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J Reinbolt

Publications and source records attributed to J Reinbolt.

At least 55 records · Page 3Linked to original sources
PubMed1985

The complete amino acid sequence of cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae.

The crystallizable cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is a dimer made up of identical subunits (Mr 63 000). Its primary structure was established using peptide sequences from four different digests of the native and citraconylated enzyme with trypsin, cyanogen bromide and staphylococcal protease. The oligonucleotide sequence of the structural gene was used as a template for the final alignment of the various peptides in the correct order.

Amino Acid Sequence↗
PubMed1983

Purification and reversible subunit dissociation of overproduced Escherichia coli phenylalanyl-tRNA synthetase.

Phenylalanyl-tRNA synthetase (EC 6.1.1.20) has been purified to homogeneity from a 100-fold overproducing Escherichia coli strain carrying a hybrid pBR322 plasmid containing the pheS-pheT locus. The purified enzyme is identical to the phenylalanyl-tRNA synthetase isolated form an haploid strain. The enzyme was found to dissociate in the presence of 0.5 M NaSCN and the alpha- and beta-subunits composing the native alpha 2 beta 2 enzyme were separated by gel filtration. Neither isolated subunit showed significant catalytic activity. A complex indistinguishable from the native enzyme with full catalytic activity is recovered upon mixing the subunits. The N- and C-terminal sequences and the amino acid composition of each subunit were determined. They are compared to the available data concerning the primary structure of the subunits, as deduced from nucleotide sequencing of the pheS-pheT operon.

Amino Acid Sequence↗
PubMed1983

Localization of two antigenic determinants in histone H4.

Four overlapping synthetic peptides corresponding to the carboxy-terminal region 80-102 of histone H4 were prepared by solid-phase peptide synthesis. Their antigenic activity was analysed by inhibition of the H4-anti-H4 reaction in complement fixation and enzyme-linked immunosorbent assay. One antigenic determinant was localized in residues 88-96 of the H4 molecule. No antigenic activity was found in peptides 80-89 and 97-102. Antibodies induced by peptide 85-102 were found to bind to free H4 in solution but not to chromatin subunits, suggesting a lack of accessibility of the C-terminal region of H4 in nucleosomes. A second epitope was found to be situated in the N-terminal region 1-53 of histone H4.

Amino Acid Sequence↗
PubMed1983

Reversed-phase liquid chromatography of peptides for direct micro-sequencing.

Tryptic and cyanogen bromide peptides derived from yeast aspartyl-tRNA synthetase and from Escherichia coli ribosomal proteins were separated by reversed-phase liquid chromatography, employing volatile buffers of low ionic strength. The conditions used allow the performance of micro-sequencing without desalting or extensive lyophilization, and can therefore be applied to peptide mixtures containing hydrophobic fragments which tend to precipitate. To prevent losses of peptides, direct ultra-violet detection of the peptides was preferred, to detection by post-column derivatization with an additional stream splitting device. Preparative separations were performed with 5-10 nmol of peptide mixture; analytical runs were made with 5-10 micrograms of protein hydrolysate.

Amino Acid Sequence↗
PubMed1983

Immunochemical studies of tobacco mosaic virus--V. Localization of four epitopes in the protein subunit by inhibition tests with synthetic peptides and cleavage peptides from three strains.

Two antigenic determinants have been located in the vicinity of residues 1-10 and 34-39 of the coat protein of tobacco mosaic virus by means of ELISA and complement fixation inhibition experiments with synthetic peptides. The determinant present in residues 1-10 is also expressed at the outer surface of the subunits when they are assembled into virions. Two other determinants expressed in the dissociated protein subunit have been located in the vicinity of residues 55-61 and 80-90 by means of inhibition tests with tryptic peptides. These results bring to seven the number of antigenic determinants of the protein subunit that have been located.

Amino Acid Sequence↗
PubMed1980

30-S ribosomal subunit proteins of an Escherichia coli mutant in which assembly of the small ribosomal subunit is temperature-sensitive.

Escherichia coli 219ts2, a temperature-sensitive streptomycin-independent revertant of the streptomycin-dependent strain E. coli 209 is defective in 30-S ribosomal subunit assembly at 42 degrees C. Total 30-S ribosomal subunit proteins of this strain contain two additional components one of which (alpha) migrates below and the other (beta) to the right of protein S7 in two-dimensional polyacrylamide gel analyses carried out according to Kaltschmidt and Wittmann. These two components are also resolved from normal 30-S subunit proteins by chromatography on phosphocellulose. Tryptic fingerprinting of proteins alpha and beta identifies alpha as protein S7B, the form of S7 found in B strains of E. coli and beta as a mutant form of protein S4 produced by deletion of about 20 amino acids from the COOH terminus of the wild-type protein.

Amino Acids↗
PubMed1980

Identification of binding sites of turnip yellow mosaic virus protein and RNA by crosslinks induced in situ.

Turnip yellow mosaic virus RNA and protein could be crosslinked in situ by ultraviolet irradiation at pH 4.8 but not at pH 7.3, and by bisulphite treatment at pH 7.3. Crosslinked peptides could be located in the primary structure of the viral coat protein. Three regions were bound covalently by ultraviolet irradiation, and two of these three regions were bound also by bisulphite treatment. The yield of the crosslinking reaction could be high, indicating that almost all protein subunits of each virion reacted with the viral RNA. The crosslinked peptides contain most of the acidic and basic amino acids of the protein, often associated into pairs of opposite charge. Implications for the folding of the RNA into the virion and for models of RNA--protein interactions are discussed.

Cross-Linking Reagents↗
PubMed1979

The primary structure of ribosomal protein S7 from E. coli strains K and B.

Ribosomal proteins S7 from 30S subunits of Escherichia coli strains K and B differ extensively in their aminoacid compositions. The experimental details which led to the determination of the complete primary structures of proteins S7K and S7B are presented. Protein S7K consists of a single polypeptide chain of 177 aminoacids giving a calculated molecular weight of 19, 732, whereas protein S7B has 153 residues which amount to a molecular weight of 17,131. Aminoacid sequences were determined by a combination of automated Edman degradation of the intact proteins in a modified Beckman sequenator and sequencing of peptides obtained by digestion with trypsin. Staphylococcus aureus protease, thermolysin and pepsin, either by solid-phase Edman degradation or by dansyl-Edman degradation. Additional information about the primary structure was derived from peptides resulting from chemical cleavages of the protein by 2-(2-nitrophenyl-sulphenyl)-3-methyl 3' bromoindolenine at its tryptophanyl bonds and by cyanogen bromide at its methionyl bonds leading to large fragments. The mutational event occurring between S7B and S7K was characterized. Protein S7K contains an additional sequence of 24 aminoacids at its C-terminal end. The aminoacid sequence of both proteins S7K and S7B was compared to the published sequences of the other ribosomal proteins of Escherichia coli and predictions for the secondary structure of these proteins were made.

Amino Acid Sequence↗
PubMed1975

Determination of the complete amino-acid sequence of protein S4 from Escherichia coli ribosomes.

After digestion of protein S4 with trypsin, all 32 tryptic peptides were isolated. Their amino acid compositions were analyzed and the sequence of the amino acids within the tryptic peptides was determined by means of a solid-phase peptide sequenator and by exopeptidases. Alignment of the tryptic peptides was established by analyzing and partially sequencing peptides isolated after digestion of the S4 protein with chymotrypsin, thermolysin and a glutamic-acid-specific protease. Further information about the alignment of peptides came from treatment of S4 with CNBr and with a lysine-modifying reagent.

Amino Acid Sequence↗