PubMed Health⌕ Search

Biomedical subjects

D H Levin

Publications and source records attributed to D H Levin.

At least 37 records · Page 2Linked to original sources
PubMed1976

Regulation of protein synthesis in rabbit reticulocyte lysates: characteristics of inhibition of protein synthesis by a translational inhibitor from heme-deficient lysates and its relationship to the initiation factor which binds Met-tRNAf.

In heme-deficient reticulocyte lysates a translational inhibitor which regulates protein synthesis is formed or activated. To define the mechanism of action of the translational inhibitor (RI), RI was partially purified. We have utilized the isolated RI to examine its relationship to the translational inhibitor formed in situ in heme-deficiency, some quantitative aspects of inhibition of protein synthesis, and the relationship of RI concentration to the initiation factor (IF-MP) which forms a ternary complex with Met-tRNAf and GTP (IF-MP-Met-tRNAf-GTP). The results demonstrate that the activity of isolated RI is related to the in situ heme-deficiency inhibitor by several criteria: (a) the biphasic kinetics of inhibition manifested by RI in lysates containing optimal levels of hemin are very similar to those observed in heme-deficiency, i.e., an initial period in which several rounds of protein synthesis proceed at the control rate followed by an abrupt decline in the rate of protein synthesis. (b) Both inhibitions are accompanied by the disaggreagation of polyribosomes with a concomitant increase in 80S ribosomes. (c) Both inhibitions are reversed by IF-MP. The isolated RI blocked protein synthesis in lysates at temperatures ranging from 15 degrees to 30 degrees. Although the rate of protein synthesis was a function of the temperature of incubation, the number of rounds of protein synthesis prior to shut-off was essentially the same at various temperatures. When RI was added to lysates, at increasing intervals after the start of incubation, the period of synthesis before shut-off (lag) progressively decreased. The inhibition of protein synthesis by RI was immediately reversed by the addition of IF-MP. The extent of reversal increased with increasing concentrations of IF-MP; at low levels of RI almost complete reversal of inhibition by IF-MP was obtained. However, at high levels of RI which did not appreciably increase the degree of inhibition of protein synthesis, equivalent amounts of IF-MP were less effective in reversing inhibition. These results suggest that the inhibition of protein synthesis by the isolated inhibitor involves the initiation factor IF-MP.

Cell-Free System↗
PubMed1975

Association of a cyclic AMP-dependent protein kinase with a purified translational inhibitor isolated from hemin-deficient rabbit reticulocyte lysates.

In the absence of added hemin, protein synthesis in rabbit reticulocyte lysates proceeds at maximal linear rates for several minutes and then ceases abruptly. Inhibition involves the action of a translational inhibitor whose formation is regulated by hemin. Addition of the isolated inhibitor to hemin-supplemented lysates produces an inhibition of protein chain initiation similar to that observed in heme-deficiency. The inhibitor has been purified over 300-fold and contains a protein kinase activity that copurifies with the inhibitory function. With calf thymus histone II as the phosphate receptor, the inhibitor-associated protein kinase requires ATP as the phosphorylating agent. Cycle AMP stimulates kinase activity 5- to 8-fold; the concentration of cycle AMP required for halfmaximal activity is 4 X 10-8 M. Preincubation of the inhibitor in the presence of cyclic AMP significantly reduces cyclic AMP-dependent phosphorylation and inhibitory activity. The corresponding protein kinase activity from hemin-supplemented lysates displays reduced cyclic AMP-dependency and little or no inhibitory activity. These findings suggest that the protein kinase activity associated with the purified translational inhibitor is involved in the mechanism of inhibition of initiation observed in hemedeficient reticulocyte lysates.

Animals↗
PubMed1973

Protein initiation in eukaryotes: formation and function of a ternary complex composed of a partially purified ribosomal factor, methionyl transfer RNA, and guanosine triphosphate.

A protein factor contained in a 1 M KCl extract of L-cell ribosomes and partially purified by chromatography on DEAE-cellulose forms a specific ternary complex with rat-liver Met-tRNA(f) and GTP. The complex is measured by its quantitative retention on nitrocellulose membranes. Complex assembly is optimal at 100 mM KCl and 0.2 mM MgCl(2), and is independent of mRNA and of ribosomes. The GTP requirement can be replaced over 65% by its methylene analogue GDPCH(2)P, indicating that GTP hydrolysis is not involved. Complex formation is inhibited by 10 muM aurintricarboxylic acid, but is unaffected by 100 muM pactamycin, 100 muM fusidic acid, or by excess uncharged methionine tRNA(f). The ternary complex is relatively stable and appears at the void volume during filtration on Sephadex G-100. At 1-3 mM MgCl(2) and in the presence of other factors, the ternary complex is implicated in protein initiation by (i) its capacity to bind to the 40S ribosomal subunit to form a 48S complex; and (ii) the subsequent association of the 48S complex with a 60S subunit to form a functional "80S complex."

Adenosine Triphosphate↗
PubMed1972

Formation of a mammalian initiation complex with reovirus messenger RNA, methionyl-tRNA F , and ribosomal subunits.

Previous data demonstrated that reovirus mRNA, synthesized in vitro with the particulate RNA transcriptase of reovirus cores, efficiently directs the synthesis of polypeptides in vitro. The present studies indicate that all of the three size classes of reovirus mRNA produced in vitro can form protein initiation complexes with rat liver [(36)S]Met-tRNA(F) and incubated 40S and 60S ribosomal subunits, which had been washed in 0.5 M KCl of mouse fibroblast L-929 cells. Mild prior treatment of the mRNA with HCHO was required to expose the initiator region. The initiation complex reacted quantitatively with puromycin to form a puromycin peptide, whose electrophoretic properties were identical to methionyl-puromycin formed in response to poly(A,G,U) or the initiator codon AUG. The complex was relatively stable and specific for [(35)S]Met-tRNA(F); rat liver [(35)S]Met-tRNA(M) was unreactive unless the supernatant factors EF T(1) and EF T(2) were also present. However, the addition of fusidic acid, at a concentration that did not affect complex formation with [(35)S]Met-tRNA(F), completely inhibited Met-tRNA(M) utilization. Exogenous ribosomal factors and GTP were not required unless the separated 40S and 60S subunits were further treated with 1 M KCl. The data suggest that reovirus mRNA contains AUG initiator codons that form a complex with Met-tRNA(F) at a puromycin-reactive site on ribosomes.

Animals↗
PubMed1971

Asynchronous synthesis of the complementary strands of the reovirus genome.

The mechanism of replication of the double-stranded RNA genome of reovirus has been analyzed by tracing the fate of the parental double-stranded RNA genome and by determining whether the complementary strands, which comprise the progeny double-stranded RNA, are synthesized simultaneously or sequentially. The results indicate that the parental double-stranded RNA is conserved as the original duplex molecule within a subviral particle throughout the viral replicative cycle. The complementary strands, which form the progeny double-stranded RNA, are produced asynchronously. Minus strands are synthesized on preformed plus-strand templates, whereas plus strands appear to be synthesized on double-stranded RNA templates.

Centrifugation↗
PubMed1971

Mechanism of reovirus double-stranded ribonucleic acid synthesis in vivo and in vitro.

The complementary strands of reovirus double-stranded ribonucleic acid (ds RNA) are synthesized sequentially in vivo and in vitro. In both cases, preformed plus strands serve as templates for the synthesis of the complementary minus strands. The in vitro synthesis of dsRNA is catalyzed by a large particulate fraction from reovirus-infected cells. Treatment of this fraction with chymotrypsin or with detergents which solubilize cellular membranes does not alter its capacity to synthesize dsRNA. The enzyme or enzymes responsible for dsRNA synthesis remain sedimentable at 10,000 x g after these enzyme or detergent treatments, indicating their particulate nature. Pretreatment of this fraction with ribonuclease, however, abolishes its ability to catalyze dsRNA synthesis, emphasizing the single-stranded nature of the template and its location in a structure permeable to ribonuclease. In contrast, the newly formed dsRNA is resistant to ribonuclease digestion at low salt concentrations and hence is thought to reside within a ribonuclease-impermeable structure.

Animals↗
PubMed1970

Properties of RNA transcriptase in reovirus subviral particles.

Subviral particles containing reovirus RNA transcriptase have been isolated from extracts of virus-infected mouse fibroblast cells. The purified particles which lacked the outer protein capsomeres of the mature virion had a buoyant density of 1.43-1.44 g/ml in CsCl and contained all of the double-stranded RNA genome of the intact virus. The particles were free of nuclease activity. RNA synthesis required all four ribonucleoside triphosphates and was dependent on magnesium or manganese; optimal activity required potassium or ammonium ions. In the presence of a ribonucleoside triphosphate regenerating system, reaction rates were linear for 20 hr. RNA yields of 40-fold in excess of input template could be obtained. Completed RNA chains were released from the subviral particles. In the course of RNA synthesis, the double-stranded RNA template was fully conserved. The RNA products formed in vitro displayed profiles in sucrose gradients similar to those found for in vitro reovirus mRNA. The RNA products were single-stranded and did not self-anneal. Over 90 percent of the transcriptase products could be annealed with template double-stranded RNA. The annealed products migrated in acrylamide gels as double-stranded RNA, indicating efficient in vitro transcription.

Centrifugation, Density Gradient↗
PubMed1970

The reovirus replicative cycle: conservation of parental RNA and protein.

The fate of parental reovirions in the viral replicative cycle has been analyzed using CsCl density centrifugation. After penetration of L-cells, reovirus is converted from a particle of density 1.39 g/cm(3) to a subviral particle of density 1.41 g/cm(3). This alteration in density is temporally correlated with the hydrolysis of viral coat proteins and is qualitatively similar when particles are labeled in their RNA or protein. Ten hours after infection, when synthesis of progeny virus is underway, the parental RNA and protein are again found at density 1.39 g/cm(3). These data demonstrate conservation of the parental RNA and protein in the subviral particle throughout the replicative cycle.

Centrifugation, Density Gradient↗