Distribution of copper-64 in control mice and in mice bearing ascitic Krebs tumor cells. (73/97)

Three to 20 hr after an i.p. injection of 64Cu (half-life, 12.8 hr) into mice bearing Krebs ascites cells, a high amount of the radioisotope was recovered in the ascites cells themselves. In the control group, the radioisotope was mainly present in the liver. Similar amounts of 64Cu were recovered in regenerating as well as in normal liver, whereas in the liver of mice bearing ascites cells, this amount was lower by 40 to 50% regardless of the ascitic volume. Thus, the copper metabolism seems to be disturbed at the hepatic level in mice bearing ascites cells. The distribution of 64Cu was 'analyzed in DNA, RNA, and proteins from cellular lysates fractionated by CsCl gradient. There was a uniform pattern of distribution in the macromolecules from ascites cells, while 64Cu' was preferentially associated with the protein fraction from liver. Further experiments indicated that, in vivo, 64Cu was bound to the DNA of ascites cells.  (+info)

DL-threo-beta-fluoroasparagine inhibits asparagine-linked glycosylation in cell-free lysates. (74/97)

The effect of beta-fluoroasparagine on N-linked glycosylation was examined in a cell-free translation system in which glycosylation is coupled to protein synthesis. The threo-isomer markedly inhibited glycosylation at a concentration of 1 mM, and this effect was blocked by L-asparagine, indicating that glycosylation was inhibited secondary to incorporation of the asparagine analog into protein. The erythro-isomer, at similar concentrations, was not incorporated into protein and had no effect on glycosylation. threo-beta-Fluoroasparagine is highly toxic to some mammalian cells in culture. Our observations suggest that its toxicity may be due in part to the failure of the fluoroasparagine-containing protein to become glycosylated. The data suggest that this analog will be useful for examining the structural determinants for glycosylation.  (+info)

Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases. (75/97)

Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6. In 90 min between 0.7 and 1.0 mol of phosphate/mol of protein S6 was incorporated by the catalytic subunit of cyclic AMP-dependent protein kinase. Of the other proteins, S3 and S7 from the small subunit and proteins L6, L18, L19 and L35 from the large subunit were predominantly phosphorylated by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates by either kinase; (3) proteins S7 and L29 were almost exclusively phosphorylated by the cyclic AMP-dependent protein kinase; (4) protein S6 and most of the other proteins were phosphorylated about two or three times faster by the cyclic AMP-dependent than by the cyclic GMP-dependent enzyme.  (+info)

Isolation and characterization of a ribonuclease activity specific for double-stranded RNA (RNase D) from Krebs II ascites cells. (76/97)

We have extensively purified from Krebs II ascites cells, although not until homogeneity, a ribonuclease which preferentially cleaves natural or synthetic double-stranded RNA substrates (RNase D); this specificity is also supported by its sensitivity to inhibition by 10(-5) M ethidium bromide. It does not degrade RNA-DNA hybrids and is, therefore, clearly distinct from previously characterized RNases H (Cathala, G., Rech, J., Huet, J., and Jeanteur, Ph. (1979) J. Biol. Chem. 254, 7354-7361). It shows no requirement for a divalent cation and is inhibited by all kinds of nucleic acids regardless of their secondary structure. It acts exclusively as an endonuclease, as shown by the analysis of degradation products, and yields 5'-phosphate termini. This enzyme is able to introduce discrete nicks into purified HeLa 45 S preribosomal RNA as well as into HeLa heterogenous nuclear RNA packaged within naturally occurring nuclear ribonucleoprotein particles. It is, therefore, an interesting candidate for an RNA-processing enzyme.  (+info)

Affinity labelling and characterization of the ppp(A2'p)nA-dependent endoribonuclease from different mammalian sources. (77/97)

The ppp(A2'p)nA-dependent endoribonucleases from a number of different mammalian sources have been investigated. The enzyme from reticulocyte lysates shows optimal activity of 50-150 mM KCl and requires the presence of Mg2+. Whilst the enzyme is inactivated after passage of reticulocyte lysates through Sephadex columns in the absence of ATP, it retains full activity provided ATP is included in the column buffer. The activity of the partially purified nuclease was unaffected by the addition of reticulocyte RNase inhibitor, which, in contrast, effectively inhibited other endogenous endonucleases. The ppp(A2'p)nA-dependent Rnase co-purified with a ppp(A2'p)nA-binding protein and with a protein which could be specifically covalently labelled with an oxidised radioactive analogue of ppp(A2'p)nA. This covalent labelling could be carried out either with the partially purified RNase or in crude extracts from rabbit reticulocytes, mouse Krebs and Ehrlich ascites tumour cells and human lymphoblastoid (Daudi) or HeLa cells. In each case the affinity labelled protein migrated to a position corresponding to a apparent molecular weight of about 85 000 on electrophoresis on dodecylsulphate/polyacrylamide gels. In all cases labelling could be prevented by the addition of an excess of unlabelled ppp(A2'p)nA but not, for example, by a similar excess of the biologically inactive dimer ppp(A2'p)'A. It is concluded that the RNase and ppp(A2'p)nA binding activities are likely to reside in the same molecule.  (+info)

An RNA-dependent nucleoside triphosphate hydrolase from Krebs-II ascites tumor cells. Detection and preliminary characterization. (78/97)

A novel enzymatic activity, RNA-dependent, NTPase, was isolated from Krebs-II ascites tumor cells. This activity is associated with ribosomes and can be detached from them by washing in KCl solutions of a higher than 0.3 M concentration. The enzyme hydrolyzes all the four nucleoside triphosphates to the corresponding nucleoside diphosphates and orthophosphate. The rate of NTP hydrolysis increases about 10-fold in the presence of natural RNAs and synthetic polyribonucleotides [except poly(G)]. Natural DNAs, both double and single-stranded, are poor cofactors, although pol(dA) and poly(dT) stimulate, to a certain extent, the rate of ATP hydrolysis. Possible involvement of RNA-dependent NTPase in protein biosynthesis is discussed.  (+info)

Unusual requirements for optimum translation of polio viral RNA in vitro. (79/97)

The translation of poliovirion RNA (polio RNA) in an in vitro fractionated system was much less efficient than that of encephalomyocarditis virion RNA (EMC RNA). In contrast, when polio and EMC RNAs were added to postmitochondrial cell lysates (S10), they were translated with equal efficiency. However, this equality was observed only when high concentrations of S10 were employed; at lower concentrations, polio RNA translation was reduced relative to that of EMC RNA. These results suggest that both the fractionated and S10 systems are limiting in a component that is required for the optimal translation of polio RNA. The elongation rates for EMC and polio RNA translation in the fractionated system were found to be similar, indicating that this component acts at an initiation step. Various components, including excess ribosomal salt wash and postribosomal supernatant of cell lysate, were added to the fractionated system in an effort to identify the slow step more precisely. Of these, only excess ribosomal salt wash specifically stimulated polio RNA translation, suggesting that one or more initiation factors is necessary in unusually large amounts for this mRNA. Various purified initiation factors were tested for the ability to enhance polio RNA translation. Of these, only purified eukaryotic initiation factor 4A had a specific effect. This suggests that polio RNA, in contrast to other mRNAs tested (EMC, reoviral, and globin), may have an unusually low affinity for this initiation factor. The significance of these results is discussed in terms of the methods picornaviruses have evolved for reprogramming the translational machinery of the host cell.  (+info)

Stimulation of de novo methylation following limited proteolysis of mouse ascites DNA methylase. (80/97)

The ability of mouse Krebs II ascites cell DNA methylase to add methyl groups to native, unmethylated DNA (de novo activity) is stimulated by limited proteolysis. The affinity of the enzyme for DNA is not altered by this treatment but the rate of reaction is increased so that 40% or more of methylatable sites are methylated within 4.5 h. The activation is associated with a decrease in size of the enzyme to 6.2 S.  (+info)