Vol. 80
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COMMUNICATIONS T O T H E EDITOR T H E INCORPORATION OF VALINE-1-CI4 INTO CYTOCHROME c BY RAT LIVER MITOCHONDRIA'
Sir:
supernatant fluid obtained after centrifuging a 1 : 3 homogenate a t 100,000 X g for 1 hour).
TABLE I Protein biosynthesis in cell-free systems has been investigated by studying the incorporation of INCORPORATION O F v A L I S E - l - c ' 4 I X T O CYTOCHROME C labeled amino acids into the total protein of subThe reaction mixtures for experiments 1 and 2 consisted of cellular structures (e.g., microsomes,* mitochon- potassium succinate (250 pmoles), L-histidine buffer of pH 7.4 (1.0 mmole), MgClz (125 pmoles), cytochrome c (0.09 dria,3 n ~ c l e i , ~and ) by measuring increases in pmole), adenosine 5'-phosphate (AMP) (200 pmoles) , ULenzymic a ~ t i v i t y .However, ~ no cell-free system has ~ a 1 i n e - l - C(10.8 ~ ~ pmoles containing 19 X lo8 c.p.ni.), been available for the study of the biosynthesis of orthophosphate (250 wmoles), soluble fraction ( 5 mi.), and a discrete protein that can be isolated in purified t h e 3 t o 6 X washed mitochondria obtained from 24 g. of form. This report describes the incorporation of rat liver, in a final volume of 25 ml. Incubated at 37' for 20 rnin. In t h e zero time experiment, the reaction mixture labeled amino acids into cytochrome c b y rat liver was treated immediately with acid. T h e final concentramitochondria. Studies on the incorporation of tions of the inhibitors used were: D N P (3.0 X lo-' M ) , labeled amino acids into the cytochrome c of rat K F (0.01 X ) , sodium arsenate (0.002 M ) . I n experiment 3, C P (313 ptnoles), and adenosine 5'-diphosphate (.lDP) liver slices have been reported.6 (50 pnioles), were substituted for succinate, AMP, cytoAfter incubation of mitochondria in the presence chrome c, and orthophosphate. T h e amount of cytochrome of an ATP-generating system and ~ ~ - v a l i n e - l - C l *c isolated from each flask was about 0.15 pmole, corrected cytochrome c was isolated by salt fractionation' and for cytochrome c additions where these were made. Sp. activity, column chromatography.8 The eluted material was E x p t . c.p.m./mg. cy.c. Conditions washed with trichloroacetic acid containing un1 Complete system 875 labeled DL-valine. The specific radioactivity of the 4- D S P 78 preparation remained constant after extensive anaerobiosis 66 washing with trichloroacetic acid, exhaustive zero time experiment 0 dialysis, paper electrophoresis, and after removal 2 Complete system 509 of the porphyrin group.g The porphyrin fraction + F-,arsenate, D S P , niiaerobiosis 0 was not radioactive. The isolated cytochrome - soluble fraction 1-33 showed one band on paper electrophoresis and con3 Complete system -395 tained 0.4427, F e ; its absorption spectra and ex- CP 7 tinction coefficients were identical to those reported The presence of labeled valine in the peptide for highly purified preparationslO and for the crystalline protein.11 However, the present state of uncer- chain of cytochrome c is suggested by the retention tainty about the purity of cytochrome c prepara- of the label throughout the purification procedures tions,I2 makes it difficult to assess unequivocally employed, and by the ATP dependence of the incorporation process. Further evidence for this view the homogeneity of our preparation. Table I shows the dependence of ~ a 1 i n e - l - C ' ~was obtained by treating a partial acid hydrolysate incorporation on the presence of .ITP generated of the labeled cytochrome with S-fluoro-2,4-dinitroeither by oxidative phosphorylation or by creatine benzene and fractionating the products by a difphosphate (CP). Replacement of valine by DL- ferential extraction procedure13 which separates 1 y ~ i n e - l - Cor ' ~ of C P by D-3-phosphoglycerategave n'(a)-(2,4-dinitrophenyl)-amino acids from 2,4similar results. The relatively small extent of dinitrophenylpeptides, and by paper chromatogvaline incorporation in the presence of 2,4dinitro- raphy. The major portion of the radioactivity phenol (DNP) or in the abseiice of added C P may appeared in fractions usually associated with dibe ascribed to endogenous substrate level phos- iiitrophenylpeptides, and distinct froin dinitrophorylation. Conditions known to inhibit such phenylvaline. Although the evidence presented strongly favors phosphorylation completely suppressed incorporation (expt. 2 ) . Valine incorporation also depends the view that isolated mitochrondria can incoron the presence of a soluble liver fraction (the porate labeled amino acids into cytochrome c, the (I) Aided by a grant (A-428-CS) from t h e G. S. Public Health immediate role of ,ITP in the process is unknown; Service a n d t h e Muscular Dystrophy Associations of America. e.g., the possibility that energy is needed only for B i d . ChPTn., 195, 649 (1952); 1'. C . Zamecnik P. Siekevitz, J . (2) amino acid transport into mitochondria has not a n d E B. Keller, zhzd., 209, 337 (1954). been excluded. I t is hoped that further studies will (3) M, V. Simpson, J. R. McLean, G . L . Cohn a n d I. l i Brandt. yield information on this point and on the question Fedci.ation Proc., 16, 219 ( l 9 5 i ) . I1) V. G. Allfrey, A . E. 11irskg ani1 S. Osawa, J . Gru. P h y s i d . . 40, of the relationship between amino acid incorpora~451(1957). tion and the dc novo synthesis of cytochrome c. (31 E. 1:. Gale, H a m e y L e c f x u r s , 51, 25 (1957). J. B. Marsh and D . L. Drabkin. J . B i d Chein., 224, 909 ( 1 9 5 7 ) . ( 7 ) D. Keilin and E . F. Hartree, Biochu?rz. J . , 39, 289 (1945). ( 8 ) S. Paleus a n d J. B. Neilands. i l r t ~C h r m Scand., 4 , 1024 (19,jO). I,!)) K r.,Paul. Acta C k m . S ~ - a i i ~4i .,. 239 ( I 9,iij) (10) E. hlargoliah B i o c h r w . J . . 56, T,2U (1!2:14) (11) U. Hagthara, 1. Rlorikdwa, I . Seziihu. T. IIc n u h i , S n l i w e . 178, G 3 O (1950). (I?) S. I'al6us and H . l h e o r c l l , . I L f L : i-hcm. > c u i t d . , 11, !IO5 ( 1 U . i i ) . (1%)
DEPARTMEST OF BIOCHEMISTRY YALEUNIVERSITY KETVHAVES,CONS.
HAROLD AI. BATI~S iU.CRADDOCK MELI-IS\'. SIMPSON
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