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Biomedical subjects

W Rychlik

Publications and source records attributed to W Rychlik.

27 records · Page 2Linked to original sources
PubMed1986

Structural analysis of the messenger RNA cap-binding protein. Presence of phosphate, sulfhydryl, and disulfide groups.

The messenger RNA cap-binding protein (CBP) was isolated from human erythrocyte, rabbit erythrocyte, and rabbit reticulocyte lysate by affinity chromatography on 7-methylguanosine 5'-triphosphate-Sepharose. The specific activity of binding to capped oligonucleotides was similar for the human erythrocyte and rabbit reticulocyte CBPs. Isoelectric focusing of human and rabbit preparations revealed that each was composed of up to five species. The pI values of human and rabbit CBPs ranged from 5.7 to 6.5. The predominant form in erythrocytes had a pI of 6.3 while in reticulocytes, two major species, having pI values of 5.9 and 6.3, were present. Labeling of rabbit reticulocytes with [32P]orthophosphate revealed that the pI 5.9 but not the pI 6.3 form contained phosphate. All of the phosphate was found in phosphoserine residues. The amino acid compositions of human erythrocyte and rabbit reticulocyte CBPs were quite similar. Both proteins had 7 tryptophanyl and 6 cysteinyl residues. Labeling with [1-14C]iodoacetic acid under native and denaturing conditions provided evidence that 2 of the cysteinyl residues are present in the reduced form and 4 in disulfide bridges. Species of CBP with faster or slower electrophoretic mobilities could be generated by treatment of the protein either with O2 in the presence of a catalyst or with dithiothreitol. The predominant form of the untreated protein migrated between these two forms.

Animals↗
PubMed1983

Localization of the elongation factor Tu binding site on Escherichia coli ribosomes.

Fluorescent techniques were used to study binding of peptide elongation factor Tu (EF-Tu) to Escherichia coli ribosomes and to determine the distances of the bound factor to points on the ribosome. Thermus thermophilus EF-Tu was labeled with 3-(4-maleimidylphenyl)-4-methyl-7-(diethyl-amino)coumarin (CPM) without loss of activity. In the presence of Phe-tRNA and a nonhydrolyzable analogue of GTP, 70S ribosomes bind the CPM-EF-Tu [Kb = (3 +/- 1.2) X 10(6) M-1] causing a decrease of CPM fluorescence. Binding of CPM-EF-Tu to 50S subunits was at least 1 order of magnitude lower than with 70S ribosomes, and binding to 30S subunits could not be detected. Reconstituted 70S ribosomes containing either S1 labeled with fluoresceinmaleimide or ribosomal RNAs labeled at their 3' ends with fluorescein thiosemicarbazide were used for energy transfer from CPM-EF-Tu. The distances between CPM-EF-Tu bound to the ribosomes and the 3' ends of 16S RNA, 5S RNA, 23S RNA, and the closest sulfhydryl group of S1 were calculated to be 82, 70, 73, and 62-68 A, respectively.

Binding Sites↗
PubMed1980

Wheat germ protein kinase affects the translation of Brome Mosaic virus ribonucleic acid in vitro.

Wheat germ protein kinase inhibits in vitro translation of Brome Mosaic virus (BMV) RNA 1 and 2, without affecting the translation of RNA 4. Inhibition of formation of BMV polypeptides 1a and 2a is due to the arrest of initiation of polypeptide synthesis. It was found that protein kinase inhibits the formation of the 80S initiation complex with BMV RNA 1 and 2, without affecting the formation of the initiation complex with BMV RNA 4. Inhibition of protein synthesis by wheat germ protein kinase is accompanied by the phosphorylation of two ribosome-associated polypeptides, with molecular weights of 32 000 and 76 000, respectively. Both polypeptides are readily dephosphorylated by the enzyme(s) present in the cell-free extract. Their dephosphorylation is accompanied by restoration of the translational capacity of the system.

Mosaic Viruses↗
PubMed1980

Purification and characterisation of adenosine-3',5'-phosphate-independent protein kinase from wheat germ.

cAMP-independent protein kinase was isolated from the wheat germ and purified to electrophoretic homogeneity. The molecular weight of enzyme was approximately 20,000, Km for ATP was (1 +/- 0.2) x 10(-5) M. V was 215 nmol phosphate mg enzyme-1 min-1, and the isoelectric point was at pH 9.2. The enzyme promotes phosphorylation of casein and crude wheat germ ribosomes.

Chromatography↗
PubMed1979

Isolation of wheat ribosomes free of high molecular weight inhibitors of the natural messenger translation.

The cell-free extract from wheat germ contains an inhibitor interfering with translation of a natural template (BMV RNA). The inhibitor affects neither the translation of poly(U) nor the aminoacylation of tRNA. It exhibits the activity of protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). The inhibitor is found in lipoprotein aggregates which can be separated from ribosomes on Sepharose 2B column. Ribosomes purified on the Sepharose are several times more active in translation of BMV RNA than those isolated by conventional methods.

Chromatography, Gel↗
PubMed1978

Protein synthesizing system from wheat germ: efficient translation of synthetic and natural messages.

Optimum conditions for translation of eukaryotic, prokaryotic and synthetic templates in wheat germ cell-free extract were determined. 1. Translation of eukaryotic message (BMV RNA and TMV RNA) was at optimum at the same concentrations of K+, Mg(2+), HEPES and spermine. In optimal conditions the efficiency of translation was high, for BMV RNA being equal to 220 pmoles and for TMV RNA, to 280 pmoles of leucine incorporated per 1 microgram of template. Prokaryotic template (Qbeta RNA) was translated under different ionic conditions. 2. Translation of synthetic template [poly(U)] was at optimum at fairly higher concentrations of K+ and Mg(2+) than those optimal for natural template translation. 3. Efficient translation of natural and synthetic templates depends on complete removal of inhibitors found in the wheat germ cell-free extract. Action of these inhibitors could be mimicked by adenine nucleotides.

Adenine Nucleotides↗