 ~1 for 2 days at O-4%, and the immunoprecipitate was removed by centrifugation. Rocket immunoelectro- phoresis was performed on the supernatant with both trypsinized fatty acid synthetase and native fatty acid synthetase as reference standards. The agarose gel contained anti-fatty acid synthetase immunoglobulins (16 pg/ml). Sample wells contained 8 ~1 of the following antigens: fatty acid synthetase, 1 = 50 pg/ml, 2 = 25 pg/ml, 3 = 12.5 pgiml, 4 = 6.3 pg/ml, 5 = 3.1 pg/ml; trypsinized fatty acid synthetase, 6 = 3.5 pg/ml, 10 = 6.9 pg/ml, 11 = 13.9 pg/ml, 12 = 27.8 pg/ml, 13 = 55.5 @g/ml; wells 7 and 9 contained 8 ~1 of the supernatant from reaction between fatty acid synthe- tase subunits and anti-thioesterase I immunoglobulins, initial fatty acid synthetase concentration 200 pg/ml, well 8 contained 16 ~1 of the same solution.

4

FIG. 4. Double diffusion of cytosol and purified fatty acid synthetase against anti-fatty acid synthetase anti- bodies, in the presence of anti-thioesterase I antibodies in the agarose. The concentrations of fatty acid syn- thetase in cytosols from the lactating mammary glands of four individual rats were determined by rocket immunoelectrophoresis. The fatty acid synthetase in the cytosol was dissociated into subunits (see Experi- mental Procedures) and applied to the antigen wells at O-4%. The agarose gel contained anti-thioesterase I immunoglobulins (2.4 mg/ml), well 1 contained puri- fied fatty acid synthetase, 9 S subunits (0.5 mg/ml). Well 2 contained the same cytosol sample as well 3, but a higher concentration of fatty acid synthetase 9 S subunits (2 mgiml). All wells held 12 ~1 of solution.

125,000; this is identical in size to that of one of the major products of limited trypsin- ization of the fatty acid synthetase (see Ref. (8) and later section of this paper). (ii) The component of the fatty acid synthetase not recognized by anti-thioesterase I immunoglobulins gives a reaction of ap- parent identity with trypsinized fatty acid synthetase when these antigens are dif- fused against anti-fatty acid synthetase immunoglobulins. (iii) Experiments on limited proteolysis of fatty acid synthetase with trypsin, chymotrypsin (22) and elas- tase (K. N. Dileepan and Smith, unpub- lished results) have revealed the thio- esterase I domains to be particularly vulnerable. (iv) Cytosol from lactating rat mammary gland contains proportionately less of the component not recognized by anti-thioesterase I antibodies. Thus when the experiment shown in Fig. 1 was per- formed with various cytosols (from different animals) containing an identical amount of fatty acid synthetase (determined by rocket immunoelectrophoresis), cytosols

384 SMITH AND STERN

240,000

-225.000

\205,000

-127,000

-125,000

-I 10,000

-95,000

35,000

17,500

0.5 I 2 5 15 50

Time (mid

TOTAL THIOESTERASE

0 lo 20 so 40 50

Time (mid FIG. 5. Time course of trypsinization of [1,3W]diisopropylphosphate-labeled fatty acid synthetase.

(A) Sodium dodecyl sulfate-polyacrylamide gel electrophoretograms, stained with Coomassie blue. (B) Fate of radioactive label.

showed no immunoprecipitin line outside give a fatty acid synthetase concentration the immunoprecipitin ring. When the four-fold that required to show the immuno- concentration of cytosol was increased to precipitin line with purified fatty acid

SUBUNIT STRUCTURE OF FATTY ACID SYNTHETASE 385

synthetase, a line was visible on the edge of the immunoprecipitin ring (Fig. 4). Using rocket immunoelectrophoresis, we esti- mated that, in cytosol, the amount of antigen recognized by anti-fatty acid synthetase but not recognized by anti- thioesterase I antibodies, contributed less than 1% of the fatty acid synthetase pro- tein. The results of these experiments suggest that the small amount of nicked polypeptides associated with the purified fatty acid synthetase were produced during the isolation and/or storage of the multi- enzyme.

From this study we conclude that the fatty acid synthetase consists of two 240,000 M, polypeptides, each containing a single thioesterase I domain. The only polypeptides present which do not contain a thioesterase I domain are attributable to nicked fatty acid synthetase.

Limited Proteolysis Approach

Fatty acid synthetase, labeled in its two thioesterase I domains with [1,3J4C]- diisopropylphosphofluoridate was subjected to limited trypsinization and the products examined at various intervals by sodium dodecyl sulfate-polyacrylamide electro- phoresis (Fig. 5). Radioautography was used to identify the radioactive polypep- tides; these were then cut out from the gel and their radioactivity was assayed. Two high molecular weight polypeptides (ap- proximately 225,000 and 205,000) were formed from the 240,000 molecular weight subunit. Neither of these polypeptides was radioactive, indicating that both lacked the thioesterase I active site. Since the i nioesterase domain was released as both the intact 35,000 M, polypeptide and its 17,500 nicked halves, it is evident from this result that at least one of the fatty acid synthetase subunits contains a thioesterase I domain in a terminal location. However, not all of the fatty acid synthetase mole- cules follow this relatively simple course of proteolysis. Radioautography revealed that a radioactive polypeptide of approximately 127,000 M, was also produced; in some experiments this was incompletely resolved from a polypeptide of about 125,000 M,. The amount of the radioactive 127,000 M,

polypeptide increased initially, and then declined as proteolysis was allowed to con- tinue (Fig. 5B). Thus, the question arose as to whether the observations resulted from proteolysis of identical subunits by several alternative pathways, or whether they resulted from proteolysis of nonidentical subunits, each by its own characteristic pathway. Figure 6 shows the two struc- tures, one a homodimer, one a heterodimer which we have considered as models for the fatty acid synthetase. In both models, the thioesterase I domain is a 35,000 M, region, with a site near the center susceptible to tryptic attack. This is a necessary assump- tion since the isolated thioesterase (35,000 M,) has been shown to be susceptible to tryptic attack under nondenaturing condi- tions, giving rise to two polypeptides of approximately equal molecular weight (9). Both models contain polypeptide regions of molecular weight 125,000 and 95,000 which are stable to trypsin under non- denaturing conditions. This is a necessary assumption, since when trypsinization is allowed to continue to completion (under nondenaturing conditions), only the 125,000 and 95,000 M, polypeptides remain in addition to the thioesterase I fragments. (In Fig. 5, trypsinization was incomplete; some of the 110,000 M, polypeptide had not yet undergone cleavage to the 95,000 M, polypeptide.) Since the original subunits are of equal size (approximately 240,000 M,) and each contains a thioesterase domain (35,000), it follows that the 125,000 and 95,000 M, polypeptides must originate from the same subunit. Thus, the only hetero- dimer structure compatible with these data is one in which the thioesterase I domain is situated at a terminus on one subunit and between the 125,000 and 95,000 M, regions on the other subunit (Fig. 5). The critical test for the homodimer and hetero- dimer models is the number of radioactive transient polypeptides which one could predict would be produced on limited trypsinization of the [ 1 ,3-14C]diisopropyl- phosphate-labeled multienzyme. The homo- dimer model predicts only one such polypeptide whereas the heterodimer model predicts three. Experimental evidence shows clearly that only one is formed, so this heterodimer model must be rejected.

386 SMITH AND STERN

I siie of tryptic attack

~thloesterase I active site

FIG. 6. Hypothetical linear models for possible homodimeric and heterodimeric fatty acid synthetase showing major points of attack by trypsin, under nondenaturing conditions. (A) The homodimer model accommodates all of the observed transient and stable polypeptides observed on trypsinization. This homodimer model predicts that only one labeled, transient polypeptide (M, = 127,000) will be produced in addition to the 35,000 and 17,500 species derived from the thioesterase domain, when fatty acid synthetase labeled with [1,3W]diisopropylphosphofluoridate is trypsinized. (B) One of two possible heterodimer models which predicts that trypsinization will generate three labeled, transient polypeptides, in addition to the 35,000 and 17,500 species derived from the thioesterase domain. For clarity, the nonradioactive polypeptide produces are not shown. An alternative heterodimer model, in which the orientation of the thioesterase domain on the lower polypeptide is reversed, would also predict the formation of three labeled, transient polypeptides.

Finally, the homodimer model also correctly unit is susceptible to limited tryptic attack predicts the number and size of the non- at three identical locations and each subunit radioactive polypeptides formed by limited contains a thioesterase I domain at a terminal trypsinization. locus. Theoretically, our results do not

In conclusion, our results indicate the exclude the possibility that, although each mammalian fatty acid synthetase is a dimer subunit has a terminal thioesterase I domain of polyfunctional polypeptides. Each sub- and a site susceptible to tryptic attack at

SUBUNIT STRUCTURE OF FATTY ACID SYNTHETASE 387

the same distance from this domain, the intervening polypeptide may contain heter- ologous sequences. Neither do our results exclude the possibility that the thioesterase I domain is C terminal on one polypeptide and N terminal on the other. We feel that these alternative structures are rather less probable and that the most likely structure for the multienzyme is that of a homodimer. Since these experiments were completed, a paper has been published by Guy et aE. (231, who used limited elastase digestion of the rabbit mammary gland fatty acid synthetase to provide evidence for subunit identity. Recent experiments in our laboratory have shown that the 4’-phosphopantetheine moiety is located on the 95,000 M, polypep- tide produced by limited trypsinization (10). Thus, the use of limited proteolysis appears to be a promising tool in mapping out the domain structure of this polyfunctional polypeptide enzyme.

ACKNOWLEDGMENTS

We are grateful to Dr. Richard Perham of the Department of Biochemistry, University of Cambridge, England, for allowing S.S. to carry out part of this work in his laboratory. Our particular thanks to Dr. Peter Lackman, of the MRC unit, Cambridge, for his help with the immunochemical studies.

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