BIOCHIMICA ET 3ZCG?IW3SCA ACTA 445 BBA jjzp FATTY ACID BIOSYNTHESIS III, INTRACELLULAR SITE OF ENrSZYMESTN LACTATION-~AE~IT MAMMARY GLAND I, SubcelIular fractions of lactating-rabbit mammary glands have been char- lacterised by the use of “marker” enzymes and these fractions were then examined for the presence of enzymes involved in fatty acid synthesis. 2. Acetate : CoA bgase (EC 6.2.1 .I) was found to be confined to the particle-free supernatant fraction, whereas acetyl-CoA carboxylase (EC S.4,x.a) was distributed between the m~crosomal and the particle-free supernatant fractions, Fatty acid syn- thetase was almost entirely situated in the particle-free supernatant fraction. &tuch lower levels of acetyl-CoA carboxylase and no fatty acid synthetase were observed in the mitochondrial fraction The implications of these findings are discussed. 3* Acetyl-CoA carboxylase is the rate-limiting step in fatty acid synthesis in this tissue under the conditions used. The intracellular site of individual. enzymes involved in fatty acid biosynthesis has been investigated for several tissues. However, few attempts have been made to characterise these sites by the use of “marker” enzymes** so as to determine the arigjn in the tissue of these enzymes. Many early experiments (see review by WAKIL~) showed that fatty acid bio- synthesis from acetate uia the malonyl-CoA pathway could be carried out by particle- free preparations from a number of tissues. Using subcellular fractions from rat liver, microsomes stimuIated the incorporation by particle-free supernatant of acetyl-GA, Abbreviation: INT, z-tP-iadophen~~)-3-ip-Ilitrophenylf-5-phenyltetrazoIium chloride. * Present address, Department of Biochemistry, Hadassah Medical School, P.O. Box rzpz, Tha Hebrew University, Jerusalem, Israel. ** The term “marker” enzymes refers to these enzymes whose intraceBufar site of activity has been established in a number of tissues.
Transcript
BIOCHIMICA ET 3ZCG?IW3SCA ACTA 445
BBA jjzp
FATTY ACID BIOSYNTHESIS
III, INTRACELLULAR SITE OF ENrSZYMES TN LACTATION-~AE~IT
MAMMARY GLAND
I, SubcelIular fractions of lactating-rabbit mammary glands have been char- lacterised by the use of “marker” enzymes and these fractions were then examined for the presence of enzymes involved in fatty acid synthesis.
2. Acetate : CoA bgase (EC 6.2.1 .I) was found to be confined to the particle-free supernatant fraction, whereas acetyl-CoA carboxylase (EC S.4,x.a) was distributed between the m~crosomal and the particle-free supernatant fractions, Fatty acid syn- thetase was almost entirely situated in the particle-free supernatant fraction. &tuch lower levels of acetyl-CoA carboxylase and no fatty acid synthetase were observed in the mitochondrial fraction The implications of these findings are discussed.
3* Acetyl-CoA carboxylase is the rate-limiting step in fatty acid synthesis in this tissue under the conditions used.
The intracellular site of individual. enzymes involved in fatty acid biosynthesis has been investigated for several tissues. However, few attempts have been made to characterise these sites by the use of “marker” enzymes** so as to determine the arigjn in the tissue of these enzymes.
Many early experiments (see review by WAKIL~) showed that fatty acid bio- synthesis from acetate uia the malonyl-CoA pathway could be carried out by particle- free preparations from a number of tissues. Using subcellular fractions from rat liver, microsomes stimuIated the incorporation by particle-free supernatant of acetyl-GA,
Abbreviation: INT, z-tP-iadophen~~)-3-ip-Ilitrophenylf-5-phenyltetrazoIium chloride. * Present address, Department of Biochemistry, Hadassah Medical School, P.O. Box rzpz, Tha Hebrew University, Jerusalem, Israel. ** The term “marker” enzymes refers to these enzymes whose intraceBufar site of activity has been established in a number of tissues.
446 S. SMITH, D. J. EASTER, R. DILS
but not of malonyl-CoA, into fatty acids a. Though no evidence’ could be obtained for the presence of acetyl-CoA carboxylase (acetyl-CoA : CO, ligase (ADP), EC 6.4.1.2) in the microsomal fraction, their stimulative effect could be imitated by the addition of purified acetyl-CoA carboxylase (cited in ref. 3.). More recently, the role of the microsomal fraction in esterifying the products of fatty acid synthesis has been in- vestigated3. The products of fatty acid synthesis have been found to inhibit acetyl-CoA carboxylase4,5.
WAKIL~ has shown that a mitochondrial fraction from pigeon, rat or beef liver can synthesise fatty acids from acetyl-CoA by elongation of existing fatty acids. This pathway does not involve malonyl-CoA. However, H~LSMANN and coworkersg-s have claimed that fatty acid synthesis could take place via the malonyl-CoA pathway in mitochondria from several tissues.
We have investigated the intracellular origin of the enzymes involved in fatty acid biosynthesis in lactating-rabbit mammary gland.
MATERIALS
NaHl*CO, was obtained from the Radiochemical Centre, Amersham, England, and Triton X-100 from Lennig Chemicals Ltd., Jarrow, England. z,5-Diphenyloxa- zole and r,4-bis-2-(5-phenoxazolyl)-benzene were purchased from Nuclear Enter- prises Ltd., Edinburgh. Bovine serum albumin, glutathione, ATP, CoA and pyridine nucleotides were obtained from Sigma Chemical Co., London, or from Boehringer and Soehne, Mannheim, Germany. The dye INT and yeast RNA were obtained from British Drug Houses, Poole, England; indoxyl acetate from Koch-Light Laboratories Ltd., Colnbrook, England, I-naphthyl th~idine-5‘-monophosphate from Sigma Chemical Co., London, and the dye Fast-Blue R.R. from G. T. Gurr Ltd., London.
METHODS
Pre+ration of acetyl-CoA This was freshly prepared for each set of assays using a modification of the
method of OCHOA~. 20 mM CoA, 0.33 mM KHCO, and 0.33% (v/v) redistilled acetic anhydride were incubated at o0 for 5 min. After acidification to pH 2 with HCI, the mixture was brought to room temperature. The yield was 95-1000/; based on disap- pearance of free sulfhydryl groupslO.
Preparation of malonyl-CoA This has been described previouslyll.
This has been described previously rlsl).* Where indicated in the text particu- late fractions were washed by resuspending in 20-25 ml 0.3 M sucrose, centrifuging at the same speed as for their preparation and the washings added back to the ma- terial still to be fractionated.
* The conditions for centiifugation were based on those of SEDGWICK AND H~~BSCHER~~ for rat liver.
Bioch&. ~~0~~~5. Acta, 125 (1966) 445-455
INTRACELLULAR SITE OF FATTY ACID BIOSYNTHESIS 447
Preparation of subcellular fractions from rat liver
Rat liver was homogenised in approx. 15 ml per g wet weight of 0.3 M sucrose in a Potter-Elvehjem Teflon-glass homogeniser at 600 rev./min for twelve “up and down” strokes. After straining through cotton wool, cell fractions were prepared as described previously for lactating-rabbit mammary gland 11-13.
Characterisation of subcellular fractions
For this, “marker” enzymes were assayed as follows: Succinate dehydrogelzase (Succinate : INT oxidoreductase EC 1.3.99.1) was as-
sayed by the method of PENNINGTON’~, except that 0.2 mM EDTA was added to the incubation system and 0.1-0.3 ml of methanol was added to each cuvette to prevent cloudiness due to water. This enzyme was regarded as a marker for mito- chondrial membranes14.
Isocitrate dehyclrogenase (Ls-isocitrate : NADP oxidoreductase (decarboxylating), EC 1.1.1.42) was assayed by the method of OCHOA’~ as a marker for soluble intra- and extramitochondrial fractions 16.
6-Phosphogluconate dehydrogenase (6-phospho-D-gluconate : NADP oxidoreduc- tase (decarboxylating) EC x.1.1.44) was assayed by the method of GLOCK AND MCLEAN I7 as a marker for the particle-free soluble fractions 17.
Phosphodiesterase I (thymidine-5’-monophospho-I-naphthyl ester r-naphthyl- hydrolase, EC 3.1.4.1.) was used as a possible microsomal marker enzyme”‘, and was assayed by coupling the liberated I-naphthol with Fast Blue RR. It was found neces- sary to employ a reference curve using I-naphthol as a standard (SMITH AND DILS, unpublished results).
Ali-esterase (Carboxylic ester hydrolase, EC 3.1.1.1) was used as a microsomal marker for liver’@ and was assayed by a modification of the method of UNDERHAY et al.ls.
Cystine reductase 2o (NADH: L-cystine oxidoreductase, EC 1.6.4.1) and glucose- 6-phosphatase a1 were assayed as possible microsomal markers in mammary gland. The latter enzyme was assayed by the method of H~~BSCHER AND WESTON.
Assays of enzymes involved in fatty acid synthesis
Acetate: CoA ligase (AMP) (EC 6.2.1.1). The formation of acetyl-CoA from acetate was assayed by the hydroxamate method of LIPMANN AND TUTTLE~~. Incu- bations contained (final volume 1.0 ml) : 150 mM potassium phosphate (pH 6.6), IO mM ATP, 3.3 mM MnCl,, 0.15 mM CoA and 200 mM of hydroxylamine hydrochloride neutralised to pH 6.6 with NaOH, and protein. After incubating for 30 min at 37”. the reactions were stopped with 2 ml FeCl, reagent (0.37 M FeCl,, 0.2 M trichloroacetic acid and 0.66 M HCl). Denatured protein was removed by centrifugation and A,,,,, of the supernatant determined. Control assays with no added CoA showed no enzyme activity; a control value, obtained by omitting acetate from the incubation medium, was subtracted in all cases.
Acetyl-CoA carboxylase (acetyl-CoA :CO, ligase (ADP), EC 6.4.1.2). Assay meth- ods for this enzymeZ4-27 are frequently based on [14C]bicarbonate fixation by acetyl- C~Ainto[~~C]malonyl-CoA.Details of the assaywere establishediusingthe fractionpreci- pitated at o-25 y0 saturation with (NH,) $0, from the particle-free supernatant. Incu-
448 S. SMITH, D. J. EASTER, R. DILS
bations contained (final volume x.0 ml) : IOO mM potassium or sodium phosphate pH 7.25-7.35, IO mM ATP, 3.3 mM MnCl,, 0.2 mM acetyl-CoA, 25 mM potassium citrate, I mg bovine serum albumin, IO mM GSH, IO mM NaH14C0 3 (I PC /pmole)and enzyme fraction (zo-IOO ,ug protein). Two control assays were always used, one without added protein, the other with no added acetyl-CoA. After incubation for IO min at 37O in stoppered centrifuge tubes, the reaction was terminated with 0.1 ml of 5 M perchloric acid. Malonic acid (0.1 ml of 0.1 M) was added, the tubes vigorously shaken for I min, and centrifuged at maximum speed in an MSE “Minor” bench centrifuge, with stoppers removed. 0.1 ml of 0.1 M KHCO, was added, the tubes reshaken to flush out remaining radioactive bicarbonate, and recentrifuged. The flushing-out procedure was repeated. The level of unreacted [%]bicarbonate was reduced to less than IO-~% of the original level, with complete recovery of added malonic acid.
r.o-ml portions of the flushed incubation mixtures were dissolved in IO ml of Triton X-Ioo-xylene phosphor (I : 2, v/v) and counted at 70-75 y0 efficiency 28 using a Nuclear Chicago Three-Channel Automatic Liquid Scintillation Spectrometer.
The xylene phosphor used in these experiments was 0.6% of z,5-diphenyloxa- zole (w/v) and 0.12 o/0 of r,4-bis-z-(5-phenyloxazolyl)-benzene (w/v) in A.R. xylene.
The specific activity (S.A.) of the enzyme is expressed as mpmoles bicarbonate incorporated per mg protein per h.
Fatty acid syntketase: Incubations contained (final volume 2.6 ml): 385 mM potassium phosphate (pH 6.6), 0.35 mM malonyl-CoA, 0.15-0.38 mM NADPH, 2 mM GSH and protein. The reaction was followed by observing the decrease in A 34,,m,,. A control assay omitting malonyl-CoA was used.
Protein. This was determined as described previouslyll. RNA. This was estimated by the method of SCHNEIDER~~, using yeast RNA as
a standard.
RESULTS
Characterisation of sub-cellular fractions of lactating-rabbit mammary gland Nuclear fraction: Because of the large amount of cell debris and connective
tissue which was unavoidably included in a mammary-gland homogenate, the nuclei were excluded from this study. The nuclei-free homogenate contained no detectable DNA.
Mitochondrial fraction : 80 y. of the succinate dehydrogenase activity (Table I) was recovered in the 5 - 104 x g. min sediment, which was therefore regarded as the mitochondrial fraction. The particle-free supernatant fraction contained relatively little activity (2%). The rather large SD. of the recovery is due to the fact that in one preparation only 56% recovery was obtained. Subcellular fractions from this preparation were stored for 8 days at -20’ before assay of succinate dehydrogenase, whereas the other three preparations were assayed fresh. The succinate dehydrogenase :activity in the mitochondrial fraction was not removed by washing (see Table II). The distribution of succinate dehydrogenase we have obtained is in contrast to that reported by H~LSMANN AND Dow 8, whose mitochondria seem to have suffered some damage as judged by the cytochrome oxidase (cytochrome c:O, oxidoreductase, EC 1.9.3.1) distribution. The mitochondrial preparations were not without isocitrate de- hydrogenase (Table I) which is thought to be a soluble intra- and extra-mitochondrial
KNTRACELLULAR SITE OF FATTY ACID BIOSYNTHESIS 449
TABLE I
DISTRIBUTION OF PROTEIN, RNA AND MARKER ENZYMES IN LACTATING-RABBIT MAMMARY GLANDS
All values given are for unwashed fractions and represent percentage total activity (or protein or RNA) recovered. The recovery column expresses the total activity (or protein or RNA) re- covered as a percentage of the activity (or protein or RNA) found in the nuclei-free homogenate. Where sufficient results are available, results are expressed as mean -& S.D. The figure in paren- theses represents the number of experimental fractionations performed.
dehydrogenase 3.1 $: 2.6 2.9 * r.1 1.8 + 0.9 92.4 ic 3.9 94.2 i 7.3 (5)
Protein 15.3 i: 2.4 ‘5.4 + 3.8 19.3 -& 2.6 49.8 & 6.r 98.6 f 7.5 (8)
enzyme. Furthermore, we found, as did LOWENSTEIN and co-workersso, that the mitochondrial fraction showed an increase (2.8 fold) in isocitrate dehydrogenase ac- tivity after exposure to hypotonic sucrose, indicating that during preparation the mitochondria have not been damaged in such a way as to have lost all their soluble intramitochondrial enzymes. The mitochondrial fractions referred to in the text were stored in 0.3 M sucrose. The mitochondrial fraction contained only 8% of the RNA, 13% of the phosphodiesterase I and 3.1 o/o of the 6-phosphogluconate dehydrogenase activity recovered. Most of the 6-phosphogluconate dehydrogenase activity associated with this fraction was removed after one wash (see Table II). A large proportion of the phosphodiesterase I activity could be removed after two washes (Table II).
~o~~~s~~e fruc~~o~~ This fraction sedimented at 2.6 - xo6 x g * min and con- tained some of the marker enzymes for both mitochondrial and microsomal fraction (Tables I and II).
Microsomal fraotion: Little or no glucose 6-phosphatase, cystine reductase or NADf nucleosidase activities were detected in this fraction. The RNA distribution (Table I) was similar to that obtained by SLATER AND PLANTEROSE 31 in lactating-rat mammary gland. The RNA:protein ratio in the microsomal fraction was not de- creased on washing. Phosphodiester~e I is the only suitable microsomal marker we have found for this tissue as yet. The activity of this enzyme was confined exclusively to the particulate fractions and that of the microsomal fractions was not decreased by successive washing procedures (see Table II). The microsomal fraction contained small amounts of succinate dehydrogenase, isocitrate dehydrogenase and 6-phos- phogluconate dehydrogenase (Table I). Almost all of the latter enzyme could be removed by a single washing (Table II).
Particle-free ss@ernatant fraction : 6phosphogluconate dehydrogenase activity, which was taken as the index of soluble extr~itochondrial enzymes, was recovered almost exclusively in the 6.2 . 10s x g = min supernatant (Table I). This fraction was relatively free from contamination by mitochondrial membranes and contained no phosphodiesterase I, the microsomal marker.
Biochim. Biophys. Acta, 125 (1966) 445-455
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INTRACELLULAR SITE OF FATTY ACID BIOSYNTHESIS 4.51
Intracellular site of enzymes of fatty acid synthesis
Acetate:CoA .&use (AMP) : This enzyme has been found in the mitochondria of certain tissuess2, but in two mammary gland preparations was mainly confined to the particle-free supernatant fraction (see Table II for one of these experiments).
Acetyl-CoA carboxylase: Using four separate preparations, this enzyme was located predominantly in the microsomal and particle-free supernatant fractions (Table II). In six out of seven separate preparations, the specific activity of the mi- crosomal enzyme was 3.5-6.0 times higher than that of the particle-free supernatant enzyme. In the seventh preparation the specific activities were approximately equal. A typical distrubution of this enzyme is given in Table II.
Marker studies showed that the microsomal acetyl-CoA carboxylase activity could not be explained by contamination of this fraction with particle-free super- natant, since using five separate preparations, the level of phosphogluconate dehy- drogenase activity associated with the microsomal fraction was only 1.8 f o. 9%
(Table I) and could be completely removed by a single washing (Table II). However, the activity of the microsomal acetyl-CoA carboxylase was only partially removed by the washing procedure in four separate experiments. The actual amount of activity removed by washing varied considerably from one preparation to another.
The distribution of succinate dehydrogenase shows that any contamination of the microsomal and particle-free supernatant fractions by mitochondrial membranes cannot account for the acetyl-CoA carboxylase of these fractions (Tables I and II). When mitochondrial fractions were assayed for acetyl-CoA carboxylase, the activity was consistently low, though high acetyl-CoA substrate blanks were noted. If these blank values were not substracted, the total acetyl-CoA carboxylase activity of this fraction would, at the most, double.
On repeated washing of the microsomal fraction, the microsomal marker en- zyme, phosphodiesterase I, behaved differently to acetyl-CoA carboxylase (Table II). Whereas acetyl-CoA carboxylase could be removed by this procedure, phosphodies-
terase I could not. A comparison of the cofactor requirements for optimum activity of the acetyl-
CoA carboxylase(s) of the microsomal and particle-free supernatant fractions has re- vealed only minor differences so far.
Fractionation of the particle-free supernatant acetyl-CoA carboxylase by am- monium sulphate precipitation showed that 80% of this activity could be recovered in the o-25 o/o saturation fraction, with an r8-fold increase in specific activity.
Acetyl-CoA carboxylase distribution in rat liver : In view of the unexpected finding of acetyl-CoA carboxylase of high specific activity in the microsomal fraction of lactating-rabbit mammary gland, two subcellular fractionations of rat liver were car- ried out (see METHODS).
The distribution of acetyl-CoA carboxylase however was quite different from that found in mammary gland (compare Tables II and III). No activity was detected in the microsomal fraction and most of the activity was recovered from the particle- free supernatant fraction. This was as might have been expected, because this enzyme was first purified from pigeon-liver particle-free supernatant 33 and the work of ABRA- HAM et al.a suggested that the microsomal fraction of rat liver is devoid of acetyl-CoA carboxylase activity.
Fatty acid synthetase : Two mammary-gland preparations have been assayed for
B&him. Bio#ys. Acta, 125 (1966) 445-455
452 S. SMITH, D. J. EASTER, R. DILS
TABLE III
SUBCELLULAR DISTRIBUTION OF ACETYL-CO.4 CARBOXYLASE AND MARKER ENZYMES IN RAT LIVER
Each particulate fraction was washed once and the washings added back to the material to be subsequently fractionated. IO-50-,~g portions of protein were used in the assay of acetyl-CoA carboxylase. o/0 figures represent per cent nuclei-free homogenate activity (or protein) recovered. S.A. is specific activity in mpmoles bicarbonate incorporated per mg protein per h.
Fraction Protein Acetyl-CcA carboxylase Succinate Carboxylic
Mitochondrial fraction ‘4.4 7.8 3.9 62.5 Composite fraction 12.0 3.4 2.0 14.5 Microsomal fraction 25.3 0 5.8 Particle-free supernatant 46.0 40.3 Si.0 2.4 -__- Per cent of the NFH
activity recovered 97.7 94.9 85.2
100
5.3 II.7 72.0
4.9
Phosfiho- gluconate dehydro- genase (%!
___- 100
0
0
0
9s
93.9 98
synthetase activity. In both experiments the majority of the activity was located in the particle-free supematant. The results of one of these experiments are shown in Table II.
In the presence of malonyl-CoA, the rate of oxidation of NADPH was not af- fected by the addition of acetyl-CoA (Fig. I).
Fig. I. Spectrophotometric assay of fatty acid synthetase. Assays contained (in a total volume of 2.6 ml) : 385 mM K,HPO,-KH,PO, (pH 6.6) and 3.1 mg particle-free supernatant protein but noGSH. Additions were: a, 0.38 mM NADP+; b, 0.38 mM NADPH; c, 0.38 mM NADH + 0.35 mM malonyl-CoA; d, 0.38 mM NADPH + 0.35 mM malonyl-CoA; e, 0.38 mM NADPH + 0.35 mM malonyl-CoA + roo antibiotin units* of avidin; f, 0.38 mM NADPH + 0.35 mM malonvl-CoA + 0.1 mM acetyl-CoA.
* I antibiotin unit gives 50% inhibition of growth of La&bacillus arabinosus in the presence of I mpg biotin (see ref. 34).
A possible explanation might be that a malonyl-CoA decarboxylase (malonyl- CoA carboxy-lyase, EC 4.1.1.9) is present in the mammary-gland preparation. This might supply sufficient acetyl-CoA to condense with malonyl-CoA under the condi- tions used in this assay. It is unlikely that the decarboxylation is achieved by reversal of the acetyl-CoA carboxylase, as avidin did not inhibit the oxidation of NADPH
Biochim. Biophys. Acta, 125 (1966) 445-455
INTRACELLULAR SITE OF FATTY ACID BIOSYNTHESIS 453
in the absence of acetyl-CoA (Fig. I). In the presence of malonyl-CoA, NADH was oxidised at a much slower rate than NADPH (Fig. I). The disadvantage of the spec- trophotometric method is that, due to the presence of dehydrogenases in the particle- free supernatant, the NADPH which has been oxidised can be quickly reduced (Fig. I). This resulted in the observed reaction rate being linear with time only for a short period, i.e. while the level of NADP+ was still fairly low.
An attempt was made to assay the synthetase by the incorporation of [3H] CoASAc into fatty acids in the presence of malonyl-CoA and reduced pyridine nu- cleotide. Though synthetase activity was recovered almost exclusively in the particle- free supematant fraction, the specific activity of this enzyme was unexpectedly low. Since this low specific activity is unlikely to be due to loss of tritium label by ex- change 1135, it may be due to a large isotope dilution of the [3H]CoASAc by unlabelled acetyl-CoA formed by decarboxylation of malonyl-CoA. This experiment at least showed that both acetyl-CoA and malonyl-CoA are required for the synthetase.
The rate-limiting step in fatty acid synthesis: The activity of the acetyl-CoA carboxylase was measured in a “microsomal + supernatant” fraction which was also assayed for synthetase activity. This fraction could convert 164 mpmoles HCO,- per mg protein per h to malonyl-CoA and could oxidise 1424 m,umoles NADPH (712 mpmoles malonyl-CoA incorporated) per mg protein per h.
The lowest specific activity of acetate : CoA ligase (AMP) in the “microsomal+ particle-free supematant” fraction from a number of tissue preparations was 430 mpmoles acetyl-CoA formed per mg protein per h; the highest specific activity of acetyl-CoA carboxylase observed in any preparation of this fraction was only 164 m,umoles HCO,- incorporated per mg protein per h.
Since it is difficult to simultaneously assay all the enzymes involved in fatty acid synthesis on fresh material, a comparison was made of their activities in fresh nuclei-free homogenate. This fraction could convert (per mg protein for h) 290 rnp- moles acetate to acetyl-CoA, carboxylate 115 mpmoles acetyl-CoA and oxidise (in the presence of malonyl-CoA) 538 mpmoles NADPH.
These results suggested that acetyl-CoA carboxylase was the rate-limiting en- zyme in the conversion of acetate to fatty acid in this tissue under the conditions used. This is in agreement with GANGULY 36, who found that acetyl-CoA carboxylase was the rate-limiting step in a number of tissues.
DISCUSSION
Our results do not support the theory of H~~LSMANN AND Dow8 that in mam- mary gland, the fatty acid synthetase activity associated with the particle-free super- natant originates in the mitochondria. Whereas fatty acid synthetase was located almost entirely (126 o/o and 85 o/O, two experiments) in the particle-free supernatant fraction, very little succinate dehydrogenase (o-5 %, seven experiments) was found in this fraction.
The distribution of acetyl-CoA carboxylase in this tissue is quite different from that found in liver (compare Tables II and III). Whereas in liver, acetyl-CoA carbo- xylase can be recovered almost exclusively from the particle-free supernatant frac- tion, in mammary gland, most of the activity is associated with the unwashed mi- crosomal fraction. The microsomal fraction in mammary gland has the highest acetyl-
Biochim. Biophys. Acta, 125 (1966) 445-455
454 S.SMITH, D.J. EASTER, R.DILS
CoA carboxylase specific activity and contains only 7% of the total succinate de- hydrogenase activity. The mitochondrial and composite fractions, although richer in succinate dehydrogenase activity than the microsomal fraction, have a comparatively low acetyl-CoA carboxylase activity. It is therefore unlikely that the acetyl-CoA carboxylase activity of the microsomal fraction is derived from disrupted mitochon- dria. On the other hand, it does seem possible that the acetyl-CoA carboxylase activity found in the particle-free supematant might have originated in the microsomes. Washing the microsomal fraction released much of the acetyl-CoA carboxylase ac- tivity, but very little protein and no phosphodiesterase I or RNA, and no phospho- diesterase I was found in the particle-free supernatant. This would seem to indicate that the acetyl-CoA carboxylase activity of the microsomal fraction must be com- paratively loosely associated. It is therefore impossible to say on the basis of these marker enzyme studies whether the acetyl-CoA carboxylase of the microsomal and particle-free supernatant fractions represents two separate enzymes or whether the microsomal enzyme has been partially solubilised during the preparation of the sub- cellular fractions.
Acetate : CoA ligase is located almost entirely in the particle-free supernatant fraction and so this fraction possesses all the enzymes necessary to convert acetate to fatty acid via the malonyl-CoA pathway. However, the microsomal fraction has been shown to stimulate fatty acid synthesis from acetate in this and other tissues. This effect has been locahsed at the level of acetyl-CoA carboxylation3~37 and is thought to result from the ability of the microsomal fraction to prevent a feedback inhibition of fatty acid synthesis by removal of the products37+. We have, in fact shown with rabbit mammary-gland preparations, under the conditions used for fatty acid synthesis from acetate, that the microsomal fraction does esterify the inhibitive end products (previous paper). However, it now seems that in this tissue an alterna- tive or additional explanation may be valid, namely that the addition of the microso- ma1 fraction to the particle-free supernatant system increases the concentration of acetyl-CoA carboxylase, the rate-limiting enzyme in fatty acid synthesis from acetate.
As no acetyl-CoA carboxylase is present in rat liver microsomes, the microsomal stimulation of fatty acid synthesis in this tissue cannot be explained on a similar basis. In this case, the relief of feedback inhibition by esterification of the end products seems at present the most credible theory of action. The microsomal stimulation of fatty acid synthesis in lactating-rabbit mammary gland may be explained in either (or both) ways and the relative importance of each mechanism remains to be demon- strated. The intracellular distribution of enzymes involved in fatty acid synthesis in the iactating-rabbit mammary gland was, to some extent, unexpected. After activa- tion of acetate to acetyl-CoA in the particle-free supernatant, one of the sites of acetyl- CoA carboxylase is microsomal. Though fatty acid synthetase is predominantly in the soluble fraction, the subsequent esterification of the synthesised fatty acids (see pre- vious paper) is again microsomal. The significance of these findings is not yet clear.
ACKNOWLEDGMENTS
We thank Professor A. C. FRAZER for his interest and encouragement in this work. The Medical Research Council of Great Britain provided a Research Scholarship (D. J.E.) and a Scholarship and Fellowship (S.S.) in support of this work. Professor
Biochirn. Biophys. Acta, 125 (1966) 445-455
INTRACELLULAR SITE OF FATTY ACID BIOSYNTWESIS 455
J. GANGULY of the Indian Institute of Science, Bangalore, India, provided useful discussions on the assay and role of acetyl-CoA carboxylase.
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