660
BIOCHEMICAL SOCIETY TRANSACTIONS
CO MMUN I CATI 0NS Ribosomal Peptidyltransferase Activity in Rat Skeletal Muscle ALEXANDRA VON DER DECKEN The Werner-Gren Institute for Experimental Biology, Norrtiillsgatan 16, S-113 45 Stockholm, Sweden The enzyme peptidyltraiisferase is an integrated part of the ribosome. It catalyses in the ribosome the peptidization between aminoacyl-tRNA and initiator methionine-tRNAOP" or peptidyl-tRNA. The antibiotic puromycin is a competitive inhibitor of aminoacyl-tRNA and an acceptor for the initiator methionine or peptide provided these are attached to their tRNA and positioned at the ribosomal P-site (Petska et al., 1972); methionyl- or peptidyl-puromycin is released; their concentration is a measure of peptidyltransferase activity (Petska et al., 1972; Baliga et al., 1973). Rats were fed for 6 consecutive days a diet containing the high-quality protein casein supplemented wit11 3 g of methionine/kg. The protein concentration was either 20 or 3 % (w/w). Skeletal-muscle ribosomes were isolated (von der Decken & Omstedt, 1972). Peptidyltransferase activity was determined (Baliga et al., 1973).Between 0.1 1 and 0.15pmol of peptidyl-tRNA per mol of ribosome were accessible as a substrate for peptidyltransferase. The activity at 20°C was 7.7 and 6.8pmol/min respectively for ribosomes of protein-fed and protein-restricted rats. The rate decreased to 6.7 and 6.5pmol/min respectively when diphtheria toxin and NAD were added as an inhibitor of elongation-factor 2. In conclusion, the decrease in peptidyltransferase activity reported here after restricted protein supply was insufficient to account for the lower rate of protein synthesis observed previously (von der Decken & Omstedt, 1972; Omstedt & von der Decken, 1972).Nevertheless, peptidyltransferase activity must be considered as one of the factors iniluencing the capacity of polyribosomes to synthesize proteins. I thank the Swedish Metlical Research Council (project no. 4266) for support.
Baliga, B. S.,Schechtman, IM. G., Nolan, R. D. & Munro, H.N. (1973) Biochim. Biophys. Actu 312,349-357
Omstedt, P. T. & von der .Decken, A. (1972) Br. J. Nutr. 27,467-474 Petska, S., Goorha, R., Rosenfeld, H., Neurath, C. & Hintikka, H. (1972) J . B i d . Chem. 247, 42584263
yon der Decken, A. & Omstedt, P. T. (1972) J. Nutr. 102, 1555-1562
Phosphorylation of Ribosomes in Adenovirus Infection G. ERIC BLAIR* and [VAN HORAKf *National institute for Medical Research, Mill Hill, London NW7 1A A , U.K., and tlnstitut fur Virologie und Immunobiologie der Universitat Wiirzburg, Versbacher Landstrasse 7, 0-8700 Wiirzburg, Federal Republic of'Germany
Previous studies of polypeptide phosphorylation in adenovirus-infected HeLa cells have demonstrated at least five proteins phosphorylated as a specific consequence of virus infection (Russell (Ir. Blair, 1977). Two of these phosphoproteins were found in association with ribosomes. The larger protein (mol.wt. lo5) could be dissociated from whereas the smaller protein ribosomes by washing in a medium containing O.~M-KCI, (mol.wt. 2.6 x lo4) remained bound to ribosomes under these ionic conditions. Since the larger protein was also found in nuclear and cytoplasmic fractions of the infected cell, it was identified as a phosphorylated derivative of a virus-coded polypeptide 1977
568th MEETING, ABERDEEN
661
(also of mol.wt. lo5), previously shown to be present in mRNA-nucleoprotein complexes isolated late in infection (Lindberg & Sundquist, 1974). We now report that the smaller protein is a phosphorylated derivative of the ribosomal protein designated as protein S6 (Wool & Stoffler, 1974; Kaerlein & Horak, 1976). HeLa cells, either mock-infected or infected with 50 plaque-forming units of adenovirus type-5/celI, were incubated for 20h at 37°C. Both cultures were labelled for 2.5h with 2mCi of [32P]Pi. Ribosomes were isolated and ribosomal proteins were extracted as previously described (Russell & Blair, 1977; Kaerlein & Horak, 1976). Equivalent amounts of ribosomal protein obtained from either mock-infected or infected cells were separated by two-dimensional polyacrylamide-gel electrophoresis (Kaltschmidt & Wittmann, 1970). Radioactively labelled phosphoproteins were located by radioautography. Only one labelled ribosomal phosphoprotein from adenovirus-infected cells was detected. This protein co-migrated with the ribosomal protein S6. A trace amount of label was noted in protein S6 of mock-infected cells. In vaccinia-infected HeLa cells, two phosphorylated ribosomal proteins were detected in addition to phosphorylated protein S6 (Kaerlein & Horak, 1976). These were the phosphorylated derivatives of proteins S2 and P3, an unidentified phosphoprotein bound to the 40s ribosomal subunit which might be virus-coded. Thus the pattern of ribosomal-protein phosphorylation induced by two DNA viruses is different. The origin of the protein-S6 phosphorylation in adenovirus-infected cells is not clear. Whether the phosphorylation is catalysed by a virus-coded or virus-induced protein kinase, or results from the virus-induced inhibition of a phosphoprotein phosphatase, remains unknown. Similarly, the function of ribosomal-protein phosphorylation is unknown (Wool & Stoffler, 1974). It is tempting to speculate that phosphorylation of ribosomes may have some role in the preferential translation of virus mRNA known to take place in virus-infected cells. G. E. B. was the recipient of a short-term European Molecular Biology Organization fellow-
ship. Kaerlein, M. & Horak, I. (1976) Nuture ( b n d o n ) 259, 15&151 Kaltschmidt, E. & Wittman, H. G. (1970) Anal. Biochern. 36,401-412 Lindberg, U. & Sundquist, B. (1974) J. Mol. Biol. 86,451-468 Russell, W. C. & Blair, G. E. (1977) J. Gen. Virol. 34, 19-35 Wool, I. G. & Stoffler, G. (1974) in Ribosomes (Nomura, M., Tissieres, A. & Lengyel, P.,eds.), pp. 417-460, Cold Spring Harbor Laboratory, Cold Spring Harbor
Translation of Mouse Myeloma Messenger Ribonucleic Acid in a Messenger-Dependent Cell-Free Protein-Synthesizing System STEWART A. LAIDLAW and ALAN R. WILLIAMSON Institute of Biochemistry, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
Polyribosomes were extracted from two IgG2,*-secreting tissue-culture cell lines (Pl-17 and 5563), grown in RPMI-1640 medium, by using a specially adapted method (J. N. Bennett, L. C. Fitzmaurice & A. R. Williamson, unpublished work). From these, a poly(A)-containing RNA fraction was isolated by slight modifications of standard methods (Lee et al., 1971; Aviv & Leder, 1972). Both polyribosomes and poly(A) RNA were added toa messenger-dependentcell-free protein-synthesizingsystem derived from rabbit reticulocytes (Pelham & Jackson, 1976). The translation products were analysed on polyacrylamide slab gels (Laemmli, 1970). Comparison of products of polyribosome- and poly(A) RNA-directed synthesis indicated no specific loss of any
* Abbreviation: IgG2., immunoglobulin G2=. Vol. 5
BIOCHEMICAL SOCIETY TRANSACTIONS
CO MMUN I CATI 0NS Ribosomal Peptidyltransferase Activity in Rat Skeletal Muscle ALEXANDRA VON DER DECKEN The Werner-Gren Institute for Experimental Biology, Norrtiillsgatan 16, S-113 45 Stockholm, Sweden The enzyme peptidyltraiisferase is an integrated part of the ribosome. It catalyses in the ribosome the peptidization between aminoacyl-tRNA and initiator methionine-tRNAOP" or peptidyl-tRNA. The antibiotic puromycin is a competitive inhibitor of aminoacyl-tRNA and an acceptor for the initiator methionine or peptide provided these are attached to their tRNA and positioned at the ribosomal P-site (Petska et al., 1972); methionyl- or peptidyl-puromycin is released; their concentration is a measure of peptidyltransferase activity (Petska et al., 1972; Baliga et al., 1973). Rats were fed for 6 consecutive days a diet containing the high-quality protein casein supplemented wit11 3 g of methionine/kg. The protein concentration was either 20 or 3 % (w/w). Skeletal-muscle ribosomes were isolated (von der Decken & Omstedt, 1972). Peptidyltransferase activity was determined (Baliga et al., 1973).Between 0.1 1 and 0.15pmol of peptidyl-tRNA per mol of ribosome were accessible as a substrate for peptidyltransferase. The activity at 20°C was 7.7 and 6.8pmol/min respectively for ribosomes of protein-fed and protein-restricted rats. The rate decreased to 6.7 and 6.5pmol/min respectively when diphtheria toxin and NAD were added as an inhibitor of elongation-factor 2. In conclusion, the decrease in peptidyltransferase activity reported here after restricted protein supply was insufficient to account for the lower rate of protein synthesis observed previously (von der Decken & Omstedt, 1972; Omstedt & von der Decken, 1972).Nevertheless, peptidyltransferase activity must be considered as one of the factors iniluencing the capacity of polyribosomes to synthesize proteins. I thank the Swedish Metlical Research Council (project no. 4266) for support.
Baliga, B. S.,Schechtman, IM. G., Nolan, R. D. & Munro, H.N. (1973) Biochim. Biophys. Actu 312,349-357
Omstedt, P. T. & von der .Decken, A. (1972) Br. J. Nutr. 27,467-474 Petska, S., Goorha, R., Rosenfeld, H., Neurath, C. & Hintikka, H. (1972) J . B i d . Chem. 247, 42584263
yon der Decken, A. & Omstedt, P. T. (1972) J. Nutr. 102, 1555-1562
Phosphorylation of Ribosomes in Adenovirus Infection G. ERIC BLAIR* and [VAN HORAKf *National institute for Medical Research, Mill Hill, London NW7 1A A , U.K., and tlnstitut fur Virologie und Immunobiologie der Universitat Wiirzburg, Versbacher Landstrasse 7, 0-8700 Wiirzburg, Federal Republic of'Germany
Previous studies of polypeptide phosphorylation in adenovirus-infected HeLa cells have demonstrated at least five proteins phosphorylated as a specific consequence of virus infection (Russell (Ir. Blair, 1977). Two of these phosphoproteins were found in association with ribosomes. The larger protein (mol.wt. lo5) could be dissociated from whereas the smaller protein ribosomes by washing in a medium containing O.~M-KCI, (mol.wt. 2.6 x lo4) remained bound to ribosomes under these ionic conditions. Since the larger protein was also found in nuclear and cytoplasmic fractions of the infected cell, it was identified as a phosphorylated derivative of a virus-coded polypeptide 1977
568th MEETING, ABERDEEN
661
(also of mol.wt. lo5), previously shown to be present in mRNA-nucleoprotein complexes isolated late in infection (Lindberg & Sundquist, 1974). We now report that the smaller protein is a phosphorylated derivative of the ribosomal protein designated as protein S6 (Wool & Stoffler, 1974; Kaerlein & Horak, 1976). HeLa cells, either mock-infected or infected with 50 plaque-forming units of adenovirus type-5/celI, were incubated for 20h at 37°C. Both cultures were labelled for 2.5h with 2mCi of [32P]Pi. Ribosomes were isolated and ribosomal proteins were extracted as previously described (Russell & Blair, 1977; Kaerlein & Horak, 1976). Equivalent amounts of ribosomal protein obtained from either mock-infected or infected cells were separated by two-dimensional polyacrylamide-gel electrophoresis (Kaltschmidt & Wittmann, 1970). Radioactively labelled phosphoproteins were located by radioautography. Only one labelled ribosomal phosphoprotein from adenovirus-infected cells was detected. This protein co-migrated with the ribosomal protein S6. A trace amount of label was noted in protein S6 of mock-infected cells. In vaccinia-infected HeLa cells, two phosphorylated ribosomal proteins were detected in addition to phosphorylated protein S6 (Kaerlein & Horak, 1976). These were the phosphorylated derivatives of proteins S2 and P3, an unidentified phosphoprotein bound to the 40s ribosomal subunit which might be virus-coded. Thus the pattern of ribosomal-protein phosphorylation induced by two DNA viruses is different. The origin of the protein-S6 phosphorylation in adenovirus-infected cells is not clear. Whether the phosphorylation is catalysed by a virus-coded or virus-induced protein kinase, or results from the virus-induced inhibition of a phosphoprotein phosphatase, remains unknown. Similarly, the function of ribosomal-protein phosphorylation is unknown (Wool & Stoffler, 1974). It is tempting to speculate that phosphorylation of ribosomes may have some role in the preferential translation of virus mRNA known to take place in virus-infected cells. G. E. B. was the recipient of a short-term European Molecular Biology Organization fellow-
ship. Kaerlein, M. & Horak, I. (1976) Nuture ( b n d o n ) 259, 15&151 Kaltschmidt, E. & Wittman, H. G. (1970) Anal. Biochern. 36,401-412 Lindberg, U. & Sundquist, B. (1974) J. Mol. Biol. 86,451-468 Russell, W. C. & Blair, G. E. (1977) J. Gen. Virol. 34, 19-35 Wool, I. G. & Stoffler, G. (1974) in Ribosomes (Nomura, M., Tissieres, A. & Lengyel, P.,eds.), pp. 417-460, Cold Spring Harbor Laboratory, Cold Spring Harbor
Translation of Mouse Myeloma Messenger Ribonucleic Acid in a Messenger-Dependent Cell-Free Protein-Synthesizing System STEWART A. LAIDLAW and ALAN R. WILLIAMSON Institute of Biochemistry, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
Polyribosomes were extracted from two IgG2,*-secreting tissue-culture cell lines (Pl-17 and 5563), grown in RPMI-1640 medium, by using a specially adapted method (J. N. Bennett, L. C. Fitzmaurice & A. R. Williamson, unpublished work). From these, a poly(A)-containing RNA fraction was isolated by slight modifications of standard methods (Lee et al., 1971; Aviv & Leder, 1972). Both polyribosomes and poly(A) RNA were added toa messenger-dependentcell-free protein-synthesizingsystem derived from rabbit reticulocytes (Pelham & Jackson, 1976). The translation products were analysed on polyacrylamide slab gels (Laemmli, 1970). Comparison of products of polyribosome- and poly(A) RNA-directed synthesis indicated no specific loss of any
* Abbreviation: IgG2., immunoglobulin G2=. Vol. 5
