THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 2, Issue of January 25, pp. 880-886, 1982 Printed in U.S.A.

Purification and Characterization of S-Acyl Fatty Acid Synthase Thioester Hydrolase which Modifies the Product Specificity of Fatty Acid Synthase in the Uropygial Gland of Mallard*

(Received for publication, June 22, 1981, and in revised form, September 24, 1981)

Linda Rogers, P. E. Kolattukudy, and Mertxe deRenobales From the Znstitute of Biological Chemistry and Biochemistry/Biophysics Program, Washington State University, Pullman, Washington 99164

A thioesterase present in the uropygial gland of mal- lard functions in conjunction with fatty acid synthase to produce short chain acids in this gland (deRenobales, M., Rogers, L., and Kolattukudy, P. E. (1980) Arch. Biochem. Biophys. 205,464-477). This enzyme, capable of releasing short chain acids from fatty acid synthase, was purified to near homogeneity. This S-acyl fatty acid synthase thioester hydrolase is a monomer with Mr = 29,500 and its amino acid composition is signifi- cantly different from those of other thioesterases. It catalyzes hydrolysis of CoA esters maximally at pH 6.5-8.0 and it regenerates the overall activity of phen- ylmethanesulfonyl fluoride-treated synthase. With CoA ester substrates, this enzyme showed preference for Clz and Cl0 whereas Ce, Cs, C14, C16, and C18 showed 30-50% of the rate obtained with Clz. This specificity was significantly different from that of two other thioesterases from the same gland. The acyl fatty acid synthase hydrolase was inhibited by reagents directed against active serine and histidine, strongly suggesting that it is a serine esterase. This enzyme was also highly sensitive to thiol-directed reagents. The acyl fatty acid synthase hydrolase activity, but not the CoA esterase activity, was inhibited by high ionic strength. This hydrolase was nearly as effective in regenerating the overall activity of phenylmethanesulfonyl fluoride- treated fatty acid synthase from rat mammary gland as a similar hydrolase obtained from the mammary gland. On the other hand, with the avian fatty acid synthase, the avian hydrolase was 20-30 times more efficient than the hydrolase from the rat mammary gland. The occurrence of ample amounts of acyl fatty acid syn- thase hydrolase specifically in the glands which pro- duce short chain acids strongly suggests that this hy- drolase is involved in the production of the short chain acids in these specialized glands.

Fatty acid synthase from vertebrates generates palmitic acid from acetyl-coA and malonyl-CoA (1). The chain length of the product is consistent with the chain length specificity of the chain terminating thioesterase, which is a segment of the multifunctional fatty acid synthase. Specialized tissues such as mammary glands and uropygial glands of certain waterfowl generate short chain acids (2 ,3) . Evidence has been

* This work was supported in part by Grant GM-18278 from the United States Public Health Service. This is Scientific Paper 5960, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, WA 99164. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

presented that in such cases an ancillary thioesterase hydro- lytically releases the growing acyl chains from the synthase and such a thioesterase has been purified from the mammary glands of rats (4) and rabbits (5). In the uropygial gland of mallard, two thioesterases (A and B) other than that con- tained in fatty acid synthase were found and only thioesterase B was found to be capable of hydrolyzing acyl fatty acid synthase (6). In this paper we report that the acyl fatty acid synthase hydrolase activity which hydrolytically releases acyl chains from the synthase can be resolved from thioesterase B. Purification of this enzyme to homogeneity and the charac- teristics of this enzyme are also described. Although thioes- terases are widely distributed in animal tissues (7-lo), the present enzyme is only the second case of an enzyme which can use acyl fatty acid synthase as a substrate and thus modify the product specificity of fatty acid synthase. This class of enzyme will be referred to as S-acyl fatty acid synthase thioester hydrolase (acyl fatty acid synthase hydrolase) to distinguish such enzymes from the more widespread thioes- terases which hydrolyze CoA esters. The present results show that such modification of the product specificity of fatty acid synthase is not unique to mammary glands as previously thought.

MATERIALS AND METHODS’

RESULTS

Resolution of Thioesterase B a n d S-Acyl Fatty Acid Syn- thase Thioester Hydrolase-Since thioesterase B from the mallard uropygial gland is only the second case of an enzyme which can hydrolytically remove the acyl chains from fatty acid synthase, we attempted to purify this enzyme. The gel filtration of the protein fraction containing both thioesterase B (CoA ester hydrolase) and acyl fatty acid synthase hydro- lase on a Sephadex G-100 column showed that the bulk of the CoA ester hydrolase activity was resolved from the protein fraction which caused chain shortening and regeneration of the overall activity of PMSF2-treated fatty acid synthase. The latter two activities were associated with the same protein

’ Portions of this paper (including “Materials and Methods,” Figs. 1, 3, and 4, and Table I) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biolog- ical Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 81M-1484, cite authors, and include a check or money order for $2.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

* The abbreviations used are: PMSF, phenylmethanesulfonylfluo- ride; DTE, dithioerythritol; FAS, fatty acid synthase; TE 11, thioes- terase I1 from rat mammary gland; DTNB, 5,5-dithio-bis(2-nitroben- zoic acid); BSA, bovine serum albumin; SDS, sodium dodecyl sulfate.

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Acyl Fatty Acid Synthase Hydrolase 88 1

fraction. Addition of the fraction containing the highest CoA esterase activity to the fatty acid synthase did not cause the formation of detectable amounts of fatty acids shorter than CI6 whereas the fraction containing high activity for reversing the PMSF inhibition caused production of Cl0, Cl?, and CI4 fatty acids from malonyl-CoA by fatty acid synthase (data not shown). The latter fraction also caused the production of short chain branched acids from methylmalonyl-CoA by fatty acid synthase. These results clearly showed that thioesterase B and acyl fatty acid synthase hydrolase are distinctly different proteins.

Purification of Acyl Fatty Acid Synthase Hydrolase-The protein fraction containing both CoA ester hydrolase and acyl fatty acid synthase activities obtained from the Sepharose 6B was subjected to ion-exchange chromatography on DEAE- Sephacel. A large portion of protein containing CoA ester hydrolase activity did not bind to the column; however, this protein did not have any acyl fatty acid synthase hydrolase activity. Since high ionic strength tended to inactivate the acyl fatty acid synthase hydrolase activity, a pH gradient was used to elute the proteins. Acyl fatty acid synthase hydrolase, which emerged at pH 5.0, was clearly resolved from the major CoA ester hydrolases and this protein fraction had a low CoA ester hydrolase activity (Fig. 1). SDS gel electrophoresis showed a major band a t about 30 kilodaltons and numerous minor bands on either side. When this protein fraction was applied to a CM-Sephadex column at pH 5.6, a protein fraction was not retained and this protein had no acyl fatty acid synthase hydrolase activity. Upon application of a pH gra- dient, the acyl fatty acid synthase hydrolase activity was eluted (Fig. 1). SDS gel electrophoresis of this protein showed one major band at 30 kilodaltons and eight minor bands all representing proteins larger than 30 kilodaltons. When this protein fraction was subjected to gel filtration on a Sephadex G-75 column, two major protein fractions were observed and the acyl fatty acid synthase hydrolase activity was contained in one of them; the protein peak representing higher molecular weight did not show any enzymic activity. Since the enzyme activity profile coincided with the protein profile of the second peak, the enzyme appeared to be near homogeneity. SDS gel electrophoresis showed only one major band, strongly sug- gesting that the acyl fatty acid synthase hydrolase was near homogeneity. A typical purification result is shown in Table I.

Molecular Weight a n d Amino Acid Composition-SDS gel electrophoresis with proteins of known molecular weight showed that acyl fatty acid synthase hydrolase from the uropygial gland of mallard had M, = 29,500 k 760 whereas under the same conditions the thioesterase I1 from the mam- mary gland of rat showed M , = 32,500 (Fig. 2). In Table 11, amino acid composition of the acyl fatty acid synthase hydro- lase is compared with that of the thioesterase segment of fatty acid synthase and that of thioesterase I1 from rat mammary glands. All three showed comparable amounts of most amino acids. The thioesterase segment from fatty acid synthase showed a much higher content of alanine, methionine, and tyrosine and a lower amount of phenylalanine than the present enzyme (14). Thioesterase I1 of rat mammary gland showed a much larger content of alanine and methionine than the present enzyme (4).

Catalytic Properties-The rate of hydrolysis of dodecanoyl- CoA increased in a rectilinear manner with increasing protein concentration from 2 to 8 pg of protein but below 2 pg the substrate apparently inhibited the enzyme. Addition of bovine serum albumin relieved this inhibition and under these con- ditions linear increase of the rate of hydrolysis was observed up to 4 pg of protein (Fig. 3A). Regeneration of the overall

7 -

6 -

5 -

I- 4 - I 12

c - s 3-

2 2- LL J a

W -1 0 I

I I I I 0 0.2 0.4 0.6

RELATIVE MIGRATION

FIG. 2. Molecular weight determination of purified acyl fatty acid synthase hydrolase. A, SDS gel electrophoresis of purified acyl fatty acid synthase hydrolase from the uropygial glands of mallards. B, molecular weight determination of purified duck uropy- gial gland acyl fatty acid synthase hydrolase (DUG-hydrolase) and partially purified rat mammary gland thioesterase I1 (RMG ?‘E In by SDS electrophoresis. Enzyme purifications and electrophoresis were done as described in the text.

TABLE I1 Antino acid composition of the acyl fatty acid synthase hydrolase

from the mallard uropygial gland, the thioesterase segment of goose uropygial gland fatty acid synthase, and rat mamntap

gland thioesterase I1 __-__ ”~___

Goose fatty acid Acyl fatty acid synthaw” Rat mammary

acid synthase hydrolase thioesterase gland”1’E I1 segment

resiclucs/rnol enzvrnc _ _ ~ ~ .

Asx 24 26 28 Thr 11 14 13 Ser 18 21 I7 Clx 29 32 27 Pro 15 13 17 Cly I7 22 19 Ala 14 29 23

Val 14 10 14 Met 1 4 5 Ile 20 15 18 Leu 30 34 36

I’he 18 8 18

His 12 10 10 1 1 10 a

cys 4 6 3

TYr (i 1 1 6

Lys 17 15 21

TrP N.D.‘ 4 3

” From Kef. 14. From Kef. 4. ‘ N.D., not determined.

activity of PMSF-treated fatty acid synthase showed a recti- linear relationship with the amount of acyl fatty acid synthase hydrolase added up to 4 pg under the present assay conditions (Fig. 3B). The rate of hydrolysis of dodecanoyl-CoA was linear with time up to 10 min of incubation and the recovery of the activity of PMSF-treated fatty acid synthase also showed linear rates for 5-10 min. Hydrolysis of dodecanoyl-CoA by acyl fatty acid synthase hydrolase showed a rather broad pH

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882 Acyl Fatty Acid Synthase Hydrolase

optimum giving maximal activity between pH 6.5 and 8.5; further increase in pH resulted in a decline in the activity (Fig. 4). This pH profde was in contrast to the profiies shown by thioesterases A and B from the uropygial gland of mallard; the former showed maximal activity between 7 and 9 whereas the latter showed maximal activity at around pH 8.5 (Fig. 4). Increasing substrate (dodecanoyl-CoA) concentration resulted in increasing rates up to about 40 p~ and subsequent increases began to show saturation, but with much higher substrate concentration (>80 p ~ ) inhibition became a problem. With low protein concentrations, bovine serum albumin had to be used to get maximal activity. Under such conditions, a typical substrate saturation pattern was observed and from linear double reciprocal plots, a K, of 80 p~ was calculated for dodecanoyl-CoA. However, in complex reaction mixtures con- taining bovine serum albumin which competes for binding of CoA esters, K, does not have the normal meaning. Operation- ally for routine assays, 80 p~ dodecanoyl-CoA was used to avoid substrate inhibition and serum albumin was avoided whenever possible. For determining substrate specificity, the substrate coccentration was optimized for maximal activity.

Inhibitors-Acyl fatty acid synthase hydrolase was severely inhibited by thiol-directed reagents, N-ethylmaleimide andp- hydroxymercuribenzoate; only about 5 p~ concentration was sufficient to achieve 50% inhibition (Table 111). Because of this stringent requirement for SH group(s), the presence of SH compounds in the medium was necessary to maintain maximal activity of the enzyme. Diisopropylfluorophosphate, a commonly used active serine-directed reagent, was not in- hibitory even after treatment with 5 mM concentration for 30 min. A similar enzyme from the rat mammary gland showed a half-life of 260 min in the presence of 5 mM diisopropylfluo- rophosphate (4). However, phenylmethanesulfonyl fluoride, an organic phosphate (fospirate) and an organic phosphoro- thioate (chlorpyrifos) severely inhibited the enzyme at fairly low concentrations, giving 50% inhibition at 10-50 p ~ . There- fore, it appears clear that this enzyme has an active serine a t its active site. In support of this conclusion, diethylpyrocar- bonate, a relatively specific reagent for histidine, severely inhibited this hydrolase. Thus, the catalytic mechanism of

TABLE I11 Inhibitors of the thioesterase activity of acyl fatty acid synthase

hydrolase from the uropygial gland of mallard Thioesterase activity was measured radiochemically as described

in the text, except that prior to addition of substrate, the remainder of reaction mixture was incubated for 30 min at 30 "C.

Inhibitor Concentration Inhibition B

N-Ethylmaleimide ( p ~ ) 1 16 5 46

10 65 50 90

p-Hydroxymercuribenzoate ( p ~ ) 5 31 10 95

Diethylpyrocarbonate (ELM) 50 54 100 95

Diisopropylfluorophosphate (mM) 1 0 2 0 5 0

Phenylmethanesulfonyl fluoride 0.05 47 (mM) 0.1 80

0.5 96 O,O-Dimethyl-O-(3,5,6-trichloro-2- 0.01 87

pyridy1)-phosphate (fospirate) 0.1 100 (KIM)

0,O-Diethyl-O-(3,5,6-trichloro-2- 0.01 38 pyridy1)-phosphorothioate (chlor- 0.1 95 pyrifos) (mM)

this acyl fatty acid synthase hydrolase is quite similar to that used by the thioesterase segment of fatty acid synthase.

Substrate Specificity-Since acyl fatty acid synthase with defined acyl chains cannot be prepared for determination of the chain length specificity for the acyl fatty acid synthase hydrolase activity, CoA esters were used as model substrates (Fig. 5). Dodecanoyl-CoA and decanoyl-CoA were hydrolyzed most rapidly while other fatty acyl-CoA from Cg to CIS showed similar rates (30-50% of that obtained with Clz). Thioesterase B from the uropygial gland showed a somewhat similar sub- strate specificity; this enzyme catalyzed decanoyl-CoA most rapidly and longer and shorter CoA esters showed progres- sively lower rates. In contrast, thioesterase A showed very little preference for the chain length of acyl-CoA except that somewhat lower rates were observed with the shorter CoA esters.

Differential Effects of Ionic Strength on the Hydrolysis of CoA Esters and Acyl Fatty Acid Synthase by Acyl Fatty Acid Synthase Hydrolase-With the preparations containing both

300

200

IO0

h

TE-A

ACYL- FAS-

30

15

6 8 IO 12 14 16 18

I

I

CHAIN LENGTH FIG. 5. Chain length specificity for the hydrolysis of CoA

esters by thioesterase A (TE-A), thioesterase B (TE-E) and acyl fatty acid synthase (FA@ hydrolase. Substrate saturation patterns were determined for each CoA ester with and without 1 mg/ ml BSA; maximal rates for each substrate are shown. Assays were done spectrophotometrically. Purified acyl fatty acid synthase hydro- lase and partially purified thioesterases were used.

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Acyl Fat ty Acid Synthase Hydrolase 883

thioesterase B and acyl fatty acid synthase hydrolase, high ionic strength inhibited only the latter but not the CoA esterase activity. Because of the presence of thioesterase B, it was not possible to determine whether the two activities of acyl-FAS hydrolase were differentially affected by ionic strength. Therefore, the effect of ionic strength on purified acyl fatty acid synthase hydrolase was determined (Fig. 6). Regeneration of the overall activity of PMSF-treated synthase was severely inhibited even at 0.1 M KC1 whereas CoA ester hydrolase activity was not affected by this ionic strength (Fig. 6); even 0.3 M KC1 had no effect on the thioesterase activity whereas the S-acyl fatty acid synthase thioester hydrolase activity was more than 90% inhibited (data not shown). The rat mammary gland enzyme was similarly affected by the ionic strength (Fig. 6). Since fatty acid synthesis is not affected by this ionic strength, it is highly likely that the interaction between the synthase and acyl fatty acid synthase hydrolase was inhibited by high ionic strength.

Specificity of Acyl Fatty Acid Synthase Hydrolases for Acyl Fatty Acid Synthase from Rat and Auian Sources-To test whether acyl fatty acid synthase hydrolase from the avian source can use acyl fatty acid synthase from mammalian sources, PMSF-treated fatty acid synthase from rat mammary gland was used as the substrate for the avian hydrolase (Fig. 7 ) . The avian hydrolase regenerated the activity of the PMSF- treated synthase from the rat. Direct comparison between the

)UG-HYDROLASE

"A

01 0 2

KC1 (MI FIG. 6. Effect of ionic strength on the regeneration of overall

activity of PMSF-treated fatty acid synthases. Effect on PMSF- treated goose fatty acid synthase (0) by the purified S-acyl fatty acid synthase thioester hydrolase from the duck uropygial gland (DUG- HYDROLASE) and rat mammary gland (RMG TE II) and on the dodecanoyl-CoA hydrolase activity (A) of the acyl fatty acid synthase hydrolases. Synthase activity was assayed spectrophotometrically. Control 100% activity was 2.0 m o l of NADPH oxidized/min for acyl fatty acid synthase hydrolase activity and 2 nmol of decanoyl-CoA hydrolyzed/min for CoA hydrolase activity.

acyl fatty acid synthase hydrolase (TE 11) from the rat mam- mary gland and that from the uropygial gland of mallard was not possible because each had a different specific activity for CoA ester hydrolysis. However, based on equal CoA ester hydrolase activity, the two acyl fatty acid synthase hydrolases were almost equal in their effectiveness in regenerating the overall activity of the PMSF-treated mammalian fatty acid synthase. On the basis of the amount of protein used, the mammalian hydrolase was somewhat more efficient than the avian hydrolase in using the mammalian acyl fatty acid syn- thase as substrate. In any case, the avian enzyme was remark- ably efficient in its ability to function with the mammalian synthase.

A comparison between the ability of the two acyl fatty acid synthase hydrolases to regenerate the overall activity of PMSF-treated avian fatty acid synthase revealed significant differences. At very low levels of the hydrolase, the avian hydrolase was much more effective than the mammalian hydrolase (Fig. 7). The mammalian enzyme was, however, capable of hydrolyzing avian acyl fatty acid synthase but this activity required substantially more mammalian hydrolase than the avian hydrolase. The overall activity of PMSF- treated avian synthase recovered by 2.9 pg of the avian hydro- lase was equal to that recovered by 84 pg of the mammalian hydrolase. Even if one takes into account the small difference in the degree of purity of the preparations used. it is clear that the avian enzyme was far more active than the mammalian hydrolase in regenerating the activity of PMSF-treated avian synthase. Comparison of the effectiveness of the two hydro- lases on the basis of equal amounts of CoA ester hydrolase activity also clearly showed that the avian hydrolase was far more efficient than the mammalian hydrolase in hydrolyzing avian acyl fatty acid synthase.

DISCUSSION

The acyl fatty acid synthase hydrolase isolated from the uropygial glands of mallards is only the second case of a thioester hydrolase which functions in conjunction with fatty acid synthase to bring about alteration in the products gen- erated by the synthase. Both in the mammary glands and in the uropygial glands of ducks, this relatively small hydrolase replaces the resident thioesterase of the synthetase and thus the short acyl chains are released. Unlike the present acyl fatty acid synthase hydrolase, the thioesterase segment of the synthase, once cleaved from the synthase, is not capable of recovering the overall activity of PMSF-treated synthase. Therefore, it is apparent that the noncovalent interaction of the present hydrolase with the synthase is more efficient than that of the thioesterase segment of .the synthase which ob- viously depends on its covalent attachment for its function. This apparent competitive advantage of the acyl fatty acid

RMG-FAS - 0.05 - GOOSE- FAS

RMG TEII DUG-HYDROLASE

RMG T E E

DUG - HYDROLASE

20

THIOESTERASE ACTIVITY (nmoleslmin 1 FIG. 7. Functional compatibility of the avian and mamma- rat mammary gland (RMG) fatty acid synthase and PMSF-treated

lian fatty acid synthases and acyl fatty acid synthase hydro- goose uropygial gland fatty acid synthase. Assays were done spectro- lases. Effectiveness of acyl fatty acid synthase hydrolases isolated photometrically as described in the text, except that 60 pg of PMSF- from duck uropygial gland (DUG-HYDROLASE) and fat mammary treated synthase was used. The DEAE-purified acyl fatty acid syn- gland (RMG TE In in restoring the overall activity of PMSF-treated thase hydrolase from mallard was used in these experiments.

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884 Acyl Fatty Acid Synthase Hydrolase

synthase hydrolase over the resident thioesterase of the syn- thase allows the small protein to have access to the short acyl chains that are formed on the synthase. The thioesterase domain in the synthase from the uropygial gland, as in the synthase from other vertebrates (15, 16), is linked to the rest of the synthase by a flexible region as deduced from the finding that proteases such as trypsin, elastase, and clostripain release the thioesterase domain (14).3 Attempts to obtain the other domains by proteolysis have resulted in only limited success, presumably because the other domains of the syn- thase are more tightly organized without ready access to proteolytic enzymes. These observations suggest that the thioesterase domain, attached to the synthase via a flexible stretch of peptide, can be readily displaced by the acyl fatty acid synthase hydrolase. Generation of the overall activity of PMSF-treated synthase by the acyl fatty acid synthase hy- drolase was inhibited by moderately high ionic strengths which did not inhibit either fatty acid synthase or the CoA ester hydrolyzing activity of the acyl fatty acid synthase hydrolase from the duck uropygial gland and rat mammary gland. Therefore, it appears likely that such ionic strengths interfered with the ionic interaction between the synthase and the hydrolase in both the avian and mammalian systems.

The mechanism of acyl chain release by the acyl fatty acid synthase hydrolase most probably involves active serine as indicated by the severe inhibition by PMSF, an organic phos- phate and an organic phosphorothioate. This enzyme was relatively insensitive to diisopropylfluorophosphate just as the mammary gland hydrolase (4). Inhibition by diethylpyrocar- bonate gave supporting evidence for the presence of the charge relay system characteristic of serine esterases. Whether the present hydrolase releases pantetheine-bound or cysteine- bound acyl groups is not known. Since scavenging of free CoA by ATP:citrate lyase did not affect removal of acyl chains from the synthase: it is highly unlikely that release of fatty acids by this hydrolase involves a CoA ester intermediate.

There is ample evidence that the present acyl fatty acid synthase hydrolase is involved in the production of the short chain acids in vivo in the gland. This enzyme is present at measurable levels only in the gland of mallard but not in the uropygial gland of goose which does not generate such acids (6). We have isolated a similar enzyme from the uropygial gland of Peking duck which also generates short chain acids5 Even though it is difficult to accurately determine the amount of the hydrolase present in the gland, it is estimated that there is approximately 1 mol of hydrolase/mol of the synthase peptide (250 kilodaltons) in the uropygial gland of mallard. In vitro, the amount of acyl fatty acid synthase hydrolase re- quired to bring about near maximal regeneration of the activ- ity of PMSF-treated synthase indicated that only 1 mol of hydrolase was required/mol of synthase peptide (250 kilodal- tons). This observation suggests a rather strong interaction between the hydrolase and the synthase in contrast to the results obtained with a similar hydrolase from rat mammary glands (4). In support of this conclusion was the finding that the hydrolase from the mallard uropygial gland was nearly as effective as the hydrolase from the rat mammary gland in releasing acyl chains from the synthase from the rat whereas the mallard hydrolase was far more effective with avian fatty acid synthase.

The discovery of thioesterase I1 which could prematurely release acyl chains from acyl fatty acid synthase in the mam- mary gland suggested the possibility that this acyl fatty acid

A. J. Poulose and P. E. Kolattukudy, unpublished results. A. J. Poulose, L. Rogers, and P. E. Kolattukudy, unpublished

L. Rogers and P. E. Kolattukudy, unpublished results. results.

synthase hydrolase is the product of advanced evolution, generated to provide short chain acids in the milk and this mechanism of chain length modification was suggested to be unique to mammary glands (4). However, with the discovery of a somewhat similar acyl fatty acid synthase hydrolase in the uropygial gland of the mallard, it is clear that this basic mechanism of production of short chain acids is not unique to mammals. The present observation that the two acyl fatty acid synthase hydrolases from the avian and mammalian sources can function in conjunction with the synthases from both sources and the result that the hydrolases from both sources are similarly affected by ionic strength suggest that much of the basic mechanisms involved in the interaction between the hydrolase and synthase were conserved.

Thioesterases which catalyze hydrolysis of CoA esters are widely distributed in animals (7-lo), plants (17, 18), and microbes (19-21). However, the function of such thioesterases is not clear. In the uropygial glands of mallards, there appears to be at least four different thioesterases: the thioesterase segment of fatty acid synthase (6), the present scyl fatty acid synthase hydrolase, thioesterase A, and thioesterase B. The function of the fist two appears to be clear whereas the function of the other two remains obscure. Each thioesterase appears to have different size and substrate specificity. Prelim- inary studies indicate that all four use similar catalytic mech- anisms. The present acyl fatty acid synthase hydrolase at a wide range of concentrations did not show a precipitin line with rabbit antibodies prepared (14) against the isolated thioesterase domain of fatty acid synthase and the thioester- ase activity was not affected by the antibody preparation. Thus, the present enzyme is immunologically quite different from the thioesterase segment of the synthase. More infor- mation on the structure of the four thioesterases is required before a meaningful comparison can be made to determine whether these thioesterases arose from the same ancestral gene.

Biochemical studies on specialized glands such as the mam- mary gland and the uropygial gland reveal the strategy used in the production of unusual metabolic products. A single new protein, acyl fatty acid synthase hydrolase, allows these spe- cialized tissues to generate short chain fatty acids using the catalytic activity of the synthase. Malonyl-CoA decarboxylase in the uropygial gland of goose allows this gland to generate multiple methyl branched acids utilizing acetyl-coA carbox- ylase and fatty acid synthase, two highly evolved complex enzymes present in all tissues (22-24). This strategy of using a minimum number of additional proteins to drastically alter the nature of products generated by the highly evolved com- mon enzymes would provide an evolutionary advantage. How the expression of genes coding for these ancillary enzymes is controlled during differentiation is not known.

Acknowledgments-We thank Dr. Gurusiddaiah for assistance in the amino acid analysis, Cynthia McGaffey for technical assistance, and Richard Hamlin for maintaining the birds.

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Waxes (Kolattukudy, P. E., ed) pp. 94-141. Elsevier, New York 4. Libertini, L. J., and Smith, S. (1978) J. Biol. Chem. 253, 1393-

1401 5. Knudsen, J., Clark, S., and Dils, R. (1973) Biochem. J. 160, 683-

691 6. deRenobales, M., Rogers, L., and Kolattukudy, P. E. (1980) Arch.

Biochem. Biophys. 205,464-477 7. Knauer, T. E. (1979) Biochern. J. 179,515-523 8. Lee, K. Y., and Schulz, H. (1979) J. Bwl. Chem. 254,4516-4523

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9. Berge, R. K., and Farstad, M. (1979) Eur. J. Biochem. 96, 393-

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13. Hirs, C. H. W. (1967) Methods Enzymol. 11, 197-199 14. Bedord, C. J., Kolattukudy, P. E., and Rogers, L. (1978) Arch.

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Proc. Natl. Acad. Sci. U. S. A. 73, 1184-1188

Supplemental Materlal TO:

PURIFICATION AND CHARACTERIZATION OF S-ACYL FATTY SYNTHASE THIOESTER NYDROLASE WHICH MODIFIES THE PRODUCT SPECIFICITY OF FATTY ACID

SYNTHASE IN THE UROPYGIAL GLAND OF MALLARD

by

Llnda Rogers, P . E . XolattukUdy and Mertxe deRenObaleS

MATERIALS AND METHODS

MATERIALS

Mallard ducks IAna5 lac rhynchosl vere purchased from the Ted-Mar

a hlqh-energy breeder ratron. The uropygial glands of domesrlc whlte geese llcensed game farm, ~i~up,"Wa.sh~ngton. and maintained I" outdoor cages on

(a 5. domestlcu~l rarsed by the Hutterlan Brethren, M a r l m , Washmgton, were exclsed immediately after klllmg the blrd. frozen ~n llquld N2 and

Anlmdl R E S O U ~ C ~ Center, Washington State Unlverslty. Sepharose 18 and 68, Stored at -80 ' . Lactating female rats were Supplled by the Laboratory

Cod derIvatlves, phenylmerhanesulfonyl fluoride, and 5,5'-dlthlo-bls- DEAE-Sephacel. CM-Sephadex, G-75 and G-100 Sephadex. dlthloerythr-ltol, acyl-

12-nlt10benzol~ acldl were purchased from Slqma Chemcal Company. Low molecular welght standard proteln mlxture was purchased from BloRad

malonyl-Cal and Ornnlfluor were from New England Nuclear. Laboratorles. Richmond, Callfarnla. [2-"Clmalonyl-CaA, [methyl-"Clmethyl-

RESOLUTION OF KYL-FAS HYDROLASE FROM THIOESTERASE 8

Birds were kllled by exiangulnatlon and the glands were excised. After removing the adherlnq tlssue and fat, the gland tlsSUe was hornagenlred ~n 0.1 M sodlum phosphate buffer, pH 7.0, contalnlng 1.0 ml4 DIE and 0.25 M sucrose, (1.25 ml buffei1qlandl. The membranes were removed from the

m l n a t 105,000 g : each t m e the pellet was dlscarded. The flnal 105.000 g homogenate by cenrrifugatlon tvlce for 20 m l n at 27,000 9, and twlce for 90

vlth 0.1 W sodlum phosphate buffer, pH 7.0. contalnlng 1.0 mM EDTA and supernatant was applled to a Sepharase 6B column 13 x 90 Cm) equlllbrated

0.5 rn DTE and eluted at a flow rate of 0.4-0.5 mllmln vlth the same buffer; 15 mln fracrlons were collected. Fractions were assayed spectrophata- metrzcaliy for fatty acid synthase actlvity and acyl -FA5 hydrolase actlvlty as lndxcared below. The thxoesterase actlvlty was assayed radlochemlcally. Fracrlons contalnlng rhloesterase and acyl-FAS hydrolase actlvltles were

Bwchem. Biophys. 189,382-391 18. Joyard, J., and Stumpf, P. K. (1980) Plant Physiol. 65, 1039-1043 19. Barnes, E. M., Jr., and WaM, S. J. (1968) J. Biol. Chem. 243,

20. Bonner, W. M., and Bloch, K. (1972) J. Biol. Chem. 247, 3123-

21. Spencer, A. K., Greenspan, A. D., and Cronan, J. E., Jr. (1978) J.

22. Buckner, J. S., Kolattukudy, P. E., and Rogers, L. (1978) Arch.

23. Kim, Y. S., and Kolattukudy, P. E. (1978) Arch. Biochem. Bio-

24. Buckner, J. S., and Kolattukudy, P. E. (1976) in Chemistry and Biochemistry of Natural Waxes (Kolattukudy, P. E., ed) pp 148-184, Elsevier, New York

2955-2962

3133

Biol. Chem. 253,5922-5926

Biochem. Biophys. 186,152-163

PhyS. 190,585-597

PURIFICATION OF ACYL-FAS HYDROLASE FROM THE UROPYGIAL GLANDS OF MLLIRDS

The concentrated rhlaesterase-contalnIng fractlon obtalned from the Sepharose 68 gel fxltratlan Step described above was diluted 1:5 with water contalnlng 0.5 mM DIE and reconcentrated. 5 0 that the flnal buffer rnedlvm contalned 0.02 M sodlurn phosphate, 0.5 mM DTE and 0.2 mM EDTA. The flnal volume Was less than 113 the bed volume of the OCAE column used I" the next Step. A DEAE-Sephacel column 15 m l bed vollqland) Was rqulllbrated ~n

centrated protein from the 68 qel flltratlon Was applled to the DERE columrl 0.02 M sodium phosphate buffer, pH 6.0, contalnlnq 0.5 mI4 DTE. The can-

and the column was washed wlth 0.02 M sodlum phosphate buffer. p~ 6.0. con- talnlng 0.5 mM OTE untll the unbound protelns were removed. After the absorbance at 280 nm returned to base llne, the e l u t l o n buffer was changed to 0.1 M mOnobas1c sadxum phosphate contalnlng 0.5 TVI DTE. The column was

were assayed for acyl-FAS hydrolase spP=trophotometrlcally and far thlo- eluted at 0.3 m l l m l n . and 4-5 ml fractlons were collected. The fractlans

These fractions were pooled, the pH was ad3usted to 6 . 8 by the addltlon of esterase radlochemlcally. The former eluted over a pH range of 5.15-4.95.

ultraflltratlon a s before. The concentrate was diluted 1:5 Wlth H 0 con- 0.1 M dlbaslc sodium phosphate contalnlng 0.5 mM DTE and concentrated by

talnlng 0.5 ml4 DTE and concentrated by 'ultraflltratron. The r e s u l ~ ~ n g proteln solution was applled t o a CM-Sephadex column ( 3 mi bed volume1 gland) equllibrared wlth 0.02 n cltrate-sodlum phosphate buffer. pH 5.6, contalnlng 0.5 mM DTE. Sample volume was less than 2 5 % of the column bed volume. After cernovlng the unbound protelnr by washlng vlth the same buffer, a llnear gradlent of 0.02 n cltrate-phosphate. pll 5.6, to 0.1 M 50dlum phosphate, pH 7.0, was applLcd ( 1 3 ml total vol/rnl column bed volumel. The pH remdlned at 5.6 for % 4 0 - 4 5 % of the gradlent volume an& then rose to 7.0. The fractions Contalnlng the acyl-FAS hydrolase a c r l v l t y were pooled and concentrated as before. The concentrated enzyme was appllrd to a Sephadex 6-75 column 11.3 x 90 col equlllbrated vlth 0.1 M sodlurn

vlth the same buffer at 0.25 r n l l m l n COlleCtlng 2 nl fractrons. Al: of the phosphate buffer, pH 7.0, contalnlnq 0.5 mM DTE a?d the proteins were rlutrd

above procedures were done at 4 * .

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886 Acyl Fatty Acid Synthase Hydrolase

0 2 10 '0

x 8

E

0.5 2

0 1

o a w v)

50 70

FRACTION NUMBER

.F~gure I . Typlcal protein and enzyme elution proflles in procedures used

Sepharose 68 g e l flltratlon chromatography of 105.000 g supernatant of gland for the pvrlflcatlon O f acyl-FAS hydrolase. From top to bottom: 68.

wlth acyl-FAS hydrolase actlvlty. DEAE. DEAE-Sephacel chromatography of the homogenate. ThlOeSteraSe actlvity lpattern not shown1 co-chromatographed

protem from the Sepharose 68 step; pH of the eluant 1s Shown by the dashed llne. CM, CM-Sephadex chromatography Of the proteln from the DEAE-Sephadex step: p~ of the eluant 13 shown by the dashed llne. G-75, Sephadex G-73 gel fllrrarlon of the protein from CM-Sephadex step. Enzyme assays are described I" text.

TABLE I

Puizfxcatmn of S-acyl fatty acld synthase thmester hydrolase from duck uropygial glands

Actxvity

Total Protein Total SpeClflC "moles nmoles/mm/

s tep I " 9 l NADPHlmln mg Yield

Sepharose 68 170 DEAE-Sephacel 7.5 CM-Sephadex 2.5 Sephadex G-75 1.1

24900 146 100 4490 598 18 2025 810 1230

8.1 1120 4.9

Glands 127.6 41 from 12 mallards yielded 930 mg proteln I" the 105,000 g

measured mtll the fatty acxd synthase was removed by the Sepharose 68 Step. supernatant but the amount Of acyl-FAS hydrolase actlvlty could not be

ACtrvlty 1s expressed a s the recovery of the overall actlvity of PMSE-treated synthase.

PURI€ICATION OF GOOSE FATTY ACID SYNTHASE

gel filtratlon a5 previously descrlbed (11). The speclflc actlvity and The synthase was purlfled from frozen uropygial glands by Sepharose 48

electropharetlc purlty Of fhls enzyme were comparable to that Isolated from fresh t ~ s u e .

PREPARATION OF PMSE-TREATED FATTY ACID SYNTHASE

Fatty acld synthase from the uropygral gland of goose was lnacfivated 96-988 Wlth PMSF as previously described 16) . Thl5 enzyme Could be stored frozen several months wlrhout loss of actlvlty. Partially purlfled rat mammary gland fatty acld synrhace was treated w ~ t h PMSF a s descrlbed before 141. ThlS enzyme was more sensltzve to PMSF treatment than was the synthase from the uropyglal gland and therefore the repeated treatment Wlth PMSE used Wlth the enzyme from the g0oe.e could not be used.

ENZYME ASSAYS ____ described 1111. Acyl-€AS hydrolase was assayed specrrOphotometrlcally by determming the ~ncrease ~n malonyl-CoA dependent NADPB ondation by PMSF- treated goose fatty acid synthase (61. The assay mixture contamed 25 Yg PMSF-treated synthase, 15 nmole acetyl-coA, 30 "mole malonyl-CoA. 66 m o l e NADPH and acyl-FAS hydrolase in 0.3 ml Of 0.1 M sodlum phosphate, pH 7.0. contalnlng 1.0 mM DTE. NADPH oxldatlon was moasured by following the decrease I" absorbance at 340 nm for 5 mln at 30°C; the Control rate obtalned without acyl-EAS hydrolase was subtracted. The assays lnvOlvlng

mammary gland ITE 11) wlth PMSF-treated goose fatty acld synthase or PMSE- combination of the acyl-€AS hydrolases from the uropyglal gland and rat

treated rat mammary gland fatty acld synthase were done slrnllarly. except 60 ug of elther PMSF-treated synthase was used.

Spectrophatametrlc assay far fatty acld synthase was done as prevrausly

The chaln-shortening ability of the acyl-FAS hydrolase was measured by

CoA (for n-fatty acld syntheslsl or 15 nmole Imethyl- l"Clmethylmalonyl-CoR lncubatlng 180 "mole NADPH, 20 nmole acetyl-coA and 20 nmole i2-'*Clmalonyl-

and acyl-FAS hydrolase I" a total volume of 1.0 ml of 0.02 M sodurn Ifor branched chaln fatty acld synthesis), 100 p q goose fatty acld synthase,

phosphate buffer. pH 7.0, containing 0.5 mM DTE for 1 h at 30'. The

esters were prepared and sublected to radx gas-lwud chromatography a5 reaction was stopped wlth 6N HC1 and the fatty acids were extracted: butyl-

previously described ( 6 1 .

Thloesterase activlty was measured Spectzophofomefrrcally and by a

DTNB, 0.1 mM CI2-COA and 2-10 y g protein in a total Volume Of 0.3 ml Of radiochemlcal assay. The reaction mlxture for the former contalned 0.1 M

measured. The concentratmn and cham length of the COR ester were varled 0.1 M sodium phosphate buffer. pH 8 . 0 . and absorbance change at 412 nm Was

when assaying for substrate speclflcity; when used, BSA was added at a flnal COncentratrOn of 1 rnglml. The reactlon mlxture for the radiochemical assay contained 20 nmole Of 11-14CIC12-CoA, and the enzyme 110-20 ug acyl-FAS hydrolase, 50-80 ug thiOeStera6e A 01 5-15 y g thloesterase 81 I" a total volume of 0.25 ml of 0.1 M sodium phosphate buffer, pH 7.0. Contblnlng 0.5 mM DTE. The reaction was started by addition of the substrate. and after 6 mi" of lncubarion at 3OoC. 25 y l 35% perchloric acid and 0.25 ml 958 ethanol were added. The free fatty acid was recovered by extraction with hexane 12 x 1.5 mll. The spectrophotometrlc assay gave lower actlvlty than that observed wxth the radiochemzcal assay wlth the pnrlfled acyl-FAS hydrolase, most probably because of the extreme sensitivity Of this enzyme to thiol-duected reagents. The spectrophotometric assay was used only I" the chain length specifzcity studies.

ELECTROPHORESIS

Sodrum dodecyl Sulfate disc gel electrophoresls Was performed previously descrlbed (11). u s m q a 12% polyacrylamide renolvlng gel.

DETERMINATION OF PROTEIN AND PADIOACTIVITY

ethanol in toluene cdntaining 4 gll Omnifluor and assayed for radioactlvlty Aliquots Of substrates and pxoducts were dlSsO1Yed In 15 m l of 30%

I" a Packard Trl-Carb llquld sc1ntlllat10n counter, Model 3255. COuntlng efficlency was 8 0 % for I'c as determmed wlth [l*C1t~luene standard. Protein was derermmed bv the method of Lowry et. al. 112) With bovine serum albumin as standard.

AMINO ACID ANALYSIS

Purlfled acyl-€AS hydrolase was hydrolyzed wlth 6N HC1 for 24 h, under vacuum, at 110'. Cysteme and methlonlne were determlned after performlc acld oxldatlon Of protein samples 1131. Analyses were done at the Bloanalytzcal Laboratory, Wdshinqton State U n ~ ~ e r s i t y , On a Beckman Model 121 MB amlno acld analyzer

PARTIAL PURIFICATION OF FAT " M A R Y GLAND FATTY ACID SYNTHASE AND THIOESTEMSE I1

m o l e Ci2CoA hydrblyredlrnLnlrni.

PARTIAL PURIFICATION OF THIOESTERASES A AND B FROM MALLARD UROPYGIAL GLAND

gel filrratmn on Sepharose 68. and therefore the pooled thmesterase ThlOeSteraSeS A and B were not resolved from the acyl-FAS hydrolase by

Flgure 4. Effect Of pH on thioesterase actlvlty Of purlfled acyl-FAS hydro-

chemically uslng CIZ-CoA as substrate a s described I" tent: ln the Case of lase and partially purlfled thioesterases A and 8. Assays were done radlo-

TE-0. 1 rnglrnl BSA was included in the reactlon mlxtule.

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L Rogers, P E Kolattukudy and M deRenobalesgland of mallard.

which modifies the product specificity of fatty acid synthase in the uropygial Purification and characterization of S-acyl fatty acid synthase thioester hydrolase

1982, 257:880-886.J. Biol. Chem. 

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