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

Bruce

Lyon

Memorial

Research

SMITH2

AND ALAN

STERN

Laboratory, Children’s Hospital Medical Oakland, Calijornia 94609 Received

April

4, 1979;

Center,

51st and Grove

Streets,

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 complexes 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 complex 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 subunits. On the other hand, the multienzyme Mycobacterium smegmatis appears to be an oligomer of identical polyfunctional polypeptide chains (4). In animals, the fatty acid synthetases are found as dimers of approximate M, 0.5 x lo6 (see Ref. (5) for review). 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. 379

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 immunoglobulin 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-

0003-9861/79/12037309$02.00/0 Copyright

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All

of

rights

Press,

Inc.

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 prepared in 0.1 M NaCl/O.Ol M sodium phosphate buffer (pH 7.2)/5 mM EDTAlO.01 M sodium azide for Ouchterlony 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 Coomassie brilliant blue (17). Quantitative immunoprecipitin reactions. Reactions, carried out as described previously (12), were allowed to take place at 0-4°C for 2 days. Every precaution 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. Agarose gels (1%) containing anti-fatty acid synthetase immunoglobulins in sodium barbital/Tris/glycine buffer, 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 electrophoresis. 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]diisopropylphosphojluoridate-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 sulfatepolyacrylamide electrophoresis. The following molecular 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 dithiothreitol (19). That dissociation was complete, was confirmed 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

Immunochemical

AND DISCUSSION

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 thioesterase I component of the fatty acid synthetase. These antibodies react specifically 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 immunoglobulins in agarose containing various amounts of anti-thioesterase I immunoglobulins (Fig. 1). Undissociated fatty acid synthetase, trypsinized fatty acid synthetase, and thioesterase I yere included in other antigen wells. In the absence of antithioesterase I immunoglobulins, fatty acid synthetase subunits gave a sharp immunoprecipitin line. As expected, lines of partial identity (spurs) were formed when either trypsinized fatty acid synthetase or thioesterase 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 diameter of the rings decreased as the concentration of anti-thioesterase I immuno-

SUBUNIT

STRUCTURE

OF FATTY

381

ACID SYNTHETASE

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 synthetase. 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 immunoglobulins, indicating that all of the enzyme had been precipitated within the immunoprecipitin 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 synthetase subunits, a distinct immunoprecipitin line, formed by the anti-fatty acid synthetase immunoglobulins, persisted outside the immunoprecipitin ring. Clearly the fatty acid synthetase contained some polypeptide which was not recognized by anti-thioesterase I antibodies. This precipitin 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 removed by centrifugation, and the super75

.

verus

wsw

mti-TE 1

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 antithioesterase 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 immunoglobulins 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 acid synthetase protein would remain in the synthetase immunoglobulins. At equivsupernatant after treatment with antialence, anti-thioesterase I immunoglobulins thioesterase I immunoglobulins. Alternaprecipitated a total of 22 pg of complex tively, if the thioesterase I domains were from 8.4 pg fatty acid synthetase. This present, one on each subunit, both subunits corresponds to approximately 3 antibody would be precipitated by the anti-thiomolecules per fatty acid synthetase subunit. esterase I immunoglobulins and the amount In comparison, anti-fatty acid synthetase of non-antibody protein remaining in the immunoglobulins precipitated 72 pg of supernatant would necessarily be very complex from 8.4 pg fatty acid synthetase small, since the fatty acid synthetase 9 S subunits, equivalent to 12 antibody preparation consisted of more than 95% molecules per fatty acid synthetase subunit. 240,000 M, polypeptides, as determined The supernatants remaining after titration by sodium dodecyl sulfate-polyacrylamide of fatty acid synthetase to equivalence, electrophoreis (8). Thus, quantitation of the with anti-thioesterase I immunoglobulins, amount of antigen remaining in the supergave 16 pg antibody-antigen precipitate natant, that was recognized by anti-fatty when mixed with anti-fatty acid synthetase acid synthetase, would be expected to immunoglobulins. distinguish between these two possibilities. Since the number of anti-thioesterase I To this end, we used the technique of antibody molecules associated with the rocket immunoelectrophoresis. fatty acid synthetase 9 S subunits at Fatty acid synthetase 9 S subunits were equivalence was close to the minimum titrated to equivalence with anti-thiorequired for lattice formation, it was esterase I immunoglobulins and the preconceivable that some soluble anti-thiocipitate was removed by centrifugation. esterase I-fatty acid synthetase subunit Portions of the supernatant were analyzed complexes might have been formed which by rocket immunoelectrophoresis, using subsequently were precipitated by anti- both fatty acid synthetase and trypsinized fatty acid synthetase immunoglobulins. To fatty acid synthetase as standards (Fig. 3). test this possibility, fatty acid synthetase Trypsinized fatty acid synthetase was labeled on its two thioesterase I domains included, since the immunoprecipitin line with [3H]diisopropylphosphofluoridate (1.8 formed by the component of the fatty acid mol inhibitor/m01 fatty acid synthetase synthetase not recognized by anti-thiodimer) was dissociated into 9 S subunits and esterase I immunoglobulins gave an aptreated with anti-thioesterase I immunoparent reaction of identity with that formed globulins. The immunoprecipitate contained by trypsinized fatty acid synthetase, when 99.3% of the radioactivity. This indicated these antigens were diffused against anticlearly that both thioesterase I domains fatty acid synthetase immunoglobulins were precipitated with anti-thioesterase I (Fig. 1). In practice, only a slight difference antibodies and ruled out the possibility was detected in rocket heights when equal that soluble antibody-antigen complexes amounts of fatty acid synthetase and remained after the reaction. However, trypsinized fatty acid synthetase were since not all of the protein in the fatty acid compared. The results showed that when synthetase 9 S subunit preparation was 9 S subunits were treated with anti-thioprecipitated by anti-thioesterase I anti- esterase I antibodies, the amount of protein bodies, the experiments did not rule out the remaining in the supernatant which was possibility that both the thioesterase I recognized by anti-fatty acid synthetase domains resided on a single subunit. Such antibodies, corresponded to only 3.5% of the an arrangement would allow only one sub- initial fatty acid synthetase protein. This unit to react with anti-thioesterase I result demonstrated clearly that the antiimmunoglobulins while both would be gen in the fatty acid synthetase preparation recognized by anti-fatty acid synthetase which was recognized by anti-fatty acid immunoglobulins. If this were the case, synthetase, but not by anti-thioesterase I then one would predict that half of the fatty immunoglobulins, was not a subunit lacking

SUBUNIT

STRUCTURE

OF FATTY

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 synthetasederived impurity. Present evidence indicates the former possibility to be the more likely: (i) The purified fatty acid synthetase contains as its major “impurity” a component of molecular weight approximately

ACID SYNTHETASE

383

4 FIG. 4. Double diffusion of cytosol and purified fatty acid synthetase against anti-fatty acid synthetase antibodies, in the presence of anti-thioesterase I antibodies in the agarose. The concentrations of fatty acid synthetase 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 Experimental 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 purified 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 trypsinization of the fatty acid synthetase (see Ref. (8) and later section of this paper). (ii) The 123456789lOlll2l3 component of the fatty acid synthetase not recognized by anti-thioesterase I FIG. 3. Rocket immunoelectrophoresis of fatty acid immunoglobulins gives a reaction of apsynthetase-derived antigen not recognized by anti- parent identity with trypsinized fatty acid thioesterase I antibodies. Fatty acid synthetase, 9 S synthetase when these antigens are difsubunits (20 pg), was reacted with anti-thioesterase fused against anti-fatty acid synthetase I immunoglobulins (4.2 mg) in a volume of 100 ~1 for immunoglobulins. (iii) Experiments on 2 days at O-4%, and the immunoprecipitate was limited proteolysis of fatty acid synthetase removed by centrifugation. Rocket immunoelectro(22) and elasphoresis was performed on the supernatant with both with trypsin, chymotrypsin tase (K. N. Dileepan and Smith, unpubtrypsinized fatty acid synthetase and native fatty acid the thiosynthetase as reference standards. The agarose gel lished results) have revealed esterase I domains to be particularly contained anti-fatty acid synthetase immunoglobulins (16 pg/ml). Sample wells contained 8 ~1 of the following vulnerable. (iv) Cytosol from lactating rat antigens: fatty acid synthetase, 1 = 50 pg/ml, 2 = 25 mammary gland contains proportionately pg/ml, 3 = 12.5 pgiml, 4 = 6.3 pg/ml, 5 = 3.1 pg/ml; less of the component not recognized by trypsinized fatty acid synthetase, 6 = 3.5 pg/ml, anti-thioesterase I antibodies. Thus when 10 = 6.9 pg/ml, 11 = 13.9 pg/ml, 12 = 27.8 pg/ml, the experiment shown in Fig. 1 was per13 = 55.5 @g/ml; wells 7 and 9 contained 8 ~1 of the formed with various cytosols (from different supernatant from reaction between fatty acid syntheanimals) containing an identical amount of tase subunits and anti-thioesterase I immunoglobulins, fatty acid synthetase (determined by initial fatty acid synthetase concentration 200 pg/ml, rocket immunoelectrophoresis), cytosols well 8 contained 16 ~1 of the same solution.

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

2

I

5

15

50

Time (mid

TOTAL

0

lo

20

so

THIOESTERASE

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 the immunoprecipitin ring. When the concentration of cytosol was increased to

give a fatty acid synthetase concentration four-fold that required to show the immunoprecipitin line with purified fatty acid

SUBUNIT

STRUCTURE

OF FATTY

synthetase, a line was visible on the edge of the immunoprecipitin ring (Fig. 4). Using rocket immunoelectrophoresis, we estimated that, in cytosol, the amount of antigen recognized by anti-fatty acid synthetase but not recognized by antithioesterase I antibodies, contributed less than 1% of the fatty acid synthetase protein. 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 multienzyme. 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 electrophoresis (Fig. 5). Radioautography was used to identify the radioactive polypeptides; these were then cut out from the gel and their radioactivity was assayed. Two high molecular weight polypeptides (approximately 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 molecules 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,

ACID

SYNTHETASE

385

polypeptide increased initially, and then declined as proteolysis was allowed to continue (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 structures, 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 assumption since the isolated thioesterase (35,000 M,) has been shown to be susceptible to tryptic attack under nondenaturing conditions, 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 nondenaturing 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 heterodimer 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 heterodimer models is the number of radioactive transient polypeptides which one could predict would be produced on limited trypsinization of the [ 1 ,3-14C]diisopropylphosphate-labeled multienzyme. The homodimer 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 predicts the number and size of the nonradioactive polypeptides formed by limited trypsinization. In conclusion, our results indicate the mammalian fatty acid synthetase is a dimer of polyfunctional polypeptides. Each sub-

unit is susceptible to limited tryptic attack at three identical locations and each subunit contains a thioesterase I domain at a terminal locus. Theoretically, our results do not exclude the possibility that, although each subunit has a terminal thioesterase I domain and a site susceptible to tryptic attack at

SUBUNIT

STRUCTURE

OF FATTY

the same distance from this domain, the intervening polypeptide may contain heterologous 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, polypeptide 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. REFERENCES 1. KNOBLING, A., SCHIFFMAN, D., SICKINGER, M., AND SCHWEIZER, E. (1975) Eur. J. Biochem. 56, 259-367. 2. STOOPS, J. K., AWAD, E. S., ARSLANIAN, M. J., GUNSBERG, S., AND WAKIL, S. J. (19’78) J. Biol. Chem.

253, 4464-4475.

3. PACKTER, N. M., AND ALAM, A. (1978) Biochem. Sot. Trans. 6, 195-197. 4. WOOD, W. I., PETERSON, D. O., AND BLOCH, K. (1978)

J. Biol.

Chem.

253, 2650-2656.

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5. SMITH, S. (1976) in The Immunochemistry of Enzymes and Their Antibodies (Salton, M. R. J., ed.), pp. 125-146, Wiley, New York. BLOCH, K., AND VANCE, D. (1977) Annu. Rev. Biochem.

9. 10. 11. 12. 13.

46, 263-298.

LIBERTINI, L. J., AND SMITH, S. (1978) J. Biol. Chem. 253, 1393-1401. LIN, C. Y., AND SMITH, S. (1978) J. Biol. Chem. 253, 1954-1962. DILEEPAN, K. N., LIN, C. Y., AND SMITH, S. (1978) Biochem. J. 175, 199-206. LIBERTINI, L. J., AND SMITH, S. (1979) Arch. Biochem. Biophys. 192, 47-60. SMITH, S., AGRADI, E., LIBERTINI, L. J., AND DILEEPAN, K. N. (1976) Proc. Nat. Aead. Sci. USA 73, 1184-1188. SMITH, S. (1973) Arch. Biochem. Biophys. 156, 751-758. FAHEY, J. L., AND HORBETT, A. P. (1959) J. Biol. Chem.

234, 2645-2651.

14. OUCHTERLONY, 0. (1978)in Handbookof Immunodiffusion and Immunoelectrophoresis, pp. 3123, Ann Arbor Science Pub., Ann Arbor, Mich. 15. MANCINI, G., CARBONARA, C. O., AND HEREMANS, J. F. (1965) Immunochemistry 2, 235254. 16. SMITH, S., AND ABRAHAM, S. (1971) J. Biol. Chem.

246, 6428-6435.

17. WEEKE, B. (1976) in a Manual of Quantitative Immunoelectrophoresis (Axelser, N. H., Krill, J., and Weeke, B., eds.), Vol. 2, Suppl. 1, pp. 15-46, Universitetsforleget, Oslo. 18. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 19. SMITH, S. (1971) Biochim. Biophys. Acta 251, 477-481. 20. MARTIN, R. G., AND AMES, R. N. (1961) J. Biol. Chem. 236, 1372- 1379. 21. SUMNER, J. B., AND GRAELEN, N. (1938) J. Biol. Chem. 125, 33-36. 22. AGRADI, E., LIBERTINI, L. J., AND SMITH, S. (1976) Biochem. Biophys. Res. Commun. 68, 894-900.

23. GUY, P., LAW, S., AND HARDIE, G. (1978) Febs. Lett.

94, 33-37.