ARCHIVES OF BIOCHEMISTRY AND RIOPHYSICS 166, 751-758 (1973) Studies on the Immunological Cross-Reactivity and Physical Properties of Fatty Acid Synthetases’ STUART SMITH The Bruce Lyon Memorial Research Laboratory, Children’s Hospital Medical Center of :Vorthern California, Fiftq-First arid Grove Streets, Oakland, California 94609 Received December 20, 1972 Fatty acid synthetase enzymes were purified from the liver, mammary gland, and adipose tissue of rats and the liver and mammary gland of mice. The enzymes from the liver and mammary gland of the same species have similar molecular weights and and dissociate into subunits at comparable rat,es. Rabbit antisera were prepared against the fatty acid synthetase from the lactating rat mammary gland. Cross-react,ivity between different fatty acid synthetases was determined by immunodiffusion and immunoprecipitin tests. ?Jo differenres in im- munological cross-reactivity could be detected in liver, mammary gland, and adipose enzymes from the same species; fatty acid synthetases from the rat and mouse gave reactions of incomplete identity. Partially purified fatt,y acid synthetases from pigeon liver and rabbit, mammary gland did not react with the antiserum. It is concluded that the immunochemical approach is useful in determining the degree of resemblance between fatty acid synt’het,ases from different species. Within a given species, the liver and mammary gla.nd fatty acid synthetases seem to be very similar. if not identical, proteins. The enzymes involved in the synthesis of of some animals the FAS is responsible for fatty acids from nwtyl- and mnlonyl-CoA t’he synthesis of medium-chain-length fatty in animals (a) and some microorganisms acids, whereas in the liver the FAS syn- (3, 4) are arranged in the form of a multi- thesizes only long chain fatty acids (11). enzyme complex. A number of these com- A number of workers have examined the plexes have been purified to homogeneity, properties of these enzymes to determine from birds (5, 6), fish (7), and mammals mhethcr the different functional roles could (8-10) and some of their propert’ics reviewed be related to some basic struct,ural dif- (2, 9), but as yet no attempt has been made ference in the FAS complexes (9, 10 to rationalize sny differences or similarities 12, 13). on a phylogenet,ic basis. Within some species, In recent years, it has been established it has been established that the fatty acid that the immunological cross-reactivity of a synthetase (FAS)z clnzymes can fulfill dif- series of proteins is closely related to the ferent physiological functions in different sequence resemblance, or more precisely tissues. For example, in t’hc mammary glands to the native three-dimensional st’ructure 1 This study was reported in part at, the eighth of the proteins (14, 15). Consequently, the Meeting of the Federation of European Biochemi- immunochemical approach has been used cal Societies in Amsterdam, August, 1972 (1). both to establish the evolutionary trend of Financial support for this work was provided by the structure of classes of proteins (16) and Grant GB-32087 from the National Science Foun- to demonstrate differences in the structure dation and Public Health Service Research Grant of enzymes obtained from different tissues CA 11736 from the National Cancer Institute. of the same species (17, 18). 2 Abbreviations used : FAS, fatty acid synthe- The object of this study was to deter- t,ase; DTT, dithiothreitol. mine, on the basis of immunological cross- 751 (Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
Transcript
ARCHIVES OF BIOCHEMISTRY AND RIOPHYSICS 166, 751-758 (1973)
Studies on the Immunological Cross-Reactivity and Physical
Properties of Fatty Acid Synthetases’
STUART SMITH
The Bruce Lyon Memorial Research Laboratory, Children’s Hospital Medical Center of :Vorthern California, Fiftq-First arid Grove Streets, Oakland, California 94609
Received December 20, 1972
Fatty acid synthetase enzymes were purified from the liver, mammary gland, and adipose tissue of rats and the liver and mammary gland of mice. The enzymes from the liver and mammary gland of the same species have similar molecular weights and and dissociate into subunits at comparable rat,es.
Rabbit antisera were prepared against the fatty acid synthetase from the lactating rat mammary gland. Cross-react,ivity between different fatty acid synthetases was determined by immunodiffusion and immunoprecipitin tests. ?Jo differenres in im- munological cross-reactivity could be detected in liver, mammary gland, and adipose enzymes from the same species; fatty acid synthetases from the rat and mouse gave reactions of incomplete identity. Partially purified fatt,y acid synthetases from pigeon liver and rabbit, mammary gland did not react with the antiserum.
It is concluded that the immunochemical approach is useful in determining the degree of resemblance between fatty acid synt’het,ases from different species. Within a given species, the liver and mammary gla.nd fatty acid synthetases seem to be very similar. if not identical, proteins.
The enzymes involved in the synthesis of of some animals the FAS is responsible for fatty acids from nwtyl- and mnlonyl-CoA t’he synthesis of medium-chain-length fatty in animals (a) and some microorganisms acids, whereas in the liver the FAS syn- (3, 4) are arranged in the form of a multi- thesizes only long chain fatty acids (11). enzyme complex. A number of these com- A number of workers have examined the plexes have been purified to homogeneity, properties of these enzymes to determine from birds (5, 6), fish (7), and mammals mhethcr the different functional roles could (8-10) and some of their propert’ics reviewed be related to some basic struct,ural dif- (2, 9), but as yet no attempt has been made ference in the FAS complexes (9, 10 to rationalize sny differences or similarities 12, 13). on a phylogenet,ic basis. Within some species, In recent years, it has been established it has been established that the fatty acid that the immunological cross-reactivity of a synthetase (FAS)z clnzymes can fulfill dif- series of proteins is closely related to the ferent physiological functions in different sequence resemblance, or more precisely tissues. For example, in t’hc mammary glands to the native three-dimensional st’ructure
1 This study was reported in part at, the eighth of the proteins (14, 15). Consequently, the
Meeting of the Federation of European Biochemi- immunochemical approach has been used
cal Societies in Amsterdam, August, 1972 (1). both to establish the evolutionary trend of
Financial support for this work was provided by the structure of classes of proteins (16) and
Grant GB-32087 from the National Science Foun- to demonstrate differences in the structure dation and Public Health Service Research Grant of enzymes obtained from different tissues CA 11736 from the National Cancer Institute. of the same species (17, 18).
2 Abbreviations used : FAS, fatty acid synthe- The object of this study was to deter- t,ase; DTT, dithiothreitol. mine, on the basis of immunological cross-
751
(Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
752
reactivity, whether any structural differences could be established between FAS’s from different tissues of the same species.
EXPERIMENTAL PROCEDURE
Maferials. Acetyl CoA was prepared from acetic anhydride by the method of Simon and Shemin (19) and malonyl CoA by the method of Lynen (20). These CoA derivatives were purified by chromatography on a column of DEAE-cellulose (21 X 2.5 cm) which had been equilibrated with 0.01 M LiCl in 0.003 M HCl. Material was eluted from the column with a concave gradient of LiCl (0.0-0.20 M) in 0.003 M HCl. The fractions con- taining acetyl CoA (or malonyl CoA) were lyoph- iliaed, the residue dissolved in a minimum volume of water, and the salt removed by passage through a column (31 X 2 cm) of Sephadex C-15. The concentration of acetyl CoA (or malonyl CoA) was determined by measuring the extinction at 260 and 232 nm and by measuring the release of sulfhy- dry1 groups, with 5,5-dithiobis(2-nitrobenzoate), following mild alkaline hydrolysis.
Other cofactors were obtained from either Cal- biochem, Sigma, or Boehringer and Soehne. Com- plete and incomplete Freund’s adjuvant was ob- tained from Difco, and Agarose (electrophoresis grade) from Nutritional Biochemicals. hlannex DEAE-cellulose was purchased from hlann, Ani- line blue-black from Matheson, Coleman and Bell, and [1-‘“Clacetic anhydride from New England Nuclear Corporation.
Animals. Lactating rats of the Long-Evans strain, lactating mice of the C3H strain, and lactating rabbits of the New Zealand White variety were used as the sources of mammalian fatty acid synthetases. Dr. C. S. Nicoll of the Department of Physiology-Anatomy, University of California, Berkeley, kindly supplied a well-fed pigeon for preparation of the pigeon liver fatty acid synthetase. Rats and mice were fed the ap- propriate Purina Chow and supplied with water ad Zibitum. Three days before sacrifice, the lactat- ing rats and mice were transferred to a high- glucose, fat-free diet (21). This procedure has the effect of increasing the level of hepatic fatty acid synthetase (21), so good yields of both the liver and mammary FAS’s could be obtained from the same animals. In some experiments, male rats and mice were used as sources of the hepatic or adipose fatty acid synthetases. These animals were fasted 2 days and refed the high-glucose, fat-free diet for 3 days prior to sacrifice. Lactating rabbits were supplied with water ad Z&turn and fed a rabbit chow obtained from Dean’s Animal Feed, BeI- mont, CB. All animals were killed by cervical dislocation.
Purification of fatty acid synthetases. The fatty
acid synthetases were isolated from lactating rat mammary gland, lactating mouse mammary gland, rat liver, rat adipose, and mouse liver by a pro- cedure which was originally developed for the rat mammary gland enzyme (9). Three or more prep- arations of each enzyme have been made by this procedure.
Measuremenl of fatty acid synthetase activity. Assay systems consisted of 0.1 M potassium phos- phate (pH 6.6), 1.5 X IO-’ M NADPH, 5 X 10-5 M acetyl CoA, 5.4 X 10-j M malonyl CoA, and enzyme in a total volume of 0.5 ml; the reaction was meas- ured spectrophotometrically (9). One unit of enzyme activity is defined as the amount of en- zyme catalyzing the malonyl-CoA-dependent oxidation of 1 nmole NADPH/min at 30°C. In experiments where the chain-length of the prod- ucts was determined, [l-‘4C]acetyl CoA was used, and the radioactive fatty acids isolated as de- scribed by Abraham et al. (22).
Protein determinations. The method of Lowry et al. (23) was used with the modification described previously (9).
JfoZecuZar weight determinations. Samples were dissolved in 0.25 M potassium phosphate buffer (pH 7.0) containing 1 X 1O-3 M EDTA and 1 X IF3 M DTT. Equilibrium sedimentation was car- ried out in double-sector cells with a column height of 3 mm, using a Beckman Spinco model E ultra- centrifuge. The rotor temperature was maintained at 22°C. For determination of the molecular weight of the mouse liver and mouse mammary gland en- zymes, the ultracentrifuge was equipped with a uv monochromator. An ultracentrifuge fitted with interference optics was used for determination of the molecular weight of the rat liver enzyme. Equilibrium states were established by the menis- cus depletion procedure (24), and the weight average molecular weights calculated (25), as- suming a value for the partial specific volume of 0.740, as determined for the rat mammary gland enzyme (9).
Sucrose density gradient cenlrifugation. Dissocia- tion of the enzymes into their component subunits was monitored by sucrose density gradient cen- trifugation as described by Smith (13).
Amino acid analysis. Samples were dialyzed against distilled water for 24 hr at O-5°C and lyophilized. Hydrolysis was carried out in sealed, evacuated glass tubes for 22 hr with 6 N HCl at 110°C. A Beckman 120 amino acid analyzer was used to determine the amino acid composition (26). Cysteine content was determined as cysteic acid, after performate oxidation and hydrolysis (27) and also by titration of the native enzyme with 5,5’-dithiobis(2-nitrobenzoate) in 6 M urea (12). Tryptophan was determined spectrophotometri- tally (28). The cysteine values obtained by titra-
FATTY ACID SYNTHETASES 753
tion with 5,5’-dithiobis(%nitrobenzoate) were integrated into the overall composition by deter- mining the tryptophan content on the same preparation.
Preparation of antiserum. Homogeneous prep- arations of lactating rat mammary gland fatty acid synthetase dissolved in 0.25 M potassium phos- phate buffer (pH 7.0) containing 1 X low3 M EDTA and 1 X 1O-3 M DTT were centrifuged at 100,OOOg for 10 min to remove agglutinated protein. These preparations were used to immunize young (‘2- 2% kg) New Zealand White rabbits. Approxi- mately 10 mg of enzyme, dissolved in 1 ml buffer, was emulsified with 1 ml complete Freund’s a,djuvant and then injected subcutaneously into the rabbits. Booster injections of 5 mg enzyme in 1 ml buffer, emulsified with 1 ml incomplete Freund’s adjuvant were administered at 3-wk intervals to maintain the titer of antiserum.
Batches of antiserum were collected and the T-globulin fraction isolated by precipitation m-ith ammonium sulfate (0-33r; ). The y-globulin frac- tion was dissolved in 0.01 x potassium phosphate (,pH 8.0), dialyzed against t’he same buffer over- night, and stored at -20°C. The potency of the antiserum varied considerably from one rabbit to another.
Immunodiflusion. Double diffusion analyses (29) were carried out on 1:; agarose gels prepared in 0.05 M barbital buffer (pH 8.6), on microscope slides. Diffusion proceeded for twenty-four hours at 2O”C, then nonagglutinated proteins were re- moved by exhaustive washing with 0.89yc NaCl. The immunodiffusion plates were next washed in distilled water for 24 hr and then stained by dip-
ping in 0.57” Aniline blue-black (in 27; aqueous acetic acid) for 3 min. Excess stain was washed out with 2%, acetic acid.
Zmmunoprecipitin reactions. Reaction mixtures (0.5 ml) consisted of 1 mg anti-FAS r-globulin and various amounts of antigen in 0.04 M potassium phosphate (pH 7.0). Tubes were stored for 4 days at 0-2°C to ensure complete precipitation of the antibody-antigen complexes. Bgglutinated pro- tein was then collected by centrifugation at 40,OOOg for 15 min at O”C, washed with 1.5 ml ice-cold 0.89c< NaCl, recentrifuged, and dissolved in 0.5 N NaOH (0.3 ml). The protein content was then determined as described by Lowry et al. (23), except that NaOH was omitted from Reagent A and the final volume of the assay mixture was 1.95 ml. Blank values were always determined by separately omitting antibody and antigen.
RESULTS
General Properties of PAS’s
The FAS’s from mouse liver, mouse mam- mary gland, rat liver, rat mammary gland, and rat adipose tissue were purified to high specific activity, usually in the range 8W 900 units/mg. Each FAS, when subject,ed to sucrose density gradient centrifugation, gave a single protein peak corresponding approximately to a 13 S species. Homo- geneity of the liver and mammary gland enzymes was confirmed by the rectilinear nature of the plots of --In c against r2 in the sedimentation equilibrium studies (see Fig.
36 0 36 5 310 37 5 50 0 50 5 510 500 50 5 510
r* P r*
FIG. 1. Sedimentation equilibrium of native fatty acid synthetases. (A) Rat liver FAS, initial pro- tein concentration 0.5 mgjml, interference optics. Molecular weight = 474,000 f 10,000 (mean f SE, 3 determinations). (B) Mouse liver FAS, initial protein concentration 0.5 mg/ml, uv scanner. Molecular weight = 586,000 (single determination). (C) Mouse mammary gland FAS, initial protein concentration 0.5 mg/ml, uv scanner. Molecular weight = 594,000 (single determination).
754 SMITH
1 and Ref. 9). Under optimum cofactor conditions, all the purified enzymes syn- thesized predominantly pslmitic acid from acetyl CoA, malonyl CoA, and NADPH (Table I).
Molecular Weights of FAS’s
The molecular weight value obtained for the rat liver enzyme (474,000) is some- what lower than that reported previously (540,000) by Burton et al. (8) and is very close to the value obtained for the rat mam- mary enzyme (478,000) by Smith and Abra- ham (9). In this study, the molecular weights for the liver and mammary gland enzymes were very similar for each particular species.
TABLE I
CHAIN LENGTH OF FATTY ACIDS SYNTHESIZED BY Va4RIOUS FAS’S~
Rat mammary gland Rat liver Rat adipose Mouse mammary
gland Mouse liver
I -
Distribution($ ;adioactivity n
6 FIG. 2. Ouchterlony immunodiffusion analyses
of partially purified FAS’s. Center wells con- tained 0.25 mg anti-(rat mammary gland) FAS r-globulin. (A) Outer wells contained 1 unit FAS activity; (PiL) pigeon liver, (MoL) mouse liver, (RaL) rat liver. (B) Outer wells contained 4 units FAS activity; (RaM) rat mammary gland, (RbM) rabbit mammary gland, (MOM) mouse mammary gland.
Dissociation into Subunits
The rat mammary gland FAS was previ- ously shown (12) to be a cold labile enzyme which undergoes reversible cold-induced dissociation into half-molecular weight sub- units. First order rate constants for the
under a variety of conditions (13). When dissociation process have been measured
the rat gland, rat liver, mammary mouse mammary gland, and mouse enzymes liver were aged under similar conditions, it was found that the mouse enzymes dissociated slower than the rat enzymes (Table II). TV.,. . . . ^ __
TABLE II
DISSOCIATION OF FATTY ACID SYNTHETASES INTO SUBUNITP
FAS First order rate constants in (days)-’
0°C 20°C
Rat mammary gland 0.4 0.03 Rat liver 0.3 0.02 Mouse mammary gland 0.02 0 Mouse liver 0.03 0
a FAS’s were stored at a protein concentration of 1 mg/ml in 0.25 M potassium phosphate buffer (pH 7) containing 1 mM EDTA and 1 mM DTT. The extent of dissociation was determined at var- ious intervals by sucrose density gradient cen- trifugation.
With enzymes irom both species, however, dissociation was more rapid at 0°C than at 20°C.
Immunodijksion Studies
In a preliminary survey, carried out with partially purified preparations [purified to the calcium phosphate gel stage (9)], the cross-reactivity of fatty acid synthetases of liver and mammary gland enzymes was studied (Fig. 2). It can be seen that neither the pigeon liver nor the rabbit mammary gland enzymes showed any reaction in the immunodiffusion test. The rat and mouse liver and mammary gland enzymes gave precipitin lines with some evidence of spur formation. In a more detailed study, the cross-reactivity of a number of homogenous rat and mouse FAS preparations was com- pared (Fig. 3). In Fig. 3A, it can be seen that a reaction of complete identity is ob-
FATTY ACID SYNTHETASES 755
b D
FIG. 3. Ouchterlony immunodiffusion analyses of homogeneous FAS’s. Center wells contained 0.32 mg anti-(rat mammary gland)FAS r-globulin, outer wells contained 45 pg FAS. The antibodies used in this study were prepared from a different rabbit than those used in the experiments de- scribed in Fig. 2. Identification as in Fig. 2; (RaA) rat adipose tissue.
t,ained between the rat’ liver, mammary gland, and adipose enzymes. Similarly, re- actions of complete identity are observed with the mouse liver and mammary gland enzymes (Fig. 3B). Figures 3C and 3D demonstrate all the possible combinations of the five homogenous antigens placed in adjacent wells. It can be seen that reac- tions of partial identity are obtained wher- ever FAS’s from different species are pres- ent in adjacent wells. The spur formation is always found to point toward the well containing the mouse enzymes, which are least relat’ed to the parent antigen, the rat mammary gland enzyme. Identical results were obtained when antibodies prepared against t’he rat liver FAS were used in place of those raised against the rat mammary gland enzyme.
Partially purified FAS’s (calcium phos- phate gel stage) from livers of pigeon, mouse, and rat and from mammary glands of rabbit, mouse, and rat were also subjected to immunoelectrophoresis (11). 21’0 precipitin line was obtained with either the pigeon
liver or rabbit mammary gland preparations. The mouse liver, rat liver, mouse mammary gland, and rat mammary gland preparations all gave a single precipitin line at the same dist,ance from the antigen well, indicating that the PAS antigens had about, the same electrophoretic mobility.
Immunoprecipitin Reactions
The immunoprecipitin titration curves for the rat liver, mammary gland, and adipose PAS’s followed very similar profiles (Fig. 4). The titration curves for mouse liver and mammary FAS’s also exhibited similar profiles, reaching equivalence point at a somewhat higher antigen/antibody ratio than in the case of the rat enzymes. All antigen was precipitated up to equiv- alence point as evidened by the absence of FAS activity in the supernatant. At equiv- alence point, the precipitates obtained with the rat enzymes contained 1.6-1.8 pg anti- body/l pg antigen, whereas those obtained with the mouse enzymes contained l&1.1 pg antibody/l Fg ant’igen. If the difference in molecular weights of the rat and mouse enzymes is taken into account, then the composition of the precipitates approxi- mates 0.81 mg antibody/l nmole rat en- zyme and 0.62 mg antibody/l nmole mouse enzyme.
Amino Acid Analysis
A comparison of the previously reported analyses of the amino acid composition of the rat liver (8) and rat mammary gland (9) fatty acid synthetases reveals con- siderable difference in the sulfhydryl con- tent of the enzymes. The analysis of the rat liver enzyme did not include the basic amino acids. In view of the fact that the rat liver and mammary gland enzymes ap- peared to be antigenically identical, it was decided to carry out a more complete anal- ysis of the rat liver FAS amino acids. The results are presented in Table III; for com- parison, previously published data for the rat mammary and pigeon liver enzymes are included. When the compositions are compared on a mole% basis, the differences in composition of the rat liver and mam- mary gland enzymes were very small and within the experimental error of the pro-
756 SMITH
120 I I I I I
I I 40 60
I a0
i 100
FAS protein added, ,xg
FIG. 4. Immunoprecipitin reactions of various FAS’s. (O---O) RaL, (O---O) RaM, (t--m) RaA, (O--O) MoL, (a- - -0) MOM. Identification as in Figs. 2 and 3. The same preparation of antibodies was used in experiments reported in Figs. 3 and 4; each tube contained 1 mg anti-(rat, mam- mary gland) FAS r-globulin.
cedure. In our hands, the reproducibility of the analytical procedure, including per- formate oxidation, hydrolysis, and chro- matography, was no better than f 5%. The differences in percent composition of the pigeon liver FAS, compared to the rat enzymes, were greater than the limits of experimental error in the case of several amino acids.
DISCUSSION
Immunochemical procedures have been used successfully to demonstrate structural differences between fructose-l, 6-diphospha- tases from different tissues of the rabbit (17) and to demonstrate similarities be- tween pyruvate carboxylases from dif- ferent tissues of the rat (30). A correlation between cross-reactivity, measured by the immunoprecipitin technique, and amino acid sequence has been demonstrated for a series of lysozymes (31). Furthermore, t’he degree of compositional difference can often provide a rough guide to the number of sequence differences (32-34).
It would appear that the liver and mam- mary gland FAS enzymes of the rat have identical amino acid compositions, but differ slightly from the pigeon liver enzyme. With proteins such as the FAS’s, that con-
tain several thousand amino acid residues, small compositional differences probably correspond to a large number of sequential differences. The immunochemical approach is invaluable, as it provides an amplification of the differences. Thus, the pigeon liver en- zyme, which shows only small compositional differences from the rat enzymes, is anti- genitally quite distinct; it does not cross- react with the rat’ enzymes. Furthermore, the immunochemical procedures were ca- pable of revealing structural differences be- tween more closely related species, the rat and mouse. The techniques did not, how- ever, reveal any differences in cross-reac- t.ivit,y of FAS’s from different tissues of the same species. In addition to being anti- genitally identical, the liver and mammary gland FAS’s of a particular species have identical molecular weights and dissociate into their component, subunits at comparable rates. However, when the comparison is made between FAS enzymes of different species (rat and mouse), differences are ob- served with all these parameters. Based on these results, it would seem that, within a given species, the FAS’s of liver and mam- mary gland are very similar, if not identical proteins. It has recently been demonstrated that the fatty acid synthetases purified from
a Mean of three analyses on the same enzyme preparation, except for cysteine value which is the mean of two determinations on the same en- zyme preparation by amino acid analysis and two determinations on different enzyme preparations by the 5,5’-dithiobis(2-nitrobenzoate) procedure.
b Taken from Smith and Abraham (9). c Taken from Hsu el al. (5).
the livers of normal, fasted and fasted-refed rats are also antigenically indistinguishable (35, 36).
Immunochemical procedures have been used in this study to demonstrate structural differences in the FAS’s of two closely re- lated members (rat and mouse) of the myo- morph suborder of rodents, a member of the related lagomorph order (rabbit), and an avian species (pigeon). These differences presumably result from amino acid replace- ments which have taken place during evolu- tion. The immunochemical approach to es- t’ablishing structural and evolutionary rela- tionships between enzyme homologues may prove to be particularly advantageous in the case of large molecular weight proteins, where physicochemical techniques such as sequencing would be impracticable.
The conclusion that, within a given spe- cies, the liver and mammary gland FAS’s are probably identical proteins, has some sig- nificance in terms of the function of these enzymes in their respective tissues. The mammary gland and liver FAS’s have rather different physiological roles in most mam- maIs, the former being responsible for syn- thesis of medium chain fatty acids and the latter for long chain (11). A number of workers have studied the properties of vari- ous mammary gland FAS’s, in the hope of revealing some unique structural properties which might have some functional signifi- cance (9, 10, 37, 38). Evidence is accumu- lating which points to some other factor being involved in regulation of chain-termi- nation in fatty acid synthesis (39).
ACKNOWLEDGMENTS
I am extremely grateful to Pauline Sweet for her expert assistance in the immunological study, to Bert Martin for performing bhe amino acid analyses, and to Ruthe Wayner of the Department of Biochemistry, University of California, Berke- ley, for carrying out the analytical ultracentrifuge studies. I thank Dr. S. Abraham for his interest and encouragement.
REFERENCES
1. SMITH, S. (1972) Abstr. Commun. Meet. Fed. Eur. Biochem. Sec. 8, 1124.
2. KUMAR, S., PHILLIPS, G. T., AND PORTER, J.
W. (1972) Znt. J. Biochem. 3, 15-32. 3. LYNEN, F. (1961) Fed. Proc. Fed. Amer. Sot.
