THEJOURNAL OF BIOLOGICAL

CHOMISTRY
Vol. 256, No. 12, h u e ofJune 25, pp. 6282-621M,1981
Prrntedin U.S.A.

Acetoacetyl-CoA Reductase Activityof Lactating Bovine Mammary

Fatty Acid Synthase*
(Received for publication, June 19, 1980, and in revised form, January 12, 1981)

Peter F. DoddsS, M. Guadalupe F. Guzmang, Stephen C. Chalberg, Gregory J. Anderson, and

Soma Kumarj
From the Department of Chemistry, Georgetown University, Washington, D. C. 20057

Fatty acid synthase, purified from lactating bovine
mammary gland, utilizes coenzyme A esters of aceto-
acetic, 3-hydroxybutyric, and crotonic acids as sub-
strates for its partial reactionsat micromolar concen-
trations. The NADPH:acetoacetyl-CoA reductase had a
K,,, of 5 PM acetoacetyl-CoA and a V,, of about 4 pmol
of NADPH oxidized min” mg“. In contrast, theX,,, for
the model compound, acetoacetyl pantetheine was 820
PM and that of S-acetoacetyl-N-acetylcysteaminewas
over 40 mM. The reduction of acetoacetyl-CoA was
observed with the enzyme from rat tissues also but not
with those from avian tissues or yeast. With the bovine
mammary enzyme, the reaction was found to oxidize 2
mol of NADPH for every mol of acetoacetyl-CoA con-
sumed. Butyrate was the major product of reduction.
The reductase activitywas susceptible to inhibition by
several sulfhydryl reagents; it was lost when the syn-
thase was dissociated into one-half molecular weight
subunits or when the incubation mixture was depleted
of CoA. It was competitively inhibited by acetyl-coA,
butyryl-CoA,methylmalonyl-CoA, and Z-methylcro-
tonyl-CoA. These results aswell as its use as a primer
in fatty acid synthesis by the enzyme suggest that the
acetoacetyl group from acetoacetyl-CoAis transferred
to the enzyme, presumably to its4’-phosphopantheine
prosthetic group. The acyl group is then expected to
remain attached to the enzymewhile it is reduced,
dehydrated, andreduced again toform a butyryl group
which can either undergo chain elongation, if malonyl-
CoA is present, or be released from the enzyme by
hydrolysis or transfer to free COG.
Fatty acid synthase in eukaryotes is a multifunctional en- zation of theacetoacetyl-CoAreductase activity.
from animal tissues and are nonidentical in that from yeast
(1).
Of the partial reactions involved in the synthesis of fatty
acids, the transferof acetyl andmalonyl groups to the enzyme
and their subsequent condensation to form acetoacetyl-s-en-
zyme have been demonstrated by direct assays as has the
removal of the completed chain from theenzyme (4, 5). The
partial reactions involving the reduction of the 3-ketoacyl
intermediate to 3-hydroxybutyrylgroup, its dehydration, and
subsequent reduction to form butyryl group have been in-
ferred from individual reactions observedby using the appro-
priate thiol estersof N-acetylcysteamine asmodel substrates
(3, 6). Because of the obligatorycovalentlinkage of the
intermediate acyl chains to the prosthetic group during the
chain elongation process, the reactions with the model com-
pounds necessitated the use of high concentrations of the
substrates aswell as of the enzyme. While the employmentof
model substrates was useful in obtaining a n insight into the
mechanism of fatty acid synthesis, they were unsuitable for
the physicochemical characterization of the individual reac-
tions.
We have reported previously that bovine mammary syn-
thase, in contrast to thepigeon liver and yeast enzymes with
which the pioneering work on fatty acid synthesis was done,
possessed a high crotonyl-CoA reductase activity which was
dependent on the structural integrity of the complex (7, 8).
This enabled us to determine many features of this partial
reaction regarding structural requirements in the substrate
and its kinetics (9, IO). An examination of the other partial
reactions catalyzed by this enzyme revealed the presence of
an extremely activeacetoacetyl-CoA reductase activity anda
much lower 3-hydroxybutyryl-CoA dehydratase activity. This
report describes the resultsof work aimed at the characteri-
’

zyme complex, classified as Type 1 (l),which catalyzes the

seven or moreindividual reactions involved in the sequential EXPERIMENTAL PROCEDURES‘
addition of two carbon units,derived from malonyl-CoA, to a
primer acetyl-coA or butyryl-CoA (1, 2 ) . Currently, there is RESULTS
general agreement that the enzyme consists of an aggregateof Reduction of COA-S-ACAC~ by Fatty Acid Synthase-The
two polypeptide chains which are identical in the enzyme CoA-S-AcAc reductase activity and the fatty acid synthase
activity copurified through all the stepsof purification follow-
* This work was supported by National Institutes of Health Grant ’Portions of this paper (including “Experimental Procedures,”
AM 16086. The costs of publication of this article were defrayed in Figs. 2, 6 , and 8, and Tables I1 and VII) are presented in miniprint at
part by the payment of page charges. This article must therefore be the end of this paper. Miniprint is easily read with the aid of a
hereby marked “advertisement” in accordance with 18 U.S.C. Section standard magnifying glass. Full size photocopies are available from
1734 solely to indicate this fact. the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda,
$ Recipient of a Wellcome Research Travel Grant. Presentaddress, MI) 20014. Request Document No. 80M-1240, cite authorb), and
Department of Biochemistry, Physiology, and Soil Science, Wye include a check or money order for $4.80 per set of photocopies. Full
College, Near Ashford, Kent TN25 5AH, England. size photocopies are also included in the microfdm edition of the
8 Recipient of Mexican Government Predoctoral Fellowship. Pres- Journal that is available from Waverly Press.
ent address, Quimica Hercules s. A. de C. V., Sara Avenida 4553, ’The abbreviations and trivial name used are: AcAc, acetoacetyl;
Mexico D. F., Mexico. NAcCyst, N-acetylcysteamine; diamide, azodicarboxylicacid bis-N,N-
1 To whom correspondence should be addressed. dimethylamide.

6282

This is an Open Access article under the CC BY license.

 Acetoacetyl-CoA
Reduction Synthase
Acid
by Fatty 6283

ing the fist ammoniumsulfateprecipitation in amanner at varying concentrations are shown in Fig. 1B.The K , for
similar to thecopurification of crotonyl-CoA reductase activ- this substrate exceeded that for CoA-S-AcAc by a factor of
ity (9). The ratio of the two activities ranged throughoutfrom over 100. With NAcCyst-S-AcAc, the often used model sub-
1:3.2 to k3.6 (fatty acid synthase activity:CoA-S-AcAcreduc- strate, the plateau region was not reached even at 40 mM
tase). A very active CoA-S-AcAc:NADH oxidoreductase pres- concentration (data notshown). In this respect,the mammary
ent in the cytosol (15) was removed during purification. Fig. enzyme differs from the pigeon liver enzyme which has a K,,,
lA shows the effect of varying CoA-S-AcAc concentration. of 30 mM ( 6 ) .
The inhibition by the substrate at concentrations in excess of The effects of pH on the reduction of CoA-S-AcAc and
50 p ~ observed
, with the mitochondrial CoA-S-AcAc:NADH NAcCyst-S-AcAc are shown in Fig. 2. A marked difference is
oxidoreductase (131, was not observed with this enzyme. The seen in the pH profiles obtained with the two AcAc deriva-
reaction was totally irreversible even at a pH as high as 9.0 tives.
and using 100 PM each of ~(-)-3-hydroxybutyryI-CoAand The reduction of CoA-S-AcAcwas highly specific with
NADP'. The activities obtained on using AcAc-pantetheine respect to NADPH. NADH was less than one-tenth as effec-
tive as NADPH.
Stoichiometry of the CoA-S-AcAc Reduction Reaction-
The results of the experiment using three procedures to de-
termine the ratioof NADPH oxidized to CoA-S-AcAcreduced
are summarized in Table I. They show that approximately 2
mol of NADPH were oxidized for every mole of CoA-S-AcAc
used up in the reaction. This suggests thatthe reaction
proceeds from the first reduction, which uses 1mol of NADPH
to produce 1mol of 3-hydroxybutyryl derivative, through the
dehydration reaction to the subsequent reduction of crotonyl
group using the second mole of NADPH to form 1 mol of
butyryl group. Under the conditions used, the reaction ap-
pears to level off when about 80% of the CoA-S-AcAc present
was consumed.
0,200 0.500 The above methods were unsuitable for determining the
stoichiometry of NAcCyst-S-AcAc reduction because the re-
action mixture needed 20 to 40 pmol of the substrate/ml of
the incubation mixture of which less than 1 pmol was con-
sumed. An alternative procedure was, therefore, adopted in
order to identify the product of the reaction. "H from
["HINADPH, continuously regenerated from [1-"Hlglucose
and NADP', was incorporated into theproducts of the reac-
tion using one or the other of the AcAc thiol esters as sub-
strate. The products of the reaction were then analyzed by
radio-gas chromatography. The chromatograms obtained are
shown in Fig. 3. Both chromatograms show an unidentified
radioactive peak with an adjusted retention timeof 12-13min
which cochromatographs with neither D-3-hydroxybutyric
acid nor crotonic acid. Of the 188,000 dpm of radioactivity
extracted from the incubation containing CoA-S-AcAc, 70%
cochromatographed with but+@ acid and only 1%cochro-
- 2 0 2 4 6 8 1 0 matographed with 3-hydroxybutyric acid. In contrast, of the
II [AcetoacetylPantetheine] ( m M" ) 209,000 dpm of radioactivity extracted from the incubation
FIG. 1. The reduction of acetoacetyl-CoA and acetoacetyl using NAcCyst-S-AcAc, nearly 10 times more radioactivity
pantetheine by fatty acid synthase. The standard assay conditions was associated with 3-hydroxybutyric acid than with butyric
were used for the indicated concentrations of ( A ) CoA-S-AcAc; ( B ) acid.
AcAc-pantetheine. The enzyme had a specific activity of 850 units Effect o f SH Inhibitors" number of SH reagents in-
mg". hibited CoA-S-AcAc reduction. An inhibition of 95% or more
TABLE I
Stoichiometry of the reduction of acetoacetyl-CoA
Acetoacetyl-CoA consumed and the ratios of NADPH/CoA-S-AcAc as shown by the enzymic assay and the assays of Mg2+ enolate. The
reaction was carried out as described under "Experimental Procedures" using the concentrations of NADPH, CoA-S-AcAc, and enzyme
indicated.
Addltinnf Mg2' enolate
Experiment NADPH I

Enzyme Czit;- NADPH oxidized Enzymic assay

pH 7.95 pH 8.16
_____
I+! PM PM nmol nrnol" ratio nmol" ratio nmot' ratio
50 1 20 150 82 39 2.1
2 20 50 150 88 37 2.3 41 39 2.2 2.1
3 30 100 300 151 100 1.5 85 81 1.9 1.8
4 40 100 300 2.2 178 79100 1.8 84 2.1
Mean 1.9
" CoA-S-AcAc consumed.
6284 Acetoacetyl-CoA Reductionby Fatty Acid Synthase
1 TABLEI11
Transacylase activity of fatty acid synthaseusing various
coenzyme A derivatives
The rate of exchange of the acyl group of the different CoA
derivatives with [3H]CoA catalyzed by the synthase (specificactivity,
800-900) was determined a t 2-3 “C asdescribed under “Experimental
Procedures.”
Substrate Acyl exchange
nmol min” mg“ enzyme
Acetyl-coA 8170
Butyryl-CoA 8840
Acetoacetyl-CoA 7200
~(-)-3-Hydro~ybutyryl-CoA 450
Trans-crotonyl-CoA 1120

TABLE
IV
Trans-crotonyl-CoA and acetoacetyl-CoA reductase activities of
fatty acid synthasefrom various tissues
Each of the activities of the various enzymes were determined
under their respective optimum conditions described in the text.
Activities“
I Crotonyl- CoA-S- :{:
0 4 8 12 16 20 24 28 32 Source Of enzyme Synthase CoA re- B/A AcAc re-
TIME (rnin) (A) ductase ratlo ductase (c)
(B)
nmol nmol nmol
Cow mammary 733 746 1.02 3438 4.70
Rat liver 1041 434 0.42 1645b 1.31
Rat adipose 1137 440 0.39 1346 1.18
Rat mammary 1176 514 0.44 1441 1.33
Pigeon liver 2015 16 0.01 29 0.01
Goose uropygial 940 22 0.02 28 0.03
Chicken liver 2300 20 0.01 56 0.02
Yeast 2644‘ 21 0.01 48 0.02
Each activity is expressed as nanomoles of NADPH oxidized/
(I

min/mg of enzyme.
* This enzyme sample had a fatty acid synthase specific activity of
1256.
Assayed under conditions given in Ref. 30.

of the crotonyl-CoA reductase activity of bovine mammary

fatty acid synthase was lost under the same circumstances.
The results of the experiments designed to investigate the
effect of dissociation and reassociation of fatty acid synthase
TIME ( m i d on CoA-S-AcAc and NAcCyst-S-AcAc reducing activities are
presented in Table 11. It can be seen that fatty acid synthase
FIG. 3. The products of reduction of acetoacetyl thiol esters.
3H-labeled products of the reduction of CoA-S-AcAc (A) and NAc- and CoA-S-AcAc reductase activities are almost completely
Cyst-S-AcAc ( B ) were analyzed by radio-gas chromatography as absent from the dissociated enzyme. NAcCyst-S-AcAcreduc-
described under “Experimental Procedures.” The dotted line repre- ing activity was also lowered but about 25% was consistently
sents theradioactive trace and the solid line is the signal output from retained. Reassociation of the complex results in nearly com-
the flame ionization detector. The two chromatograms which contain plete recovery of all the activities.
different amounts of carrier butyric acid (adjusted retention time, 4- Acyl CoA:Fatty Acid Synthase Acyltransferase-The re-
5 min) and 3-hydroxybutyric acid (16-17 min) were obtained using
different size aliquots of the extract and were run at different atten- sults obtained by employing the new assay for the exchange
uations. The small unidentified mass peaks are impurities present in of the various acyl groups from their respective CoA esters to
the carrier 3-hydroxybutyric acid. free CoA are shown inTable 111. The rates of the exchange of
acetyl, butyryl, and acetoacetyl groups are high and are similar
in value to the acetyl exchange reported for rat mammary
was obtained with about 100 p~ N-phenylmaleimide or N - fatty acid synthase
(5). It is noteworthy that although the
methylmaleimide; 75 p~ iodoacetamide inhibited the reaction relative rates of acyl transfer do not quantitatively agree with
by 33%, and 50 p~ CoA-S-AcAc reduced the inhibition to the relative rates of the enzyme reaction, the exchange of
varying extents. It has not been established yet whether this ~(-)-3-hydroxybutyryl group which wasdehydrated at a slow
inhibition is dueto theblockage of the 4“phosphopantetheinyl rate (Fig. 8 and Table VII) is also the slowest with respect to
SH group of the enzyme (22, 23) or of an essential SH group the exchange reaction.
of the reductase site(s) of the enzyme complex. Crotonyl-CoA and CoA-S-AcAcReductase Activities of the
Effect of Dissociation and Reassociation on CoA-S-AcAc Enzyme Complex from Various Sources3-Table IV shows
Reduction-Kumar et al. (6) have shown that pigeon liver the activities present in the fattyacid synthase isolated from
fatty acid synthase, when dissociated into one-half molecular
weight subunits, becomes totally incapable of synthesizing The data were derived from M.S. thesis, Georgetown University.
fatty acids but loses only 30% ofits crotonyl-NAcCystreducing Permission to use the datais gratefully acknowledged to Alice Fisher,
activity. Maitra and Kumar(€9, however, found that over 70% the author.
 Acetoacetyl-CoA Reductionby Fatty Acid Synthase 6285

/ I

I I I I I
0.0 0.1 0.2 0.3 a4 ao 0.1 0.2 43 a4

11 [Acetoacetyl -CoA] ( pM") 11 [Acetoacetyl-CoA] ( p6 ' )

5. I

00
.oo .02 .04 .06 .00 .10 U
0.02 0.04 0.06 0.08 0.10
11 [Acetoacetyl -Con] ()I M")
(COA-S-ACRC)-~ ~01-1

FIG. 4. Inhibition of CoA-S-AcAc reduction by acetyl-coA, p ~ A-A,

; 50 p ~ C,. methylmalonyl-CoA: t-"., 0 p M ; M,
butyryl-CoA, methylmalonyl-CoA, and 2-methylcrotonyl-CoA. 7.5 p ~ W,
; 10 p ~ C;k - U , 20 p ~ D,
. 2-methylcrotonyl-CoA:
The standardconditions were employed to assay the reaction except M, 0 p ~M ; ,18 p ~ A-A,
; 36 p ~ M; ,
90 p ~ The
.
for the concentrations of CoA-S-AcAc used. A, acetyl-coA. H, 0 lines which extend to the right or top border were drawn using an
p ~ W,; 1 p ~H ; ,5 p ~M; ,
20 p ~ A-A,
; 50 p ~ B,. additional data point which had to be excluded from the figure
butyryl-CoA M, 0 p ~M ; I p ~H
, ; ,
5 p ~[3"--13,20
; because of the scale used.

various tissues and yeast. The ratios of the two activities to was bound at the same sites as the natural substrates (10).
the synthase activity are also given. It can be seen that the When similar studieswere carried outon CoA-S-AcAc reduc-
bovinemammary gland hasthehighest ratios. Boththe tase activity, it became apparent that effect
theof the presence
reductase activities are present to significant extents in the of malonyl-CoA was complex. This is because, a t p H 7.6 (the
enzyme from rat tissues. Previous work using cruder enzyme pH of the assay mixture),CoA-S-AcAc acts as a primer (see
preparations from rat tissues gave conflicting results (21, 24). below) for the malonyl-CoA-dependent fatty acid synthesis.
As reported in earlier studies, ofboththese reductase activitiesMalonyl-CoA was, therefore, replaced with methylmalonyl-
were low in the enzymeisolated from yeast and avian tissues CoA, a structural analogue which has no activity as a chain
(3, 25). elongator with this enzyme (10). The results, shown inFig. 4,
Effects of Acetyl-, Butyryl-, Methylmalonyl-, a n d 2-Meth- A-D, demonstrate that all four compounds inhibit the reduc-
ylcrotonyl-CoA on CoA-S-AcAc Reduction-The normal sub- tion of CoA-S-AcAc in a competitive manner. Replots of the
strates for the synthesis of fatty acids are acetyl-coA and slopes of the Lineweaver-Burkplots against the concentration
malonyl-CoA. Butyryl-CoA has been shown betoan excellent of inhibitor showed that the inhibition by each of the com-
alternativeprimer to acetyl-S-CoA (26). Thesesubstrates pounds was that of a simpler linear competitive nature. It is
were effective inhibitors of trans-crotonyl-CoA reductase ac- apparent from the graphs that at the lowest concentrations of
tivity of the enzymewhich established that the crotonyl group these CoA esters there is often a measurable increase in the
6286 Acetoacetyl-CoA Reductionby Fatty Acid Synthase
Vmax of the reaction. This effect has been observed earlier in
the studies of the inhibition of crotonyl-CoA reductase as well
(10). The values of K, for the reduction of AcAc-S-CoA,
obtained from Dixon plots from which theratesat zero
inhibitorconcentration were omitted,are:acetyl-coA, 4.0
pM; butyryl-CoA, 4.5 p M ; methylmalonyl-CoA, 5.5 PM; and 2-
methylcrotonyl-CoA, 4.5 p ~These . compare with K, values of A
7 pM and 6 p~ for acetyl-coA andbutyryl-CoA, respectively,
obtained when they were used to inhibit crotonyl-CoA reduc-
tase activity (10).
Use of CoA-S-AcAc as Primer in Fatty Acid Synthesis-
The reduction of CoA-S-AcAc to butyrate can be visualized
to involve the sequential production of AcAc-S-enzyme, 3-
hydroxybutyryl-S-enzyme, crotonyl-S-enzyme, and, finally,
butyryl-S-enzyme as intermediates. If the butyryl group, so
formed, is bound to theenzyme at the same site(s) where fatty
acid synthesis occurs, CoA-S-AcAc wouldbe expected to serve
effectively as a primer inthe malonyl-CoA-dependent synthe-
sis of fatty acids by the enzyme. This was ascertained by
determining the extent of incorporation of [2-14C]malonyl-
CoA into fatty acids using CoA-S-AcAc as primer. For com-
parison, acetyl-coA, propionyl-CoA, and butyryl-CoA were
also used as primers in separate incubations. The results
presented inFig. 5 and Table V show that the extent of
incorporation of malonyl-CoA into fatty acids using CoA-S-
AcAc is comparable to thecorresponding values obtained with
TIME (MINI
the other primers. As expected, hexanoic is the acid formed
with the shortest chain when CoA-S-AcAc (or butyryl-CoA)
is used as primer. This excludes the possibility of the fatty
acids having been synthesized using as primer acetyl-S-en-
zyme which was formed by the decarboxylation of malonyl-
CoA (27). The formation of only odd chain acids, beginning
with pentanoic acid when propionyl-CoA was used as primer,
lends further support to the absence of decarboxylation of
malonyl-CoA to produce acetyl-coA oracetyl-S-enzyme.
Effect of CoA on the CoA-S-AcAcReductase Reaction-In
view of the pronounced effect CoA had on the crotonyl-CoA
reductaseactivity (LO) of the synthase,experiments were
undertaken to determine whether CoA had a similar effect on
the CoA-S-AcAc reductase activity as well. Data presented in
Fig. 6 show that CoA alters markedly the pH profile of the
reaction, as it does the crotonyl-CoA reductase activity, and
that its stimulatory effect is more pronounced at the subop-
timal pH values.
Effect of the Depletion of CoA from the Incubation Mix-
ture-For the depletion of CoA (28) generated by the possible
transfer of the AcAc group from its CoA derivative to the
enzyme prior to its reduction,phosphotransacetylase and
acetyl phosphate were included in the incubation mixture

TABLEV
Formation of individual acids from the different acyl CoA esters
TIME ( M I N )
used as primers
The reaction mixture contained in 0.5 ml the ingredients listed in FIG. 5. Products of fatty acid synthase reaction using CoA-
the text and 14.4 pg of fatty acid synthase of specific activity, 720. S-Ac or COG-S-AcAcas primer and [2-14C]malonyl-CoA.The
The details of the extraction and analysis of the products are described fatty acids produced by the enzyme were extracted and analyzed by
in the text.With propionyl-CoAas primer, the radioactive peaks were radio-gas chromatography. Thesolid lines represent the outputs
located between the mass peaks of acids with one more and one less from the flame ionization detector, and the dottedlines are the
carbon atoms. outputs from the proportional counter. The numbers inparentheses
indicate the number of C atoms in the saturated fatty acids. The
Total Distribution of ["C]malonyl-CoA incor-
nyl-CoA in- porated intofatty acid chain lengths arrows indicate the attenuation changes. A , CoA-S-Ac; B , CoA-S-
AcAc.
coworation c, c, Ca c,,, c,.' c,, c,,, C,"
nmol min"
mg" 76 containing Tris-HC1 buffer as themedium. The dataon CoA-
Acetyl-coA 315 18 7 4 5 5 7 49 S-AcAc and crotonyl-CoA reductase activities are shown in
6
Acetoacetyl-CoA 264 0 10 8 10 9 20 42 2 Table VI. It can be seen that depletion ofCoA virtually
Butvrvl-CoA 366 0 12 9 10 10 16 42 2 eliminates both reactions. Pantetheine, mimicking the effect
Propionyl-CoA 400 38 40 CoA,
of not onlyrestores
the activity
but like CoA itstimulates
 Acetoacetyl-CoAReduction. by Fatty Acid Synthase 6287
TABLE
VI specifically with acetyl-coA and malonyl-CoA as the only
Inhibition of crotonyl-CoA and acetoacetyl-CoA reductase carbon sources. The failure to detect the presence of any
activities of the synthase on the depletion of CoA and its intermediate between the substratesand the products and the
restoration by pantetheine absence of any reaction with the intermediates of the p-
The synthase had a specific activity of 790. For both the reductases, oxidative process was clarified whenthe investigations in the
the control system contained 100 mM Tris-HC1,pH 6.9, at 37 “C. The laboratories of Lynen and Porter showed that the intermedi-
concentrations of phosphotransacetylase, acetyl phosphate, pante-
theine, and CoA were 5 pg/ml, 75 mM, IOO~M,and 7.5p ~respectively.
,
ates were linked to the sulfhydryl of the 4’-phosphopante-
Reductase reaction rate“
theine prosthetic group of the complex (22, 23). The occur-
Additions to control
rence of some of the partial reactions involved inthe growing
Crotonyl- Acetoacetyl. acyl chain was demonstrated using model substrates consist-
CoA CoA
ing of thiol esters of acetoacetic, 3-hydroxybutyric, or crotonic
None 140 2200 acids with NAcCyst (3, 6). The reduction of acetoacetyl and
Acetyl phosphate 103 1897
Phosphotransaeetylase 107 2454
crotonyl and the dehydration of 3-hydroxybutyryl groups
Phosphotransacetylase -+ acetyl phos- 6 0 required relatively high concentrations of these model com-
phate pounds, as much as 100 t o 500 times the concentrations of
1036
Phosphotransacetylase -t acetyl phos- 337 CoA esters used in the presentstudy (6). The failure to detect
phate and pantetheine any reaction with the corresponding CoA esters at micromolar
Pantetheine + acetyl phosphate 2877 584
levels was attributed to the specificity of the acyltransferase
Pantetheine 96 1 3030 activities of the enzymecomplex for acetyl and malonyl
Coenzyme A 1133 3170 groups. It was also shown that the use of these model sub-
Reaction rates are givenas nanomoles of NADPH oxidized min”
strates resulted in only a single reaction step of the entire
mg” of protein. normal reaction sequence ( 3 , 6 ) .
The present study reveals that fatty acid synthase isolated
from lactating bovine mammary gland as well as from different
rat tissues differs from the enzyme from yeast and avian
tissues in that it is able to reduce acetoacetyl-CoA and cro-
tonyl-CoA at micromolar concentrations (Table IV). Of the
mammalian enzymes tested, the one from bovine mammary
gland had the highest of these partial reactions. The enzyme
from the different tissues of rat exhibited similar reactivities
indicating the absence of organ-specificdifferences in the
enzyme. This conclusion is supported by the similarity of
immunological cross-reactivities previously reported by Smith
(29).
The results presented in the present communication are
consistent with a mechanism for the reduction of CoA-S-AcAc
in which the AcAc group is transferred from CoA to become
covalently bonded to the enzyme, presumably to the pros-
FIG. 7. Inhibition of acetoacetyl-CoA reductase activity by thetic SH group of the enzyme. It would then become indis-
the depletion of CoA and its restoration by CoA or pantetheine. tinguishable from the AcAc group formed on the enzyme by
The incubation conditions werethose describedin Table VI. I, control the condensation of acetyl and malonyl groups during the
CoA-S-AcAcreductase reaction;2, control assay plus complete phos- synthesis of fatty acids. Such a transfer, if it does occur with
photransacetylase reactionsystem plus 100 p~ pantetheine;3, control
assay plus complete phosphotransacetylase reaction.0.2 pmol of CoA NAcCyst-S-AcAc, appears to do so only to a slight, extent as
was added where indicated. judged from the formation of a small amount of butyrate (Fig.
3B).The evidence for the postulation of a transfer of the AcAc
both the activities. The acutal traces of the recorder pen group from its CoA ester is: 1) the low K, ( 5PM) obtained for
CoA-S-AcAc, which is of the same order as the values for
showing the effects of the addition of CoA or pantetheine are
presented in Fig. 7. Pantetheine was also found to relieve CoA-S-Ac and malonyl-S-CoA, e.g. 8 PM and 18 p ~ respec- ,
tively. These have been established to have their acyl groups
entirely the inhibition of the fattyacid synthase reaction due
transferred to the enzyme (22, 23). In contrast, the Km for
to the depletion of CoA (28) using, in the present study, the
phosphotransacetylase reaction (data not shown). AcAc-S-pantetheine is over 100 times higher while the value
Reversible Dehydration of 3-Hydroxybutyryl-CoA-The for NAcCyst-S-AcAcis even higher. 2) The consumption of 2
results of the assay in which the dehydration of 3-hydroxy- molecules of NADPH/molecule of CoA-S-AcAc reduced (Ta-
butyryl-CoA was coupled to the NADPH-dependent reduc- ble I)and the formation of butyrate (Fig. 3A) from this
tion of crotonyl-CoA, a reaction of the synthase well charac- substrate indicate the occurrence of two reductive reactions
terized in this laboratory, are shown in Fig. 8. The reaction which could not have occurred without the intervening de-
was highly specific for the CoA derivative of ~(-)-3-hydrox- hydration reaction. These reactions wouldbeexpected to
ybutyrate. The activity with the corresponding L(i-)-isomer occur in this sequence, by means of the reactions involved in
wasonly one-tenth as much. Table VI1 shows the results the synthesis of fatty acids, only if the AcAc group is bound
using the more direct optical assay. The rateof the reversible to theprosthetic group. 3) The rate of acyl exchange between
dehydration is verylow compared to the rateof the reduction CoA-S-AcAc and free CoA (Table 111) is comparable to the
of COA-S-AcAc. rates of exchange of the acetyl and butyryl groups. 4 ) The
inhibition of the reduction of CoA-S-AcAc competitively by
DISCUSSION
methylmalonyl-CoA, CoA-S-Ac, and butyryl-CoA, the acyl
When the nature of the biosynthesis of long chain acids was groups all of which are known to bind to theprosthetic group
first elucidated, using fatty acid synthase from yeast (3) and of the enzyme (22, 23), and bytiglyl-CoA, a competitive
pigeon liver (251, it was found that the synthesis occurred inhibitor of crotonyt-CoAreductase reaction (IO),lend further
6288 Acetoacetyl-CoA
Reduction
support to the binding of the AcAc group to the prosthetic
group. 5) The susceptibility of the reduction of CoA-S-AcAc
to SH reagents and the protection afforded by the substrate
to this inhibition is consistent with, although it does not prove,
the binding of the acyl group to the prosthetic SH group. 6)
The use of CoA-S-AcAc as primer in the synthesis of fatty
acids, producing hexanoic and longer chain acids, indicates its
reduction to butyryl-S-enzyme followed byfurther chain elon-
gation. 7) Finally, the total loss of the reductase activity on
the depletion ofCoA from the reaction mixture and its res-
toration by the addition ofCoA or pantetheine would be
difficult t o account for on any basis other than by assuming
the transfer of the AcAc group to the enzyme and, following
its reduction, the transfer of the butyryl group back to CoA.
In contrast to the rates of reaction with CoA-S-AcAc and
trans-crotonyl-CoA, the reversible dehydration of 3-hydrox-
ybutyryl-CoA wasslow (Table VII). The dehydration was
significant only when it was coupled to the reduction of the
crotonyl group formed (Fig. 8). This low rate of dehydration
could be the result of a low rate of transfer of the acyl group,
which must precede its dehydration, or low rate of dehydra-
tion itself or both. If the actual dehydration reactionwas slow,
the rateof reduction of the CoA-S-AcAc wouldhave been no
faster or the product would have been 3-hydroxybutyrate. As
a matter of fact, the CoA-S-AcAc reduction was 15 times
faster than thereaction involving 3-hydroxybutyryl-CoA. This
would suggest that the rate of transfer of the acyl group to
the prosthetic SH must be responsible for the slow rate of
reaction. This is supported by the datapresented in Table 111.
The new assay described in this study for determining the
rates of acyl exchange offers some advantage over the assay
method used in previous studies (6, 30). The latter assayed
the enzyme-catalyzed transfer of 14C-labeledacetyl or malonyl
groups from its CoA derivative to pantetheine. One is not
certain that the rate of transfer of the acyl group from the
enzyme to pantetheine, a model substrate, represents the rate
of the acyl transfer to and from CoA, the natural acyl carrier,
even though the concentration of pantetheine employed is 35
times the concentration of the acyl-CoA (6).The assay devel-
oped in the present study was patterned after the pyrophos-
phate exchange reaction for aminoacid activation (31) and
employs equimolar concentrations of ['HICoA and acyl-CoA.
The real advantage of the present assay is in its avoidance of
radiolabeled acyl-CoAs, many of which are unavailable com-
mercially and aretedious to prepare in pureform (9).It must
be mentioned that although the exchange of the acyl group is
enzymatically catalyzed, the actual involvement of the pros-
thetic SH group, rather than the OH or SH group of the
loading site, remains to be established. The involvement of
the loading site only in the exchange reaction measured in
this as well as the previous studies might account for the
extremely fast rates of reaction (5, 6) obtained for substrate
acyl groups.
A phylogenetic distinction between fatty acid synthase ob-
tained from the mammalian tissues on the one hand and the
enzyme from the avian tissues and yeast,on the other,is clear.
In addition to thecatalysis of the two reductase reactions,the
mammalian enzyme produces relatively large amounts of bu-
tyryl-CoA besides palmitic acid and is capable of utilizing
Synthase
byAcid
Fatty
butyryl-CoA efficiently as primer (32). The physiological sig-
nificance of thesereactions of the enzyme to the tissues
concerned remains obscure.
Acknowledgments-We thank Dr. 0. Gabriel forhelp with and the
use of the radiochromatogram scanner and Chas. J. Schmidt & Co.,
Baltimore, MD, for its valuable assistance in obtaining lactating
bovine mammary tissues.
REFERENCES
1. Bloch, K., and Vance, D. (1977) Annu. Rev. Biochem. 46, 263-
298
2. Volpe, J. J., and Vagelos, P. R. (1973) Annu. Rev. Biochem. 42,
21-60
3. Lynen, F. (1961) Fed. Proc. 20,941-955
4. Nixon, J. E., Phillips, G. T., Abramovitz, A. S., and Porter, J. W .
(1970) Arch. Biochem. Biophys. 138,372-379
5. Smith, S.,Agradi, E., Libertini, L., and Dileepan, K. N. (1976)
Proc. Natl. Acad. Sci.U.S. A . 73, 1184-1188
6. Kumar, S., Dorsey, J. A,, Muesing, R. A., and Porter, J. W . (1970)
J. Biol. Chem. 245,4732-4744
7. Maitra, S.K., and Kumar, S.(1974) J. Biol. Chem. 249, 111-117
8. Maitra, S.K., and Kumar, S.(1974) J. Biol. Chem. 249, 118-125
9. Strom, K. A., Galeos, W . L., Davidson, L. A,, andKumar, S.
(1979) J. Biol. Chem. 254,8153-8158
10. Strom, K. A,, and Kumar, S . (1979) J . Biol. Chem. 254, 8159-
8162
11. Burton, D. N., Haavik, A. G., and Porter, J. W . (1968) Arch.
Biochem. Biophys. 126, 141-154
12. Stoops, J. K., Ross, P., Arslanian, M. J., Aune, K. C., Wakil, S.J.,
and Oliver, R.M. (1979) J. Biol. Chem. 254, 7418-7426
13. Noyes, B. E., and Bradshaw, R. A. (1973) J. Biol. Chem. 248,
3052-3059
14. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82.70-77
15. Nandedkar, A. K. N., andKumar, S. (1969) Arch. Biochem.
Biophys. 134, 563-571
16. Majerus, P. W . ,Alberts, A. W . ,and Vagelos, P. R. (1965) J. Biol.
Chem. 240,618-621
17., .Pullman, M. E. (1973) Anal. Biochem. 54, 188-198
18. Warburg, O., and Christian, W . (1957) Methods Enzymol.3,451-
454
19. Bradford, M. M. (1976) Anal. Biochem. 72,248-254
20. Decker, K. (1963) in Methods ofEnzymatic Analysis(Bergmeyer,
H. V., ed) pp. 425-428 Academic Press, New York
21. Brady, R. O., Bradley, R. M., and Trams, E. G. (1960) J . Biol.
Chem. 235,3093-3098
22. Lynen, F., Oesterhelt, D., Schweizer, E., and Wellecke, K. (1968)
in Cellular Compartmentalization and Control of Fatty Acid
Metabolism (Gran, F. D. ed) pp. 1-24, Academic Press, New
York
23. Phillips, G. R., Nixon, J. E., Dorsey, J. A., Buttenvorth, P. H. W . ,
Chesterton, C. J., and Porter, J. W . (1970) Arch. Biochem.
Biophys. 138,380-391
24. Robinson, J. D., Bradley, R. M., and Brady, R. 0. (1963) Bio-
chemistry 2, 191-194
25. Wakil, S.J. (1961) J. Lipid Res. 2, 1-24
26. Lin, C. Y., and Kumar, S.(1971) J. Biol. Chem. 246,3284-3290
27. Katiyar, S. S .,
Briedis, A. V., and Porter, J. W . (1974) Arch.
Biochem. Biophys. 162,412-420
28. Linn, R. C., Stark, M. J., and Srere, P. A. (1980) 1.Biol. Chem.
255, 1388-1392
29. Smith, S. (1973) Arch. Biochem. Biophys. 156, 751-758
30. Lynen, F. (1969) Methods Enzymol. 14, 17-33
31. Stulberg, M. P., and Novelli, G. D. (1962) Methods Enzymol. 5,
703-707
32. Abdinejad, A,, Fisher, A. M., and Kumar, S.(1981) Arch. Bio-
chem. Biophys., in press
 Reducfion by Fatty Acid Synthase
Acetoacetyl- CoA
Supplemental Materialt o . Acetoacetyl-CoA Reductase of Lactatlng Bovlne hlammary Fdtfy
ACtivlty
A d d Synthase by F. Dadds. M . Cuadalupe F. Guman. Stephen C. ChaIberg,
Peter
Gregory
Anderson Soma Kumdr.
and
EXPERIMENTAL PROCEDURES
Materials
Fatty acld synthase from lactatlng bavlne mammary gland was prepared by
the modifled procedure Of Strom et a l . 19). and rat llver by the method of
Burton et dl. and from rat GiZTdymal tl5sUe by the method Of Stoops
s.I 1 2 K T h(11)
esynthase purified from rat mammary gland. pigeon liver. goose
uropyqlal gland. chicken liver and yeast were generous glfts from Drb. Stuart
Smith, John W. Porter, P. C. Kolattukudy, Vasudev C . Joshi and Sallh J. Wakil.
respectively. Fresh bovlne m a n n a r y synthase had a synthase activity of 600-
1000 unlts per mq protein 191. It decllned somewhat on prolonged storage.
The preparation and assay of acetoacetyl pantetheine were according to
the procedures described by Noyes and Bradshaw 1131. For the preparatzon of
pantetheine, 100 mq Of pantethlne was dissolved ~n 25 ml Of H20. A 5 nl
aliquot of thls wa9 removed and brought to pH 8.5 with 5 mM NaOH, then reduced
wlth approxlmtely 300 mg of ptasniumborohydride added in small portions over
a period Of 30 m m . After adlusting the pH to 1.8 wlth O.IN HC1, al~quotswere
removed and the free sulfhydryl content determined by the method of Ellman 114).
From this the pantethine ConCentratlon Of the stock solution was calculated.
P a m e t h e m e "Sed in enzymnatlc assays was obtained by reducing an aliquot of the
32908 gas chromtoqraph fltted Wlth a glass column I180 x 2 m m, I.D.1 contaln-
m g 10% SP-216-PS on 100/120 mesh Supelcoport Isupelco, Inc.1 was Used. Hellurn
J.
at 25 ml/min was used as carrier gas. The column ove was held at 115'C for 2
min and then the temperature was increased at 8'C m i n - ? to 195-Cwhere it was
held Until the completion of the run. The effluent qas was spllt With about
10% going to the flame ionlzation detector and the remamder to a Packard 894
gas proportional counter, where It was oxrdiaed to carbon diaxlde and wafer
vapor in a copper oxide furnacg. The water vapor was then reduced to hydrogen
gas by steel w o e 1 in a second furnace. The tritium content of the hydrogen
gas thus prodmeed was determined in the gas flow proportional tube. The out-
puts from this and the f l a w ionlzation detector were mnitored on a dual pen
recorder (Linear Instruments). Peak lntegratmn of the radmactlve trace was
performed uslnq an Autolab Mlnlgratoe (Specfro-Physics).
Analysis of PzOduCtS of the InCorpoIatlOn of 12-14C1Malonyl-CoR
The assay mixture contained. 2 n 0.5 ml. 100 mn potasrlwn phosphate, pH
7.0. 0.1 EDTA, 5 IRM dithlothreitol, 5 mM NADPH, 0.5 mM prlmr acyl-CoA.
1 & 12-1$lmalonyl-CoA I1 uCl/umol) and about 15 ug Of fatty acid synthase.
The appropriate inqredlents were premcubated at 37- far 10 m l n and the re-
action inltlrted by the additron of t h e primer followed lmmedlately by that of
malonyl-Cod. After 12 min the reactlon was stopped by the addlrlon of 10 . )I
of 10 M KOH. A 10 nl aliquot of a standard mixture of c a ~ r l e racids contalnrng
even number of carbon atoms of 4 to 18 and of 13 were added. The tubes were
heated at 95' for 2 h, Cooled, acldlfled with 20 u1 Of 6.5 M H2S04 and ex-
tracted four times wlth 2.5 ml of pentane. Malonic acid is totally excluded
from thlS extract. A one-ml aliquot was used for d e t e m m l n g the total l n -
corporatmn Of radmactlnty. The remainmg w a s Sufflclent for duplicate de-
terminations of the composition. Each allquot was reduced ~n volume under NZ
to 10 "1 at -10' to -15' (to minlrnlze the loss of bUtyrlc and hexanoxc acids]
and taken up in a 50 "1 syringe. A 10 111 aliquot used for washings was taken

-
stock pantethlne 501Yt10n with an equimolar a m u n t of dithiothreitol. Since UP in the same syringe and the combined mlxture used for gas chromatography.
the lncubatlon mixture COntalnS a Vast excess Of dithiothreitol, the pante- The analysla was as descrzbed for the products Of CoA-5-AcAc redoctmn exce t
theine concentration was calculated assumxng complete reduction of pantethine. that Water produced In the CUO furnace WEIS absorbed zn a P p , trap and the lfC
content of the CO was measured without prior passage through the Second
DL-3-hydroXybutyriC acid (si-) resOlYed and the 02Aester-B O f 01-1 and (steel Y O 0 1 1 f d e e . The losses I" Cq and Cg acids were determlned relative
Ll+l isomerlc aclds were prepared and assayed a3 descrrbed previously (15). to C13: acid from the m 5 8 trace and appropriate corrections applled to the
All the other substrates and Cofactors were obtained from conmercial source^. areas of the radmacrlve peaks.
The concentration Of the solutions of the various COA esters was determlned
SpeCtTOphotDmetrlCllly from their respective w l a r absorbancies at 260 nm (9). Dissocxat1on and ReasSoCiatlOn of Fatty ACld Synthase
Determlnatlon of Enzyme Activities and Protein Concentrations. Samples Of fatty acid synthase solUtiOn5 Were dlalyzed against 120
Volumes Of a SolutlOn consistlng Of 10 mH TZ11-35 mM glyckne. pH 8.5, 1 mM
Fatty acid synthase actlvity refers to the malonyl-CoA and acetyl-CoA dlthiothreltol, 0.1 ny( EDTA for 24 h wlth One change of buffer 16). Thls
dependent Synthesis Of fatty acids and unless othewlse Stated employed the treatment dissociates the bovine mammary enzyme 1 8 1 . For reassoc~atron,the
standard Spectrophotometric OT radiochemical assays (7,9!. For the study of diSSoClated enzyme was filtered through Sephadex G - 5 0 , equllibrated with 0 . 2 M
the reductlo" of COAL-S-AcAc,the standard incubation mixture contained. in a Pota981Um phosphate buffer, pH 7.0, containrng 1 mM dlthmthreitol and 0.1 mM
total Volume Of 1.0 ml, 0.1 M potassium phosphate buffer. pH 7.6, 1 mM dithio- EDTA 181. The proteln peak was collected and adlusted to Obraln 10 m~ dithlo-
threitol, 50 yM COR-S-AcAc. 125 UM NADPH and a b u t 5 LJg of the enzyne. After threitol.
temperature equilibration at 37-for 10 mi" the reaction was initiated by the
addltlon of the enzyme and the lnitial veloclty determined from the decrease
in absorbance at 3 4 0 n
m.
Tvo methods were employed for the assay of reversible 3-hydroxybutyryl-CoA
dehydratase activltles. In the first, the formation Of CrOtOnyl-COA was fol-
lowed by mnitorlng the change In A280 according to the procedure of Majerus
g. 1161. Th16 reaction Could be assayed in the reverse direction by fol-
lowing the disappearance (due to hydration) of crotony1-coA a190 from changes
~n A280. In the second method the dehydration Of 3-hYdroXybUtyryl-CoA was
coupled to the NADPH dependent-reduction by the enzyme of the Crotonyl-COA
formed. For thls the assay mixture. in a f i n a l V D l W 1 ml, contained 50 mI4
blclne, pH 8 . 3 , 1 mn dithlothreltol, 125 YM NADPH, 100 YM EDTA, 5 P q enzyme
and the m d x a t e d concentrations of Dl-l-3-hrdroxybutyrvl-c~A.
Far the assay of the transacylase reaction laCy1-COA:fatty acid synthase
acyltransferase1 an assay was developed which did not involve the use of la-
belled acyl-CoA's. The princlple of the assay depends On the transfer Of the
acyl group from the acyl-CoA to free trltisted coenzyme A aecordmq tothe
equatlonsi
Acyl-S-COA + Enr P Acyl-Enr + CoA (11
Acyl-Enz t 13HICoA-SH ~ A c Y ~ - S - [ ~ H I CtO AEnz 12)
Net: Acyl-S-CoA + 13HlCoASH dAcyl-S-13HlCoA + CoASH 13)

TABLE VI1

dehydratase actlvtv
Reversible Dl-~-~-hydroxybuty~~lyl-Col\

Protem concentration was determined by the method of Warburg and Chrlsti-

an (18) or alternately by the dye bindrng method using pmre fatty acld synthase The dehydratlon Of Dl-I-B-hydroxybutyryl-CoA and hydratlon of trans-crotonyl-
as standard (191. Unless Other activity IS stated the specific activity of the CoA were measured from the appropriate changes in absorptmn a m nm. 1.0 m l
enzyme is glven In terms Of malonyl-CoA and acetyl-coA dependent NADPH oxida- Of the incubation contalned 10 ug of fatty acld synthase.
tlon. One Unlt Of enzyme is the amount that oxidizes 1 -1 of NRDPH per m n -
Ute under standard assay conditions. Actlvlty
Experiment
Stoichiometry of CoA-S-AcAc Reduction NUmber Evffer substrate (nmol.m~n-l.mg-l~

The reactlon was Carried Out in a Cuvette containing, ~n d total Volume 1 0.1M K-phosphate 3-fOHlbutyry1-C~A 30
Of 1 m l , 0.1 M potassium phosphate buffer, pH 6.8, 1 dlthiothreltol. 0.1 mM pH 6 . 8 125 U M
EDTA and the amounts of COA-S-AcAC, NADPH and the enzyme Shown in Table 1. The
amount of NADPH consumed at the completion Of the reaction was determined from 2 0.1M K-phosphate 3-IOH)butyryl-CoA 45
the change In absorption at 340 nm and assuming c 6.22 x 103M-1cm-1. Three
I pH 6.8 125 :IM
allquots were removed from each cuvette for independent determinations of the
concent-"10n of COA-S-ACAC r e m r n l n g . The flrst, an enzymic derermlnation, 3 0.1M K-phosphate Crotonyl-CoA 105
used L~i-h',droxyacyl-CoA:Nl\g+ OXldoreduCtase IE.C.1.1.1.351 ,sing the method pH 6.8 100 Y M
descrrbed by Decker 1201. For the Other two, allqUOtS were added to a solution
Of 0.1 M TI15 buffer contalnlng 0.1 mM EDTA and 20 mM MgC12 at pH 7.95 or pH 4 0.1M BlClne Crotonyl-CoA 80
8.16. The dbSorbance of the resultant Mq-enolate complex w a s measured at 303 pH 8 . 3 80 bM
nm. The E303 at pH 7.95 f r his complex was found to be 15.7 X 103M-lcm-l and
at pH 8.16 to be 20.9 X 10SM-EcmK1.
The Products Of ReduCtlOn Of COA-S-ACAC and NACCySt-S-AcAC
A system generatrng 13HINADPX, slmllar to that descrlbed by Brady et a l .
(211, vas used to enable the ldentiflcdtlon of the products of the reduzioy
of acetoacetyl th101esters. Incubations contalned, ~n d t o t a l volume of 0.1 ml,
0.1 M POtassrUm Phosphate buffer pH 7.6: 5 mW dlthlothrertol, 4 mM MgCl2: 4 mM
ATP: 120 vM NADP , 4 mM f 1 - 3 H 1 giuco3e ISpeClflC acltivlty 5 mCi/mmol): 0.3 rng
hexokinase IE.C.2.7.1.II glucose-6-phosphate dehydragenase'lE.C.1.1.1.491 mix-
ture (Boehrrnger): 0.5 mM CoA-S-AcAc or 20 mM NACCySt-S-AcRC, and 30 u q of
fatty a c l d synthase. The Control contained no ACAC thlol ester. The reactmn
mixture w a s prelncubated at 37'C for 5 m l n to e n s ~ r ethe reduct-on of N m P + and
the reactlo" was lnltlated by rhe addltlon of AcRc thlol ester and fatty acld
synthase. After 1 5 min the r e a c t i o n was stopped by the additlon of 50 "1 of
10 M KOH and heated to 90- for 1 0 mrn to hydrolyse the thiol esters. Carrier
butyrrc and 3-hydmxybutyr1~a c i d s were added, the mixture acldrfled and ex-
rrdcted, u s r n g small a l l q w t s , Into 1 2 ml of diethylether containlng 108 n-
pentane by volume. The extract, containlng over 95% of each acld was drled
over anhydrous Na2SOb and a portlon concentrated very slowly to 1 6 ul under a
Stream of N2 I" a salt-rce bath l-lO'l. Thls fraction combined With 10 91
warhlngs was used for analysls by gas-lrquld chromatography. A Perkln-Elmer
6290 Acetoacetyl-CoA Reduction by Fatty Acid Synthase

Flg. 2 The e f f e c t s o f pH on t h er e d u c t l o " o f a c e t o a c e t y l derivatives by f a t t y

= a p t
or the bu f e r used. o"--o, 5 0 UM COR-S-ACAC i n 100 mn
Lho8phate;f0"0, 50 irM CaA-S-AcAc i n 100 llLY TriS-HC1: O?:?r rM4
NACCVSt-S-ACAC ~n 103 ;nH P h o w h a t e ; and A- b . 2 0 mn NAcCYet-S-AcAc

250

" 200

-
'@

: r
rl
IC 150
E

-g
I

2 100

' 50
1
L I
100 200 300
1 J

0.01 0.02 0.03 0.04

[D-3-Hydro~vbutyrvl-COA] !Jfl-'