Bovine adrenal steroid hydroxylase system III. Reconstitution of adrenal iron-sulfur protein

Bi,,chimica et Biophysica Acta, 336 (1974) 309-317

((~,Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands B: ~- 36632

13 V I N E A D R E N A L S T E R O I D H Y D R O X Y L A S E SYSTEM I

R E C O N S T I T U T I O N O F A D R E N A L I R O N - S U L F U R PROTEIN*

K rSUKO SUHARA, KAZUKO KANAYAMA "°, SHIGEKI TAKEMORI and SAYUKI KATAGIR! £, .Trtment of Chemistry, Faculty o f Science, Kanazawa Uni~'rsity, lshikawa 920 (Japan) ft eived August 14th, 1973)

S; MMARY I. The ape adrenal ferredoxin has been prepared by treatment of native at~enal ferredoxin with trichloroacetic acid. 2. The recombination reaction o f the apoprotein with Fe'" and sulfide in the presence o f 2-mercaptoethanol was greatly enhanced by the presence o f a high concentration o f urea, up to 75','-,, recovery. 3. Crystals o f the reconstituted protein were prepared followed by chromatography on DEAE-cellulose. 4. The reconstituted adrenal ferredoxin was homogeneous on ultracentrifugation and on disc electropboresis and was indistinguishable from the native material in terms o f its catalytic functions, absorption spectrum, molecular weight and the iron content. 5. A m e t h o d has been described for measuring the labile sulfide in adrenal ferredoxin. The sulfide content was the same in both preparations.

INTRODUCTION

As described in the first paper o f this series [I ], functionally active iron-sulfur pu,,~ein (adrenal ferredoxin), an oxidation-reduction intermediate between an N , D P H - d e p e n d e n t adrenal ferredoxin reductase and a cytochrome P-450 in the st~ ,rid hydroxylase system of adrenocortical mitochondria, has been obtained in cr talline form. The iron and labile sulfide o f adrenal ferredoxin could be separated from the p~ ein moiety by treatment o f t h e native material with the iron-chelating agent. , . , ' di ridyl [3], or with trichloroacetic acid [4]. The apoprotein thus prepared could re mbine with Fe z+ and sulfide to form adrenal ferredoxin as reported previously b, ,imura ['~]. However the reconstitution appeared to be incomplete. " The preceding papers in this series are ref. I (I) and ref. 2 (If).

" Present address: Department of Biochemistry, Kanazawa Medical University, lshikawa (Ja

a).

310 The purpose o f the present paper is to give the improved p r o c e d u r e for th~ reeonstitution of adrenal ferredoxin from the apoprotein, f o r m i n g the basis fo: further studies o f the function and the chemical and physical characteristics o f tht iron-sulfur protein, MATERIALS A N D METHODS

N A D P +, N A D P H , glucose 6-phosphate a n d glucose-6-phosphate d e h y d r o genase were purchased from Boehringer und Sohne, G m b H , M a n n h e i m . Epinephrin~ and N,N'-dimethyl-p-phenylenediamine hydrochloride were obtained from Wak~ Pure Chemicals Ltd, Osaka and deoxycorticosterone from Sigma Chemical Co. St. Louis. All other chemicals were of the highest commercially available grades Crystalline adrenal ferredoxin (A4,4 ,m/A27~, nm: 0.86) [I], N A D P H - a d r e n a l fcrredoxiJ reductase [2] and cytochrome P-450 [5] were prepared from bovine adrenal corte,~ as described previously. Porcine adrenal ferredoxin (.4414~m/A276nm: 0.81) wa~ prepared from the adrenal cortex by a m e t h o d similar to that o f bovine adrenai ferredoxin [1 ]. Crystalline chroroplast ferredoxin (A420 ~m/,42,,6 ,,i: 0.49) was prepared from spinach leaves according lo the m e t h o d o f Buchanan and A r n o n [6]. Crystalline metapyrocatechase was prepared from Pseudontonas putida as described previously [7]. Crystalline cytochrome c was prepared from bovine heart by the m e t h o d of Hagihara et al. [8]. Superoxide dismutase was prepared from bovine erythrocytes by the method o f McCord and Fridovich [9]. Sedimentation velocity analyses were performed with a Spinco Model E analytical ultracentrifuge at 20 C using a boundary cell. Disc electrophoresis was performed essentially as described by Davis [10] with the use of 7.5",, polyacrylamide gel. The gel was immersed in 10'~, trichloroacetic acid for 16 h, and then stained ~ith ~,. I ",, solution of amido black in 7'!,, acetic acid. Protein was determined by the biuret m e t h o d with crystalline bovine serum albumin (Sigma) as a standard [I I]. The iron content was determined by using the o-phenanthroline m e t h o d described by Massey [12]. For the measuremen! o f the amount of labile sulfide liberated from iron-sulfur protein, a modification of the method of Fogo and Popowsky [13] was used. To 0.4 ml of the sample in a tube (15 mm . 95 ram) 1~3 ml o f I ;'~,, zinc acetate and 501d o f 12"o N a O H were added The tubes were stoppered, and shaken vigorously for 20 s. After standing at tool1 temperature for specified time, 0.25 ml o f 0.5",, N,N'-dimethyl-p-phenylenediamin. hydrochloride in 5.5 M HC! and 50itl of 23 m M FeCI3 in 1.2 M HCI were added t~ each tube. The mixtures were then shaken vigorously for 20 s. After standing ft~ 20 rain, 0.45 ml of distilled water ~as further added, and then the faint turbidity c protein precipitated in the reaction mixture was removed by centrifugation. Th clear supernatant was separated, and the absorbancc was measured at 670 nm ~vi! the reagent blank as a reference. Under these conditions, I / , m o l e o f Na:S gave a absorbancc of 11.4 at 670 nm in this system. The adrenal ferredoxin-dependent reduction o f cytochromc c ~as assayed b the method described previously [I ]. The adrenal ferredoxin-dependent f o r m a t i o n of superoxide anion was assayc with epinephrine as an indicator by measuring increase in absorbance at 480 nm ~ described by Misra and Fridovich [14]. The reaction mixture II m l ) c o n t a i n e d 5

311 t,r .~les o f p o t a s s i u m p h o s p h a t e buffer ( p H 7.8), I(10 nmoles of EDTA, 200 nmoles o~ :pinephrine, 40 n m o l e s o f N A D P H , 500 pmoles of adrenal ferredoxin reductase a~ a p p r o p r i a t e a m o u n t s o f adrenal ferredoxin. T h e activity o f t h e steroid 1 lp~-hydroxylation reaction was assayed at 25 C ~ a deoxycorticosterone as substrate by measuring fluorimetricaily the a m o u n t of c, i ¢ o s t e r o n e f o r m e d [15]. T h e reaction mixture (0.5 ml) contained 25,umoles of p assium p h o s p h a t e buffer (pH 7.2), 100 nmoles of deoxycorticosterone, 370 pmoles t, y t o c h r o m e P-450, 5C0 pmoles of adrenal ferredoxin reductase, 8,umoles of KCi, 8 /~ ~les o f MgCi2, 5 8 0 n m o l e s o f N A D P +, 4 . 5 p m o l e s of glucose 6-phosphate, 1.5 u .s o f glucose-6-phosphate dehydrogenase and appropriate amounts of adrenal f, ¢doxin. T h e reaction was initiated by the addition of glucose-6-phosphate d ydrogenase and after 15 rain terminated by the addition of I ml of 0.5 M H2SO~. R ~ULTS I¢ , o n s l i m t i o n

of a d r e n a l . l e r r e d o x i n

Adrenal ferredoxin crystals ~65 rag) were dissolved in 50ml of 1 0 r a m p~tassium p h o s p h a t e buffer (pH 7.4) containing IC0 mM 2-mercaptoethanol. To this solution 10,',, trichloroacetic acid was added over a 30-rain period with stirring at 0 C to a final c o n c e n t r a t i o n o f 40.. During the addition, the adrenal ferredoxin color gradually faded and the white precipitate of the apoprotein appeared. After slanding for additional 15 rain with stirring at 0 C, the suspension was centrifuged at 30000 g for 15 rain. The precipitate was dissolved in 2 0 m l of 100ram TrisHCI buffer (pH 9.0) c o n t a i n i n g IG0 m M 2-mercaptoet.hanol and 8 M urea. The solution was i n c u b a t e d anaerobically in a T h u n h e r g tube for 40rain at room temperature. At the end of the period, each 1 ml of both 100 mM FeSO4and 100 mM Na,S were aerobically introduced into the apoprotein solution (50 ml). After standing at r o o m t e m p e r a t u r e for an additional 30 rain, the mixture was diluted with 40 ml o f 100 m M T r i s - H C I buffer (pH 8.5), dialyzed against 2 I of 10 mM Tris-HCl buffer (pH 8.0) for 3 h and was further dialyzed overnight against 21 fo l0 rum potassium p h o s p h a t e buffer (pH 7.4). The dialyzed solution was centrifuged to remove in,,oluble malerials and put o n t o a DEAE-cellulose column (I,3 cm 10 cm) equilibrated with l0 m M p o t a s s i u m p h o s p h a t e buffer (pH 7.4). After washing the column ~i~h 20 ml o f the same buffer, the brown color fraction of the reconstituted adrenal fe~ .edoxin was eluted with the p h o s p h a t e buffer containing 5(30 mM KCt. The black c, c( 7-~ tl

,red substances c o n t a i n i n g ferrous sulfide remained on the upper part o f the zmn. The ~yicld ofreconstituted adrenal ferredoxin from starting malerials ~as about ., at this stage. The eluate was 3-fotd diluted with the phosphate buffer and was 3 applied onto a DEAE-cellulose column (2.8 cm 3- 26cm) equilibrated with the

s~ t! tt •' o~ cc b~ 5(

: buffer c o n t a i n i n g 170 l~M KCI. The c o l u m n was v, ashed with about 400 m] of ,ame buffer c o n t a i n i n g 170 m M KCI and adrenal ferredoxin was then cluted with buffer c o n t a i n i n g 3~]0 m M KCI. Most of the unreacted apoproteins were rated du i n g this stage o f c h r o m a t o g r a p h y . The combined effluent ~as dialyzed night against the p h o s p h a t e buffer and the diaIyzate was adsorbed onto a DEAE:lose c o l u m n (0.7 cm 3 cm) equilibrated with the phosphate buffer. The dark ,n part was slowly eluted in a small volume with the phosphate buffer containing 11M (NFI4)2SO.~ (pH 7.4). Poundered ~NH4).,SO4 was added to the concentrated

312 adrenal ferredoxin solution until the solution became stightly turbid. The resultin small a m o u n t s o f precipitate ~ e r c centrifuged olt'. Poundered (NH.t),S04 ~;ts l'urthe ddded until the ~olution b e c a m e slightly turbid. After .,,funding o~crnight, the b r o ~ l cryst;Jis ~ere collected by centrifugation and dissoJ,,ed in a minimal ~,oll.inlc o|" th~ p h o s p h a t e buffer. Adrenal ferredoxin ~ a s further recr)stalli/ed its abo~c. -[he p u r i l index (.-I~., ,,~ .42-0 ,,,, in the oxidized form) o f the t~ ice-recryslallb'ed prcpar~ttion x~a found to be 0.78. The cryst:l!s are sho,an in Fig. I.

El¢'¢'tr,vJhore/ic and ul/rac~'mrifi~.~a/ protwrth'~ In order to compare the elcctrophoretic patterns of the ape ;Jlld reconstitute, ~tdrenal ferredoxin, the samples ~ere anat~zed b) disc electroohorc.,,i~ on pol) acr~l,'lmide get. As ~,ho',vn in Fig. 2A ;ind B. the :.|po and reconslituted prep~lrati,m ~crc essentially honlogeneous. Vvhcn the apo adrenal fcrredo\in ~;is mixed ~ l l either reconstituted or native prep~lration, the electrophoretic pauern of the mixtttr ga~e a single shm'p h;md (Fig. 2('). These results indicate th;~t the electrophorcl~ mohitities are essentially the same for the a p o and holo adrenal ferredoxin unde these conditions.

Fig. I. Crystals of the reconstituted adrenal ferredoxin. The photograph was taken at 5 ~C. 1. s~:ale ir the photograph is O.05 ram.

31~

A

B

C

la~ 2. ] - I ¢ ¢ t r o p h o r e t i c p~Lttcrn,~ o f arh+ a d n : n i t l l~rt'+do~,in i ~). r~:+t~n,ditu|~.,d a d n : n a l l'~rrcdoxin ~l]] i~+d th~.'Jr r n i x l u r + , ' ( ' ) o r ] pol.~ acr.~lamide [z~.'l i|t p+l ~+O. A. ",~,mpl+ o f ilpt:, ~.tdlu~lld l¢r+¢do~m +I~ -g.L ]*'~,,g~ '~,,il.S a p p l i e d I o ;I '...'Oltlnln (() 7~111 4~-~ Cn~l [h,.' cl,:~:tn~pl~.t~r~:li¢ rim ~:~, pcrf,~rm~..d ~ l l h ;~ ¢¢ur.,liillt c u r r c n l o f I.~ I l I A p~.'r IllbL' t'~r 2.5 h in a ~¢lrigcr~t,~r.

Fig. 3 ~ho~'s the ~,edimenlalion p~Hlen'rJ,, of th~ ~i]~ :u]d r'c,:~,u~titutcd ~idJ~.'n.~l ferredoxin. Both pre.p~r~lion,, ~ho~ud onl'~ one s~mm~trical D:~I,- rh~.. ~lhw,, nf .~':.... f o r l h e a p o

and

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13

• U l t r a c e n t r i f u g a t i o n P a t t e r n s ()f the ;tpo (.&') a n d r e c o n s t i t u t e d (B) adrcm~t f~.'r~L',h,~;~ [ ,rein was an ~4.2 m g m l sltltJli~m in _~O nl'kl pol;i~,~il.ml p h o s p h a t e hLJffcr, l'q | "~ 4. ~;~mr,~u~.; ,. ,' H~iothreitol. T h e r c c o n s t i t t l t c d p r ~ l c i n ~,;t ~, a 7.5 trig lllt ,,olution it] ~(I ,11~,| t",O[,~ls",,,lll] pl.~ '~l', ,' . pl'J 7,4. Phot(~grz~ph~ ~k'rc l;tk~.'ll 4x 111hl afh:r a l ( a i n i n g ;! r o t o r spL'~.'d o i 5~' ~ ( ) ic. u,J~,

314

Comparison of elution volume from Sephadex G-IO0 column The molecular sizes o f the apo a n d reconstituted adrenal ferredoxin wen determined by the m e t h o d o f gel filtration using Sephadex G-100 c o l u m n and comparec with that of the native preparation. The ratio o f the elution v o l u m e o f the protein to that o f blue dextran, Vo/Vo, was f o u n d to be 1.71 for apoprotein and i.74 fo reconstituted protein, respectively. These values corresponded to t h a t for nativ, adrenal ferredoxin (1.71). These findings including sedimentation analysis sugges that both apo and reconstituted adrenal ferredoxin have similar molecular size as th, native one, and no aggregation occurs.

Spectral properties The absorption spectra o f apo and reconstituted adrenal ferredoxin ar compared in Fig. 4. In the reconstituted p r e p a r a t i o n , the peaks were at 320, 414 an, 455 nm. its absorbance ratio o f 414 nm/276 n m was 0.78, which was slightly Iow¢ than that o f the native one (0.86). The spectrum of a p o p r o t e i n exhibited a m a x i m u n in the ultraviolet region, but no absorption in the visible region.

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Fig. 4. Abso:'ption spectra o f apo and reconstituted adrenal ferredoxi•s in 50 m M potassium phosi~hat¢ buffer, pH 7,4. The protein concentration was 1.0 mg:ml, I: reconstituted adrenal fcrredoxitl; I I: apo adrenal ferredoxin.

h'olt conte/iL

Th~ iron content of reconstituted adrenal ferredoxin was estimated to be ! 1:~ ngatoms of iron per mg o f protein on biuret basis. The value is in g o o d agreeme t with that of native adrenal ferredoxin (120 n g a t o m s of iron per m g protein) [I ]. TI apoprotein contained no significant a m o u n t s o f iron (less than 5 n g a t o m s o f irt per mg o f protein).

Labile ~ul]ide content The value of 58 nmoles o f labile sulfide per m g of protein was obtained und the reported assay conditions [16]. However, as shown in Fig, 5, extension o f t incubation time with the alkaline zinc acetate solution over 2 h resulted in release a maximal a m o u n t o f sulfide: 1 2 0 n m o l e s of labile sulfide per m g protein t reconstituted adrenal ferredoxin. The molar ratio o f iron to sulfur a p p r o a c h e d one. T

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,,. 5, Effect o f t h e i n c u b a t i o n on labile sulfide analysis, F o r conditions see Materials and Methods. -O, reconstituted a d r e n a l ferredoxin: &---&, ap o adrenal ferredoxin; - - : , , chloroplast feroxin; A - - - ~ . , a p o c h l o r o p l a s t ferredoxin.

ne results were o b t a i n e d with native adrenal ferredoxin. A similar p h e n o m e n o n was ,o observed with porcine adrenal ferredoxin in which 116 nmoles of sulfide and ! 12 a t o m s o f iron per m g o f protein were estimated. The freshly prepared bovine and I ,rcine a p o adrenal ferredoxin and metapyrocatechase ['/], a non-home iron cont.,ning enzyme, as s t a n d a r d s yielded essentially no detectable labile sulfide. The Llantitative estimation of" the a m o u n t o f labile sulfide in the same way ~as also made ~ ah spinach chloroplast ferredoxin which had been reported to contain two gatoms o f iron and two moles oflabile sulfide per mole of protein [I 7]. As shown also in F!g. 5, in contrast to adrenal ferredoxin, almost all o f the detectable labile sulfide in chloroplast fcrredoxin was rapidly released when added to the alkaline zinc acetate solution.

('¢,/nparison of catalytic activities hel,'een native and reconstituted adrenal ferredoxins The activity c,f a d r e n a l ferredoxin in the reconstituted steroid I I/~-hydroxylase system was estimated in the presence of a N A D P H generating system, N A D P H adrenal ferredoxins reductase, c y t o c h r o m e P-450 and deoxycorticosterone. As shown in Fig. 6, the native a n d reconstituted adrenal ferredoxin functioned equally well in the hydroxylation o f deoxycorticosterone. T h e function o f the native and reconstituted preparations was also examined by measuring the stimulation o f the rate o f adrenal ferredoxin-dependent cytochrome c reducLon in the presence o f N A D P H - a d r e n a l ferredoxin reductase and N A D P H . The activity o f the reconstituted adrenal ferredoxin was identical with that of the native one (620 moles o f c y t o c h r o m e e reduced per rain per mole oFadrenal ferredoxin). The

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, reconstituted adrenal ferredoxin; H ,

native

316 apoprotein did not replace the holoprotein either in c y t o c h r o m e c reduction or in I l#-hydroxylase system. When adrenal ferredoxin was reduced with N A D P H and adrenal ferredoxin reductase, the superoxide anion, 02% was generated as j u d g e d by accumulation o! adrenochrome in the presence o f epinephrine a n d O, (Fig. 7). N o O2- generation was observed in ape adrenal ferredoxin. As shown in Fig. 8 the conversion o f epinephrine tc adrenochrome was greatly inhibited by superoxide dismutase from bovine erythrocytes I

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Fig. 7. The adrenal ferredoxin-dependent f o r m a t i o n o f a d r e n o c h r o m e . F o r c o n d i t i o n s see Materials and Methods. ~ . . . C , reconstituted adrenal ferredoxin; H , native a d r e n a l ferredoxin. Fig. 8. Inhibition o f a d r e n o c h r o m e f o r m a t i o n by superoxide dismutase. F o r c o n d i t i o n s see Material~ and M e t h o d s . . ' 2 - - - . . , reconstituted adrenal ferredoxin; H , native a d r e n a l ferredoxin.

The concentration o f superoxide dismutase at 50 ~% inhibition was found to be 5 nM for both native and reconstituted adrenal ferredoxin. Since each a d r e n o c h r o m e corresponds to !.39 0 2 - as reported [14], the rate of generation o r superoxide anion can be calculated to be 90 per min per mole of adrenal ferredoxin. The value i,~ consistent with that of Ch)stridium pasteurianum ferredoxin [14]. DISCUSSION

Results of the present studies give the improved procedure for the reconstitulion of adrenal ferredoxin. Judging from the homogeneity, molecular weight, absorption spectrum, iro~ and sulfide content and catalytic functions, the reconstituted adrenal ferredoxin, a~ well as the native one, may be suitable for critical analysis of its catalytic mechanisn~ and studies o f the physical and chemical properties o f the iron-sulfur protein. When the ape adrenal ferredoxin was incubated with Fe 2÷ and sulfide in th, presence of 2-mercaptoethanol, the yield o f the reconstituted preparation wa considerably low (20'~). The reconstitution of ad renal ferredoxin was greatly enhance¢ by the presence of 8 M urea: 7 5 ~ reconstitution was achieved. This observation i consistent with the concept that urea alters the c o n f o r m a t i o n of the protein so that i facilitates binding of the added Fe 2÷ and sulfide. The effect of urea on the activatio~ is in good agreement with our previous observations on reconstitution of iron containing enzymes such as m a m m a l i a n liver homogentisicase [18] and m i c r o b b metapyrocatechase [7]. The second point worthy o f c o m m e n t is the tendency o f m a n y a u t h o r s to detec labile ~bitide indifference toward the time required for its release from their specifie¢

317 i~ ,n-sulfur p r o t e i n . In fact, the r e p o r t e d values o f detectable sulfide in adrenal fi .redoxin v a r y b e t w e e n 58 a n d 100 n m o l e s / m g o f protein [3, 16]. Recently, Siegel al. [19] r e p o r t e d t h a t the total a m o u n t o f released labile sulfide o f N A D P H - s u l f i d e !uctase d e p e n d e d u p o n the i n c u b a t i o n time with an alkaline zinc acetate solution. As p r e s e n t e d in this paper, the m o l a r ratio o f iron to sulfide in adrenal I redoxin was a p p r o x i m a t e l y t w o when analyzed without incubation after alkaline c r e a g e n t was a d d e d . E x t e n s i o n o f the incubation time resulted in release o f l i t i o n a l d e t e c t a b l e sulfide a n d the m o l a r ratio o f iron to sulfide approached one. ese results suggest t h a t t h e reaction o f sulfide in some iron-sulfur proteins with an aline zinc reagent m i g h t be time dependent. In the latter case, a serious considera~ on the time f a c t o r s h o u l d be essential. KNOWLEDGEMENTS T h e a u t h o r s g r a t e f u l l y a c k n o w l e d g e the help o f Professor R. Sato, institute for |' ,~tein R e s e a r c h , O s a k a University, for supplying the adrenal glands. They are also i~ .lebted to Dr Y. K i s h i d a , D e p a r t m e n t of Biology, K a n a z a w a University, for i= ~otomicrography a n d to Miss H. K a w a k a m i , Cancer Research Institute, K a n a z a w a t miversity, f o r u l t r a c e n t r i f u g e experiments. R~FERENCES I Suhara, K., Takcrruari, S. and Katagiri, M. (1972) Biochim. Biolahys. Acta 263, 272-278 2 Suhara, K.. Ikeda, Y.. Takemori, S. and Katagiri, M. (1972} FEBS Lett. 28, 45-47 3 Kimura, T, {1968) in Structure and Bonding {Jorgensen, C. K., Neilands, J. B., Nybolm, R. S., Reinen, O. and Williams, R. J. P, ,eds). VoL 5, pp. !-40, Springer-Verlag, Berlin 4 Kimura, T. and Huang, J. J. (1970) Arch. Biochem. Bioph,~s. 137, 357-364 5 Hashimoto, S.. Suhara, K., Takemori, S. and Katagiri, M. (1971) Seikagaku 43. 535 6 Buchanan, B. B. and Amon, D. I. (1971) in Methods in Enzymology (Cotowick, S. P. and Kap|an, N, O., eds), Vol. XXlli, pp. 419--423, Academic Press, New York 7 Takemori, S., Komiyama, T. and Katagiri, M. (1971) Eur. J. Biochem. 23. 178-184 8 Hagihara, B,, Tagawa, K.. Morikawa, I.. Shin. M. and Okunuki, K. 11958) J. Biochem. Tokyo 45. "/25-735 ' McCord, J. M, and Fridovich, l. (1969) J, BioL Chem. 244, 6049-6055 ,0 Davis, B. J. (1964) Ann. N,Y. Acad. Sci. |21,404-427 I1 Layne, E. (1957) in Methods in Enzymology {Colowick. S. P. and Kaplan, N. O.. eds), Vol. Ill, i~P. 450-45 l, Academic Press, New York l? Massey, V. (1957) J. Biol. Chem. 229, 763-770 l, Fogo, J. K. and Popowsky, M. (|949) Anal. Chem. 21,732-734 1.~ Misra, H. P. and Fridovich, I. 0971) J. Biol. Chem. 246. 6886-6890 [ Rosenthal. O. and Narasimhulu, S. ii969) in Methods in Enzymology (Colowick, S. P. and Kaptan, N. O., eds), Vol. XV, pp. 604-605, Academic Press, New York I Kimura, T. and Suzuki, K. (1967) J. BioL Chem. 242, 455491 I Tagawa, K. and Arnon, D. I. (1968) Biochim. Biophys. Acta 153, 602-613 T l'akemori, S., Furuya. E., Mihara, K. aad Katagiri, M. (1'-)68) Eur. J. Biochem. 6. 4[1 ~418 i qiegeL L. M., Murphy. M. J. and Kamin, H. {1973) J. Biol. Chem. 2.48. 251~264