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Biomedical subjects

B Hardesty

Publications and source records attributed to B Hardesty.

At least 109 records · Page 6Linked to original sources
PubMed1978

GTP hydrolysis during methionyl-tRNAf binding to 40 S ribosomal subunits and the site of edeine inhibition.

Three lines of evidence are presented indicating that GTP hydrolysis associated with eukaryotic peptide initiation occurs in the absence of 60 S subunits when methionyl-tRNAf is bound to 40 S ribosomal subunits. An enzyme fraction required for binding of methionyl-tRNAf to 40 S subunits and peptide initiation, tentatively equated with eIF-(4 + 5), has GTPase activity and appears to be responsible for hydrolysis of GTP in the methionyl-tRNAf.eIF-2.GTP complex. Direct analysis of the methionyl-tRNAf.40 S complex formed with with eIF-2 and [8-3H] guanine, [gamma-32P]GTP reveals bound guanine but not gamma-phosphate. Edeine, a peptide antibiotic containing spermidine and beta-tyrosine residues at its COOH terminus and NH2 terminus, respectively, blocks peptide initiation and interferes with binding of methionyl-tRNAf to 40 S ribosomal subunits. Inhibition of binding is observed when the eIF-2-mediated binding reaction is carried out with GTP but not with guanosine 5'-(beta,gamma-methylene)triphosphate or guanosine 5'-(beta,gamma-imido)triphosphate. Edeine was labeled by iodination and shown to bind with high affinity to 40 S but not to 60 S ribosomal subunits. It is suggested that edeine blocks a specific site on the 40 S ribosomal subunit to which a segment of the methionyl-tRNAf molecule is bound during the course of the initiation reaction sequence.

Animals↗
PubMed1977

Partial purification and characterization of a translational inhibitor from Friend leukemia cells.

A translational inhibitor has been isolated from unstimulated Friend leukemia cells. The inhibitor is a heat-labile, sulfhydryl reagent-insensitive protein with a molecular weight of approximately 214,000. It inhibits protein synthesis at a step of peptide chain initiation by preventing initiation factor-dependent binding of methionyl-tRNAf to 40 S ribosomal subunits. However, it does not interfere with the formation of the ribosome-independent ternary complex between the initiation factor IF-E2, methionyl-tRNAf, and GTP. The inhibitor preparation contains protein kinase activity which phosphorylates the smallest subunit of IF-E2. There appears to be a functional similarity between this inhibitor from Friend leukemia cells and the hemin-controlled repressor from reticulocytes.

Animals↗
PubMed1977

Partial reaction of peptide initiation inhibited by phosphorylation of either initiation factor eIF-2 or 40S ribosomal proteins.

Preparations of the hemin-controlled repressor (HCR) from rabbit reticulocytes contain 3':5'-cyclic-AMP-independent protein kinase activity for the smallest subunit of the peptide initiation factor eIF-2 and for proteins of reticulocyte 40S ribosomal subunits. Binding of the ternary complex formed between Met-tRNAf, GTP, and eIF-2 to 40S ribosomal subunits is shown to be inhibited by phosphorylation of either the ribosomal subunits or eIF-2. The protein kinase activity responsible for phosphorylation of eIF-2 has been separated from the activity for phosphorylation of 40S ribosomal subunits and shown to independently block the same partial reaction of peptide initiation. It appears that different enzymes are involved, each capable of regulating peptide initiation at the same step but by a different mechanism.

Animals↗
PubMed1977

Subcellular distribution of aminoacyl-tRNA synthetases in various eukaryotic cells.

The total amount, size distribution and binding of aminoacyl-tRNA synthetases to ribosomes in a variety of mammalian and avian cells was studied under standard conditions of sample preparation and assay. Aminoacyl-tRNA synthetases appear to exist in three general forms; 'free' enzyme of about 4-9 S, one or more 'enzyme complexes' of about 18-25 S, and in association with ribosomes. The aminoacyl-tRNA synthetase activity for many individual amino acids was surprisingly similar in cell types chosen to be diverse with respect to differentiation state, transformation, and growth rate. Total activity for all amino acids varied about 4-fold, based on a constant volume of cells. Embryonic tissues had a comparatively high proportion of total synthetase activity associated with ribosomes, whereas this value was relatively low for mouse liver. Distinctive distribution patterns with common and variable features were observed for individual enzymes. The only aminoacyl-tRNA synthetases found not to be associated in significant amounts with either 18-25 S enzyme complexes or ribosomes in any of the cell types examined were the enzymes for alanine, histidine, and serine. All cell types evidenced 18-25-S synthetase activity for arginine, aspartic acid, glutamine, glutamic acid, isoleucine, leucine, lysine, methionine, proline, and valine, although in quite variable porportions of the total activity observed for these amino acids. For example, of the valyl-tRNA synthetase activity not associated with ribosomes, 35% and 100% were found to sediment at 18-25 S in Friend leukemia cells and mouse liver respectively. All cells had two easily distinguishable peaks of arginyl tRNA synthetase activity at 4-9S and 18-25S respectively; however, the relative proportion of enzyme activity in the peaks differed between cell types. Phenylalanyl-tRNA synthetase was not observed to occur in an 18-25-S complex in any of the cell types examined but was bound to ribosomes in variable but generally relatively high proportions. Numerous other specific differences are described. No underlying physiological or biochemical principle has been recognized to account for the specific distribution patterns observed. However, they may reflect variations in cellular architecture that may be related to regulation of protein synthesis.

Amino Acyl-tRNA Synthetases↗
PubMed1976

Specificity of the protein kinase activity associated with the hemin-controlled repressor of rabbit reticulocyte.

Highly purified preparations of hemin-controlled repressor of rabbit reticulocyte contain a 3':5'-cyclic AMP-indenpendent protein kinase activity that phosphorylates the low-molecular-weight (about 38,000) polypeptide chain of the initiation factor that forms a ternary complex with GTP and Met-tRNAf. These preparations also phosphorylate several polypeptide components of reticulocyte 40S ribosomal subunits. However, no significant levels of phosphorylation are observed when casein, histones, Artemia salina 40S ribosomal subunits, or other initiation factor fractions are used as substrates although high levels of phosphorylation are obtained with cruder preparations of the repressor. An antibody to these highly purified preparations of repressor has been obtained from the serum of immunized goats. Preincubation with immune goat IgG results in the neutralization of the inhibitory activity of the repressor, while normal IgG has no effect. Preincubation with immune IgG also abolishes the protein kinase activity responsible for the phosphorylation of the initiation factor and reticulocyte 40S subunits. Histone phosphorylation by crude repressor preparations, on the other hand, is unaffected by preincubation with immune IgG.

Animals↗
PubMed1975

Isolation and partial characterization of a 40 S ribosomal subunit-transfer ribonucleic acid binding factor from rabbit reticulocytes.

A factor that catalytically promotes the codon-directed, GTP-independent binding of tRNA to 40 S ribosomal subunits has been isolated from the postribosomal supernatant and the ribosomal wash of rabbit reticulocytes. The factor is a heat labile, sulfhydryl reagent-sensitive protein of a molecular weight of approximately 50,000. It consists of two non-identical subunits of Mr equals approximately 30,000 and 20,000. Its basic character has been confirmed by the high ratio of basic amino acids to nonamidic aspartic and glutamic acid present in the purified protein. Formation of a factor promoted 40 S-poly(U)-phenylalanyl-tRNA initiation complex causes a shift in the Mg-2+ concentration optimum for polyphenylalanine synthesis from 8 mM to 4mM.

Amino Acids↗
PubMed1975

A simple method for the purification of reticulocyte globin messenger ribonucleic acid.

A relatively simple and inexpensive method has been developed for the preparation of highly purified rabbit reticulocyte globin mRNA. After phenol extraction, polysomal RNA was chromatographed on Sigmacell type 38 cellulose and Sepharose 4B. The resulting mRNA preparation has a purity in excess of 90%. No selective loss of either alpha or beta globin mRNA is observed.

Animals↗