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Studies on a protein isolated from livers of diabetic and fasted rats
ARCHIVES
Ok' BIOCHEMISTRY
Studies
ASD RIOPHYSICS
on a Protein Diabetic
JANET
I.ipicl
(1971)
14,343-353
Isolated and
Fasted
from
Livers
of
Rats’
M. COLLIM, MARGARET C. CRAIG, CARL JI. NEPOKROEPF, A. L. KENSAN or) JOHIZ’ W. PORTER
Melabolism Laboralory, of Physiolvgical
Velerans Chemislry,
Atltninixlralion LTni~ersil!l
Keceived -4ugust
Hoxpild, oj W’ixeonsin,
11, 1970; accepted
Madison, .Vndiwn,
November
lI~‘isconxirr, and Ihe Deparlttlenl H’iwonxitc.
53706
3, 1970
A protein that is synthesized by livers of diabetic and fasting rats has been isolated and purified. This protein is either not, synthesized or it is synthesized in trace amonnt,s by livers of normal or fasted and refed animals. This component, designated as “78” protein, accompanies t.he fatty acid synthetase through all st.eps of purificat.ion. However, it can be separated from the fat.ty acid synthetase by two successive sucrose density gradient cellt,rifugatiolis. When separat.ed in this way it, is nearly homogenous in size and charge (disk gel elect rophoresis). The 7S proteins obtained from diabetic and fast.ed livers are ident,ical wit.h respect to size, charge, and immunochemical propert.ies. This protein does not. cat,alyze any of the partial reactions of fatty acid synt,heSis, does not. bind acetyl or malonyl groups, and it. does not. exhibit acetyl-CoA carboxylaae activity. Immunological studies show it to be unrelated to the fat.ty acid synthetase. Finally, it has no effect. on the rate of fatty acid Bylit,heBiB or on acetyl-CoA carboxylase activity. Thus, on the basis of these properties, t,he 78 prot.ein is not related to the fatty acid synt.het,asc and probably not to acetyl-CoA carboxylase. The function of this prot.ein remains t.r) be determined.
In the course of studies on the effect of nutritional and hormonal fact,ors on the synthesis of the rat liver fatty acid synt.hetase Burton et al. (1) observed t.he presence of a protein that accompanied the synthetase complex through each step of purification. The content of this contaminating protein increased as animals were fasted for increasing lengths of time. It: also disappeared on refeeding fasted animals, and at 24-X hr of refeeding it was no longer detect,able. The protein contaminating fhe fat.ty acid synthetase was also present in the livers of dutbetic rats. It was suggest.ed from the st.udies of Burton et al. (1) that. the protein contaminat1 This investigation was supported in part by liesearch Grants AM01383 from the Sational IrMitute of Art,hritis and Metabolic Diseases of t.he National Institutes of Health, Unibed States Pltblic Health Service, and (i-68-38 from the Life insurance Medical Research Fund.
ing the fat.ty acid synt,het.aseobtained from livers of fasted or diabet.ic rats might be a precursor in the synthesis of t.his complex or an intermediate in its degradation. It was also suggested that this protein might be unrelated to the fatty acid synthetase. In the present paper we have investigated t,hese possibilities. Init.ially we were able to show that the contaminating protein can be separated from the fatty acid synthetase by two successive sucrosedensity gradient. cenkfugations. The protein isolated from diabetic or fasted livers and separated in this way is nearly homogenow on disk gel electrophoresis and ultracent,rifugation. In addition t,he proteins obtained from fasted and diabet.ic livers are identical in their behavior on disk gel electrophoresis, sucrose densit.v gradient centrifugation, and immunokffusion. Hence they are either identical or nearly identicd in structure.
The contaminating protein, termed “78” protein, is synt.hesized by the liver dur& fast,ing. This protein shorvs none of the parGal react,ions of t.he fatty acid synthetase complex. P\‘either does it bind acetyl or malonyl groups, nor show acetyl-&A carboxylase activit.y. Furthermore, it. is im-
munologically dist.inct from the fatty acid synthet,ase. Hence, it is unrelated by these criteria to the fat.ty acid synthetase. It also does not affect t.he rates of fat.ty acid synt.hesis or t.he carboxylation of acct.yl-CoA.
phosphate buffer, pH 6.8, wan subst.ituted for t.he final Sephadex (i-100 gel filtration sl.ep. Iso~alion f~j 78 contponenl jrottt J’ally acid x!jn fhelaae of ral lirer. Patty acid synt.het.ase was prepared by t.he procedure of Burton ef a/. (8) from animals of different nutritional and hormonal states. This prot.ein was then applied IO a slmose gradient and centrifuged, as reported in a latel section, at a rotor speed of 48,060 rpm. Light absorption af 280 nip was determined for each collected fraction, and 1hose fractions rorresponding to the slower moving protein from four to six separate gradients were combined. The resulting sollltion was dialyzed against 0.5 Y potassilml phosphate buffer, plI 6.8, precipit.ated with ammonium su1faf.e (O-8041 satllration), and again dialyzed. This protein was applied to a second sucrose gradient. and centrifuged as dcscribcd previonsly. Fractions lb20 (see Fig. 4B and 1)) were combined and used in subsequent experiments.
EXPEHIMENTAL PIIOCEI)Ul~E Jfulerials. Experimental materials were obtained from the following sources: acetic anhydride-I-“C, malonic acid-2-l”C, palmit,yi-CoAl-l%, sodium bicarbonate-‘%, and uniformly labeled L-leucine-1% from New England Nuclear; I’atrlial purificalion of acelyl-CoA carboxylase. acetyl-CoA, malonyl-CoA, CoA, and NADPH Acetyl-CoA carboxylase, from livers of rats fed II from P-L Biochemicals; 2-mercaptoelhanol and normal diet, was part ially purified (through t,he dit.hiothreit.ol from Calbiochem; and palltct.heine second ammonium sulfate R~IJ) by t hc method from the Sigma Chemical Company. All other of Suma, liingelmann, and I+Jetl (!I). This prepreagents used were of analytical grade. aration had an acet yl-CoA t!arboxylase activity Synthesis of coenzynted eslers. Acetyl-CoAof 65-V& mpmoles of CO, irlcorpIjrated!min/mg of 1-14C and caproyl-CoA were synthesized by the protein and a negligihlc activity for fat,f.y acid method of Simon and Shemirl (2), and malonylmpmoles of acetyl-(:oA incorCoA-2-14C was synthesized by the method Of synthesis, 0.0082 porated/min/ing of prof ein. Trams and Brady (3). Coenzyme-A esters were Asmy for prokin. Assays for the protein conpurified by 1he paper chromatographic systems of ceIlf,rations of soluf.i(Jns were carried out. by I.he Brodie stud Porter (4). billret. mct.hod of ( LJrnaIl, Bard:lwill, and David Treatmen/ ofl animals. ;11ale rats, weighing (10)~ about 150 g each, were used in most expcrimcnt.s. Assay for jall!l ucirl synlhclaxe n&Sly. The Animals were fasted for varying periods of time or assay tiystem uncd WUHeither the radiochemical they were fasted and refed. When animals were assay Of Hsu, Wasson, and Porter (7), as modified refed, a fat-free diet was used (5). Uniformly tJy Bllt 1erwort.h ef ul. (ll), or ii H~JC’D~ rqJhotometric labeled r,-leucine-14C was administered as an assay. The following c:ollcc~ltrlttio~lo of substra1.c intraperit,oneal injection of mJ aqueous SOhJtiOI~ of the amino acid at an appropriate t.imc prior to and enzyme, in a l-ml vol~mx, were used in f.he sacrifice (1). Animals were killed lay a blow to t.hc lat1er assays: acdyl-CIJA, 5 X 10-S M; malonylCoA, 1 x l(r’ M; NAI)l’Il, 1 x lo-’ u; IGl)TA, bnse of the skull. 8 X l(Y M; potwssirun phosphate buffer, 0.2 M Alloxan diabetes was induced in rats weighing and pH 7.0; and protein, 2 X 10eRM. The specific approximat.ely 250 g by the injection of a 5c/, acl.ivify of the fat,ty acid synthet.ase is expressed solution of alloxan monohydrate (40 mg/kg) via as millimicromoles of fat.Iy acids (mamoles of the tail vein. TW(J weeks later the animals had NAIjPII oxidized + 14) formed per minut.o per blood glucose levels of 250 mg/lOO ml or higher. milligram of protein. Ru$ers. All bnfiers cont.ained 1 mM Bl)TA and Assays for par/id readions of full!y acid synlheeit.her 1 rn~ dithiothreitol or 1 mM fl-mercaptosis. Assays for the partial reactions of fatty acid et,hanol, unless otherwise specified. synthesis were performed aa described by Kumar Purijicalicttr oj fatly acid synthelase. Rat liver snpernatant solut,ions were prepared by t,he al. 01. (12), with ooe exception. In t.hc palmitylCoA deacylase rcncf ion, the conc:cntration of met,hod of Wakil, Port.er, and Gibson (6) as modipalmityl-CoA-1-~4C was 4 p~ and the qlrantit.y of fied by Bsu el aI. (7). The fat.ty acid synthetase profein used was 1 pg. was then purified from the supernatant solut.ion Assays for lkc binding oj acelgl and rnalonyl as described by Rurton, Haavik, and Porter (8), except, that dialysis against. 0.5 .M pottwsium g,otrpx to /he ~-a//y/ ucid nynlh.e/axe. Assays for the
A PROTEIN
OF
DIABETIC
covalent binding of acet,yl and malonyl groups to protein were performed through the addition of 5 amoles of acetyl-CoA-1-“C or malonyl-CoA-2-lS IO 250 pg of prot.ein in 1 ml of 0.5 M phosphate buffer at pH 6.8. The assay mixture wae incubat,ed at 0” for 30 set and the reaction was stopped by i.he addition of 0.5 ml of 6270 perchloric acid. The protein precipitate was centrifuged and then washed three times each with 1 ml of 0.2 N acet,ic acid. The washed protein was assayed for radioactivity as described below. Assay for acelyl-Co:od carbozylase a&‘&g. The radiochemical assay method of Chang el al. (13) was used. Acet,yl-CoA carboxylase, 0.2 mg of prot.ein, W&B preincubated at 37” for 30 min at pH 7.0 with 60 rnM Tris (Cl-), 3 my glutathione, 8 mM MgClr, 0.1 rnx EDTA, 0.6 mg protein of bovine serum albumin/ml, and 5 rnx cit.rate (KC). The formation of “C-malonyl-CoA was effected by the addition of preincubated enzyme, 20 ~g of protein, to a solution containing 60 nlM Tris (Cl-), 2 mni ATP, 8 mr.i MgClz, 10 mM KH’dCO, (0.2 pCi/pmole), 0.2 mM acet.yl-CoA, 3 rnM glut,athione, 0.1 m.u EDTA, 0.6 mg protein of bovine serllm albumin, and 5 rnM citrat,e (K+). A final volume of 1.0 ml was incubated for 10 min at 37”. The reaction was terminated by the addition of 0.2 ml of 6 N HCl, and the incubation mixture wae evaporated to dryness. Residual radioactivity was det.ermined bg assay in a dioxane solution in a liquid scintillation spectrometer. Deleminalion of radioactivif;g. All measurements of radioact.ivity were ohtained wit,h a
AND
FASTED
11.41’S
345
Packard liquid scintillation spectrometer. When the radioactivity of prot,eins was to be determined, samples were dissolved in 887& formic acid and then diluted w&h 2 ml of et.hanol. Toluenebased sciut.illation solution was t.hen added to each sample. .~n.alytical rcItracen./rifugation. Ve1ocit.y sediment,ation experiments were carried out in a Spinco Model E ultracentrifuge. The rotor t,emperature was 20” and the speed was 59,780 rpm. Sedimentation coefficients were corrected for solveut viscosity and density at 20”. Sucrose density gratlienl cerrlrifugalion. Sucrose density gradient cenbrifugation was performed for 23.5 hr at 48,060 rpm in a Spinco SW5OL rotor in 5 ml of a 15-355;, sucrose gradient containing 0.5 M potassium pho5phat.e buffer, pH 6.8, 1 mM EUTA, and 1 my dithiot.hreitol. Fractions of 0.14 ml each were collected from the bott.om of the tube after centrifugat.ion. Disk gel eleclrophoresis. Acrylamide disk gel elecbrophoresis was carried out, according to t.he method of Davis (14). Five per cent acrylamide was used in the resolving gel. After electrophoresis t,he gels were fixed and stained by the procedure of Chrambach et III. (15). Prepa.ralion of f&/u acid sgnlhelase anliserur~~. The fatty acid synthet,ase used as antigen w&s isolated by t.he normal purification procedure (8) from the livers of rat.s which had been fasted for 48 hr and theu refed for the same period of time on a fat-free diet. Approximately 30 mg of fatt.y acid synt het.ase, in iucompletc Freund’s adjuv:m~,
Fro. 1. The total protein and enzyme activity of fatty acid synt.het,aae isolated from livers of control and fasted rats. Groups of four rat.s were fasted for varying periods of time, then killed, and the fatt,y acid synthetaae of livers of these animals was purified by t,he procedure of Burton et al. (8). The protein (fatty acid synt,hetase) obtained per liver is given in mg, l d; the specific enzyme activity inpmoles of product formed/min/mg of protein, O-----O; and t.he total units of fatty acid synt.hetaae per liver (specific activit,y X mg of protein), (>-.-a-.@.
346
COLLINS
FIG. 2. Sucrose density gradient. centrifugation of rat liver fatty acid synthetaee isolated from livers of control and fasted rat,s. Animals frosted for 0, 8 and 32 hr were killed, and the fatty acid synthetaae of the liver was isolated by the procedure of Burton et al. (8). The isolated enzyme was t,hen subjected to sucrose density gradient centrifugation, aa described in the text, at a rotor speed of 40,000 rpm. Samples from the bottom ho the top of the tube read from left t.o right in the Qure. Values are report.ed for light absorpt,ion at 280 nip (@---0) and enzyme activity for fat)ty acid synthesis (X-----X). A, 0 hr; B, 8 hr; and C, 32 hr of fasting. was inject,ed subcutaneously into each of two rabbit;s over the thighs and shoulders bilaterally at four sites. This procedure was repeated 3 and 8 weeks later. The rabbits were then bled at l-week intervals to obtain antiserum. The antiserum was atored at - 15” unt,il used. Im?,~unfdi~~crrion. Onchterlony micro-doublediffusiou teats wit.h antiserum and various preparations (Jf the fatty acid synthetase and 7S prot,ein were carried out, in 0.5% agarose.* RESULTS
The detection of a protein copurifying with the jatty acid synthetase obtain.edfrom fast& and diabetic alrimals. The total number of e Operation Manual, LKB 68ODA Immunodiffuosin Equipment, p. 17.
ET Al,.
milligrams of protein per liver, isolated 2~s fat,ty acid synt.hetase, from fasted rats is plot,ted as a function of time of fasting in Fig. 1. The specific act,ivity of the fatty acid synthetase isolated from livers of fasting animals and the t.ot.al unit.s of enzyme per liver are also reported in Fig. I.. The procedure used for the isolation of the fatty acid synt,het.ase in this experiment yields a homogenous protein when t.he enzyme is isolated from normal or fasted and refed animals (8). The presence of a protein contaminating the fatty acid synt,hetase is suggest.ed from these results because of the marked decline in the specific activit,y of thth enzyme during fasting, compared 1.0the tot al number of milligrams of protein isolated, which shows a slight, rise. The presence of a protein contaminating the fatty acid synt.hebase was shown via sucrose density gradient centrifugation of protein isolated from the livers of 0-, X-, and 32-hr fasted rats (Fig. 2). At, 32 hr approximat,ely 50 % of the isolated prot.ein did not cont.ain fatt,y acid synthetase activit:y. Previously we showed that this contaminating prot,ein is not detectable by sucrose denait.y gradient centrifugation upon refeeding of fasted animals for 24 hr or longer (1). Similar results were obtained when fatty acid synthet.ase was purified from livers of alloxan diabetic animals. The t.otal number of milligrams of fatt.y acid synthet,ase protein obtained per liver increased over the level of controls, but t.he specific act,ivity for fat1.y acid synthesis and t,he total units of enzyme decreased (1). Sucrose density gradient ccnt.rifugation of the fat,ty acid synthet.ase purified from livers of diabet.ic animals revealed the presence of a cont,aminat:ing protein similar to t.hat, found associated with the fatt,y acid synthetase prepared from fast,ed animals. Properties of contam.inatiq! pro,!&. The sediment,ation pat.terns obtained on ultracentrifugation of fatty acid synt,hetase prcpared from livers of diabet.ic and GO-hr fast.ed rats are shown in l;ig. 3. Two major components with s~~,,~values of 32 and 7 were observed for each preparat,ion. The fast,cr moving component is the fat t)y acid synthetase. The molecular weight of the cant ami-
A PROTEIN
OF 1)IABKTIC
ANI)
FA!!TP:I)
347
RATS
Fro. 3. Sedimentation characteristics of rat liver fatty acid synthetase isolated from livers of diabetic and GO-hr fast.ed animals. Ultracentrifugat~ion was carried out as described in the text.. Sediment&ion is from left t.o right. Photographs were taken at. 8-min intervals. (A) Fatty acid synthetase from diabet.ic animals, (B) fat,t.v acid synt,hetaee from 60-hr fasted animals. I50 A 1.20
C 600
,900 -
400.
,600. 2 lt N.3006 5F
;.200i 5
0
0
FIG. 4. Separation of the 78 component from rat liver fatty acid synthetase. Sucrose density gradient, cent,rifugations were carried out as described in t.he text and eluate fractions were combined aa indicat.ed. (A) First centrifugation of fatt.y acid synthet.aae obtained from diabet.ic animals, (B) second centrifugat,ion of protein collected as indicated from the first centrifugation, (C) the same as (.4) except that. the fatty acid synthetaer was purified from livers of animals fast.ed for 60 hr, (I)) second centrifugation of protein collected as indicat,ed from the first centrifugat.ion. Values are reported for light absorption at 280 rnb (0-O) and enzyme activity for the synthesis of fat.ty acids (X-----X).
natlng protein is 235,000, which is smaller than that of the fatty acid synthetase (mol u-t 540,000). Separation of the 7s component from the
fatty acid synthetase has been achieved as described under Experimental Procedure. The behavior of this component on t,he first and second sucrose dcnsit,y gradient cen-
34s
COLLINS
FIG. 5. TXsc gel electrophoresis of the 78 component, isolated from livers of diabetic and 60-hr fasted animals. The 7S component, 40 Bg of protein, isolated aa described in t.he text, wae applied IO a polyacrylamide gel and then subjected to electrophoresis as det.ailed in the lXxperiment.al section. The 78 component obtained from diabetic animals was applied t,o tube 1; that, from 60-hr fasi ed animals was applied t,o t.nbe 2.
t rifugation of the fatty acid synt.hetase fraction isolated from diabet,ic and 60-hr fast.ed animals is shown in Fig. 4. The 75 protein isolated by this procedure had no act.ivity for fatty acid synt,hesis while the fat.ty acid synthet.ase isolated from 4%hr refed animals bv the standard procedure had normal act ikit y. The 75 protein was also assayed for acetvl-CoA carboxylase activity. No enzyme acti& y was observed at a protein concentration of 6.75 pg/ml. The elect,rophoretic behavior on disk gel of the 78 component isolated from diabetic and fasted animals is shown in Fig. 5. Each protein migrated to the same position in the gel. Thus the proteins isolated from diabetic
ET AI,.
and fasted animals are ident,ical in elect:rophoretic behavior, aswell as in sedimentation charact,eristics, as shown in Figs. 3 and 4. Origin of tb.e7S compownf. Because of the apparent, relat.ionship between a decreasein t,ot,al fatty acid synthetase units and the appearance of the 7s protein, it was suggested t,hat this prot.ein might be composed of subunits of the fat,ty acid synthet.asc (1). If subunits, these could be intermediates in t,he synthesis or pr0duct.s of the metabolism of the complex. In previous investigations we demonstrated that radioactivity of L-leutine-14C is not, incorporated into the 75 component, during the refeeding of fasted animals (1). The incorporat,ion of L-leuciwJ4C into the isolated 7s protein by animals fast.ed for 15 hr is shown in Fig. 6. It is evident, from t,his result, that t,he 7s prokein is synthesized during fast.ing. Hence, the 7s prot,ein is peculiar t.o t.he fast,ing and diabetic states, and is not, present in a significant amount, in livers of normal or fasted and refed rat,s. Relationship of the 7S protein lo th.eJatty acid synthetase.The synt,hesisof palmitic acid from acetyl-CoA and malonyl-CoA by the fatty acid synlhetase enzyme complex involves several intermediate st.eps.Assays for t,hose intermediate react,ions of fatty acid synt,hesishave been report.ed for the pigeon liver fatty acid synthetase by Kumar et al. (12). Similar assays for these partial act,ivities were performed with rat liver fat.ty acid synt,hetase and the 7s component, in an effort to establish either a relationship, or :L nonrelationship, between these two proteins. Table I reports t,he enzyme activities of protein purified from livers of control and GO-hr fast,ed animals. A comparison of the overall fatty acid synt.hct asc nct,ivit.y for protein from the cont.rol and fasted atumnls indicates that, the protein from the fnsled animals contains only about, 10% of fat.ty acid synthetase. Sone of t.he activit,ies for the partial reactions of fat.ty acid synthesis for the preparabion from GO-hrfasted animals is significant,ly higher t,han expected on this basis. Therefore, we conclude that, the act.ivity seen in the fatt.y acid synthetuse preparations obtained from GO-hrfasted unimals is due to the presence of fatty acid
A PROTEIN
OF l~IABI:TIC!
AIW
FASTEI)
349
RAT.+
.a00
80 z0 5
E’ 8
it \ L 0 40 z E 5 F 20 8
C-J ,600 &
6.
5 E .400 B % p ,200 5 i
% lz 0
IO
20 FRACTION
Jo
40
NUMBER
FIG. 6. Incorporation of L-leucine-W into the 7s protein of rat liver during fast.ing. At. 11 hr after the start of fast.ing, four animals received an intraperit.oneal injection of uniformly labeled L-leucine-“C (0.125 ml and 12.5 pCi). The animals were killed at 15 hr of fasting. The fatt.y acid synthetase was prepared and t.he 78 protein was separated from t,he synthetase aa described in t,he text,. This figure plots the behavior of the protein on the second sucrose density gradient centrifugation of t.hat procedure. Values are reported for light absorption at 280 w (0-O) and radioactivity (X--X). (Note: samples ass:tyed for radioact.ivit.y were counted for 60 min.)
synthet.ase and not to the presence of these enzyme activities in the 75 component.. The results of assays on the 7s protein isolated from diabetic animals and on t,he fatty acid synthetase isolated from normal animals for t,he part.ial reactions of fatty acid synthesis are presented in Table II. The 75 protein had no measurable acf.ivity for the synt,hesisof fatty acids, the transacylation of acetyl- and malonyl-CoA, the deacylation of palmityl-CoA, or the reduction of S-w&oacet.yl-:V-acetyl cyst,eamine. A fundament.al activity of the fat,t,y acid synt.het,ase is its ability to bind acetyl and malonyl groups. This reaction is carried out by the dissociated complex (half-molecularweight subunit:s) as well as the fatty acid synthet,ase (12). Table III reports the results of an attempt to bind acetyl and malonyl groups (from their CoA est,ers) to the 75 prot.ein isolated from livers of OO-hr fasted rats. The conditions of the experiment. are described in the Experimental section. There was no binding of eit.her acetyl or malonyl moieties t,o the 75 protein. I~,lnau,l.odi$usio,lof thefatty acid syrl thetase and the 7s protein. Further evidence that the TS protein is unrelated to t.he fatty acid synthet.ase was obtained through immuno-
diffusion experiments. Two distinct bands, one major and one minor, were obtained on react,ion of antibody with a fatky acid synthetase preparation obtained from livers of 16-hr refed animals (Fig. 7). The possibility that the minor band is due to the 7s component while t.he major one is due to fatt\ acid synt.hetase was investigated by reaction of a&body with the purified TS component and antibody with a preparation in which the 75 compound had not. been separated from the fatty acid synthetnse. An identit J reaction was obtained between the 75 component precipitin band and the minor band of t.he unseparated preparation (E’ig. 8). So detectable cross reaction was obtained with t.he major band. This result supports the previous conclusion that the 75 component is unrelawd to the fatty acid synthet ase. Immunodiffusion was also used to provide further evidence that the 7s component from st.arved animak is identical to the 75 protein in diabetic animals. Isolated 7s components from diabetic and starved animals yielded identical precipitin bands upon reaction with antibody (Fig. 9). It, must be pointed out that in order for the 75 component to react with antibod?, there must have been some 7s component 111
350
COLLTNS TABLE
A(:TIVITIIS PLXIFIH) FROM
KSZYMIS
I
TABLE
J~.\Ts Control specific activity”
Assay -
III
OF F.\TTY .4cr11 SYNTHKT.LW LIVKRS OF CONTROL a+~)
60-Hn F.\sw:I,
_----
ET AI,.
----
-.._I_--
Fatt.y acid synthesis Acet,yl-CoA t,ransacylaac Malonyl-CoA trensacylaee Palmityl-CoA deacylase CO* exchange S-Acetoacetyl-NAC” reductasec S-n, L-a-hydroxybut.yrylNAC dehydrate” S-Crot.onyl-NAC reduct,aee*
i
--.----. I
hILHr fasted specific activitf
-I-_-.--
65.7 6.0 750. I 50. 943. 100. I 326. 29.4 1.10 0.04 2.48 x lo” 1.73 x lo2 18.3
-- -.-.-
Substrate -
-
-.-
-.-..
-.._-
mpmoksbound/m6
-
-
-. control ..-.-.-.
-
of protein
--.
-. --
-
iS Component
.-_. .-..
-.-..
.-
Acetyl-CoA 1.25 .004 Malonyl-GA 0.713 - ,001 ---. --. ._ _ ----. .---...-. a Incubations and subsequent operations were performed aa described in the text. The 7A component. was isolated from livers of 60-hr fmtRl.ed animals.
0.98
3.42
0.535
(aAll activities arc expressed as mpmoles of product formed/min/mg of protein. h M-Acet.yl cysteamine derivative. 6 The subst,rate concentration used wae 14.82 m51. d The substrate concentrat,ion used was 9.0 rnM.
6 The substrate
concentration
used wu
10.8
rnM.
TABLE
II
J~NZYMB AWIVITIKH OP CONTROL SYXTHET.SK al) OP THK 7s IS~I,.~TIXI FROM l)I.\nleTlc -.- ---.
i control 7g0t$-
Assay
---_--
F.ITTY Acre COMI~ONE:WI~ ANIMALS' ---.
specific activity”
- -----
---.
FIG. 7. Immunodiffusion of a fatt.y acid synthet.aee preparation from the livers of 16-hr refed animals. The center well (A) contained antiserum prepared ae described in the text and the peripheral wells (B, C, and 11) cont.ained a fatty acid synthetase preparation from the livers of 16-hr refed animals. The diffusion medium wae 0.5$.;, agaroae. Samples were washed wit.h 0.857; saline to remove unreacted protein and then st,ained wit.h amid0 black.
specific activity
-. ____
Fat,t.y acid synthesis 28.0 1 0 Acetyl-CoA transacylaae 408. Malonyl-CoA transacylase ! 790. Palmityl-CoA deacylaee IS-Acetoacetyl-NAC reduc(l?, 10s x tasec I ___-.-- --.._ a The strain of rats used in these experiments was different from the one used in the st.udies on fa&ing. b Specific activity is given in mpmoles of product formed min/mg of prot,ein. c The substrate concentration used wae 14.82
i ;
rnM.
the preparat.ion which was used to produce the antiserum. Alt,hough bhe 75 component was not, detectable by ultracentrifugation, gel electrophoresis and sucrosedensity gradi-
ent. centrifugation (8) in preparations of fat.ty acid synthetase isolated from animals which had been starved and refed for 4s hours, this does not. preclude its being present, in very small am0unt.s which are detectable only by highly sensitive immunological met,hods. E$ect of the 78 protein 011the activity of the fatty acid qynthetase. Since the 75 protein exhibited none of the activit,ies of fatty acid synthesis, its role as a possible inhibitor or activator of t.he fatty acid synt,het.ase was determined. The effect of the 75 component on the rat.e of synthesis of fatky acids by the fatt,y acid synt,hebaseis shown in Fig. 10A. Fat,ty acid synthetase, 10 wg of protein, was incubated for 30 min with varying amounts of 7s protein. Spectrophotometric assaysfor fatty acid synthesis were then performed. No
A PROTI
OF
DIABETIC
FIG. 8. Immurlodiffusion of fatty acid synthetase from which the contaminating 7S component, had not been removed and isolated 7S component. Well A contained antiserum. Well B comt,ained a preparation (from 60-hr fasted animals) of the fatty acid synthetase prior to its separation from t.he 7S component by successive sucrose densit:y gradient. cent,rifugat,ion. Well C contained 78 component (from 60-hr fasted animals) which had been separated from the fatty acid synthetase by sucrose density gradient centrifugation. The diffusion medium was 0.5’;:; agarose. The samples were IIO~, stained.
FIG. 9. Immunodiffusio~~ of purified 76 component from livers of 60-hr fasted animals and purified 7S component from livers of diabetic animals. Well A contained antiserum. Well B contained 7S componenl from the livers of 60-hr fasted animals and well C contained 7S component from the livers of diabetic animals. Both 7s compol1ent.s had been separated from the fatty acid syot hetase by Iwo successive sucrose density gradient centrifugations. The diffusion medium was 0.5’y0 agarose. After being washed wit.h 0.85y0 saline t.o remove extraneous protein, the samples were stained wit,h amido black.
inhibition or enhancement, of the rate of synthesis was observed. Hence, activity of t,he fat,t.y acid synthetase is unaffected by the presence of the 75 protein. #feet (d the TS protein otL the activity of acetyl-CoA carboxylase. The effect of the 75 component. on the rate of acetyl-CoA carboxylation was also test.ed (Fig. 10R). Varying amounts of the 75 component (O-O.75 pg
AND
FASTED
351
R.ATS
(J-A--’ 2
4
6
8
IC
FIG. 10. Assays for fatty acid synthetase and acetyl-CoA carboxylase activity in 6he presence of t.he 78 component. A. Fatty acid synt,hetase, 10 fig of prot,ein, was incubated for 30 min at 37” with varying amounts of 78 component. The mixt.ure was then assayed spectrophotometrically for fatt,y acid synthesis. B. Acetyl-CoA carboxylase, 20 pg of prot,ein, was incubat.ed for 10 min at 37” wit.h varying amounts of t,he 7s component. Enzyme activity was det.ermined as reported under Experimental Procedure.
protein/ml) were incubated with acetyl-CoA carboxylase (20 pg of prot.ein). The addition of 7s component had no effect on t,hihisenzyme activity. The highest. concentration of 75 component. tested represents an approximate ;iO-mole excess of this component, t.o enzyme. This calculation is based on t,he assumption t.hat. only one-tent.h of t.he protein in our preparation was acet,yl-Coa carboxylase. This is reasonable inasmuch as this preparation exhibited 10 %I of t,he activity of pure acetyl-CoA carboxylase (9). The active enzyme form was assumed t,o have a molecular weight of 4 X lo6 (16).
352
COLLINS
It is evident from the experimental results presented in this paper t,hat a 75 protein component is found in associat,ion with purified rat liver fatty acid synthet.ase isolated from livers of fasted or diabetic rats. This protein is synthesized in t,he livers of fast.ing and diabet,ic rats. The incorporat.ion of radioactivit,y of leucine int.o the 75 protein and the net, increase in its quant,it,y under t,hese conditions are evidence in support, of this conclusion. This protein disappears, or is reduced to a negligible quantit,y in liver on refeeding fasted animals or on treatment of diabetic animals with insulin. Hence, the fat,ty acid synt,het,ase purified from livers of normal or fast.ed and refed rats cont.ains eit.her traces or none of bhis contaminating protein. The 75 component, is separated from the fnt,t.y acid synthetase by two successive sucrose density gradient centrifugations. This protein is nearly homogenous on disk gel elect,rophoresis and on sucrose density gradient cenkifugation. The molecular weight of t,he TS component, is approximately 2.3s X 105. The 7s components from diabetic and fasted rats behaved identically on sucrose density gradient centrifugation and disk gel electrophoresis. lkrt,her confirmation of their identity was obtained in the immunodiffusion studies. Upon react.ion with antibody, t,hese proteins gave precipitin bands of identity. Thus, the 75 protein from diabetic animals is the same as t,hat from fast,ed animals. This observat.ion is consistent wit,h t,he close met,abolic relationship observed between diabetic and fasted states in animals. In our earlier studies (1) we suggest:ed that. t,hc contaminating protein might be a precursor or a degradat,ion product, of the fatty acid synthetase. However, our inability to secure t,he incorporation of radioacbivity of W-leucine into this protein on refeeding fasted animals suggested that it was not a precursor. To establish 1vit.h greater cer, tainty the relationship or lack of relationship of the 75 prot.ein to the fatt.y acid synt,hetase, assays were performed for the part,ial reactions of fatty acid synt.hesis. The purified 75 component, did not, exhibit act,ivity for any of these partial reactions. Keit.her did it
ET AL.
bind acetyl or malonyl groups. As a COIIsequence it: is concluded that the 75 component is unrelated to t,he fat.ty acid synthat,ase. This conclusion was confirmed by the lack of an immunochemical cross react.ion between the 7s component precipitin band and the major precipitin band due to the fatty acid synthetase. The possibility that the 78 protein might, fun&on in the regulation of fat.t,y acid synt,hesis was also considered. However, this prot,ein had 110 effect on either fatty acid s:);nthet,ase or acet.yl-CoA carboxylase activity. The fact. that the 75 protein is synthesized at a time when protein synthesis is at, a minimum suggests t,hat, this protein may have an important role in diabebic and fasted animals. Investigations direct.ed toward the identification of this role are in progress. ACKNOWLEDGMENTS We express our sppreciatiorl to Dr. William L. Kopp for his advice and the use of his equipment in the immunological studies. We are also grateful t,o Mr. Gerard0 Velasquez for assistance in the performance of the ultracentrifugatiorl analyses, and tt) Dr. Suriender Kumar for some of the assays for the partial reactions of fatty acid synthesis.
1. 2. 3. 4. 5. 6. 7. 8. 9.
11. N., COLLINS, J. M., KI~NNAK, A. L., .\w) PoRTKR, J. W., J. Bid. Ch:hem. 244, 4510 (1969). SIMON, E. J., AND 8til?dIN, I)., J. Amer. Chem. Sot. 76, 2520 (1953). TRAMS, El. G., AND Baan~, 11. O., J. Amer. Chem. Sot. 82, 2972 (1960). BRODIE, J. 1)., AND PDRTRR, J. W., Hioch.em. Biopkys. lies. Cornmw. 3, 173 (1960). KORNACKRR, M. H., AND LO\VENSTIGIN, J. M., Biochem. J. 96, 209 (1965). WAKIL, ti. J., PORlUR, J. W., AND (;IHSOS, 1). M., Biochint. Hioph~~s. Ada 24, 453 (1957). Hsu, R.. Y., WASSON, G., AND PORTER, J. W., J. Biol. Chhem. 240, 3736 (1965). BURTON, 1). X., Hanvra, A. G., AXD PORTIX, J. W., Arch. Biochent. Biophyx. 126, 141 (1968). BURTON,
Nun5.4,
S.,
I~NC;KLMANN,
15.,
AND
LYNKN,
F.,
Biochem. Z. 340,228 (1964). 10. GORNALL, A. G., B~RDA~ILL, C. J., AND I~AVID, M. M., J. Hiol. Chem. 177. 751 (1949). il.
A PHOTISIN
OF
DIAI3F;TIC
RAIJM, II., OLSON, E. B., ~~.\RGOLIS, s. .4., I\ND PORTER, J. W., Arch.. lliochem. Riophys. 116, 453 (1966). 12. Kuw4R, S., DORsRy, J. .4., h’b;SI~G, I<. A., AKD PORTRR, J. W., .I. Rid. Chesr . 246, 4732 (1970) .
13. CHANG, H-C., ~ICIDMAX, Laiu~, hf. I)., Hiockem. 911119~. 28, 682 (1967).
I.,
TM~H,
Hioph.yx.
(;., Kes.
.\SD
Cant-
AN‘11
F,4STRI)
353
II..4TS
14. I).\vIs, B. J., Ann. :V. I’. Acad. Sci. 121, 404 (1964). 15. CHR.~~ICE~, >f., .a~
(1967). 16. (hEGOLIN,
A., I~EISFELU, Z.VXRI, J., Anal.
C.,
l
KLEINSCHMIDT, A. K., Aoc. LTu/. A cad. Sci.
(1966).
E.,
lt. .4., WYCROFF, Biochem. 20, 150 WARNER,
.\SD LXNE, Cr. S. A.
It.
c.,
M. 66,
D.,
1751
Ok' BIOCHEMISTRY
Studies
ASD RIOPHYSICS
on a Protein Diabetic
JANET
I.ipicl
(1971)
14,343-353
Isolated and
Fasted
from
Livers
of
Rats’
M. COLLIM, MARGARET C. CRAIG, CARL JI. NEPOKROEPF, A. L. KENSAN or) JOHIZ’ W. PORTER
Melabolism Laboralory, of Physiolvgical
Velerans Chemislry,
Atltninixlralion LTni~ersil!l
Keceived -4ugust
Hoxpild, oj W’ixeonsin,
11, 1970; accepted
Madison, .Vndiwn,
November
lI~‘isconxirr, and Ihe Deparlttlenl H’iwonxitc.
53706
3, 1970
A protein that is synthesized by livers of diabetic and fasting rats has been isolated and purified. This protein is either not, synthesized or it is synthesized in trace amonnt,s by livers of normal or fasted and refed animals. This component, designated as “78” protein, accompanies t.he fatty acid synthetase through all st.eps of purificat.ion. However, it can be separated from the fat.ty acid synthetase by two successive sucrose density gradient cellt,rifugatiolis. When separat.ed in this way it, is nearly homogenous in size and charge (disk gel elect rophoresis). The 7S proteins obtained from diabetic and fast.ed livers are ident,ical wit.h respect to size, charge, and immunochemical propert.ies. This protein does not. cat,alyze any of the partial reactions of fatty acid synt,heSis, does not. bind acetyl or malonyl groups, and it. does not. exhibit acetyl-CoA carboxylaae activity. Immunological studies show it to be unrelated to the fat.ty acid synthetase. Finally, it has no effect. on the rate of fatty acid Bylit,heBiB or on acetyl-CoA carboxylase activity. Thus, on the basis of these properties, t,he 78 prot.ein is not related to the fatty acid synt.het,asc and probably not to acetyl-CoA carboxylase. The function of this prot.ein remains t.r) be determined.
In the course of studies on the effect of nutritional and hormonal fact,ors on the synthesis of the rat liver fatty acid synt.hetase Burton et al. (1) observed t.he presence of a protein that accompanied the synthetase complex through each step of purification. The content of this contaminating protein increased as animals were fasted for increasing lengths of time. It: also disappeared on refeeding fasted animals, and at 24-X hr of refeeding it was no longer detect,able. The protein contaminating fhe fat.ty acid synthetase was also present in the livers of dutbetic rats. It was suggest.ed from the st.udies of Burton et al. (1) that. the protein contaminat1 This investigation was supported in part by liesearch Grants AM01383 from the Sational IrMitute of Art,hritis and Metabolic Diseases of t.he National Institutes of Health, Unibed States Pltblic Health Service, and (i-68-38 from the Life insurance Medical Research Fund.
ing the fat.ty acid synt,het.aseobtained from livers of fasted or diabet.ic rats might be a precursor in the synthesis of t.his complex or an intermediate in its degradation. It was also suggested that this protein might be unrelated to the fatty acid synthetase. In the present paper we have investigated t,hese possibilities. Init.ially we were able to show that the contaminating protein can be separated from the fatty acid synthetase by two successive sucrosedensity gradient. cenkfugations. The protein isolated from diabetic or fasted livers and separated in this way is nearly homogenow on disk gel electrophoresis and ultracent,rifugation. In addition t,he proteins obtained from fasted and diabet.ic livers are identical in their behavior on disk gel electrophoresis, sucrose densit.v gradient centrifugation, and immunokffusion. Hence they are either identical or nearly identicd in structure.
The contaminating protein, termed “78” protein, is synt.hesized by the liver dur& fast,ing. This protein shorvs none of the parGal react,ions of t.he fatty acid synthetase complex. P\‘either does it bind acetyl or malonyl groups, nor show acetyl-&A carboxylase activit.y. Furthermore, it. is im-
munologically dist.inct from the fatty acid synthet,ase. Hence, it is unrelated by these criteria to the fat.ty acid synthetase. It also does not affect t.he rates of fat.ty acid synt.hesis or t.he carboxylation of acct.yl-CoA.
phosphate buffer, pH 6.8, wan subst.ituted for t.he final Sephadex (i-100 gel filtration sl.ep. Iso~alion f~j 78 contponenl jrottt J’ally acid x!jn fhelaae of ral lirer. Patty acid synt.het.ase was prepared by t.he procedure of Burton ef a/. (8) from animals of different nutritional and hormonal states. This prot.ein was then applied IO a slmose gradient and centrifuged, as reported in a latel section, at a rotor speed of 48,060 rpm. Light absorption af 280 nip was determined for each collected fraction, and 1hose fractions rorresponding to the slower moving protein from four to six separate gradients were combined. The resulting sollltion was dialyzed against 0.5 Y potassilml phosphate buffer, plI 6.8, precipit.ated with ammonium su1faf.e (O-8041 satllration), and again dialyzed. This protein was applied to a second sucrose gradient. and centrifuged as dcscribcd previonsly. Fractions lb20 (see Fig. 4B and 1)) were combined and used in subsequent experiments.
EXPEHIMENTAL PIIOCEI)Ul~E Jfulerials. Experimental materials were obtained from the following sources: acetic anhydride-I-“C, malonic acid-2-l”C, palmit,yi-CoAl-l%, sodium bicarbonate-‘%, and uniformly labeled L-leucine-1% from New England Nuclear; I’atrlial purificalion of acelyl-CoA carboxylase. acetyl-CoA, malonyl-CoA, CoA, and NADPH Acetyl-CoA carboxylase, from livers of rats fed II from P-L Biochemicals; 2-mercaptoelhanol and normal diet, was part ially purified (through t,he dit.hiothreit.ol from Calbiochem; and palltct.heine second ammonium sulfate R~IJ) by t hc method from the Sigma Chemical Company. All other of Suma, liingelmann, and I+Jetl (!I). This prepreagents used were of analytical grade. aration had an acet yl-CoA t!arboxylase activity Synthesis of coenzynted eslers. Acetyl-CoAof 65-V& mpmoles of CO, irlcorpIjrated!min/mg of 1-14C and caproyl-CoA were synthesized by the protein and a negligihlc activity for fat,f.y acid method of Simon and Shemirl (2), and malonylmpmoles of acetyl-(:oA incorCoA-2-14C was synthesized by the method Of synthesis, 0.0082 porated/min/ing of prof ein. Trams and Brady (3). Coenzyme-A esters were Asmy for prokin. Assays for the protein conpurified by 1he paper chromatographic systems of ceIlf,rations of soluf.i(Jns were carried out. by I.he Brodie stud Porter (4). billret. mct.hod of ( LJrnaIl, Bard:lwill, and David Treatmen/ ofl animals. ;11ale rats, weighing (10)~ about 150 g each, were used in most expcrimcnt.s. Assay for jall!l ucirl synlhclaxe n&Sly. The Animals were fasted for varying periods of time or assay tiystem uncd WUHeither the radiochemical they were fasted and refed. When animals were assay Of Hsu, Wasson, and Porter (7), as modified refed, a fat-free diet was used (5). Uniformly tJy Bllt 1erwort.h ef ul. (ll), or ii H~JC’D~ rqJhotometric labeled r,-leucine-14C was administered as an assay. The following c:ollcc~ltrlttio~lo of substra1.c intraperit,oneal injection of mJ aqueous SOhJtiOI~ of the amino acid at an appropriate t.imc prior to and enzyme, in a l-ml vol~mx, were used in f.he sacrifice (1). Animals were killed lay a blow to t.hc lat1er assays: acdyl-CIJA, 5 X 10-S M; malonylCoA, 1 x l(r’ M; NAI)l’Il, 1 x lo-’ u; IGl)TA, bnse of the skull. 8 X l(Y M; potwssirun phosphate buffer, 0.2 M Alloxan diabetes was induced in rats weighing and pH 7.0; and protein, 2 X 10eRM. The specific approximat.ely 250 g by the injection of a 5c/, acl.ivify of the fat,ty acid synthet.ase is expressed solution of alloxan monohydrate (40 mg/kg) via as millimicromoles of fat.Iy acids (mamoles of the tail vein. TW(J weeks later the animals had NAIjPII oxidized + 14) formed per minut.o per blood glucose levels of 250 mg/lOO ml or higher. milligram of protein. Ru$ers. All bnfiers cont.ained 1 mM Bl)TA and Assays for par/id readions of full!y acid synlheeit.her 1 rn~ dithiothreitol or 1 mM fl-mercaptosis. Assays for the partial reactions of fatty acid et,hanol, unless otherwise specified. synthesis were performed aa described by Kumar Purijicalicttr oj fatly acid synthelase. Rat liver snpernatant solut,ions were prepared by t,he al. 01. (12), with ooe exception. In t.hc palmitylCoA deacylase rcncf ion, the conc:cntration of met,hod of Wakil, Port.er, and Gibson (6) as modipalmityl-CoA-1-~4C was 4 p~ and the qlrantit.y of fied by Bsu el aI. (7). The fat.ty acid synthetase profein used was 1 pg. was then purified from the supernatant solut.ion Assays for lkc binding oj acelgl and rnalonyl as described by Rurton, Haavik, and Porter (8), except, that dialysis against. 0.5 .M pottwsium g,otrpx to /he ~-a//y/ ucid nynlh.e/axe. Assays for the
A PROTEIN
OF
DIABETIC
covalent binding of acet,yl and malonyl groups to protein were performed through the addition of 5 amoles of acetyl-CoA-1-“C or malonyl-CoA-2-lS IO 250 pg of prot.ein in 1 ml of 0.5 M phosphate buffer at pH 6.8. The assay mixture wae incubat,ed at 0” for 30 set and the reaction was stopped by i.he addition of 0.5 ml of 6270 perchloric acid. The protein precipitate was centrifuged and then washed three times each with 1 ml of 0.2 N acet,ic acid. The washed protein was assayed for radioactivity as described below. Assay for acelyl-Co:od carbozylase a&‘&g. The radiochemical assay method of Chang el al. (13) was used. Acet,yl-CoA carboxylase, 0.2 mg of prot.ein, W&B preincubated at 37” for 30 min at pH 7.0 with 60 rnM Tris (Cl-), 3 my glutathione, 8 mM MgClr, 0.1 rnx EDTA, 0.6 mg protein of bovine serum albumin/ml, and 5 rnx cit.rate (KC). The formation of “C-malonyl-CoA was effected by the addition of preincubated enzyme, 20 ~g of protein, to a solution containing 60 nlM Tris (Cl-), 2 mni ATP, 8 mr.i MgClz, 10 mM KH’dCO, (0.2 pCi/pmole), 0.2 mM acet.yl-CoA, 3 rnM glut,athione, 0.1 m.u EDTA, 0.6 mg protein of bovine serllm albumin, and 5 rnM citrat,e (K+). A final volume of 1.0 ml was incubated for 10 min at 37”. The reaction was terminated by the addition of 0.2 ml of 6 N HCl, and the incubation mixture wae evaporated to dryness. Residual radioactivity was det.ermined bg assay in a dioxane solution in a liquid scintillation spectrometer. Deleminalion of radioactivif;g. All measurements of radioact.ivity were ohtained wit,h a
AND
FASTED
11.41’S
345
Packard liquid scintillation spectrometer. When the radioactivity of prot,eins was to be determined, samples were dissolved in 887& formic acid and then diluted w&h 2 ml of et.hanol. Toluenebased sciut.illation solution was t.hen added to each sample. .~n.alytical rcItracen./rifugation. Ve1ocit.y sediment,ation experiments were carried out in a Spinco Model E ultracentrifuge. The rotor t,emperature was 20” and the speed was 59,780 rpm. Sedimentation coefficients were corrected for solveut viscosity and density at 20”. Sucrose density gratlienl cerrlrifugalion. Sucrose density gradient cenbrifugation was performed for 23.5 hr at 48,060 rpm in a Spinco SW5OL rotor in 5 ml of a 15-355;, sucrose gradient containing 0.5 M potassium pho5phat.e buffer, pH 6.8, 1 mM EUTA, and 1 my dithiot.hreitol. Fractions of 0.14 ml each were collected from the bott.om of the tube after centrifugat.ion. Disk gel eleclrophoresis. Acrylamide disk gel elecbrophoresis was carried out, according to t.he method of Davis (14). Five per cent acrylamide was used in the resolving gel. After electrophoresis t,he gels were fixed and stained by the procedure of Chrambach et III. (15). Prepa.ralion of f&/u acid sgnlhelase anliserur~~. The fatty acid synthet,ase used as antigen w&s isolated by t.he normal purification procedure (8) from the livers of rat.s which had been fasted for 48 hr and theu refed for the same period of time on a fat-free diet. Approximately 30 mg of fatt.y acid synt het.ase, in iucompletc Freund’s adjuv:m~,
Fro. 1. The total protein and enzyme activity of fatty acid synt.het,aae isolated from livers of control and fasted rats. Groups of four rat.s were fasted for varying periods of time, then killed, and the fatt,y acid synthetaae of livers of these animals was purified by t,he procedure of Burton et al. (8). The protein (fatty acid synt,hetase) obtained per liver is given in mg, l d; the specific enzyme activity inpmoles of product formed/min/mg of protein, O-----O; and t.he total units of fatty acid synt.hetaae per liver (specific activit,y X mg of protein), (>-.-a-.@.
346
COLLINS
FIG. 2. Sucrose density gradient. centrifugation of rat liver fatty acid synthetaee isolated from livers of control and fasted rat,s. Animals frosted for 0, 8 and 32 hr were killed, and the fatty acid synthetaae of the liver was isolated by the procedure of Burton et al. (8). The isolated enzyme was t,hen subjected to sucrose density gradient centrifugation, aa described in the text, at a rotor speed of 40,000 rpm. Samples from the bottom ho the top of the tube read from left t.o right in the Qure. Values are report.ed for light absorpt,ion at 280 nip (@---0) and enzyme activity for fat)ty acid synthesis (X-----X). A, 0 hr; B, 8 hr; and C, 32 hr of fasting. was inject,ed subcutaneously into each of two rabbit;s over the thighs and shoulders bilaterally at four sites. This procedure was repeated 3 and 8 weeks later. The rabbits were then bled at l-week intervals to obtain antiserum. The antiserum was atored at - 15” unt,il used. Im?,~unfdi~~crrion. Onchterlony micro-doublediffusiou teats wit.h antiserum and various preparations (Jf the fatty acid synthetase and 7S prot,ein were carried out, in 0.5% agarose.* RESULTS
The detection of a protein copurifying with the jatty acid synthetase obtain.edfrom fast& and diabetic alrimals. The total number of e Operation Manual, LKB 68ODA Immunodiffuosin Equipment, p. 17.
ET Al,.
milligrams of protein per liver, isolated 2~s fat,ty acid synt.hetase, from fasted rats is plot,ted as a function of time of fasting in Fig. 1. The specific act,ivity of the fatty acid synthetase isolated from livers of fasting animals and the t.ot.al unit.s of enzyme per liver are also reported in Fig. I.. The procedure used for the isolation of the fatty acid synt,het.ase in this experiment yields a homogenous protein when t.he enzyme is isolated from normal or fasted and refed animals (8). The presence of a protein contaminating the fatty acid synt,hetase is suggest.ed from these results because of the marked decline in the specific activit,y of thth enzyme during fasting, compared 1.0the tot al number of milligrams of protein isolated, which shows a slight, rise. The presence of a protein contaminating the fatty acid synt.hebase was shown via sucrose density gradient centrifugation of protein isolated from the livers of 0-, X-, and 32-hr fasted rats (Fig. 2). At, 32 hr approximat,ely 50 % of the isolated prot.ein did not cont.ain fatt,y acid synthetase activit:y. Previously we showed that this contaminating prot,ein is not detectable by sucrose denait.y gradient centrifugation upon refeeding of fasted animals for 24 hr or longer (1). Similar results were obtained when fatty acid synthet.ase was purified from livers of alloxan diabetic animals. The t.otal number of milligrams of fatt.y acid synthet,ase protein obtained per liver increased over the level of controls, but t.he specific act,ivity for fat1.y acid synthesis and t,he total units of enzyme decreased (1). Sucrose density gradient ccnt.rifugation of the fat,ty acid synthet.ase purified from livers of diabet.ic animals revealed the presence of a cont,aminat:ing protein similar to t.hat, found associated with the fatt,y acid synthetase prepared from fast,ed animals. Properties of contam.inatiq! pro,!&. The sediment,ation pat.terns obtained on ultracentrifugation of fatty acid synt,hetase prcpared from livers of diabet.ic and GO-hr fast.ed rats are shown in l;ig. 3. Two major components with s~~,,~values of 32 and 7 were observed for each preparat,ion. The fast,cr moving component is the fat t)y acid synthetase. The molecular weight of the cant ami-
A PROTEIN
OF 1)IABKTIC
ANI)
FA!!TP:I)
347
RATS
Fro. 3. Sedimentation characteristics of rat liver fatty acid synthetase isolated from livers of diabetic and GO-hr fast.ed animals. Ultracentrifugat~ion was carried out as described in the text.. Sediment&ion is from left t.o right. Photographs were taken at. 8-min intervals. (A) Fatty acid synthetase from diabet.ic animals, (B) fat,t.v acid synt,hetaee from 60-hr fasted animals. I50 A 1.20
C 600
,900 -
400.
,600. 2 lt N.3006 5F
;.200i 5
0
0
FIG. 4. Separation of the 78 component from rat liver fatty acid synthetase. Sucrose density gradient, cent,rifugations were carried out as described in t.he text and eluate fractions were combined aa indicat.ed. (A) First centrifugation of fatt.y acid synthet.aae obtained from diabet.ic animals, (B) second centrifugat,ion of protein collected as indicated from the first centrifugation, (C) the same as (.4) except that. the fatty acid synthetaer was purified from livers of animals fast.ed for 60 hr, (I)) second centrifugation of protein collected as indicat,ed from the first centrifugat.ion. Values are reported for light absorption at 280 rnb (0-O) and enzyme activity for the synthesis of fat.ty acids (X-----X).
natlng protein is 235,000, which is smaller than that of the fatty acid synthetase (mol u-t 540,000). Separation of the 7s component from the
fatty acid synthetase has been achieved as described under Experimental Procedure. The behavior of this component on t,he first and second sucrose dcnsit,y gradient cen-
34s
COLLINS
FIG. 5. TXsc gel electrophoresis of the 78 component, isolated from livers of diabetic and 60-hr fasted animals. The 7S component, 40 Bg of protein, isolated aa described in t.he text, wae applied IO a polyacrylamide gel and then subjected to electrophoresis as det.ailed in the lXxperiment.al section. The 78 component obtained from diabetic animals was applied t,o tube 1; that, from 60-hr fasi ed animals was applied t,o t.nbe 2.
t rifugation of the fatty acid synt.hetase fraction isolated from diabet,ic and 60-hr fast.ed animals is shown in Fig. 4. The 75 protein isolated by this procedure had no act.ivity for fatty acid synt,hesis while the fat.ty acid synthet.ase isolated from 4%hr refed animals bv the standard procedure had normal act ikit y. The 75 protein was also assayed for acetvl-CoA carboxylase activity. No enzyme acti& y was observed at a protein concentration of 6.75 pg/ml. The elect,rophoretic behavior on disk gel of the 78 component isolated from diabetic and fasted animals is shown in Fig. 5. Each protein migrated to the same position in the gel. Thus the proteins isolated from diabetic
ET AI,.
and fasted animals are ident,ical in elect:rophoretic behavior, aswell as in sedimentation charact,eristics, as shown in Figs. 3 and 4. Origin of tb.e7S compownf. Because of the apparent, relat.ionship between a decreasein t,ot,al fatty acid synthetase units and the appearance of the 7s protein, it was suggested t,hat this prot.ein might be composed of subunits of the fat,ty acid synthet.asc (1). If subunits, these could be intermediates in t,he synthesis or pr0duct.s of the metabolism of the complex. In previous investigations we demonstrated that radioactivity of L-leutine-14C is not, incorporated into the 75 component, during the refeeding of fasted animals (1). The incorporat,ion of L-leuciwJ4C into the isolated 7s protein by animals fast.ed for 15 hr is shown in Fig. 6. It is evident, from t,his result, that t,he 7s prokein is synthesized during fast.ing. Hence, the 7s prot,ein is peculiar t.o t.he fast,ing and diabetic states, and is not, present in a significant amount, in livers of normal or fasted and refed rat,s. Relationship of the 7S protein lo th.eJatty acid synthetase.The synt,hesisof palmitic acid from acetyl-CoA and malonyl-CoA by the fatty acid synlhetase enzyme complex involves several intermediate st.eps.Assays for t,hose intermediate react,ions of fatty acid synt,hesishave been report.ed for the pigeon liver fatty acid synthetase by Kumar et al. (12). Similar assays for these partial act,ivities were performed with rat liver fat.ty acid synt,hetase and the 7s component, in an effort to establish either a relationship, or :L nonrelationship, between these two proteins. Table I reports t,he enzyme activities of protein purified from livers of control and GO-hr fast,ed animals. A comparison of the overall fatty acid synt.hct asc nct,ivit.y for protein from the cont.rol and fasted atumnls indicates that, the protein from the fnsled animals contains only about, 10% of fat.ty acid synthetase. Sone of t.he activit,ies for the partial reactions of fat.ty acid synthesis for the preparabion from GO-hrfasted animals is significant,ly higher t,han expected on this basis. Therefore, we conclude that, the act.ivity seen in the fatt.y acid synthetuse preparations obtained from GO-hrfasted unimals is due to the presence of fatty acid
A PROTEIN
OF l~IABI:TIC!
AIW
FASTEI)
349
RAT.+
.a00
80 z0 5
E’ 8
it \ L 0 40 z E 5 F 20 8
C-J ,600 &
6.
5 E .400 B % p ,200 5 i
% lz 0
IO
20 FRACTION
Jo
40
NUMBER
FIG. 6. Incorporation of L-leucine-W into the 7s protein of rat liver during fast.ing. At. 11 hr after the start of fast.ing, four animals received an intraperit.oneal injection of uniformly labeled L-leucine-“C (0.125 ml and 12.5 pCi). The animals were killed at 15 hr of fasting. The fatt.y acid synthetase was prepared and t.he 78 protein was separated from t,he synthetase aa described in t,he text,. This figure plots the behavior of the protein on the second sucrose density gradient centrifugation of t.hat procedure. Values are reported for light absorption at 280 w (0-O) and radioactivity (X--X). (Note: samples ass:tyed for radioact.ivit.y were counted for 60 min.)
synthet.ase and not to the presence of these enzyme activities in the 75 component.. The results of assays on the 7s protein isolated from diabetic animals and on t,he fatty acid synthetase isolated from normal animals for t,he part.ial reactions of fatty acid synthesis are presented in Table II. The 75 protein had no measurable acf.ivity for the synt,hesisof fatty acids, the transacylation of acetyl- and malonyl-CoA, the deacylation of palmityl-CoA, or the reduction of S-w&oacet.yl-:V-acetyl cyst,eamine. A fundament.al activity of the fat,t,y acid synt.het,ase is its ability to bind acetyl and malonyl groups. This reaction is carried out by the dissociated complex (half-molecularweight subunit:s) as well as the fatty acid synthet,ase (12). Table III reports the results of an attempt to bind acetyl and malonyl groups (from their CoA est,ers) to the 75 prot.ein isolated from livers of OO-hr fasted rats. The conditions of the experiment. are described in the Experimental section. There was no binding of eit.her acetyl or malonyl moieties t,o the 75 protein. I~,lnau,l.odi$usio,lof thefatty acid syrl thetase and the 7s protein. Further evidence that the TS protein is unrelated to t.he fatty acid synthet.ase was obtained through immuno-
diffusion experiments. Two distinct bands, one major and one minor, were obtained on react,ion of antibody with a fatky acid synthetase preparation obtained from livers of 16-hr refed animals (Fig. 7). The possibility that the minor band is due to the 7s component while t.he major one is due to fatt\ acid synt.hetase was investigated by reaction of a&body with the purified TS component and antibody with a preparation in which the 75 compound had not. been separated from the fatty acid synthetnse. An identit J reaction was obtained between the 75 component precipitin band and the minor band of t.he unseparated preparation (E’ig. 8). So detectable cross reaction was obtained with t.he major band. This result supports the previous conclusion that the 75 component is unrelawd to the fatty acid synthet ase. Immunodiffusion was also used to provide further evidence that the 7s component from st.arved animak is identical to the 75 protein in diabetic animals. Isolated 7s components from diabetic and starved animals yielded identical precipitin bands upon reaction with antibody (Fig. 9). It, must be pointed out that in order for the 75 component to react with antibod?, there must have been some 7s component 111
350
COLLTNS TABLE
A(:TIVITIIS PLXIFIH) FROM
KSZYMIS
I
TABLE
J~.\Ts Control specific activity”
Assay -
III
OF F.\TTY .4cr11 SYNTHKT.LW LIVKRS OF CONTROL a+~)
60-Hn F.\sw:I,
_----
ET AI,.
----
-.._I_--
Fatt.y acid synthesis Acet,yl-CoA t,ransacylaac Malonyl-CoA trensacylaee Palmityl-CoA deacylase CO* exchange S-Acetoacetyl-NAC” reductasec S-n, L-a-hydroxybut.yrylNAC dehydrate” S-Crot.onyl-NAC reduct,aee*
i
--.----. I
hILHr fasted specific activitf
-I-_-.--
65.7 6.0 750. I 50. 943. 100. I 326. 29.4 1.10 0.04 2.48 x lo” 1.73 x lo2 18.3
-- -.-.-
Substrate -
-
-.-
-.-..
-.._-
mpmoksbound/m6
-
-
-. control ..-.-.-.
-
of protein
--.
-. --
-
iS Component
.-_. .-..
-.-..
.-
Acetyl-CoA 1.25 .004 Malonyl-GA 0.713 - ,001 ---. --. ._ _ ----. .---...-. a Incubations and subsequent operations were performed aa described in the text. The 7A component. was isolated from livers of 60-hr fmtRl.ed animals.
0.98
3.42
0.535
(aAll activities arc expressed as mpmoles of product formed/min/mg of protein. h M-Acet.yl cysteamine derivative. 6 The subst,rate concentration used wae 14.82 m51. d The substrate concentrat,ion used was 9.0 rnM.
6 The substrate
concentration
used wu
10.8
rnM.
TABLE
II
J~NZYMB AWIVITIKH OP CONTROL SYXTHET.SK al) OP THK 7s IS~I,.~TIXI FROM l)I.\nleTlc -.- ---.
i control 7g0t$-
Assay
---_--
F.ITTY Acre COMI~ONE:WI~ ANIMALS' ---.
specific activity”
- -----
---.
FIG. 7. Immunodiffusion of a fatt.y acid synthet.aee preparation from the livers of 16-hr refed animals. The center well (A) contained antiserum prepared ae described in the text and the peripheral wells (B, C, and 11) cont.ained a fatty acid synthetase preparation from the livers of 16-hr refed animals. The diffusion medium wae 0.5$.;, agaroae. Samples were washed wit.h 0.857; saline to remove unreacted protein and then st,ained wit.h amid0 black.
specific activity
-. ____
Fat,t.y acid synthesis 28.0 1 0 Acetyl-CoA transacylaae 408. Malonyl-CoA transacylase ! 790. Palmityl-CoA deacylaee IS-Acetoacetyl-NAC reduc(l?, 10s x tasec I ___-.-- --.._ a The strain of rats used in these experiments was different from the one used in the st.udies on fa&ing. b Specific activity is given in mpmoles of product formed min/mg of prot,ein. c The substrate concentration used wae 14.82
i ;
rnM.
the preparat.ion which was used to produce the antiserum. Alt,hough bhe 75 component was not, detectable by ultracentrifugation, gel electrophoresis and sucrosedensity gradi-
ent. centrifugation (8) in preparations of fat.ty acid synthetase isolated from animals which had been starved and refed for 4s hours, this does not. preclude its being present, in very small am0unt.s which are detectable only by highly sensitive immunological met,hods. E$ect of the 78 protein 011the activity of the fatty acid qynthetase. Since the 75 protein exhibited none of the activit,ies of fatty acid synthesis, its role as a possible inhibitor or activator of t.he fatty acid synt,het.ase was determined. The effect of the 75 component on the rat.e of synthesis of fatky acids by the fatt,y acid synt,hebaseis shown in Fig. 10A. Fat,ty acid synthetase, 10 wg of protein, was incubated for 30 min with varying amounts of 7s protein. Spectrophotometric assaysfor fatty acid synthesis were then performed. No
A PROTI
OF
DIABETIC
FIG. 8. Immurlodiffusion of fatty acid synthetase from which the contaminating 7S component, had not been removed and isolated 7S component. Well A contained antiserum. Well B comt,ained a preparation (from 60-hr fasted animals) of the fatty acid synthetase prior to its separation from t.he 7S component by successive sucrose densit:y gradient. cent,rifugat,ion. Well C contained 78 component (from 60-hr fasted animals) which had been separated from the fatty acid synthetase by sucrose density gradient centrifugation. The diffusion medium was 0.5’;:; agarose. The samples were IIO~, stained.
FIG. 9. Immunodiffusio~~ of purified 76 component from livers of 60-hr fasted animals and purified 7S component from livers of diabetic animals. Well A contained antiserum. Well B contained 7S componenl from the livers of 60-hr fasted animals and well C contained 7S component from the livers of diabetic animals. Both 7s compol1ent.s had been separated from the fatty acid syot hetase by Iwo successive sucrose density gradient centrifugations. The diffusion medium was 0.5’y0 agarose. After being washed wit.h 0.85y0 saline t.o remove extraneous protein, the samples were stained wit,h amido black.
inhibition or enhancement, of the rate of synthesis was observed. Hence, activity of t,he fat,t.y acid synthetase is unaffected by the presence of the 75 protein. #feet (d the TS protein otL the activity of acetyl-CoA carboxylase. The effect of the 75 component. on the rate of acetyl-CoA carboxylation was also test.ed (Fig. 10R). Varying amounts of the 75 component (O-O.75 pg
AND
FASTED
351
R.ATS
(J-A--’ 2
4
6
8
IC
FIG. 10. Assays for fatty acid synthetase and acetyl-CoA carboxylase activity in 6he presence of t.he 78 component. A. Fatty acid synt,hetase, 10 fig of prot,ein, was incubated for 30 min at 37” with varying amounts of 78 component. The mixt.ure was then assayed spectrophotometrically for fatt,y acid synthesis. B. Acetyl-CoA carboxylase, 20 pg of prot,ein, was incubat.ed for 10 min at 37” wit.h varying amounts of t,he 7s component. Enzyme activity was det.ermined as reported under Experimental Procedure.
protein/ml) were incubated with acetyl-CoA carboxylase (20 pg of prot.ein). The addition of 7s component had no effect on t,hihisenzyme activity. The highest. concentration of 75 component. tested represents an approximate ;iO-mole excess of this component, t.o enzyme. This calculation is based on t,he assumption t.hat. only one-tent.h of t.he protein in our preparation was acet,yl-Coa carboxylase. This is reasonable inasmuch as this preparation exhibited 10 %I of t,he activity of pure acetyl-CoA carboxylase (9). The active enzyme form was assumed t,o have a molecular weight of 4 X lo6 (16).
352
COLLINS
It is evident from the experimental results presented in this paper t,hat a 75 protein component is found in associat,ion with purified rat liver fatty acid synthet.ase isolated from livers of fasted or diabetic rats. This protein is synthesized in t,he livers of fast.ing and diabet,ic rats. The incorporat.ion of radioactivit,y of leucine int.o the 75 protein and the net, increase in its quant,it,y under t,hese conditions are evidence in support, of this conclusion. This protein disappears, or is reduced to a negligible quantit,y in liver on refeeding fasted animals or on treatment of diabetic animals with insulin. Hence, the fat,ty acid synt,het,ase purified from livers of normal or fast.ed and refed rats cont.ains eit.her traces or none of bhis contaminating protein. The 75 component, is separated from the fnt,t.y acid synthetase by two successive sucrose density gradient centrifugations. This protein is nearly homogenous on disk gel elect,rophoresis and on sucrose density gradient cenkifugation. The molecular weight of t,he TS component, is approximately 2.3s X 105. The 7s components from diabetic and fasted rats behaved identically on sucrose density gradient centrifugation and disk gel electrophoresis. lkrt,her confirmation of their identity was obtained in the immunodiffusion studies. Upon react.ion with antibody, t,hese proteins gave precipitin bands of identity. Thus, the 75 protein from diabetic animals is the same as t,hat from fast,ed animals. This observat.ion is consistent wit,h t,he close met,abolic relationship observed between diabetic and fasted states in animals. In our earlier studies (1) we suggest:ed that. t,hc contaminating protein might be a precursor or a degradat,ion product, of the fatty acid synthetase. However, our inability to secure t,he incorporation of radioacbivity of W-leucine into this protein on refeeding fasted animals suggested that it was not a precursor. To establish 1vit.h greater cer, tainty the relationship or lack of relationship of the 75 prot.ein to the fatt.y acid synt,hetase, assays were performed for the part,ial reactions of fatty acid synt.hesis. The purified 75 component, did not, exhibit act,ivity for any of these partial reactions. Keit.her did it
ET AL.
bind acetyl or malonyl groups. As a COIIsequence it: is concluded that the 75 component is unrelated to t,he fat.ty acid synthat,ase. This conclusion was confirmed by the lack of an immunochemical cross react.ion between the 7s component precipitin band and the major precipitin band due to the fatty acid synthetase. The possibility that the 78 protein might, fun&on in the regulation of fat.t,y acid synt,hesis was also considered. However, this prot,ein had 110 effect on either fatty acid s:);nthet,ase or acet.yl-CoA carboxylase activity. The fact. that the 75 protein is synthesized at a time when protein synthesis is at, a minimum suggests t,hat, this protein may have an important role in diabebic and fasted animals. Investigations direct.ed toward the identification of this role are in progress. ACKNOWLEDGMENTS We express our sppreciatiorl to Dr. William L. Kopp for his advice and the use of his equipment in the immunological studies. We are also grateful t,o Mr. Gerard0 Velasquez for assistance in the performance of the ultracentrifugatiorl analyses, and tt) Dr. Suriender Kumar for some of the assays for the partial reactions of fatty acid synthesis.
1. 2. 3. 4. 5. 6. 7. 8. 9.
11. N., COLLINS, J. M., KI~NNAK, A. L., .\w) PoRTKR, J. W., J. Bid. Ch:hem. 244, 4510 (1969). SIMON, E. J., AND 8til?dIN, I)., J. Amer. Chem. Sot. 76, 2520 (1953). TRAMS, El. G., AND Baan~, 11. O., J. Amer. Chem. Sot. 82, 2972 (1960). BRODIE, J. 1)., AND PDRTRR, J. W., Hioch.em. Biopkys. lies. Cornmw. 3, 173 (1960). KORNACKRR, M. H., AND LO\VENSTIGIN, J. M., Biochem. J. 96, 209 (1965). WAKIL, ti. J., PORlUR, J. W., AND (;IHSOS, 1). M., Biochint. Hioph~~s. Ada 24, 453 (1957). Hsu, R.. Y., WASSON, G., AND PORTER, J. W., J. Biol. Chhem. 240, 3736 (1965). BURTON, 1). X., Hanvra, A. G., AXD PORTIX, J. W., Arch. Biochent. Biophyx. 126, 141 (1968). BURTON,
Nun5.4,
S.,
I~NC;KLMANN,
15.,
AND
LYNKN,
F.,
Biochem. Z. 340,228 (1964). 10. GORNALL, A. G., B~RDA~ILL, C. J., AND I~AVID, M. M., J. Hiol. Chem. 177. 751 (1949). il.
A PHOTISIN
OF
DIAI3F;TIC
RAIJM, II., OLSON, E. B., ~~.\RGOLIS, s. .4., I\ND PORTER, J. W., Arch.. lliochem. Riophys. 116, 453 (1966). 12. Kuw4R, S., DORsRy, J. .4., h’b;SI~G, I<. A., AKD PORTRR, J. W., .I. Rid. Chesr . 246, 4732 (1970) .
13. CHANG, H-C., ~ICIDMAX, Laiu~, hf. I)., Hiockem. 911119~. 28, 682 (1967).
I.,
TM~H,
Hioph.yx.
(;., Kes.
.\SD
Cant-
AN‘11
F,4STRI)
353
II..4TS
14. I).\vIs, B. J., Ann. :V. I’. Acad. Sci. 121, 404 (1964). 15. CHR.~~ICE~, >f., .a~
(1967). 16. (hEGOLIN,
A., I~EISFELU, Z.VXRI, J., Anal.
C.,
l
KLEINSCHMIDT, A. K., Aoc. LTu/. A cad. Sci.
(1966).
E.,
lt. .4., WYCROFF, Biochem. 20, 150 WARNER,
.\SD LXNE, Cr. S. A.
It.
c.,
M. 66,
D.,
1751