Biochem. J. (1982) 208, 443-452 443Printed in Great Britain

Studies on acetyl-CoA carboxylase and fatty acid synthase from ratmammary gland and mammary tumours

Patricia M. AHMAD, Douglas S. FELTMAN and Fazal AHMADPapanicolaou Cancer Research Institute, 1155 N. W. 14th Street, P.O. Box 016188, Miami, FL 33101, U.S.A.

(Received 9 June 1982/Accepted 14 July 1982)

The activities of two lipogenic enzymes, acetyl-CoA carboxylase and fatty acidsynthase, were determined in two transplantable mammary adenocarcinomas (13762and R3230AC) carried by non-pregnant, pregnant and lactating rats, and in mammarytissue of control animals (non-tumour-carrying) of comparable physiological states.During mammary-gland differentiation of control or tumour-carrying animals, theactivities of acetyl-CoA carboxylase and fatty acid synthase in the lactating glandincreased by about 40-50-fold over the values found in non-pregnant animals. On theother hand, in tumours carried by lactating dams there were only modest increases(1.5-2-fold) in acetyl-CoA carboxylase and fatty acid synthase compared with theneoplasms carried by non-pregnant animals. On the basis of the Km values for differentsubstrates and immunodiffusion and immunotitration data, the fatty acid synthase ofneoplastic tissues appeared to be indistinguishable from the control mammary-glandenzyme. However, a comparison of the immunotitration and immunodiffusionexperiments indicated that the mammary-gland acetyl-CoA carboxylase might differfrom the enzyme present in mammary neoplasms.

In mammalian tissues the biosynthesis of long-chain fatty acids is catalysed by the sequentialactions of acetyl-CoA carboxylase (EC 6.4.1.2) andfatty acid synthase. Acetyl-CoA carboxylase is abiotinyl-enzyme and catalyses the formation ofmalonyl-CoA (reaction i). Fatty acid synthasecontains 4'-phosphopantetheine, which acts as theacyl carrier during fatty acid biosynthesis. Thisenzyme complex catalyses reaction (ii).

mammary-gland carboxylase into the enzymicallyactive polymer (Lane et al., 1974; Ahmad et al.,1978). Further results suggest that the carboxylaseis also regulated by a phosphorylation-dephos-phorylation cycle. Phosphorylation inactivates,whereas dephosphorylation activates, the inactivecarboxylase (Carlson & Kim, 1974; Lee & Kim,1977; Brownsey et al., 1977; Hardie & Guy, 1980).

Acetyl-CoA + ATP + HCO3- = malonyl-CoA + ADP + P (i)

Acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14H+ --palmitic acid + 8 CoA + 14 NADP+ + 7CO2 + 6H20(ii)

The carboxylase and synthase have been purifiedto apparent homogeneity from rat lactating mam-mary gland (Smith & Abraham, 1975; Ahmad et al.,1978; Ahmad & Ahinad, 1981; Ahmad et al.,1982). The properties of rat mammary-gland en-zymes are similar to those of their counterparts puri-fied from other vertebrate sources (Inoue & Lowen-stein, 1972; Lane et al., 1974; Maitra & Kumar,1974; Smith & Abraham, 1975; Tanabe et al., 1975).Acetyl-CoA carboxylase catalyses the first com-

mitted and rate-limiting step of fatty acid bio-synthesis. Like other vertebrate carboxylases, citrateconverts the essentially inactive protomer of rat

In addition, other mechanisms, such as the ratio ofthe apo- to the holo-enzyme and the rates of enzymesynthesis and degradation, are important factorsthat might influence the flux of acetyl units intolong-chain fatty acids.

Earlier studies by Sabine et al. (1966), Majerus etal. (1968) and Elwood & Morris (1968) foundaberrant regulation of fatty acid biosynthesis in anumber of hepatomas, since dietary manipulationsdid not influence fatty acid biosynthesis in liverneoplasms in a manner characteristic of liver.Neither acetyl-CoA carboxylase nor fatty acid

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synthase of hepatomas was subject to changes inenzyme concentration normally observed in hostliver after alteration of the diet (Majerus et al.,1968). Although this information has been availablefor some time, the precise mechanism of regulationof fatty acid synthesis by dietary manipulations evenin the normal liver is obscure at present. Never-theless the kinetic (affinities for substrates) andheat-inactivation (etc.) properties of a single hepat-oma acetyl-CoA carboxylase were investigated andfound to be similar to those of the enzyme from hostliver (Majerus et al., 1968). However, it is not clearfrom these studies whether the hepatoma and hostliver carboxylases are identical immunologically.

In lactating mammary gland, the conversion ofacetate and glucose into fatty acids proceeds at amuch greater rate than in mouse adenocarcinomas(Bartley et al., 1971; Lin et al., 1975). Theconversion of acetyl-CoA into long-chain fatty acidsproceeds via the reactions catalysed by acetyl-CoAcarboxylase and fatty acid synthase. Since both thecarboxylase and the synthase (especially the car-boxylase) are subject to regulation by a number ofmechanisms, the decreased capacity of mammaryadenocarcinomas to synthesize fatty acids fromacetyl-CoA could result from (a) lower amounts ofthese enzymes and/or (b) the presence of modifiedform(s) of the enzymes that are catalytically lessactive. The present paper addresses some of thesequestions. The results presented here show that,whereas fatty acid synthases of neoplastic andnormal mammary tissue appear to be identical,acetyl-CoA carboxylase of mammary neoplasmsmay be immunologically distinct from the normaltissue enzyme. Some of these data have appeared inpreliminary reports (Ahmad et al., 1976; Ahmad &Ahmad, 1979).

Materials and methods

MaterialsATP, acetyl-CoA, malonyl-CoA, CoA and

NADPH were purchased from P-L Biochemicals(Milwaukee, WI, U.S.A.). Sigma Chemical Co. (St.Louis, MO, U.S.A.) was the source for bovine serumalbumin (essentially fatty acid-free), dithiothreitol,phosphocreatine, creatine kinase (EC 2.7.3.2) and(NH4)2SO4 (enzyme grade). Freund's completeadjuvant was purchased from Difco Laboratories(Detroit, MI, U.S.A.), Ba'4CO3 (58 Ci/mol) wasfrom ICN (Irvine, CA, U.S.A.) and Omnifluor fromNew England Nuclear (Boston, MA, U.S.A.).DEAE-cellulose (DE-52) was obtained from What-man (Clifton, NJ, U.S.A.). All other chemicals wereof the highest purity commercially available. Acetyl-CoA was also prepared by the method of Simon &Shemin (1953). Its concentration was determinedenzymically (Tubbs & Garland, 1969).

AnimalsFemale rats of the Fischer strain were fed on

Purina Laboratory Chow and water ad libitum andkept in quarters that provided for a 12 h-light/12h-dark day. Pregnant rats were housed in separ-ate cages. After delivery, litters were adjusted tocontain eight pups. All animals were killed between09:00 and 10:00 h.

Mammary neoplasmsMammary adenocarcinomas R3230AC (origin-

ally obtained from Dr. Russell Hilf, University ofRochester, Rochester, NY, U.S.A.) and 13762 (fromthe Mason Research Institute Tumor Bank, Wor-cester, MA, U.S.A.) were maintained by sub-cutaneous implant of 1 mm3 pieces into the upperfore- and hind-quarters of female Fischer rats(approx. 200g). When the tumours reached 1.5-2.0cm in diameter animals were killed by cervicaldislocation. Tissue was harvested and processed asdescribed below. Time required to attain desiredtumour growth was 19-21 days for adenocar-cinoma R3230AC and 10 days for adenocarcinoma13762.

Purification of rat lactating mammary-gland acetyl-CoA carboxylase andfatty acid synthase

This was done by the method of Ahmad &Ahmad (1981) and Ahmad et al. (1982). The highlypurified acetyl-CoA carboxylase had a specificactivity of approx. 15units/mg of protein and Mr260 000 by sodium dodecyl sulphate/polyacryl-amide-gel electrophoresis. The specific activity of thefatty acid synthase was approx. 2.0-2.5 units/mg ofprotein when assayed at 370C. Both these enzymesappear homogeneous by a number of criteria(Ahmad et al., 1978; Ahmad & Ahmad, 1981;Ahmad et al., 1982). These preparations were usedfor the production of antibodies.

Preparation of tissue and partial purification ofenzymes

Tumour-bearing (host) and non-tumour-bearing(control) rats were obtained in each of threephysiological states: non-pregnant, pregnant (16-20days) and lactating (13-15 days). TumoursR3230AC and 13762 were harvested 20 and 10days -after transplantation respectively. Animalswere killed by cervical dislocation, and mammaryand/or tumour tissue was excised and then washedextensively in ice-cold 0.25M-sucrose. Each tissuewas then suspended in 2vol. of 50mM-imidazole/HCl buffer, pH6.5, containing 1mM-EDTA, 7mM-2-mercaptoethanol and 20% (v/v) glycerol, andhomogenized at 40C in a VirTis homogenizer at22500rev./min for three periods of 45s eachinterspersed with 1 min rest periods. The homo-genate was centrifuged at 2000g for 10min at 40C.

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After removal of the fat layer with a spatula, thesupernatant solution was filtered through a loose plugof cotton covered with four layers of cheesecloth.The 2000g pellet was resuspended in 1 vol. of theabove buffer, homogenized, and treated as above.The combined supernatants were centrifuged at100OOOg for 1 h. The supernatant solution from thisstep could be used for direct enzyme assays, forfurther purification (Ahmad & Ahmad, 1981) or forprecipitation with solid (NH4)2SO4 (to 50% satura-tion) and subsequent determination of acetyl-CoAcarboxylase and fatty acid synthase activities on theconcentrated protein. Enzymes obtained from thelast step were used for all immunochemical analyses,for Km determinations and for studying the effect oftemperature on catalytic activity.

Tissues were also homogenized and the enzymespartially purified by using buffers containing amixture of known inhibitors of proteolysis [EDTA,leupeptin, antipain, pepstatin A, l-chloro-4-phenyl-3-L-tosylamidobutan-2-one ('TPCK') and 7-amino-1-chloro-3-L-tosylamidoheptan-2-one ('TLCK') andin addition phenylmethanesulphonyl fluoride (whichinhibits fatty acid synthase) for acetyl-CoA carb-oxylase only]. Supplementation of buffers with theinhibitors used had no marked effect on the activitiesof acetyl-CoA carboxylase and fatty acid synthase.

Before being assayed, fractions were dialysed [toremove (NH4)2SO4J against 50 mM-imidazole/HClbuffer, pH 7.5, containing 1.0mM-EDTA and 20%glycerol for 1.5 h at 40C.

Assay ofacetyl-CoA carboxylaseThe reaction mixture in a final volume of 0.1 ml

contained 60mM-imidazole/HCl buffer, pH 7.5,24 mM-magnesium acetate, 0.4mM-EDTA, 0.10mM-EGTA, 30 mM-sodium citrate, bovine serum albumin(0.5 mg/ml), 17.5 mM-phosphocreatine, 5.6 units ofcreatine kinase (in 50 mM-imidazole/HCl buffer,pH 7.5, containing 10mg of bovine serum albumin/ml), 0.75 mM-acetyl-CoA, 25 mM-NaH'4CO3(800d.p.m./nmol) and 7.2mM-ATP. The reactionwas carried out at 370C for 5 min and terminated bythe addition of 0.025 ml of 4 M-HCl. Samples(0.05ml) were transferred to scintillation vials andevaporated to dryness in a forced air oven at 850C.The residue was dissolved in 0.5 ml of water, 5 ml ofscintillation fluid (Fricke, 1975) was added and theradioactivity was determined. One unit of enzymeactivity represents 1,pmol of malonyl-CoA formed/min at 370C.

Assay offatty acid synthaseFatty acid synthase was assayed spectrophoto-

metrically as described by Smith & Abraham(1975), except that incubation temperature was370C. All values were recorded for NADPHoxidation occurring in the presence of malonyl-CoA.

One unit of enzyme activity represents lumol ofNADPH oxidized/min at 370C.

For both the carboxylase and synthase assays,conditions were selected giving linear rates ofcatalysis with respect to protein concentration andtime of incubation.

Immunological procedures(a) Antibody production. Antibody against homo-

geneous fatty acid synthase was prepared in rabbitsas described by Ahmad et al. (1979). Anti-(acetyl-CoA carboxylase) and anti-biotinyl antibodies werealso raised in rabbits as described previously(Ahmad et al., 1978, 1981).The molecular weights of the polypeptides consti-

tuting acetyl-CoA carboxylase and fatty acid syn-thase are almost identical (Ahmad et al., 1978).Since examination by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the immuno-precipitates formed on the addition of anti-carb-oxylase or anti-synthase would not distinguishwhich of the antigens was precipitated, experimentswere performed to determine the specificity of theantisera prepared against these enzymes. Eachantibody removed only the corresponding antigenfrom solution, and no cross-reactivity could bedetected during immunodiffusion studies. When thecarboxylase and synthase present in the normal andneoplastic tissue extracts were precipitated by theappropriate antibodies and then examined bysodium dodecyl sulphate/polyacrylamide-gel electro-phoresis, besides the heavy and the light chains ofimmunoglobulin G, only the protein bands of M,240000-260000 were observed. These experimentsestablished the specificity of the antisera as well asthe size of the polypeptides found in tissue (normaland neoplastic) homogenates and partially purifiedenzyme preparations.

(b) Ouchterlony double-diffusion analyses.Double-diffusion analyses were performed by themethod of Ouchterlony & Nilsson (1978), withminor modifications as described in Ahmad et al.(1978). The concentration of agarose in gel layerswas decreased to 0.8% to facilitate diffusion of thehigh-molecular-weight acetyl-CoA carboxylase.

(c) Immunotitration procedures. Fatty acid syn-thase was incubated in duplicate with variousamounts of anti-(rat lactating mammary-gland fattyacid synthase) in reaction mixtures (total volume0.05 ml) containing 0.25 M-potassium phosphatebuffer, pH6.5, 10mM-dithiothreitol and 0.1mg ofbovine serum albumin. After incubation at 370C for15 min, tubes were centrifuged at 2000g for 10minat 27°C. Samples (0.03 ml) of the supernatantsolution were removed and assayed for synthaseactivity.

Acetyl-CoA carboxylase was incubated in dupli-cate with anti-(acetyl-CoA carboxylase) (total

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volume 0.05 ml) in 60mM-imidazole/HCl buffer,pH 7.5, containing 24 mM-magnesium acetate,0.4 mM-EDTA, 0.1 mM-EGTA, 30 mM-sodium cit-rate and 0.25 mg of bovine serum albumin. Afterincubation at 370C for 15 min, tubes were centri-fuged at 2000g for 10min at 270C. Samples(0.03 ml) were removed and assayed for acetyl-CoAcarboxylase activity.

Immunotitrations with biotin-binding antibodieswere performed as described previously (Ahmadetal., 1981).

The amount of antibody required to inactivate theenzyme completely was estimated by extrapolationsof the linear portions of the titration curves to zeroenzyme activity. These values were used to cal-culate the equivalence point (i.e. the amount ofantibody required to inactivate 1 munit of enzymeactivity).

Protein determinationProtein was determined by the method of Lowry

et al. (1951), with bovine serum albumin asstandard.

Results

Activities of acetyl-CoA carboxylase andfatty acidsynthase in tumours carried by virgin, pregnant andlactating animals and in the corresponding normalmammary tissue

The activities of acetyl-CoA carboxylase andfatty acid synthase were measured in mammarytissue and tumours of virgin, pregnant and lactatingrats. Likewise these activities were determined onmammary tissue derived from non-tumour-bearinganimals of similar physiological states. Acetyl-CoAcarboxylase and fatty acid synthase activities werelowest in virgin mammary gland (Tables 1 and 2).These enzyme activities reached their highest valuesin lactating gland, and during mid-lactation activi-ties of the synthase and the carboxylase wereapprox. 40-50-fold higher than found in the virginmammary tissue. Similar results have been reportedfor rat mammary-gland acetyl-CoA carboxylase andfor mouse and rabbit mammary-gland fatty acidsynthases (Lin et al., 1975; Mackall & Lane, 1977;Short et al., 1977).

The carboxylase and synthase activities of trans-plantable tumours carried in virgin animals wereconsistently higher than the activities in mammarytissue of non-pregnant animals, but markedly lowerthan those found in the lactating mammary gland.These results are in accord with the previous obser-vations (Abraham & Bartley, 1974) indicating thatmammary tumours synthesized long-chain fattyacids from acetate and glucose at a much lower ratethan did the lactating mammary gland. The data ofTables 1 and 2 also show that the physiological

Table 1. Acetyl-CoA carboxylase activity of mammarygland and mammary tumours

The lOOOOOg supernatant solutions derived fromvarious tissues were assayed directly for acetyl-CoAcarboxylase activity as described in the Materialsand methods section. In the case of the tissueobtained from non-pregnant animals, before assaysthe carboxylase present in the extracts was con-centrated by precipitation with (NH4)2SO4 (50%saturation). Each determination was made induplicate, with the numbers of determinations(animals) in parentheses. Agreement between valueswas + 15%.

Activity (munits/mg of protein)Physiological, A

state of Mammary Tumour Tumouranimal gland R2320AC 13762

Non-pregnant 1 (3) 4.6 (3) 2.4 (16)Pregnant 16.7 (3) 3.8 (3) 3.3 (3)*Lactating 53 (8) 8.4 (7) 4.0 (2)

* See Table 2 for explanation.

Table 2. Fatty acid synthase activity of mammary glandand mammary tumours

Fatty acid synthase activity was measured in tissueextracts prepared as described in the legend to Table1 and assayed as described in the Materials andmethods section.

Activity (munits/mg of protein)Physiological r A

state of Mammary Tumour Tumouranimal gland R2320AC 13762

Non-pregnant 3 (3) 12 (4) 13.5 (16)Pregnant 27 (3) 16.6 (3) 8.2 (3)*Lactating 123 (9) 30 (7) 24 (4)

* Tumour 13762 grew more slowly and with lessfrequency (all animals were injected at three sites) in allpregnant animals than in non-pregnant animals.

stimuli inducing the carboxylase and the synthase indifferentiating mammary gland had little effect onthe same enzymes of tumours grown in pregnant orlactating animals. The activities of acetyl-CoAcarboxylase and fatty acid synthase were similar inthe differentiating glands of animals, whethertumour-bearing or control (results not shown). Thusthe presence of a tumour did not influence theinduction of the carboxylase and the synthase duringhost mammary-gland differentiation.

Similarities and differences between neoplastic andnormal tissue acetyl-CoA carboxylase andfatty acidsynthase

Determination of Km values. In an attempt tocompare acetyl-CoA carboxylase and fatty acid

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synthase enzymes from mammary adenocar-cinomas and mammary gland, the kinetic propertiesof the enzymes carried through identical puri-fication steps were determined. The Km values for allthe substrates, determined by double-reciprocalplots, are summarized in Table 3 and are similar foradenocarcinomas and normal mammary-gland en-zymes with one exception, i.e. the Km value forNADPH of the normal tissue synthase was about2-3-fold lower than that found with the tumourenzymes. The reason for this difference is not clear.Nevertheless, on the basis of kinetic measurementsboth the acetyl-CoA carboxylase and the fatty acidsynthase of neoplasms appear to be similar to theenzymes present in the control tissue.Heat inactivation. The effect of heat on the

inactivation of acetyl-CoA carboxylase obtainedfrom different sources was also investigated. Whenthe tumour and host mammary-gland enzymes werekept at 340C for 0min before assay, no change inactivity was observed. However, preincubation at370C caused activation (30-40%) of the neoplastictissue enzyme but inactivation (10-20%) of theenzyme from normal mammary gland. Tempera-tures exceeding 370C caused the same amount ofinactivation for the enzyme from both neoplastic andcontrol tissues, with exposure to 45 0C causingalmost complete inactivation of all enzyme pre-parations investigated.

Immunodiffusion studies. The immunologicalcross-reactivity of acetyl-CoA carboxylase and fattyacid synthase from different sources was studied byimmunodiffusion (Figs. 1 and 2). For fatty acidsynthase (Fig. 2), complete fusion of the precipitinlines between tumour enzyme and control enzymewas observed, with no sign of spur formation. Such areaction is characteristic of complete identity be-

tween neoplastic fatty acid synthase and controltissue enzyme. However, different results wereobtained when the immunodiffusion experimentswere performed with acetyl-CoA carboxylase. The

Fig. 1. Immunodiffusion analysis of rat mammary-glandacetyl-CoA carboxylase with anti-(lactating rat mam-

mary-gland acetyl-CoA carboxylase)Agarose (0.8%) plates in 50mM-imidazole/HClbuffer, pH 7.5, containing 0.15 M-NaCI containedthe following (in mg of protein): centre well,anti-(lactating rat mammary-gland acetyl-CoA car-boxylase) (0.4 mg); wells 1, 3 and 5, lactatingmammary-gland extract (0.068 mg); well 2, tumour-R3230AC extract (0.081 mg): well 4, tumour-13762extract (0.091 mg). Extracts were obtained asdescribed in the Materials and methods section.These studies were performed on at least threedifferent preparations of antigen or antiserum, withidentical results.

Table 3. Km valuesfor acetyl-CoA carboxylase andfatty acid synthase ofnormal and neoplastic tissuesKm values were calculated from double-reciprocal plots of initial velocity versus substrate concentrations on twodifferent occasions. The experimental variation was about 1% and the fatty acid synthase reactions were started withmalonyl-CoA.

Acetyl-CoA carboxylaseKm value

Substrate Enzyme source ... Mammary glandAcetyl-CoA 6.2 x 10- MATP 6.6 x10-4MHC03- 10.5 x10-3MFatty acid synthase

Substrate Enzyme source ... Mammary glandAcetyl-CoA 1.4 x 10-5MMalonyl-CoA 2.0 x 10-5MNADPH 3.8 x 105M

Tumour 137627.0 x 10-SM8.0 x 10-4M7.7 x 1O-'3m

Km value

Tumour 137621.8 x 10-M2.5 x 10 M9.0x 10 SM

Tumour R3230AC6.7 x 10-SM7.4x 10-4M

6.25 x 10 3M

Tumour R3230AC1.4 x 10-SM2.4 x 10-5M

11.7 x 10-5M

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Table 4. Immunotitration of acetyl-CoA carboxylasepartially purifiedfrom control and neoplastic tissues with

anti-(rat mammary-gland acetyl-CoA carboxylase)Acetyl-CoA carboxylase was obtained from diffe-rent tissues and then partially purified up to the509-satn.-(NH4)2SO4 precipitation step. Immuno-titrations and assays were performed as detailed inthe Materials and methods section. All titrationswere done in duplicate. Values obtained fromdifferent batches of tissue agreed within 10%. Inexperiments where lactating-mammary-gland andtumour tissue extracts were combined, equal partsof each were added to give the units of activityspecified.

Fig. 2. Immunodiffusion analysis of rat mammary-glandfatty acid synthase with anti-(lactating rat mammary-

glandfatty acid synthase)Agarose plates and enzyme extracts were preparedas described in the legend to Fig. 1. The sampleswere placed as follows (in mg of protein): centrewell, anti-(lactating rat mammary-gland fatty acidsynthase) (0.14mg); wells 1, 3 and 5, mammary-gland extract (0.068mg); well 2, tumour-13762extract (0.091mg); well 6, tumour-R3230AC ex-tract (0.081mg). At least three different prepara-tions of antigen or antiserum were used, withidentical results.

enzymes from both neoplastic tissues formed pre-cipitin lines of partial identity when immunodiffusedagainst anti-(rat mammary-gland acetyl-CoA car-boxylase).

Immunotitrations of acetyl-CoA carboxylase andfatty acid synthase of control mammary gland andmammary adenocarcinomasTo estimate the quantity of acetyl-CoA car-

boxylase and fatty acid synthase, it is essential that astoicheiometric relationship exist between antigenand the corresponding antibody. In independentexperiments this requirement was satisfied for boththe enzymes by using anti-(rat mammary-glandacetyl-CoA carboxylase) and anti-(rat mammary-gland fatty acid synthase) respectively. Both thecarboxylase and the synthase from the control andthe neoplastic tissues were subjected to identicalpurification procedures before initiation of immuno-chemical titrations. Fig. 3 represents a typicalexample of immunochemical titrations of both thecarboxylase and the synthase derived from normaltissue and both mammary adenocarcinomas: in all

Enzyme sourceMammary gland I

(lactating)Mammary gland C

(pregnant)Tumour R3230AC ITumour 13762 1Mammary gland I+tumour R3230AC

Mammary gland 1+tumour 13762

Activitytitrated(munits)

1.24, 1.15, 0.99

Antiserumrequired(,g/munit)

10.3, 12.6, 11.2

).963, 0.805 9.25, 12.7

1.1,0.98,0.931.2, 1.21.0,0.89

1.09,0.917

8.6. 8.9, 9.35.4, 6.49.4, 9.6

7.7, 9.9

Table 5. Immunotitration offatty acid synthase partiallypurifiedfrom control and neoplastic tissues with anti- (rat

mammary-glandfatty acid synthase)Fatty acid synthase was obtained from differenttissue extracts, and immunotitrations and assays forfatty acid synthase activity were performed, asdescribed in the Materials and methods section.Agreement between tissue extracts prepared fromdifferent animals (tumour-bearing and non-tumour-bearing) was within 5%. All titrations were done induplicate.

Enzyme sourceMammary gland

(lactating)Mammary gland

(pregnant)Tumour R3230ACTumour 13762Mammary gland+tumour R3230AC

Mammary gland+tumour 13762

Activitytitrated(munits)1.03, 1.1

1.2, 0.978

1.4, 1.30.97, 1.01.18, 1.25

1.02, 1.10

Antiserumrequired

(ug/munit)11.9, 12.0

12.25, 11.1

11.3, 11.511.4, 11.611.6, 11.2

11.9, 10.6

cases the amount of enzyme removed is pro-portional to the amount of antibody added. Neitheracetyl-CoA carboxylase nor fatty acid synthase wasremoved by non-immune serum.

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100

° 80

c) 60CIO

E 40

20

0

Anti-(acetyl-CoA carboxylase) antibody (,ug)

100

Tumour R3230AC Tumourl3762 Mammary gland

(1 .40munits) (1.00munit) (1.03 munits)

80

>~ 40-

20-

0 6 12 18 0 6 12 0 6 12

Anti-(fatty acid synthase) antibody (ug)

Fig. 3. Immunotitration curves of (a) acetvl-CoA carboxylase and (b) fatty acid synthase from lactating rat mammarygland and mammary tumours R3230AC and 13762

Extracts were prepared, and immunotitrations and assays were performed, as described in the Materials and methodssection. Only points observed to have between 45 and 100% activity remaining were used to construct a line, whichon extrapolation intercepted the axis representing zero enzyme activity. The equivalence points were calculated fromthe amount of antibody required to inactivate 1 munit of enzyme activity. Optimal enzyme amounts had to be usedsuch that four or five points satisfied this requirement each time a titration was performed. Values betweenpreparations as well as duplicate determinations of the same preparation showed excellent agreement.

A summary of the immunotitration data on

acetyl-CoA carboxylase and fatty acid synthase ofdifferent tissues is presented in Tables 4 and 5.Despite the fact that the activities of fatty acidsynthase in mammary glands of pregnant andlactating animals are much higher than those foundin tumours, the amount of anti-(rat mammary-glandfatty acid synthase) needed to neutralize an equival-ent amount of activity in all these instances remainedconstant. Immunotitration experiments were alsoperformed on normal mammary-gland enzyme towhich had been added the enzyme derived frommammary adenocarcinomas. Addition of theseproteins did not displace the neutralization point(Table 5). These results indicate that the catalyticefficiency per fatty acid synthase molecule isidentical irrespective of the origin of tissue used in

these studies. Thus the changes in fatty acidsynthase activity are actually determined by chang-ing quantities of enzyme protein.

Whereas the immunotitration (Table 5) andimmunodiffusion data (Fig. 2) supported the iden-tical nature of neoplastic and normal tissue syn-thases, some differences were found to exist betweenthe carboxylases of neoplastic tissues and normalmammary gland. The carboxylase activity obtainedfrom both tumours 13762 and R3230AC was

neutralized by lower amounts of anti-(acetyl-CoAcarboxylase) than were needed to neutralize an

equivalent amount of enzyme activity derived fromlactating gland (Table 4). No such difference was

detected when the immunotitration experiments were

performed with the enzyme derived from mammarytissue of pregnant, lactating or involuting animals

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(a)

Tumour R3230AC Tumour 13762 Mammary glandI-; (0.981 munit) (1.25munits) (1.54 munits)

>~~~ ~~~~\ * \ \1.00

ItI \ II6 12 0 6 12 0 8 16

449

P. M. Ahmad, D. S. Feltman and F. Ahmad

1-11 0~~~~~~~~~~~~~~

l.-, , l,

CZ 60 -

40-

20-

0 0.2 0.4 0.6 0 0.2 0.4 0.6 0 0.2 0.4 0.6 0.8 1.0

Anti-(biotinyl-bovine serum albumin complex) antibody (pg)Fig. 4. Immunotitration curves of acetyl-CoA carboxylase from various tissues against anti-(biotinyl-bovine serum

albumin complex) antibodyExtracts were prepared as described in the Materials and methods section. Portions were preincubated as follows: (i)in buffer (50mM-imidazole/HCl, pH7.5, containing 1.OmM-EDTA and 20% glycerol) containing 0.5M-NaCl at270C for 30min to obtain the protomer (0); or (ii) in buffer (as above) containing 25mM-potassium citrate for10min at 270C to obtain the polymer (a). Samples of the protomer and polymer were immediately taken forimmunotitration and assayed for acetyl-CoA carboxylase activity by the methods described in the Materials andmethods section. Immunotitrations were performed on two different enzyme preparations with similar results. Eachdetermination was run in duplicate, with agreement within 5%.

Table 6. Immunotitration ofacetyl-CoA carboxylase ofdiferent tissues with anti-(biotinyl-bovine serum albumin complex)antibody

Acetyl-CoA carboxylase was obtained from tissues as described in the Materials and methods section.Immunotitrations (anti-biotinyl antibody) and assay conditions for acetyl-CoA carboxylase are also described in theMaterials and methods section. All titrations were done in duplicate. Agreement between batches was within 8%.Sedimentation coefficients were determined by layering extracts on 5-20% (w/v) linear sucrose gradients (4.6 mlvolume) in 5OmM-imidazole/HCl buffer, pH7.5, containing l.OmM-EDTA and either 0.15M-NaCl or 25mM-potassium citrate (see Ahmad et al., 1981). After centrifugation at 45000rev./min for 1.5h at 25°C in an SW50Lrotor of a Beckman L2-65B ultracentrifuge, fractions (15 drops each) were collected manually, and samples wereassayed for acetyl-CoA carboxylase activity. Sedimentation coefficients relative to thyroglobulin were calculated.

Enzyme sourceMammary gland (lactating)Tumour 13762Tumour R3230AC

Activity titrated(munits)

0.51, 0.31, 0.330.2, 0.350.33, 0.36, 0.43

Sedimentation coefficientAntiserum required AA I

(ug/munit) -Citrate +Citrate2.07, 2.1, 1.95 17 422.0, 1.9 16 303.9, 3.5, 4.0 16 24

(results not shown). Thus results presented in Fig. 1and Table 4 indicate that acetyl-CoA carboxylase ofnormal mammary tissue may differ from the enzymepresent in mammary adenocarcinomas.

Effect of citrate on the hydrodynamic charac-teristics and immunotitration of acetyl-CoA car-boxylase with anti-biotinyl antibodies

In the presence of citrate, the enzymically inactiveprotomeric form of mammalian carboxylases is

converted into catalytically competent polymer.This change in the hydrodynamic behaviour ofacetyl-CoA carboxylase is accompanied by altera-tions in its conformation that affect the biotinylgroup of the enzyme. In a previous study theligand-induced changes affecting the biotinyl groupof rat mammary-gland acetyl-CoA carboxylase wereinvestigated by using biotin-binding antibodies(Ahmad et al., 1981). These antibodies react withbiotin and give an indication of its location.

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450

Control of lipogenesis 451

By this technique it has been found that thebiotinyl group in the polymeric form of the car-boxylase is inaccessible; when the protomergenerated by high salt concentration is employed,however, the anti-biotinyl antibody reacts rapidlywith the biotin and inactivates the enzyme.To determine whether the conformation around

the biotinyl group of neoplastic-tissue carboxylasewas similar to that observed with the normal-tissueenzyme, immunotitration studies were performedwith protomeric (NaCl-induced) and polymeric(citrate-induced) forms of these enzymes, and atypical neutralization profile for the carboxylaseobtained from different tissues is given in Fig. 4.Only the protomeric form of the carboxylase reactedwith the anti-biotinyl antibody with attendant loss ofcatalytic activity.A summary of the immunotitration data and the

sedimentation coefficients determined for the proto-mer and polymer are presented in Table 6. On theaddition of 25 mM-citrate, the carboxylase isolatedfrom tumours acquired an intermediate-size poly-meric form (s 24-30 S), whereas the enzyme fromthe normal tissue sedimented in the (normally found)larger polymeric form (s >40 S).

Almost identical amounts of anti-biotinyl anti-body were required to inactivate 1 munit of acetyl-CoA carboxylase activity from mammary gland asfrom tumour 13762. The carboxylase isolated fromtumour R3230AC, however, required about 1.5-2times the amount of anti-biotinyl antibody toinactivate 1 munit of its activity.

Discussion

The results given in the present paper show thatfatty acid synthase of both the tumours examinedmanifests several distinctive properties identical withthose of the enzyme from normal or host mammarygland. In fact, in all the properties investigated (Kmvalues for substrates, immunotitration and immuno-diffusion data etc.) the tumour synthase is prac-tically indistinguishable from the normal mam-mary-gland enzyme. The results of immunotitrationstudies also show that the tissue extracts preparedfrom mammary adenocarcinomas and mammarygland of different stages of differentiation (non-pregnant, pregnant and lactating) contain antigenwhose reactivity per unit of synthase activity isconstant. Therefore, with respect to fatty acidsynthase at least, both the tumours and mammarytissue contain antigenically equivalent enzymes ofcomparable catalytic efficiency. These results there-fore support the notion that in relation to lactatinggland the decreased capacity of mammary adeno-carcinomas to synthesize fatty acids from acetyl-CoA is due to a diminution in synthase quantityrather than the presence of an enzyme with altered

catalytic properties. Lin et al. (1975) arrived atsimilar conclusions while investigating mouse mam-mary tumours.

Whereas the identical nature of fatty acid syn-thase of tumours and normal mammary tissue couldbe readily established, such was not the case whencomparisons between the properties of acetyl-CoAcarboxylases of neoplastic and normal tissue originswere made. During immunotitration studies withanti-(acetyl-CoA carboxylase) the tumour enzymerequired somewhat lower amounts of antibody forneutralization of its activity (Table 4). Examinationof the immunodiffusion data (Fig. 1), heat treatmentand behaviour in the presence of citrate (Table 6)suggests that these tumours contain a modified formof acetyl-CoA carboxylase. A number of mechan-isms are known that can alter the activity ofmammalian acetyl-CoA carboxylase: proteolysis aswell as dephosphorylation (Swanson et al., 1967;Iritani et al., 1969; Guy & Hardie, 1981). However,activation by heating at 370C of acetyl-CoAcarboxylase of neoplasms was not prevented whenthe experiments were performed in the presence ofinhibitors of proteolysis or fluoride. The latter isknown to inhibit phosphoprotein phosphatases.Therefore the nature of the difference betweenacetyl-CoA carboxylases of mammary gland andmammary adenocarcinomas is not yet understood.

The induction of these lipogenic enzymes occur-ring in differentiating mammary gland was notobserved in both the transplantable tumours whenthey were carried by pregnant or lactating animals.Thus these investigations showed for the first timethat the physiological factors that caused inductionin differentiating mammary gland had little effect onacetyl-CoA carboxylase and fatty acid synthase ofmammary adenocarcinomas. Identification of thebiological factors inducing these enzymes in normalgland is of obvious interest. It might then be feasibleto determine whether the effects of these modulatorsare modified or lost during tumorigenesis.

This work was supported by Grants CA-15196 andRR-05690 from the National Institutes of Health, U.S.Public Health Service. We thank Dr. Norman H. Altmanfor histopathological examination of tumours used inthese investigations and Mrs. Santosh Gupta for tech-nical assistance. The assistance of Miss Melissa Harrell intyping the manuscript is gratefully acknowledged.

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