Protein synthesis in plant mitochondria

445

PRELIMINARY NOTES

Protein synthesis in plant mitochondria Although it has been reported that plant mitochondria can incorporate amino acids into protein z, the maj or site for such incorporation is considered to be the microsomes ". The present study has, however, revealed that mitochondria from the seedlings of Vigna sinensis Linn. (Savi) is a far more active centre than the microsomes in incorporating glutamate. After obtaining the mitochondria ~, the supernatant fluid was spun at lO5 ooo × g for 3 h in the Spinco Model L centrifuge to sediment the microsomes. Both mitochondria and microsomes were washed oncO. The radioactivity in protein was measured according to STACHIEWIEZAND QUASTEL 4 with some additional steps. The incorporation of radioactivity by the microsomes was negligible, nor could it be enhanced by the addition of mitochondria or mitochondria plus a mixture of protein amino acids with glutamine, asparagine, ATP, GTP and the pH- 5 enzyme 5 (Table I). The supernatant fluid after the sedimentation of mitochondria was also inactive. Further, when the whole homogenate of the seedlings, freed from the cell debris, was incubated with [z4C3glutamate, radioactivity was maximal in mitochondria and negligible in microsomes (Table II). The nature of incorporation was unaltered whether the disintegration of the seedlings was carried out in the presence of 0. 5 M sucrose alone or plus o.15 M potassium phosphate buffer (pH 7.4) or the buffer plus EDTA. Likewise, the absence or presence of light during germination, variation in TABLE I [14C]GLUTAMIC ACID INCORPORATION BY THE ISOLATED PARTICLES The i n c u b a t i o n s were carried o u t for 2 h at 37 ° in o.o5 M p o t a s s i u m p h o s p h a t e buffer (pH 7.o), 0. 4 3/I sucrose, 2 m M MgC12 and i m M ATP. Radioactivity Enzyme

Additions

(counts/rain]rag (counts~rain/rag mitochondrial protein)

Mitochondria Microsomes Mitochondria + microsomes Mitochondria Microsomes Mitochondria Mitochondria Microsomes + Mitochondria

183 I35

} ~

+ microsomes + pH-5 enzyme 5 pH-5 enzyme + microsomes + p H - 5 e n z y m e

Mitochondria Mitochondria + s u p e r n a t a n t * * Mitochondria + E D T A - t r e a t e d s u p e r n a t a n t * * *

microsomal protein)

182 G T P (z mM) + a m i n o acids* (o.I mM)

I3o

5

145 IO 15o 184 94 7°

* Mixture of all the protein a m i n o acids except glutamic acid together w i t h glutamine and asp.a.ragine. S u p e r n a t a n t left over after s e d i m e n t a t i o n of mitochondria. This should c o n t a i n b o t h microsomes and soluble enzymes. *** The h e a v y metals p r e s e n t h a v e been chelated b y the addition of equivalent a m o u n t s of EDTA.

Biochim. Biophys. Acta, 53 (1961) 445-446

440

P R E L I M I N A R Y NOTES

the period of germination from 48-I2O h and of incubation from 30 min-2 h could not bring about any improvement. Though centrifugation at 2o ooo × g has been adopted as the routine procedure for sedimenting mitochondria, it has been found that more than 80 % of the active particles could be sedimented at IOOOO x g. 5-times washed mitochondria were about 2.5 fold as active as once washed mitochondria. TABLE

iI

DISTRIBUTION OF [14C]GLUTAMIC ACID IN DIFFERENT CELLULAR FRACTIONS S e e d l i n g s w e r e h o m o g e n i z e d i n 0.5 M s u c r o s e , s t r a i n e d a n d c e n t r i f u g e d a t I o o o x g for IO m i n . T h e s u p e r n a t a n t w a s b r o u g h t t o p H 7.o, i n c u b a t e d w i t h ~ 1 4 C l g l u t a m i c a c i d i n t h e p r e s e n c e of 2 m M A T P a n d z m M G T P for 2 h a t 37 °. T h e r e a c t i o n w a s s t o p p e d b y c h i l l i n g a n d t h e p a r t i c l e s fractionated centrifugally.

Radioactivity (countslmin/mg protein)

Fraction

Mitochondria M i t o c h o n d r i a l fluffy l a y e r Microsomes M i c r o s o m a l fluffy l a y e r Soluble supernatant

55 z I I 2

The incorporation of [14C]glutamic acid into the mitochondrial protein was sluggish at the beginning and increased even up to 5 h. The percentage of incorporation varied considerably and in general, increased with the decrease in the concentration of glutamate. The lowest recorded value was o.13% (at o.o2 M glutamate) and the highest 5.5 % (at o.2 mM, ignoring endogenous glutamate). Radioactive incorporation due to terminal exchange has been found to be negligible compared to the total incorporation 6. D-[14C]Glutamic acid was not incorporated. The presence of EDTA in the disintegration medium has been found to be beneficial to mitochondrial activity. The addition of microsomes, pH-5 enzyme or the supernatant fluid over the mitochondria was inhibitory to some extent. MgC12 was ineffective while the amino acid mixture brought about some inhibition. ATP and GTP were feebly stimulatory while UTP, CTP and I T P were ineffective. We are grateful to the National Institute of Sciences of India for a fellowship to one of us (H.K.D.), to the Ministry of Scientific Research and Cultural Affairs for some grants, to Dr. S. K. RoY of the Bose Institute, Calcutta, for germination facilities and to Professor B. C. GUHA for his kind interest in the work.

Department of Applied Chemistry, University College of Science and Technology, Calcutta (India) 1 2 3 4 5

G. G. H. E. E. F.

H. K. DAS S. C. RoY

C. WEBSTER, Plant Physiol., 29 (1954) 202. C. WEBSTER, Plant Physiol., 30 (1955) 351. K. DAs AND S. C. R o Y , Sci. and Culture (Calcutta), 25 (1959) 317 . STACHIEWIEZ AND J. H . QUASTEL, Can. J. Biochem. and Physiol., 37 (1959) 687. n . KELLER AND P. C. ZAMECNIK, J. Biol. Chem., 221 (1956) 45. SANGER, Biochem. J., 39 (1945) 507 •

Received August 2nd, 1961 Biochim. Biophys. Acta, 53 (1961) 4 4 5 - 4 4 6