~ 2 2 P R E L I M I N A R Y N O T E S

the r~,xtuction ot enzyme-b0und flavin by N A D P H , as sugg~.~ted by the decrease in absorbancy at 455 rr~ on addit ion of NADPH. Under anaerobic conditions, a NQ, also bound by the enzyme, wiLl accept electrons from the reduced flavin to form N Q H . and then NQHz. I f it is a~sumed tha t the NQH- is very labile to oxygen and rapidly reoxidizable to NQ by oxygen, pr, ,bably because of its special l inkage to the enzyme protein, it ~ill be unders tandable t ha t NQ mediates, in the presence of oxygen, the aerc~bic oxidat ion of N A D P H wi thout in termediate format ion of NQFI t. The semiquinone form of #-benzoquinone m a y not be oxidizable by oxygen for teas,ms still to be investigated. The part icipat ion of a radical, mtmodehydroascorbate , has alst~ been ,uggested ~n the ascorbate-s t imulated oxidat ion of NADH by adrenal micro.~me.~ ta

Institute for Protein Resem, ch, Osaka University,

Kita-ku, Osaka (Japan)

H IROKO N I S H I B A Y A S H [

-T-~ . . . . . . ~ M U R A

RYo SATO

' I{. S~T,~. H. N~st i tn~,Y^SXt ~,,~D 1". ( ' ~ U ~ A . Bicbvh,m. Biophy~+ A a a , 63 (x961) 55o. t R. S ^ x o , H. N~S~ltBAV^SHX ^~r , T. OMURA, in p r e p a r a t i o n . • B. I. . HORV.C};ER. J. B,ol. Che~., I83 (195o) 593. ' C. H. ~,W., I.I.~M.~, JR. AND I-1. K~.MI.N, J . Biol. Chem., 237 (x96"~) 597-

A. H. [~tttL,[] 'S AND R. C;. [..*,~z~DO,'~', J. Biol. Chem,, 237 { l ~ z ) :1652. * T. Oh~URA AND R. S^'ro, f . B,ol. Ck.e.m., in t h e p r e s s . ~C..~[ITOMA. I I . ~. PilSNER, } [ . C . l tEITZ alqO S. UDENFRll~NO, ,~f~:~. Bi~mhem. Biophy$., Ox

(195¢~) 43 t. j+ |{. [+II,,r~*:TTE, £.~. £.). ~.~RODIF~ .*.::O -~, K I,AI.)t:, _I- Phnvm~coi. ExpII+ TMto#. , 119 11957) 532-

t~ T. OStURA AND I { . SATO, f . Biol . Chem+. 237 {1962) PC1375 . t* T. O~tUR.g AND R. S^'io, ?L~:of~i*n. B#ophys. Acta, i n t h e prL~S. at L. ERNSTER, 1%|. ]~JUNt;K;RE.~ AND l,. I).~.NIELSON, B i o ~ . s m , Biophys. Res. Commt,~., 2 (106o) 88, II L, ERNST~R, L. L)ANIFLSO.N ANt) ~1. ].JUNC;GRF..~. ~io*h im. Btop~y.¢. Acta, 58 (19~Z] 171. 13 1~. KRISt:H AND H. ST.~.UDtNGI],R0 BioctWm. Z., 33Z (195E) 105.

Received l~.¢embe- xoth, x962 t3iochim. Bioph),s. A eta, 6 7 { tgf~3} 52o- 322

pN xoo3o The oxidat ion-reduct ion poter,tial of ¢ytochrome b 5 in soluble and part iculate

f o r m with reference to the role of lipid

The oxida t ion-reduct ion potential is one of the impor t an t properties of respiratory enzymes. I t generally determines the position of the enzymes in respiratory chains and contrlh, t tes to unders tanding thei- funct ion in the living cells. Conc~.rning the potent ial of cytochrome ha, a microsomal hemoprotein, several conflicting values have been reported. The potent ia l de termined first by YostltKAW^Z, m wi th dog. and rabbit-l iver preparations was shown to 1)e - -o .x3V. Later~ STRITTMATTER A.~D BALt "~ obtained the same value ~-ith a rat-liver par t iculate stmp~nsiop. The cyto- chrome from rabbit-liver microsomes was solubilized and purified by VELICII[ AND Sm~r~A3-rET< a who est imated its ox ida t ion- reduct ion potent ia l to be -i-0.07, V. In the Sympo~ium on Hemat in Enzymes held at Cazlberra e, the cause of the dis- crepancy xvau subjected to debate, bu t no clear answer was obtained. ~he object rff the present s tudy is to clarify this question.

Biochiss. Biopltys. Actao b7 [x~3) . ' i2a-524

PRELIMINARY NOTES ~2 3

Cytochrome b s was purified from beef liver essrntia]l¢ in the same way as des- cribed by STRla'r~A'mER e. The oxidation-rccduction potenIiais were est imated by the dye method with nile blue, methyl capri bhm and methylene blue, tee E o" values of which at pH 7.o are -o , I3 V, - - 0 o 6 V and .-t~.ot V, respectively. The measure- men t was carried out at pH 7 o anti at 23 ° ~.v;t,h clear solutions, the cytochrome and dye were placed in a Thumberg- type cu'¢ette tilled with nitrogen gas and stoppered with rubber through which di thioni te solution was intrc~duced from a syringe. The per cent reductions ,ff the pigment anti dye wero es t imated spectrophotometrical ly. and the l~>tenti:tl of cytochronm ba for half-reduction. E0'. was calculated With turbid .solutions. the mixture with (tye was placed in a test tttht, and covered with

T A B L E [

O X l D A ' / ' | O , ' q -R~DI .~CTIOIg P O T E ~ T I . * t L ~- O F CYTOCI- |1~OM_ b~. ,".T D I F F E R P . N T ~TAg~F,q

O F P U K I F I C A T I O N t a R I 3 C E L } U R I ' . "

"<.gage E," / V I

. ~ I i c r o s o n l e s - - o . 1 4

Lipc~c t r u ~ t m ~ n t -- o+ot Acid p r ~ ' ! ~ i t a t i o n ~- ,,.oz D E A E - c d l u l o s e cbromatt~gr~q~hy . o.oa

liquid paraffin. Dithionite ~ol-tion wa.~ added drop by Grop from a syringe, until the absorpt ion band of reduced cytochrome bx apv-ared. At this point, the per cent reduct ion of the dye was est imated by comgaring the color ir, ten.~ity with a series of s t anda rd dye solutkms, while the reduction grade of the cytochrome was observed b y the use of Zeis.~ hand ~pcctroscopO,*. These two methods gave practically identical resulLg,

In Table I are given the oxida t ion-reduct ion !~,tentia|~ of cytochrome .b.. at different stages of purification. The pigment in microsomes showed a low potential , - -o . I 4 V. as reported by previous investigators. When the microsome preparat ion was t rea ted with lipase, the potential shifted to a h~ghcr level, --o.ox V. As see,~ in Table II, t r ea tmen t of microsomal preparation with other ageats, such as chelate or peptideses, brought about no appreciable change in the E0'.

TA B I . ~ I[

E V F I ~ C T O F Ti~F .ATNE. 'q ]" O F .~ |CIgL"~OMAL P R I , ' P A R . ' ~ T t O N v , ' I T H %'AR[O1]~ AGE.MTS U P O N T | l g P O ~ ' J ~ N T I A L O F ~ T 4 f } C H R O M [ - ~ ~t~ I

Ag¢~ E," f V J

Pamcreafi¢ lipame (pur i f ied a c c o r d i n g to, Wtllst . i t t ter} . p H 8. L 37". 3 h + o . o t

Fxx] ium chelate { t % } . p } l 7,5. o;. 30 ~ i n - - o , t 3 Trypsdn ~Mochida P h a r m a c e u t i c a l Co.).

p H 8.be 23% ~4 h --o.~3 B a c t e r i a | p r o t e i n a s ~ (Nag,lse I n d Co.L

pH 7-5. z3 °. 24 b --u. 14

Bwchim. .RzopAys . 4eta, 67 {1963) 522-524

~ 4 P R E L I M I N A R Y N O T E S

In the next seri~,~ of experiments, the influence of the addi t ion of various sub- staatces to ptLdfied cytochrome b~ was examined. The results are given in Table I l I . Among the substances so far tested, only lipid which was ext rac ted from beef-liver micros-rues with ch lo io form-methano l (x : x) lowered the potent ia l to nearly the same level a,s tha t of paxticulatc-bnund microsomal pigment.

These findings suggest t ha t the much lower lx~tential of cy tochrome b~ in par- t iculate form m a y be a t t r ibu ted to some sort of combinat ion of the hemoprote in with Lipid in microsorae (presumably phos~holipid). The exact na ture of the com- bination is not clearly understood, but the crude lipase contained in the enzyme

]'ABLE 111

E F F E L T O F A D D I T I O N O F V A R I O U S S U B S T A N C E S O N r F I R P O T E N T I A U

O I ~ (~¥TOCI-IRONF. ~)l I N P U R I F I ~ ' D F O R M

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T~¢ amo,mt o/ Y~bstance add.cat ad, d¢~ seb'J~om¢¢ E,* fl.',, /. mJ~/ml

¢yl~tr,..'~¢ btt

N o n e -t- o÷o~ M i c r o s o m a l l ip id t 4 -- o. ! 2 l ~ v i n e s e r u m a l b u m i n t,'~o -k o . o - ~ |-~rol.atrn;.P.c 18o ~ O.oi S o d i u m c h o l a t e 63 -b o.ox Ce l lu lose ['Kl'.vder 40 Jr o~Ot Cel l i te i O "4- u.o z Alumini~ C>, t ~ + o , o x

prel~arati(>~l f rom hog pa~lcrea.s splits the lipid component of the par t icula te cytc,- chrome and solubilizes it. The par t ic ipat ion of mi tochondr ia l lipid in eleetron- tr~t~l~fer mcchanizm has been discussed recently ~. Our exper imental results migh t suggest an iml~wtant role of lipid in microsomal function.

The authors wish t~ thank Profe.~sor Y. OGURA for his wduable advice.

lkepartmcnt o/Biophysics and I3i6;?lemistvy, Faculty of Science, a,ut

Department of Phy'iological Ch~ni~vy a~ui Nulri#ion, Faculty of Medici,~,

University of Tokyo. Tokyo (Japan)

Y. KAWA1 Y. YONIZYAMA H. YOSHIKAWA

I H. YOSXtKAWA. l £ a k u to S~ibz~ts,,gal~u, 6 (1944) I x . z H . Y o s u [ ~ w A . J . Bio~I tem. T o k y o , 3 S ( x 9 5 t ) 1. It C . F . S T R t ' r z M A ' I U I ~ Z R A N D E . G. D A L k , PrO¢:. N a t l . Acaxt . S a i . U . S . , 3~ 1t95=] [ 9 .

S. F . VIXLICK ASD P. STRIT'rMAT'rRR, J . Bfo l . C./l#tlt., 2~tl {1956) a65. 0 j . E . FM.K. I t . LZ~tBSRG ^ N b R . K. MORTON, H a c m a t t u E n z y m e s , O x f o r d , P ~ r g a m o n P r e ~ ,

z(:~f~~, p. 477. Q P . , ~ T R I T T M A T ' g E R A.NID ~,. F . V]~LICK, J . J~06 f, Cke)lrJl,, 2 ~ 1 (][056) 2 2 3 .

T f~. FLe tscnzR , G. BRXB.R[.ZY, H . KLOUWB.q P, WD D. ~ . SLt, UCT~It~ACK, J . ~ i0 / . Chem+, 2)#7 ( I ~ z ) 3264.

Received J a n u a r y x6th, z963

B i o c l t i m . Bio~hys. Acra, 67 (a963) 5 2 2 - 5 2 4