Mechanism of pigeon liver malic enzyme subunit structure

~tiochimiea et Biophysica Acta, 336 (1974) 283-293

Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands t3A 36617 ] E C H A N I S M O F P I G E O N LIVER M A L I C E N Z Y M E S U B U N I T S T R U C T U R E

~RBARA H. NEVALDINE, ALIX R. BASSEL and ROBERT Y. HSU*

,partments o f Biochemistry and Microbiology, State Uttiversitv o.I" New York, Upstate Medical .nter, Syracuse, N.Y. 13210 (U.S.A.)

eceived August 17th, 1973)

CMMARY Malic enzyme f r o m pigeon liver ( t - m a l a t e : N A D P + oxidoreductase (decaroxylating) EC 1. I. 1.40) has a molecular weight of 259 900 :: 6000 from equilibrium ~:dimentation analysis. Its frictional ratio (~]0) and Stokes" radius (tt) are calculated to be 1.39 and 5.87 nm, respectively. The subunit molecular weight obtained by equilibrium sedimentation in 6 M guanidine hydrochloride is 64 800 and by disc gel clectrophoresis in sodium dodecylsulfate is 65 800, suggesting a tetrameric structure. Hectron micrographs o f both ufanyl acetate- and phosphotungstate-stained molecules show "square'" structures with four subunits located at the corners o f the square. The uranyl acetate-stained enzyme has dimensions of 10.2 nm 10.8 nm ,~ 7 nm, and the subunit has dimensions of 4.8 nm - 5.4 nm -~ 7 nm. Phosphotungstate causes dissociation o f the enzyme, and dimers and monomers are observed in the electron micrographs. A m i n o acid composition of this enzyme was determined and found to be significantly higher in tyrosine, tryptophan and ha,f-cystine content than the malic enzyme f r o m E s c h e r i c h i a coll.

INTRODUCTION

Pigion liver malic enzyme (L-malate:NADP ~ oxidoreductase ~decarboxylating) FC l. I. 1.40) is a bifunctional enzyme which plays a major role in lipogenesis by proiding reducing equivalents for hepatic biosynthesis o f fatty acids [I, 2]. The physiol, .,ical importance o f this enzyme prompted us to undertake a detailed investigation i t s physical, kinetic, and coenzyme-binding properties, of the component reactions talyzed by this enzyme, and the essential amino acid residue.,, at the active site. -'suits of some o f these studies have been documented [3-10]. Previous work also plied that this enzyme has a tetrameric structure [4], which is the commonly obwed active oligomeric form in vertebrates [1 l-15]. In lower organisms, ho~'ever, the ire enzyme exists as octamers, dimers, and monomers, as well a:~ tetramers [16-19]. in the present study, the subunit structure of this enzyme was determined b) ysico-~hemical m e t h o d s and electron microscopy: a preliminary report of o u r ,tings has been presented [10]. The amino acid composition of malic enzyme is also ,orted. " T o w h o m r e p r i n t requests s h o u l d be sent.

MATERIALS AND METHODS Materials Guanidin¢ hydroehloride (special enzyme grade) and Tris base {SchwarzMann); bovine serum albumin, ?,-globulin and ovalbumin (Mann standard molecular weight kit); 2-mercaptoethanol, catalase, and lactate dehydrogenase (Boehrinc~.:r); and dithiothreitol (Calbioehem) were purchased from sources designated abov ~. All other chemicals were of reagent grade. Deionized, distilled water was used throughout this work.

Enzyme Purification Pigeon liver malic enzyme was purified as described by Hsu and Lardy [3]. The purified enzyme was dialyzed overnight against 50 mM Tris-HCI buffer (pH 7.0) containing I m M ditlliothreitol and 10 ~,; Iv~v)glycerol, and stored at - 2 0 ~C before use, At this temperature, the purified enzyme remained full3 active for a m i n i m u m o f several weeks. Homogeneity of each enzyme preparation was routinely established by velocity sedimentation and disc gel electrophoresis techniques. Protein concentration was determined speetrophotometrically at 278 nm using an extinction coefficient o f 0,86 [3]. Sedimemation equilibrium studies Molecular ~eights o f native and dissociated enzyme were determined at 20 °C by the high speed sedimentation equilibrium method of Yphamis [20] using the multichannel centerpiece. These experiments were conducted in a Spinco Model E analytical ultracentrifuge equipped with RTIC temperature control and electronic speed control units. Rayleigh interference fringe patterns were recorded on Kodak Type II-O spectroscopic plates. The photographic plates were analyzed on a Nikon profile projector, The data were processed in a SEL 810A computer with a Fortran IV program as suggested by Yphantis 120]. The frictional ratio was calculated according to the e q u a t i o n / J ~ i. 19. I0~ '~ M, 2"'' ~ ( I-0.9982 ~),:l.%o.w ~ o . i~ ~) [21], using a molecular weight (MD o f 2600C0 Icf. p. 290L a partial specific volume (/:) of 0.741, and a sedimentation coefficient (.~,o.,) .o of 10.0 [3]. The Stokes" radius (~0 was calculated according to ~ (.ii/~O(3 M,. i: 4.-rN }~,~ [221. Sodium dodeo'lsuljaw polyacr.rlamide gel eh'ctrophoresis Preparation of the purified enzyme solution, electrophoresis, stainii;g, and motecurar weight calculations ~ere carried out as described by Web6rand Osborn I23]. Disc gel electro r !:oresis was carried out w;th the Canalco apparatus, The distance migrated by the various proteins on the gel column was measured using a linear transport attachment on a Gilford spectrophotometer. Amino acid attuh'Ms Amincjtacid a°na/yses were performed as described by Moore and Stein [24] on a Beckman Model 120C amino acid analyzer equipped with a Beckman Model 125 automatic digital integrator. The enzyme was dialyzed exhaustively against I m M ~ r i s - H C I buffer, pH 7,0, and aliquots ( 1.55-I.83 mg/ml) were hydrolyzed at 101 C

285 , 6 M H C I f o r 22, 47 a n d 72 h. T r y p t o p h a n content was determined spectrophotoetrically by the m e t h o d of Edelhoch [25]. Cystine and cysteine were determined as stele acid as described by Hirs [26]. After performic acid oxidation, the samples :re hydrolyzed for 20 h and a m i n o .'+cid analysi~ performed as above, 'eetron mieroscopJ'

Samples were prepared for electron microscopy by diluting the stock enzyme lution {cf. p. 288) to 5 0 - 6 0 p g / m l with 50 m M Tris-HCI buffer, pH 7.0, containing '?;i glycerol (v/v). A d r o p o f the diluted enzyme solution was placed on carbonated c o p p e r grids, and the grids were inverted on a d r o p of the above buffer. The ids were transferred to a d r o p o f 1%, aqueous uranyl acetate or 2 "~,;sodium phosphongstate, p H 7.1, tl, en drained on filter paper and examined immediately in a Siemens miskop IA at 80 kV. Microscope magnification was calibrated with a carbon grating .plica o f 2160 l i n e s / m s . S o m e enzyme preparations were fixed in 2.5°q~ glutaral:hyde in 50 m M T r i s - H C I ( p H 7.0) prior tc staining. tGSULFS

!h,h,cular w e ~ h t determination.~ The molecular ~eights o f native and dissociated matic enzyme were determined b~ the high speed equilibrium ultracentrifugation method o f Yphantis [20]. A typical linear plot o f the natural logarithm of fringe displacement [In(Y - Y0)) versus the square of the radius (r-') obtained on the native enzyme is shown ill Fig, I. Molecular weight was calculated using a partial specific xolume of 0.741 ml/g [3]. It was independent o f protein c o n c e n t r a t i o n in the range o f 0.52 to 1.20 mg/ml. An average value of 259 9C0 (253 400 to 267 COO) was obtained in eleven separate experiments. C o m p l e t e dissociation o f purified malic enzyme was achieved by treatment with 6 M guanidine hydrochh)ride in the presence of 10 mM 2-mcrcaptoethannl. Fig. 2

7O

7,I

~66

58

~4 | g3+4

436

__

438 r~(=M z J

44 0

I

,

wml 2

'., I. High +pced sedimentation eqtfilibrit, m ultrucentrifugation of nati~,e malic enzyme. The purified ~me was dialyzed against 50 rnM Tris HCI buffer, pH 7.0, containing I mM dithiothreitol hcfi~re • Centrifugation ~,~as performed al 2n C a! 13 (F00 rev.,min for 20 h. Protein concentration. 0.72 ml. +

286 7.~

"

6.4

r=f====/

Fig. 2. High speed sedimentation equilibrium ultracentrifugation of dissociated malic enzyme. The purified enzyme was dialyzed against 50 mM Tris-HCI buffer, pH 7.0. containing 6 M guanidin¢ hydrochlorid¢ and 10 mM 2omercaptoethanol before use. Cenlril'ugation was performed at 20 ~C at 32 000 rev./mJn for 29 h. Protein concentration, 0.59 mg,.ml.

shows the behavior of the dissociated enzyme during equilibrium sedimentation. The linearity of the plot suggests that the sample is homogenous and thai the subunits have the same or very similar molecular weights. For molecular weight calculations of a protein in 6 M guanidine hydrochloride solutions, it has been suggested that the partial specific volume ~, measured in dilute salt solutions should be decreased by0.010.02 [27]. The average subunit molecular weight calculated with a i~ of 0.731 was 64 800 (61 200 and 68 400 in two experiments at protein concentrations of 0.88 and

0.59 mgtml, respectively).

4.8

i= E~6 0 0

,°
4~1

G-L

6 MOBIUlr Y

Fig. 3. The subunit molecular v,reignt of marie enzyme as determined by gel electrophoresis in the presence of sodium dodccylsulfate. The open circle represents malic enzyme (M.E.). The closed circles indicate lh¢ mobilitics of the protein standards. The followin~l monomer molecular weights are used: bovine serum albumin (BSA, 68 000), L-amino acid oxidas¢ (AAO. 63 0001, calalase (CAT, 60 000), glutamic dehydrogcnas¢ (GDH, 56 000), ;,-globulin H (TG-H, 50 000), ovalbumin (OVA, 43 000), lactic dehydrogenase (LDH, 36 000L and ;,-globulin L (;,G-L, 23 500). Monomer molecular weight assigned to glutamie dehydrogenas¢ is that reported by Smith el al. |34]. The other molecular weights are as listed by Weber and Osborn [231.

287 F u r t h e r i n f o r m a t i o n c o n c e r n i n g the molecular size o f subunits was o b t a i n e d 1 electrophoresis e x p e r i m e n t s . T r e a t m e n t o f malic enzyme with 1% sodium dodecyllfate in ! ~/o 2 - m e r c a p t o e t h a n o l followed b y electrophoresis in sodium dodecyl, ;fate p o l y a c r y l a m i d e gels resulted in the a p p e a r a n c e o f a single protein band after • dning. A linear p l o t o f the e l e c t r o p h o r e t i c mobility versus log molecular weight the v a r i o u s p r o t e i n s t a n d a r d s is s h o w n in Fig. 3. The molecular weight o f the maIic zyme s u b u n i t was o b t a i n e d f r o m its mobility by interpolation o f this plot. T h e :rage m o l e c u l a r weight was f o u n d to be 65 800 (65 300 to 66 100) in f o u r deternations. A s u m m a r y o f the physical properties o f malic enzyme is ghown in Table I. , BLE

!

!YSIC&L PROPERTIES OF PIGEON LIVER MALIC ENZYME >sical properties "

Ltive e n z y m e

I.

,.. (8.6 m g m l )

Stokes" radius (~0 Molecular weight Subunit Molecular weight

Method

Value

Sedimenlation velocit) Sedimentation velocit) (boundary spreading) Pycnomet ry Amino acid composition Calculated**" Calculated*** Svedberg equation Sedimentation equilibrium

I0.0 S ~ 3.17. I0-" cm'. ~- J" 0.741 mtg" 0.737 rnl g'" 1.39 5.87 n m 280 000" 259 900

Sodium dodecylsulfatc gel electrophoresis Sedimcntalion equilibrium

65 800 64 800

" Hsu and Lardy [3l. "" of. p. 291. "" Calculated from a molecular v,eighl of 25'-) '-~)0and a ~ of 0.741 as described in Materials and Methods.

.I,nim, acid analysis The a m i n o acid c o m p o s i t i o n o f malic enzyme calculated on the basis o f a ~~)lecular weight o f 2 6 0 0 ( / 0 is s h o w n in Table II. With the exception o f threonine. "ine, valine, isoleucine, cysteic acid and t r y p t o p h a n , the n u m b e r o f residues reported ,~resenls the nearest integer to the average o f duplicate analyses after 22, 47, 72 h T drolysis. Six d e t e r m i n a t i o n s o f t r y p t o p h a n content yielded an average value o f ! 9.9 1.8 residues per molecule. The tyrosine content was also estimated at 295 and 300 T i as suggested by E d e l h o c h [25]. Duplicate determinations at each wavelength Ided values o f 78.8 + I (295 n m ) and 78.5 : 1.6 (300 nm) residues per molecule. ,:se value~ a r e in g o o d a g r e e m e n t ~ i t h that o b t a i n e d from the amino acid analysi~ , lhe acid ,lydrolysate. T h e a m i n o acid c o m p o s i t i o n o f the Escherickia colt malic y m e [16] normalized to a weight o f 2 6 0 0 0 0 is listed in the last cohlmn for comi -alive p u r p o s e s , A partial specific v o l u m e o f (').737 was calculated from the amino acid compo-

288 T A B L E II AMINO

ACID COMPOSITION

A m i n o acid

OF PIGEON

,-moles m g protein 22 h

47 h

72 h

Corrected

value L.vs His Arg Thr

(1.404 0.163 0,362 I),636 11.375

Ser

(}.3~kl

Gltt Pro Gi~ Ata ~:tl 'klct

11.7t13 I).352 <).530 0.593 (I.373

Asp

O.188 0.320

lle Lcu • }r Ph¢ H alP-(3 ',

LIVER MALIC ENZYME

0.711<1 1t.273 <1.270

0.41 I 0.163 0,365 (},639 O.33g 0.301 O.72~1 0.355 (I.52(+ O.591 ~~ (1.4__ 0.11~6

0.398 0.162 (I.357 11,649 11.355 0,292 O, 70,~ 0,35(I (I.534 (t.595 (t.452
0.404 11.I63 (I,361 0+642 0.393' 0.314" ().712 0,353 0.5311 11.5113 11.452" 0.1,~7

o.3~0

11.4__ +'+'

0.422 '~

0.701 (1,279 0.290

0.7413 11.2g<1 0277

I+ 7111 0._,~ -~-r , "~ 6 I)._, O. 1~7"

Trp

Moles o f residues per 260 O
Moles o f residues per 260 000 g of E+ con cnz} me"

I IJ 46 102

147 45 1416

181

175

II I 8S 2111 99 149 167 127 53 11'+ t97 7,",; +7tY) %',; 56

211~

7S

104 __<1 "" 125 147 225 17X

6o t30 169 41 66

29 4

•' A lotal of 92.3 ,g 1011l,g protein s~a~, rcct+~ercd. -File number of rc-+iduc+`of c+l~.ll amii~o acid, ¢~,~'ept tr.~ptol0han. ~cr¢ corrculcd to IO:V',, rccoxcr.x and ~:rc given ;is the n¢,~rc,,l integer. " Calculated fro:+] the dilla of Spina ¢t at. 116] " Xalu¢~, ¢~.lrap~ialed to / e r o Ii1]1¢.

'~ Value~, l'rt+m "12-I] h3,drolx,+i,, tt~cd l)etL'rmincd a, t'}slelC ;.l~.'itl ~+fler pcrtormic il¢id o'~idalion .X~¢rd+2t2 o ¢ lhrt-¢ dctt?rnlmatioll,,. + l)¢lermincd '+p¢ctr~phohm~clric.dl+~ h~ the mcth,,d o+ ldclhouh [211 ~ititm using the partial specific x o l u m c , o f the con~tiluent amint+ ~Icids according to ( o h n and Edsull [_,~]. Thi~ xaiue is ill re+~-,onal+Ic ~l+re,,2111cnl x~ilh the ~alue o f 1"1.741 o]+~tiltllt,d .I~)pycr:+~.n11"tl'.X [3].

Electron IiliC|'O',L'opiL.' ",ttldic~ ~A.~'I'Cpcrt'ormcd in ill1 :Lttempd to f u r t h e r delineate t h e s t l b t l n | t slrLICttlrE. Pk, l¢ I ',I+,+',~, ulc~'trovl phol ung.~tatu-stained

mulic cn/} mc

n+icrographs o f

t rattx I acetatc-,,tuincd

u r a n x l ~+.c¢tate- i.llld p h o s I++icrog1"aphs g a x e , , t l p e r J o r

a n d i n s i g n i l i c a n t dJs+ocJ;.ltioll ~,1' t h e e n z y m e . "+iOl'+ox¢r. a l i m e n t ( t e n s o f l h e unfixed, uruv~'+l :wetate++tained molecule + ~ c r c the ~,:tme u,+ lho.+e lixed v+ith ~ l u t a r a l deh.'+de. ~ h i c h should minin+ize d i s r u p t i o n o f the n~ti+e ~,11"u~.'tttre durins, ,~taininu. Therefore, the appear+lnce o f the e n / ) m e in uran,,l acet+tte-~,l~ined prcp~nration,, i~+ belicxcd lu he more repre,,entativ¢ o f the nuti,,e tet+ramer. ;tnd etectrt>n m i c r o ~ r a p h > o b t a i n e d b} (hi,,, nlethod ~ere used for most ~+I" the size deterlnin~it~on~,. Electron m i c r o g r a p h s o f u r a n ) l acetate-.~taincd enz} me shox+ed p r e d o m i n a n t l_~ +l ++sqttare" slructure 10.2 O7nm 10.8 0 . 7 n m ~ : S.D.}, that eva++ often seen tt+ be divided into f o u r subunits ,'.trran~ed at the corners o f the square
289

I ~ -~c I. t:lL~ctron m i c r o g r a p h s o f m a | i ~ e n z ~ n ~ s h o g i n g [ e t r a m e r i c struclure, Magnific~ltion f a c t o r : I ~- ~llld 3. 2.~i()|g~) 7. 4, 6 Lilld 9, .~(K1 I~R) . I. U r a i l y | a ¢ c l i l t c - s t a i n e d ,,nz.vmc+ ~1~-~1 n~ol¢cul¢,, , ",~ ~l 10 I ] - i l m sqb~zre o t i [ i m c . A , :~! t'.~tr~tlZl~r ~ i t h |'otfr "~ell~Ltltit', I~cate(| at t11¢ ct~rm:r,, ~'J" a ',(lu~lz¢. ;~li|i~';Itii~ll IHZI~ I()J) nnl, 2. P h o n p h o t t l t i g ~ t a t ¢ - ~ l ; i i n c d c n / ~ n l c . % mixi~ir¢ o f tetl;i111¢rs, ~JilllCl's. .0 Llpp;IrClll.

~ . 1~Ir~Inlu'r x i ¢ ~ d

do~n

4-1\~Id a x i s t ~ s.~111111¢[r~ ~.'Ii~| %ic~', ): J~, i~.'il';1111~-r ',ie\xc~|

'.+cx+,i t ( r ~ t ~ . ) l +tc+'t,il+-st~lii1,++'~.l ~r1,,',, 11112 ,+ht+',xin~ *.,~+ltllli+~.- :illtl r,,t+,:t~inglil;.tr f i l l ~.Ic,+lhlc-b~+ .tn~.t +,.irl +,ctit~ii-i." ',lrti+ltlr+,~. 4 ~.tllt| ~. I. '1"~.111~,i ~tC,+t,ltC-',t~.lillctl t+tl'Lll+~Cr ,;llld i;lli+'|'J'+liO~+lX£ l+h0it+~l,+t'h ~+~ ;1~., liliitl,;J illtJi~+tllll17 ~,ic't+< lt~i~,~ll 4-l't+Id ,ixi~+. f+. ? ;tlltt ~ II r ~ l l l ) l LlCu'ILtlt'.**sl{llll+tt I~l'C|+,ll
290 "double-bar" structures gave similar dimensions as the four-part square~ and probably represent tilted molecules (see model in Plate 1-8). The "'square" structure was also seen in phosphotungstate-stained preparations (Plate l-2A and 9), but the stain

caused dissociation of the enzyme, yielding a heterogeneous field containing tetrainers (Plate I-2A, end view; B, side view), dimers (Plate I-2C, end view), and monomers (Plate I-2D, end view; E, side view). The dissociation of several proteins by phosphotungstate has been reported previously [29]. The dimensions of the phosphotungstate-stained tetramer were 8.2 n m × 9,2 nm, about 20% smaller than the uranyl acetate-stained enzyme. The center-to-center spacing of the subunits in the phosphotungstate-stained tetramer was also smaller, and the difference was constant from one preparation to another. The reason for the difference in size is not known, but it ma) be related to the dissociation o f the enzyme by phosphotungstate, shrinkage or swelling o f the enzyme by the stains, or differences in the penetration o f the stains. in uranyl acetate-stained preparations where division into four subunits was clear (Plate I-IA and 4), the dimensions o f the subunits could be measured and were 4.8 ± 0 , S n m ~< 5.4 ± 0 . 3 n m ( ~ S.D.). Less frequently, a structure about li nm "< 7 nm composed o f two short rods could be seen (rectangular double-bar in plate l-3B and 6, left). If this is interpreted as a view of the tetramer perpendicular to the 4-fold axis o f symmetry, the dimensions o f a subunit would be 4.8 nm ~ 5.4 nm -. 7rim and those o f a tetramer would be 10.2nm "~ 10,Snm - 7nr~l. An 8 . 8 n m 6.2 nm double-bar structure was also seen in phosphotungstate-stained preparations (Plate l-2B) comparable to the I I nm - 7 nm double-bar structure seen in the uranyl acetate preparations. Dimers and monomers were present in phosphotungstatestained preparations (Plate I-2 and 9) comparable to the I I n m 7 nm double-bar structure seen in the uranyl acetate preparations, Dimers and m o n o m e r s were present in phosphotungstate preparations (Plate I-2 and 9), and a cylindrical ~r ellipsoidal subunit 4.4 nm ~ 3.9 nm , 6.2 nm could be measured.

DISCUSSION

The molecular weight of pigeon liver malic enzyme was determined by the high speed equilibrium sedimentatnon method of Yphantis [20]. ] h i s method gave reproducible results with a prec,sion of approx. 2 %. In eleven separate measurements, an average value of 259 900 .? 60(J0 was obtained. This value is 7 "., lower than the value of 280000 determined previously by the sedimentation diffusion ratio method [3]. but is more consistent with a tetramer composed of subunils of average moleculal weight of 65 300, and is used in all calculations in the current study. A tetrameric structure of this enzyme was implied in a previous report [4] on the basis that malic enzyme binds four equivalents of N A D P H , and at pH 12.3, it dissociates to give a single 2.35-S Schlieren peak, which is approximately, one-fourt~ that o f the 10-S value reported for the native enzyme. In the current study, the subunit molecular weight was estimated by the equilibrium sedimentation method on ar enzyme preparation dissociated in 6 M guanidine hydrochloride, and by the disc ge, electrophoresis method in the presence of sodium dodecyisulfate. In each case, a singk protein species,was obtained, yielding molecular weights of 64 800 .... 3600 and 65 800 respectively. These values are in good agreement with each other, and with the valu~

291 e" ~ected for a native enzyme o f a molecular weight o f 259 900 which is composed o f f, ~r subunits having the same or very similar molecular size. The elect-on micrographs o f malic enzyme give added evidence for the tetrata tic nature o f the enzyme. The square structure divided into four parts is most s ply interpreted as a planar molecule with four subunits arranged at the corners of a luare. The shape o f subunits cannot be assessed with certainty from the electron r~ .rographs, but an ellipsoidal subunit would explain the views seen in both urany[ a care- and phosphotungstate-stained preparations. Based on a dimension o f 4.8 nm L4 nm ~* 7 nm for the uranyl acetate-stained molecule and a partial specific ame o f 0.741, a cylindrical subunit would have a molecular weight of 112 000, too I. :e to be compatible with an average value of 65 300 found by sedimentation and c electrophoresis studies, whereas the molecular weight o f an ellipsoid would be 7 ,;e0, which is comparable with a N A D P H - b i n d i n g weight of 76 400 [41, and is sl htly larger but nevertheless consistent with the value of 65 300, if considerations a~ given for the inherent differences in these techniques. A comparison o f the amino acid composition of malic enzymes from pigeon li~:r and E. coli was made. Although differences in the total number of basic and h~drophobi¢ residues (v, hich takes into account conservative replacements) are within the range o f expected species variability, large variations are seen in the individual residues, notably tyrosine (78 versus 41 ): tryptophan (20 versus 4): and half-cystine

TAHI_IL I I I MOIE('UI

AR WI:I(IHTS

Source

E. colt Pigeon liver ChtckCn li~er Rat [i~er Pig heart ('~lOsOl \1 ili-~.-holtdrial M , , r,e tissues ( " H~ I~p 1

losol and it~.'hondrial -ter l i ~ e r ~e brain !oso|

()I- ~ , I A I . I ( " I , I N / ' f M E

N a t i v e enz3 m e

Subtmi!

Method"

.I/,

Method'"

tl) ~I ) ~! I 131

551) ~N)I)

259 900 26{t ( ~ 1 251) I~111

~l~ 121

t4) {4}

216 0 ~ 227 I~)O

(2}

Ref. .It',

Number

67 I~)O

8

16

65 gill) 56 ~)O

4

this paper

4

14 15

4

:ll

31

13~ ~3) ¢3)

FROM DIfI-ERIiN~I - ORGANISMS

4 4

201) t~l)l)

I I t3 15 32

',o c h o n d r i a l .4, f[

ttt! H/llllIt~i

S. L,

calls ~'i

t4~

M e t h o d s used are ( ! ) l q u i l i b r m m s e d i m e n t a t i o n , (2) S e d i m e n t a t i o n velocity. | 31 (}¢1 f i l t r a t i o n , ,:rose d e n s i t y c e n t r i f u g a t i o n , M e t h o d s u s e d are ((} E q u i ( i b r i u t n s e d m l e n t a t h ) n in g u a n i d i n e hydrochlorid¢. (2) Disc gel ~phoresi,~ in ",odimn d o d t x y i s u i f a t e , (3) C ~ n e t i c a n d or h ) b r i d i z a t i o n studies.

ele~

13. 4) 13, 4 )

65 000 65 (XX)

1 I

17 !7

292 (:56 versus 29). These differences should ultimately be verified by sequence analysis, and their functional significance ~if any) evaluated in future studies. During the last several years, a number of malic enzymes have been isolated and characterized. The molecular weights of the native and dissociated enzymes are summarized in Table 111. It is interesting to note that this enzyme has been found te exist as even-numbered multiples of a monomer unit in nature. The E. eoli enzyme is an octamer [16]. Most animal enzymes, including that of pigeon, chicken, rat and hamster liver; mouse heart, liver and kindey: as well as the round w o r m Ascaris suun, are tetramers [! !-15, 18, 30], In bovine brain and pig heart, both cytosol and mito. chondrial malic enzymes also have molecular weights compatible with that o f a tetrainer [31,32]. The enzyme from the tape worm Hymenolepis diminuta is a dimer [ 19] whereas monomers are seen in the bacteria Streptococcusfaecalis and Lactobacillu, easel [|7], Furthermore, subunit molecular weights of enzymes from these wideb differing sources are nearly identical. Although the compilation in Table ! il is limited the available experimental data suggest that the basic building block of malic enzymt from different organisms is a single peptide of approx. 65 000 in size. The occurrence of active monomers [17] further confirms the generally accepted notion of one active: site per monomer (or subunit), which is responsible for the overall and the c o m p o n e m decarboxylase and reductase reactions [5, 8, 9, 33], Moreover, this concept is entirely consistent with a sequential ordered, and concerted kinetic mechanism postulated in our early studies [6]. ACKNOWLEDGMENTS

The authors are indebted to Dr Charles Burger for the use of his Beckman Amino Acid Analyzer. This investigation was supported by grants from the following sources: National lnslitutes of Health, Institute of Arthritis and Metabolic Diseases (AM 13390) and Biotechnology | R R 00353). REFERENCES I 2 3 4

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