The action of serum albumin on oxidative phosphorylation in insect mitochondria

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297

and even at doses of 500/Lg per day there is little suppression of the P]O ratio. I t would appear, then, that the M1 preparation contains some material which produces the effects on phosphorylation, and since one major difference between the two preparations is the degree to which the particles have been washed, it would seem that this material is removed in the preparation of the M,' mitochondria. This material may be an essential cofactor for the thyroxine action or may be thyroxine itself; however, the hormone has been shown to be bound tenaciously by mitochondriaZ, s. Since the MI preparation contains some microsomal and other contamination, in addition to mitochondria, it is further possible that the phosphorylation effects on the mitochondria are mediated only through the presence of some other enzyme entity in the incubation system. If this is so, it would suggest that the primary action of thyroxine may be other than on the phosphorylation complex of the mitochondria. Experiments are in progress to ascertain the nature of the material present in the M1 preparation which produces the effects on phosphorylation. This work was supported in part by USPHS Grants C-3635 and C-3IO3.

Department o/Physiology, University of Cali[ornia Medical Center, Los Angeles, Calf[. (U.S.A.) x F. G. 8 G. 4 R. 5 G. 6 A.

A . S. FAIRHURST

J . M . MAHER* R . E . SMITH

I. HocI-I AND F. LIPMANN, Proc. Natl. Acad. Sci. U.S., 4o (1954) 9o9. F. MALEY, J. Biol. Chem., 224 (1957) lO29. F. MALEY AND H. A. LARDY, J. Biol. Chem., 2I 5 (1955) 377. E. S~tlTI~ A n D A. S. FAIRHURST, Proc. Natl. Acad. Sci. U.S., 44 (1958) 7o5, GOMORI, f. Lab. Clin. Med., 27 (1942) 955. H. LARDY, Recent Progr. in Hormone Research, io (1954) 143.

Received September 8th, 1958 * P r e s e n t a d d r e s s , D e p a r t m e n t of Zoology, C o l u m b i a U n i v e r s i t y , N.Y.

The action of serum albumin on oxidative phosphorylation in insect mitochondria It has been observed 1-3 that serum albumin and some other proteins added to the incubation medium greatly stimulate oxidative phosphorylation in insect mitochondria. SACKTORz was even unable to demonstrate any phosphorylation with housefly sarcosomes in the absence of albumin. The nature of this effect remains, however, unknown. In the present investigation with mitochondfia from larvae of the wax-moth, Galleria mellonella L., a pronounced stimulatory effect on oxidative phosphorylation was observed with both human and bovine serum albumins but with ncither egg albumin nor with an albumin fraction from an acetone powder of frog muscle (Table I). To explain the nature of this effect the ATP-orthophosphate exchange reaction was investigated. It was found (Table I) that a definite exchange could be observed only in the presence of human or bovine serum albumin. It was also observed that, if mitochondria were washed with 0.25 M sucrose containing 3 % serum albumin followed by washing with pure o.25 M sucrose in order to remove the remaining albumin, they catalyzed the exchange reaction (on an average 1.1/zatoms P/rag Abbreviations: ATP, adenosine triphosphate; AMP, adenosine monophosphate.

298

PRELIMINARY NOT~.S

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TABLE I EFFECT OF ALBUMINS ON OXIDATIVE PHOSPHORYLATION AND ATP-ORTHOPHOSPHATE EXCHANGE REACTION IN MITOCHONDRIA FROM Galleria mellonella LARVAE A. O x i d a t i v e p h o s p h o r y l a t i o n . I n c u b a t i o n m e d i u m : KC1, 2 o / * m o l e s ; MgC12, 6 / * m o l e s ; e t h y l e n e d i a m i n e t e t r a a c e t a t e , 2 / , m o l e s ; p h o s p h a t e buffer, p H 7.2, I o / , m o l e s ; tris ( h y d r o x y m e t h y l ) a m i n o m e t h a n e - H C 1 buffer, p H 7.2, 1 8 / , m o l e s ; AMP, o. 5 / , m o l e ; A T P , o . 2 / , m o l e ; s u b s t r a t e , 8 / , m o l e s ; glucose, I O / , m o l e s ; h e x o k i n a s e , 200 K.M. u n i t s ; m i t o c h o n d r i a (2- 4 m g protein) isolated in o.25 s u c r o s e c o n t a i n i n g o.ooi M A T P . F i n a l vol., I.O m l ; t e m p . , 25°; i n c u b a t i o n t i m e , 2o-4o m i n . O 2 u p t a k e was m e a s u r e d in a c o n v e n t i o n a l W a r b u r g a p p a r a t u s ; p h o s p h o r y l a t i o n was d e t e r m i n e d b y m e a s u r i n g t h e a m o u n t of h e x o s e m o n o p h o s p h a t e f o r m e d 4. B. E x c h a n g e reaction. I n c u b a t i o n m e d i u m : KC1, i o o / , m o l e s ; MgC12, 4 / , m o l e s ; e t h y l e n e d i a m i n e t e t r a a c e t a t e , 1. 5 / , m o l e s ; A T P , 5 / , m o l e s ; t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e - H C 1 buffer, p H 7.5, 3 4 / , m o l e s ; p h o s p h a t e buffer, p H 7.5, c o n t a i n i n g a b o u t 5 / , C zip, 3 / * m o l e s ; m i t o c h o n d r i a ( a b o u t I m g protein) isolated in o.25 M sucrose. F i n a l vol. w a s i.o ml. T h e r e a c t i o n w a s m e a s u r e d ~ over 15 rain a t a b o u t 22 °. T h e v a l u e s are corrected for A T P a s e a c t i v i t y . Oxidative phosphorylation P/O

A ddition

with succinate wifh glutamate

None B o v i n e s e r u m a l b u m i n , 4-5 m g H u m a n s e r u m a l b u m i n , 4.5 m g E g g a l b u m i n , 4.5 m g F r o g - m u s c l e a l b u m i n s , 4-5 m g

0.2 I.i 1. 4 o.i 0.2

Exchange reaction patom Piing protein]h

I.I 1.8

O.I 2.1 1.8 O.I O.I

TABLE II INHIBITION OF ATP--ORTHOPHOSPHATE EXCHANGE RgACTION IN '*ACTIVATED" MITOCHONDBIA FROM WAX-MOTH LARVA~ E x p e r i m e n t a l c o n d i t i o n s a n d i n c u b a t i o n m e d i u m a r e t h e s a m e as described in T a b l e I (B). F o r details c o n c e r n i n g p r e p a r a t i o n of " a c t i v a t e d " m i t o c h o n d r i a , w a s h i n g fluid etc. see t e x t . T h e e t h a n o l a n d t h e b e n z e n e e x t r a c t s used in E x p t . 2 were p r e p a r e d b y e x t r a c t i n g t h e h e a t - c o a g u l a t e d a l b u m i n f r o m t h e w a s h i n g fluid with a few m l of t h e c o r r e s p o n d i n g s o l v e n t s . Prior to t h e a d d i t i o n of i n c u b a t i o n m e d i u m a n d of m i t o c h o n d r i a , t h e e x t r a c t s were placed in i n c u b a t i o n t u b e s a n d t h e s o l v e n t s were c o m p l e t e l y e v a p o r a t e d . Ezpt.

I

z

Addition

Exckange tw~m P~ rag protein~l*

None W a s h i n g fluid (previously heated), 0.2 m l S u s p e n s i o n of c o a g u l a t e d a l b u m i n f r o m t h e w a s h i n g fluid, 0.2 m l S u p e r n a t a n t after c e n t r i f u g i n g off c o a g u l a t e d a l b u m i n , 0.2 m l Coagulated p u r e bovine s e r u m a l b u m i n

o. 80

None

Ethanol extract, z ml B e n z e n e extract, 2 ml

Inhibition %

0.39

5I

0.22

73

o.76 0.67

5 16

0.6 7 0.05 o.12

92 82

protein/h) without any albumin present in the incubation medium. These experiments seem to indicate that an inhibitor of the exchange reaction is present in insect mitochondria and that it can be removed b y serum albumin. To elucidate this question, albumin-washed ("activated") mitochondria as well as the sucrose-albumin washing fluid from these mitochondria were investigated. I t appeared that the washing fluid,

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299

previously heated for 5 min at IOO° in order to coagulate the albumin, strongly inhibited the exchange when added to the "activated" mitochondria (Table II) The same effect was observed with a suspension of coagulated albumin from this washing fluid but not with the clear supernatant after centrifuging off the precipitated albumin. In a next experiment the heat-precipitated albumin from the washing fluid was extracted with ethanol and benzene. It was found that both ethanol and benzene extracts were inhibitory (Table II), whereas the protein after extraction was without inhibitory effect. These experiments show that the inhibitor of the exchange reaction, and perhaps of oxidative phosphorylation, is removed from insect mitochondria by washing with serum-albumin solution. It is possible to extract this inhibitor by organic solvents after thermal coagulation of albumin from the washing fluid. It seems probable that the inhibitor present in isolated insect mitochondria is similar to a substance which Hf3LSMANN et al. e extracted from mitochrome 7 with organic solvents. This substance was found s to uncouple oxidative phosphorylation and to inhibit ATP-orthophosphate exchange in rat-liver mitochondria. We wish to thank Prof. E. C. SLATER and Dr. W. C. HULSMANNfrom the Laboratory of Physiological Chemistry, University of Amsterdam, for valuable discussions which stimulated the present investigation. The technical assistance of Miss J. RENIKOWSICA, is gratefully acknowledged.

LECH WOJTCZAK Department o[ Bioohemistry, N e m k i Institute of Experimental Biology, Warsaw (Poland)

ANNA B.

WOJTCZAK

1 B. SACI~TOE, J . Gen. Physiol., 37 (1954) 3432 S. E. LEWIS AND E. C. SLATER, Biochem. J., 58 (I954) 2o7, s K. R. REES, Biochem. J., 58 (1954) 196. 4 E. C. SLATER, Biochem. J., 53 (1953) 157. 5 S. O. NIELSEN AND A. L. LEHNINGER, J. Biol. Chem., 215 (1955) 555. 6 W . C. HULSMANN, W . B. ELLIOTT AND H. I~UDNEY, Biochim. Biophys. Acta, 27 (1958) 663. 7 B. D. POLIS AND H. W . SHMUKLER,J. Biol. Chem., 227 (1957) 419.

Received September 29th, 1958

The effect of cetyltrimethylammonium bromide on axonal conduction SCHOFFENIEIS et al. 1 have recently reported block of axonal conduction by a lipidsoluble quarternary ammonium ion (noracetylcholine dodeciodide). Failure to produce block with qumternary ammonium compounds had been held as evidence against the concept of the necessary involvement of acetylcholine and its metabolism in nerve and muscle conduction S. It has been proposed, however, that externally applied quarternary ammonium compounds do not block axonal conduction because they are, generally, lipid-insoluble and therefore unable to reach the active membrane 3, 4. Using the cationic surface-active agent CTMB, we have obtained varying degrees of conduction block in frog sciatic nerve depending upon the concentration and Abbreviation: CTMB, cetyltrimethylammonium bromide.