The differential inactivation of reduced nicotinamide-adenine dinucleotide oxidase and of succinate oxidase by freezing

i8

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 65135 T H E D I F F E R E N T I A L INACTIVATION OF REDUCED NICOTINAMIDEADENINE DINUCLEOTIDE OXIDASE AND OF SUCC~NATE OXIDASE BY F R E E Z I N G

PAT W. CAMERINO a~I~ TSOO E. KING

Laboratory for Respiratory Enzvmology and the Department of Chemistry, Oregon State University, Corvallis, Oreg. (U.S.A.) (Received July 22nd, t964)

SUMMARY

I. Freezing the Keilin-Hartree heart muscle preparation in an ice-salt mixture for 2 h in~ctivates NADH oxidase (NADH:O 3 oxidoreductase) activity more than succinate oxidase (succinate: O 3 oxidoreductase). This treatment also inactivates cytochrome c oxidase (cytochrome c: O3 oxidoreductase, EC 1.9.3. I). 2. The inactivation is correlated with the length of time that the preparation is frozen rather than with the act of freezing. The decrease in O~ uptake can be prevented if the suspension contains a certain minimal concentration of additional solute, 3. The succinate oxidase actixdty can be fully restored by the addition of soluble cyteehrome c; however, the NADH oxidase is not restored. 4. Differenc,_~ in the NADH to cytochrome c and the succinate to cytochrome c segments of the respiratory chain are discussed.

INTRODUCTION

The systematic dissociation and reconstitution of segments of tile m~mamalian respiratory chain with respect to succinate oxidase (succinate: Oz oxidoreductase) activity have been thoroughly documented1, 3. Investigations are also in progress to dissociate and reconstitute NADH oxidase (NADH:O2 oxidoreductase)Z, 4. As part of this work it became nec~ssary to ascertain tt e effect freezing t,( heart muscle particles has upon their catalytic activities in NADH or succinate oxidations by various oxidants, KEILIN AND HARTREE5 have reported that the succinate oxidase activity of th;3 pc,rcine heart preparatioi~ is inactivated by freezing in liquid air and that this activity can be fully restored by the addition of soluble cytochrome c. Other investigators6, ~ have also observed cryogenic influences o n succi~late oxidase activity. The data reported in this paper demonstrate that freezing affects NADH Biochim. Biophys. Aaa, 96 (I965) I8-27

INAC 71"~'ATION OF

NADH

A~/D SUCCINATE OXIDASE

I9

oxida ~e in a significantly diiferent manner than it d~:~ succinate oxidase. It appears t h a t ;~'eezing disrupts the transfer of electrons from c ,tochrome c to O 2 so that O~ uptake due to the oxidation of both N A D H and su:cinate is decreased. There is additional evidence that freezing disrupts electron flow from N A D H to a site prior to cytochrome c to a greater extent than it diminishes electron transport from succinate. METHODS

Materials The heart muscle preparation was made according to the method of KFnLIN AND HARTREE8 as adopted in this laboratoryL The .t~articles, obtained by centrifugatio_n, were s~aspended at a concentration of about 2'0 mg/ml in a borate.-phosphate buffer that was o.I M with respect to both Na2HPO 4 ~md HsBO 3. The ]~ADH was purchased from either Calbiochem or Sigma. Tile cytochrome c was obtai~md from Sigma (type III).

Assay procedures All experiments were performed at least in dupli,¢ate ; the results agreed wkhin experimental error. Assays were conducted at room temperature unless otherwise indi,~ated. The N A D H oxidas~ and succinate oxidase activities were determined polaro graphically using an Oxygraph t)urchased from Gilson Medical Electronics. Tile rates of O2 uptake are reported in terms of/zmoles 02 per rain per mg protein in a final volume of 2. 4 ml. The assay system contained a fir,al concentration of o~I M phosphate buffer, S0rensen type (pH 7.4) and, when apptkcable~ o.6 mM NADH and/or 83 mM succinate, levels at which the substrate conceiitr.ltion was not limiting. Unle~ specified, cytochrome c was not added to the a.~say mixture'. When cytochrome c was used, the final concentration was approx. 30/t:VI (see refi IO). The cytochrome c oxidase (cytochrome c:O, oxid(reductase, EC 1.9.3.I ) activity was assayed spectrophotometrically according to the method of SMITHll. The final volume was 2. 4 ml and the buffer concentration was o.I M phosphate (pH 7-4)The NADH-cytoehrome c reductase (NADH:cytochrome c oxidoreductase, ECI.6.2.I), succinate-cytochrome c reductase (succinate:cytochrome c oxidoreductase) and NADH-ferricymide r,:dactase (NADH:ferricyanide oxidoreductase) activities were assayed in the presence of I mM KCN according to the spectrophotometric methods routinely employed in this lab.~ratory 2,la with the exception that the concentrations of reagents common to the ~ssay for O 2 uptake were used for all determinations. These assays were perff~rmec on a C;lry model I I spectrophotometer. Protein was determined with the biuret real;,nt 13 in the' presence of 2% deoxycholate or was based on total fat-free dry solid * • It m u s t be emphasized t h a t tim values of t h e rat~ ,.,f O~ u p t a k e reported h~r,~ were obtained a t room t e m p e r a t u r e (23°-25 °) in the absence of ex. ~t-n~,~s eytochrome c unl,'.~s:~otherwise indicated. T h u s , these numerical values should n o t be v,a~pared with Qo$usually reported

in the literature without considering these differences.

Bioch;,n. Biophys. Acta, 96 (I965) I8-27

20

P . W . CAMERINO, T. E. KING

Experimental procedure :The heart muscle preparation was frozen for 2 h or according to the schedule ~ v e n in the RESULTS section. This was routinely accomplished by inserting a tube or small flask coutahaing the preparation into an ice-salt mixture. The suspension was compietely frozen within 5rain. A t the termination of the period of freezing, the: t u b e was t h a w ' ~ under cold running tap water until a small piece of frozen material remained. The container was then transferred to an ice bath and its contents permitted to melt. The mixture was red!:;persed prior to assay. RESULTS

Inactivation duringfreezing The data summarized in Fig. I indicate that freezing a suspension of the heart muscle preparation results in a decrease in both the NADH oxictase and succinate oxidase activities. It is also seen that tiffs decrease in the rate of O.0 uptake is greater during the utilization of NADH than of suecinate.

CL24 0.22

v 0.20 .x E

0.18

e 0,16

o.~ .=

0'i4

"rio ~ 0,08

~0.04 ~o 0.02

E 'I

2

3

4

Fig. I. Rate of O~ uptake by the heart muscle preparation before anti after freezing. The range and avcwage of nine experiments on five different batches of heart muscle preparation are given. 5gADFI was the substrate for t and 2; succinate for 3 and 4. Numbers 2 aml 4 were frozen for z h prior tx) thawing and assay.

The extent to which the NADH oxldase activtty is decreased detxnds upon the length of time that the suspension is frc~zen rather than the mere act of freezing and thawing. This is evident in the data oE Table I. A sample of tile heart muscle preparation was divided into a series of tubes. The contents were frozen for zo, 4 o, 60, or I2O min prior to thawing and assaying. The results are summarized in part A of Table I. In another series of determinations, the preparation was frozen Biochlm; Biophys. Aaa, 96 (t965) x8-27

INACTIVATION OF

NADH

AND SUCCINATE OXIDASE

2I

TABLE I DECREASE IN NADH OXIDASE AND (B} REPEAT~D FREEZING

ACTIVITY

WITH

A Singte period of fveexio~g

B Repeated freezing

Totat time frozen (rain)

Intewat o/ time frozen (rain)

o 20

Speri,% aaiv~ty"

o 20"

0,20 0.20

4°

o.t6

2o

o.I2

60

O, I I

20

O, I I

t 20

0.04

6o

" of a~,~y "' o f o. 19. "'"

INCREASIN=~ LENGTH

OF TI~77 OF FREEZING

Specific aaivity"

0.20 0.22

120 "tt

(A)

o.o7 O.O 4

T h e specific a c t i v i t y is g i v e n in t e r m s o f / , m o l e s O 2 p e r rain p e r m g o f p r o t e i n in 2.4 tv m i x t u r e . T h e c o n d i t i o n s of t h e a s s a y a r e g i v e n in t h e t e x t . A s a m p l e w h i c h w a s frozen a n d t h a w e d four t i m e s wi~_hin zo rain had a specific a c t i v i t y This s a m p l e w a s s u b j e c t e d to a s i n g l e I z o - m i n p e r i o d ,~f freezing,

for 20 min, thawed, assayed, and re-frozen within Io min. This procedure was performed a second and a third time; the fourth and final freezing perioc! ta:~ted 6o m i n The observations are listed in part B of Table I. Tl~e rate for a sample that had been frozen for a total of izo min prior to a single thawiI:g is atso listed. Another sample was repeatedly frozen and thawed a total of four tithes with 20 min. As indicated in the table the preparation which was repeatedly frczen and thawed showed less decrease in the rate of O 2 uptake than the one that was subjected to a ,dngle 2-h period of freezing. At lea.st 9o% of the residuzi NADH oxidase activity of the frozen suspension was sensitive to antimycin A under prior incubation .ff the enzyme with o.2 ~ug of the antibiotic per mg of protein at 4 °. Pra, ention of inactivation The decrease in the rate of 02 uptake can be pr~'~¢nted If the frozen suspension contains a certain minimal concentration of a solute A samFle of the heart muscle preparation in borate~phosphate buffer was diluted ~.4t!~ an equal volume of glass re-distilled water or borate-phosphate buffer. Other ~,~mples were diluted two-fold with EDTA, Na~HPO4-KH~PO4 buffer, Tris-HC1 1.- ffer (all a~ pHT.o) or wi*:h NaCI to produce a suspension that was o.I M with :'e~pect to the additional component. Since the original preparation had been *~..~:(e up m borate-phosphate buffer, the suspensions that resulted upon a two-fi~i,'! dilution were o.o5 M with respect to borate and to phosphate. The samples we~ frozen for ~ h prior to being thawed and assayed for NADH oxidase and suceinat,~ ~~xidase activities. The results are given in Table II. The prevention of the inactivation of NADH ,)xidase by freezing appears to require a minimal concentration of o.I M of solute which is further evidenced from tLe data of Table iII. The suspensions containing sr or:mate were also o.o5 M with Biockim B$ophys. Aria,

96 (~965) 18--27

22

P. W. CAMERINO, T. E, KING

T A B L E I2 RATE OF O~ ~3FTAKE B Y T H E HEART .MUSCLE PREPARAT;tON FROZEN IN T H E P R E S E N C E OF VARIOUS SOLUTES

Concentration of additional solute during freezing*

Specific activity °. NA DH

None o.i M b o r a t e - p h o s p h a t e (pH 7.0) o,~ M E D T A (pH 7.0) o.i M N a 2 H P O a - K H ~ P O 4 (pH 7.o) o.i M Tris-HC1 (pH 7.0) O.I M NaC1

S~¢inate

control

fro,en

¢ontrol

froren

o. t 8 0.20 o.2o 0.22 o.zc: o.2o

o.o6 0.06 o.zo oA 3 o.I8 o.t 4

o. ~6 o.I5 o.16 o.i 4 o.t5 o.x6

0,09 o.io oA 5 o.t 4 o,~ 7 o. 15

* Each suspension was o.o5 5I with respect to borate a n d 0.05 M with respect to p h o s p h a t e in addition to the solute listed here. "" pmoles O. per rain per m g protein. The conditions of freezing a n d t h a w i n g anti t h e a ~ a y are given in the text.

respect to borate and to phosphate. In the experiment with sucrose, the original borate-phosphate preparation had been centrifuged, washed once with glass redistilled water, re-centrifuged, and suspended in water prior to dilution with equal volumes of various concentrations of sucrose. I t w a s a l s o o b s e r v e d t h a t o . I M s u c c i n a t e o r o . I M E D T A ( p H 7.o) w o u l d completely protect the NADH oxidase activity of suspensions that were repeatedly

T A B L E III RATIO OF T HE ACTIVITY OF THE N A D q OXIDASE OF T I l E HEART MUSCLE PKEPARAT1ON BF.FOKS: AND AFTER FREEZING IN THE PRESENCE OF INCREASING CONCENTRAT|ONS OF SIdCROSE OR ~V SUCCINATE

Final concentration O. uptake @frozen preparation ,)~ uptake of control ° sucrose ( M ) Sucrose "" Succina!e ~"~

of succinate or

i .o 0.5 0-25 o. ! o o.o]

0.9.5 t .oo l .o2 o.9,~ ,xS8

O.OOI

0.45

0.52

o.ooot

o.38

o.39

None

0.38

o.3b

0.9.5 0.75

" The control was t h e s a m e b a t c h of t h e h e a r t muscle prepanttion in t h e presence o f tile solute without subjection to freezing, T h e conditions of frtx?zing a m t ti~awing a n d of t h e a~-ay are given in the text. "* No borate-phosphat~ buffer was present; Le. t h e s y s t e m was a p p a r e n t l y n o t buffered. "'" The suspension was 0.o5 M with respect to borate a n d to phosphate, @ Table II a n d t h e ~e×t. Biochim. Biophys. ~fcta, ,~6 (~9651 I8-~7

IXaC7 ~v.~rlO,~ o F N A D H AND SUCGINATE OXIDASE

23

frozen a n d t h a w e d f o u r t i m e s over a period of I a o r~fin. The p~otection afforded b y the solutes did not d e p e n d u p o n the concentration o f protein present in the frozen suspension when the anmunt o f t o t a l solids was varied in the te~ted range of 4.6 to 23.0 mg/ml. Once the N A D H oxidase a c t i v i t y o f the suspet~sion had been decreased bv freezing, i t could n o t : b e resto~*ed b y ~he a d d i t i o n o f a "'protective" a m o u n t of E D T A either i m m e d i a t e l y prior to t h e initiation of thawing or after this process had been completed. I t was observed t h a t t h e suspensions became d a r k e n e d upon inactivation, i.e. b y freezing in the absence o f a pro:ective c o n c e n t r a t i ~ of solute. After the frozenthawed saspenfion h a d been centrifuged in a Spinco Model-L centrifuge for 3 ° min at 5 0 o o o r~.:v./min, a clear yellow s u p e r n a t a n t fluid was obtained. This portion pos.~essed little NADH--ferricyanide reductase activi~y. Moreover, ~his s u p e r n a t a n t fluid did not contain a n y component t h a t would re.activate either the N A D H or succina~e oxidase. A t t e m p t s to obtain an increased C.:: uptake b y the frozen preparati(ms with the addition of the N A D H dehydrogenasc ( N A D H : (acceptor) oxidoredilc~a~) obtained b y the tlfiourea method 4 did not meet with significant success'. Increase in activily u p o n the addition o f cvtochrome c

It was observed, however, in agreement with the w~~rk of KEILIN AZtDHARTREE 5, t h a t the succinate oxidase activity, which is decreased b y freezing, could be comp l e t d y restore,l b y the addition of cytochrom e c to the assay mixture, In c o n t r a s t to this result for succinate oxidase, the a c t i v i t y of titc U A D H oxidase portion of TABI.E IV COMPARISON OF r i l e

D E C R E A S E I N T H E R A T E O F ()0 U P T A K E B Y F R E E Z I N G A N D T H E I N C R E A S E I N

T H I S A C T I V I T Y I N T I I E P R E S E N C E O F C Y T O C H R O M E C IN T H E A S S A Y

Heart muscle preparation

32 I*M Specifc achvitv§ ~2~qo~:h~ome c ............................................................................................ N.41)H Succiuate N.4 DH + succinale

t ~-

control

frozen

control

.(ro=en

o.I 5 o, t8

o,o5 o.oS

o,i 3 o,I 7

,.oS ~ 19

control

frozen

0,20 0.24

o. ii o.I9

O,2~ o.2 5

O. 12 0.17

2

-+

O,17 0.20

O.O() o. 10

O,I 3 0, I()

,~. I o ~ .: "

3

-,,-,-~-

O.I 4 0.21

o,o~ 0.07

o.I t O, 1 6

.o()

!8

0.(.)(~

¢, 1 4

0.3,'

(),I~

The specific activity is given in terms of/,moles O~ pt,v tin per mg of protein in 2.4 nil of test mixture. The conditions of freezing and thawing and ot ~,c ,,~say are given in the text. " The technique of fr~eziag is also of importance in the s*,~ubilization of NADH dehydrogenase by thiourea~,~*,tG, However, this method utilizes high r'o|or concentrations of thiourea and of sucrose which might prevent t;he destruction during ttis process of the cold-labile components of the system, It ran also be noted that freezing in t~he absence of thiouren does not result i~ the ,~lubili~tion of a NADH dehydrogenase. Biochit~', EI;ophys. A,zla, 96 (1965) 18-27

24

P. W. CAMERINO, T. E. K I N G

the frozen respiratory chain was increased but not fuUy restored by the addition of cytochrome c. These results are given in Table IV. This table also presents other signi~cant facts. Firstly, the observed rate of O 2 uptake in the presence of both NADH and succinate was less than the summation of the individuM rates in confirmation of the findings of Wv AxD T s o ~ L Secondly, the rate of O~ uptake in the presence of both NADH and suecinate was decreased b y freezing but was not increased by the addition of cytochrome c to a level greater than that of the restored succinate oxidase. T h e presence of I o p M cytochrome c during the freezing proce~ did not protect the respirato D, chain from a lo~ in either NADH oxidase or suceinate oxidase activity.

Site(s) of inactivation In order to establish the possible site(s) of damage to the respiratory chain by ttle process of reezing of the suspension, the NADH-cytochrome c reductase, succina~e-cytochron.',,:~ c reduetase, NADH-ferricyanide reductase, cytochrome c oxidase, and the NADH oxidase and succin,~te oxidase in the presence and absence of soluble TABLE V C O M P A R I S O N OF T I l E S P E C I F I C A C T I V I T I E S OF V A R | O U S R E G I O I ~ S OF T H E R E S P I R A T O R V C H A I N BEFORF~ A N D A F T E R F R E , ZING T H E H E A R T M U S C L E P R E P A R A T I O N

T h e c o n d i t i o n s c,f f i e e z i n g a n d o f t h a w i n g a n d o f t h e a s s a y a r e g i v e n in t h e t e x t anti t h e specific a c t i v i t y is e x p r e s s e d as p m o l e s O~ p e r rain p e r m g p r o t e i n in 2. 4 m l test m i x t u r e .

Activih' assayed"

32 p 3 I cytochrome c

Specific activity .............................. control frozen

N A I ) H -+O~ S u c c i n a t e -~ (), ( N A D H ,+ s u c c i n a t c ) -+ ()~

.

o.l 7 o.t 3 o.21

o.ob o.to o.13

N A I ) t t --~ O~ .quccinate -~ f)~ ( N A I ) H -!- s u c c i n a t e ) , * ( )o

~ .,"4-

o. zo ~). 16 o,z 5

o, Io o. 17 o. 17

N A D H -- c y t o c h r o m E c Succinat(: cytochronle c

+

o~ot~ o,o 5

o,o~ o.o 3

o.54 3.3

o.09 I.o

C v t o c h r o m e c -~ O,, N ' A D I I -> f E r r i c y a n i d e

~-

' T h e r a t e s dEtErminEd s p c c t r o p h o t o m e t r i c a l l y w e r e r e - c a l c u l a t e d in t e r m s o f t h e r a t e o f t),~ I p t a k e .

cytochrome c were assayed in the untreated preparation and the same preparation following a 2-h freezil N period. The rates, calculated for all of these activities in terms of #moles 02 per min per rag, are summarized in Table V, One of the s~gnificant observations is that tile activKy of the cytochrome c oxidase of the control greatly exceeds that of either the NADH oxidase or succinate oxidase at the level of cytochrome c, 32/~M, used. Freezing decreased the observed Biochim. Biophys, Aeia, 96 ( I 9 6 5 ) 1 8 - ~ 7

INAC'I:VATION O F

NADH

AND SUCCINATE OXIDASE

25

cytoc;xome c oxidase activity. It is also seen that ~!~'~zing decreased the NADH-~ cytochrome c reductase activity more than it did the succinatc-cytochrome c reductase.

DISCUSSION

Previous inv,;stigations have noted the heat-tability of the NADH oxidase activity x2 and the cold-lability of the succinate oxid:ase activity s-7 of he~rt muscle pa.:ticles. It is evident from the data of this paper that freezing for a period of 2 h decree~es the 02 uptake of the heart muscle preparation oxidizing either NADH or succinate. This decrease results from a damage to the respiratory chain at the level of the eytochrome c oxidase region that is common to both the NADH and succinate oxidase ch.~ms. The apparent re-activation of the succinate ox~dase activity by the addition of soluble cytochrome c is in accord with the proposatl°, 18 which postulates the bypass of the electron flax around tile endogenous c3~tochrome c. This by-pass is visualized as the reduction of the soluble cytochrome c by the reductase of ti~e respiratory chain and the re-oxidation of the reduced c3~ochrome c by the cytochrome c oxidase of the system. When cytochrome c is added to the assay of the succinatc oxidase of the frozen particles, the rate of O2 uptake is returned to the original level, which appears to be equal to the maximal residual activity of the cytochrome c oxidase, i.e., a rate, obtainable at infinite cytochrome c. This maxiInal activity is usually twice that of the rate at 32/~M cytochrome c (ref. 19). The addition of NADH to the a ~ a y of the frozen suspension in the [~-~o,w:e of succ~nate and c'.tochrome c gives no further increase in the rate of O2 uptake Ance the maximal rate of the partially inactivated cytochrome c oxidase has beea attained. The rate observed in the presence of NADH and cytochromo, c, but in the absence of succinate, for the frozen preparation is less than that of the control, due to the partial inactivation of the NADH-cytochrome e reductase that is required for the production of the ferrocytochrome c utilized in '.he by-pass. Since N A I ) H ferficyanide reductase is also diminished by this treat'nent, freezing may possibly affect the NADH dehydrogenase, itself. Howew~r, th~s cannot be said with certainty since the reduction of ferricyanide occurs at more than one site in the respiratory chain (cf. for example, ref. 12). A significant observation in this investigation i~ that the process of freezing has a more damaging affect on the oxidation of NAD f~ than of saccinate and that this difference may be due to a component situated between the respective dehydrogenases and their ]uncture with the remainder ,~ the respiratory chain. This inactivation could occur through a modification of t lip:.d-protein complex associated with the NADH-cytochrome c reductase and .:ytochrome c oxidase of the respiratory chain. The presence of such complexes h,'s been documented ~0. The differences between the two reductases, especially L, th zir lipid nature, ~Aave been pointed out since it has been demonstrated that NAD'~l-eytc.ch~me c reductase is more sensitive than is succinate-cytochrome c reduc:a.~ to inactivation by urea ~, by isooctane extractions zz, by acetone ~3, by ether ~4 ~ r.d by surface-active agents 2n. Moreover, cytochrome c oxidase, which is also inacti~'~ed by freezing, was found

Bioch~,~ B~ophys, Aria, 96 (t965) I8-27

26

P, w. CAMERINO,T. E. KING

t o b e inactivated b y isooctane .2. A n influence of low temperature, if not out-right freezing, has been associated with the lipids of the l~spiratory chain, since storage at --~5 ° to - - z o ° increases the inactivation of the NADH--cytochrome c reductase by organic solvents ~b,2e. It would appear that freezing m a y inactivate the lipid-protein complexes associated with the N A D H - c y t o c h r o m e c reductase and cytochrome c oxidase of the e!ectron-transport system. It has been established ~-m that freezing has a damaging affect: on certain 6ther lipid-protein compiexes. :It:is not known :why the length o f time t h a t the suspension:is frozen has a greater influence o n decreasing t h e rate o f O~ uptake than the act of freezing since the macrodisrupti0n of the particles should o c c u r during the initial formation of the ice crystals. Theobservation is that repeated freezing and thawing is less damaging than a single extended perit:d during which the suspension is frozen. This w o ~ d , however, be in agreenient with MERYMAN80 who points out that "tissue injury from freezing is primarily the result of the high salt and substrate concentrations produced by the removal of water to form ice," The correlation of this phenomenon in tissue and in a particulate homogenate is uncertain. On the other hand, this explanation is apparently inconsistent with the protection afforded by added solutes. The data of this paper indicate thnt the inactivation of both the N A D H and succinate oxidase systems can be prevented if the heart muscle suspension is frozen with a sufficient concentration (about o.I M) of solute. This condition m a y be of importance in attempts at storage of suspensions of respiratory particles (~![. LINNANE AND TITCHENER 81 and DI PRISCO et al.32), ttowever, it is somewhat puzzling that the nature of the solute, either a neutral salt, a buflk~r, a chelating agent, or even sucrose, does not show any observable difference. Another possible explanation for the increased damage during prolonged freezing may be associated with local "hot spots" produced bv the growing ice crystMs ~. Actually, the mechanism for the inactivation still remains to be explored.

ACKNOWLEDGEMENTS The technical assistance of Mrs. J. POTTER arid Mr. F. C. SEEFELDT is gratefully acknowledged. This work was supported by grants fl'om the National Science Foundation, the U.S. Public Health Service, the American Heart Association, and the Life Insurance Medical Research Fund. REFERENCES * D. KEILINANDT. E. KING, Nature, tSt (~958) t52(~. 2 T. E. KING, J. Biol. Chem., 238 (t962) 4037. 3 T. E. KINGAND R. L, HOWARD,J. B~ol. Chem., 237 (1902) 1686. 4 E. R, Rf~DFEARNANDT. E. KING, Natttve, 2o2 (t904) 13i 3. 5 D. KIr.ILINAND E. F. I-1AI~TaEE,Proc, Roy. So¢. London, See. B, I29 (Iq4o) 277. 6 F. LyNEN, Bioehem. Z., 264 (,94 o) z46; abstracted in Bet. Wisse~*schaf/. Bio:, 55 (I94 I) 547. 7 J. M. KLINKHAMERAND B. EICHEL, Nature, 193 (1962) 9~4. 8 D. KEILINAND E. F. HARTREE, Biochem. J , 41 (1947) 5OO. 9 T. E. KING, J, Biol. Chem,, 236 (196I) 2342, to P. W'. CAMERINOAND L. SMITt$,.J. Biol. Chem., 239 (1964) 2345. It L. S;~ITH,Amh. Biochem. ltiophys., 50 (t954) 285. Biochim, Biophys. Acfa, 96 (I065) *8-27

INACTI¥-kTION OF NADHAND SUCCINATE OXIDASE

~7

I-z R. Q, MORRIS AND T. E. KING, Biochemistry, I (x962) IOX7. I3 T. YONETAN~, J. Biol. Chem., z36 (x96x) 168o. 14 E. C. SLarER, Bioche)n. J., 45 (x949) I. i5 T. C~raONA, E. B, KEARN~Y, M, VXLLAVICENCIOAND T. P. SINGER, Biochem, Z,, 338 (I963)

2I 2~ 23 24

407 . A, G. CrtaPMa,~~ AND V. JAGA1~rrrA'rrlaN, personal communicat~,3n. C. Y. W u AND C. L. Tsou, Acta Physiol, Sinica, I9 (x954) 453, P . W . CI~Mt~RIN()X N D L. SMITlt, Federation Proc., 22 (4962) 654. L c - tTH AND P. ~V. ~AMERINO, Biochemistt3', 2 (1963) x43:~, D: E? GRESN ANVS. l~'~IScn~., BiOchiml Biophys. Acta, 7~ ~(1963) 554. E. R. REDFI~ARN ~,:~D ~['. E. KING, Biochem, J., 90 (1964) 291'. D, Dztm, E. C. SI.ATER AZ4DL, VEL~SraA Biochim. Biophys. Acta, 27 (t958) x33. S. FLB~SC~IER, A. Castr AND B. FLEISCaER, FederatSon Proc., e3 (1964) 486. W. I'. Cur~NINc,na,~t, C. L. HALL A~D F. L. CRAr~E, Abstr. ~'.,'~th lntern, Congr. Bioche,~. Nezv

25 26 27 28 -'9 3° 3~ 32

YO~'k, x9,54, p. 647E. R. REDFI~:~R.'% A. M. PtlMP_~IREY a'.~."~ G. H. FYNN, Bioch:m. Biophys. Acta, 44 (496o) 404 . A. NAS~t¢ .~,~O i. R. I,,sr~,'a~, J. Biol. Chem., 22z (~956) 5~ t~ J. L. O.'~c~.,.v, F. R, N. GtmD AND M. MELiN, J. Am. Chem. Soc., 72 (t95 o) 458. J. BORNS*t';IN, J. Biol. Chem., 205 (~953) 543 • J. E. LO~rELOCK, Proc. Roy. Soc. London, Ser. B, 447 (495;:) 427 • H. T. ~ERYMAN, A'~n. N , Y . ,4cad. Sci., 85 (4960) 5o3 • A. W. LL~NANE AND E. B. Tt'rcri~NEa, Biochim. Biophys..l:ta, 39 (t96o) 469. (3. I)I PRIScO, M. B~,~AY-ScttwARXZ a.'ZO H. J. SXgECKER, ~2iochem. Biophys. Res. Commu~,.,

16 x7 I8 19

2o

45 ('.,964) ~ 6 . 33 J- I . Sr~l,rl~NSON, .-Inn. N. Y. ,4cad. Sci., 85 (~96~) 535.

Biochi~, Biophys. Acta, 96 (~965) I8-27