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Title: Complete amino acid sequence of branched-chain amino acid aminotransferase (transaminase B) of Salmonella typhimurium, identification of the coenzyme-binding site and sequence comparison analysis

Abstract

The complete amino acid sequence of the subunit of branched-chain amino acid aminotransferase of Salmonella typhimurium was determined by automated Edman degradation of peptide fragments generated by chemical and enzymatic digestion of S-carboxymethylated and S-pyridylethylated transaminase B. Peptide fragments of transaminase B were generated by treatment of the enzyme with trypsin, Staphylococcus aureus V8 protease, endoproteinase Lys-C, and cyanogen bromide. Protocols were developed for separation of the peptide fragments by reverse-phase high performance liquid chromatography (HPLC), ion-exchange HPLC, and SDS-urea gel electrophoresis. The enzyme subunit contains 308 amino acid residues and has a molecular weight of 33,920 daltons. The coenzyme-binding site was determined by treatment of the enzyme, containing bound pyridoxal 5-phosphate, with tritiated sodium borohydride prior to trypsin digestion. Monitoring radioactivity incorporation and peptide map comparisons with an apoenzyme tryptic digest, allowed identification of the pyridoxylated-peptide which was isolated by reverse-phase HPLC and sequenced. The coenzyme-binding site is a lysyl residue at position 159. Some peptides were further characterized by fast atom bombardment mass spectrometry.

Authors:
Publication Date:
Research Org.:
North Carolina State Univ., Raleigh, NC (USA)
OSTI Identifier:
5607968
Resource Type:
Thesis/Dissertation
Resource Relation:
Other Information: Thesis (Ph. D.)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; AMINOTRANSFERASES; AMINO ACID SEQUENCE; COENZYMES; RECEPTORS; CHEMICAL COMPOSITION; ELECTROPHORESIS; LIQUID COLUMN CHROMATOGRAPHY; MASS SPECTROSCOPY; MOLECULAR WEIGHT; PEPTIDES; PLASMIDS; SALMONELLA TYPHIMURIUM; TRACER TECHNIQUES; TRITIUM COMPOUNDS; BACTERIA; CELL CONSTITUENTS; CHROMATOGRAPHY; ENZYMES; HYDROGEN COMPOUNDS; ISOTOPE APPLICATIONS; MEMBRANE PROTEINS; MICROORGANISMS; MOLECULAR STRUCTURE; NITROGEN TRANSFERASES; ORGANIC COMPOUNDS; PROTEINS; SALMONELLA; SEPARATION PROCESSES; SPECTROSCOPY; TRANSFERASES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Feild, M.J. Complete amino acid sequence of branched-chain amino acid aminotransferase (transaminase B) of Salmonella typhimurium, identification of the coenzyme-binding site and sequence comparison analysis. United States: N. p., 1988. Web.
Feild, M.J. Complete amino acid sequence of branched-chain amino acid aminotransferase (transaminase B) of Salmonella typhimurium, identification of the coenzyme-binding site and sequence comparison analysis. United States.
Feild, M.J. Fri . "Complete amino acid sequence of branched-chain amino acid aminotransferase (transaminase B) of Salmonella typhimurium, identification of the coenzyme-binding site and sequence comparison analysis". United States. doi:.
@article{osti_5607968,
title = {Complete amino acid sequence of branched-chain amino acid aminotransferase (transaminase B) of Salmonella typhimurium, identification of the coenzyme-binding site and sequence comparison analysis},
author = {Feild, M.J.},
abstractNote = {The complete amino acid sequence of the subunit of branched-chain amino acid aminotransferase of Salmonella typhimurium was determined by automated Edman degradation of peptide fragments generated by chemical and enzymatic digestion of S-carboxymethylated and S-pyridylethylated transaminase B. Peptide fragments of transaminase B were generated by treatment of the enzyme with trypsin, Staphylococcus aureus V8 protease, endoproteinase Lys-C, and cyanogen bromide. Protocols were developed for separation of the peptide fragments by reverse-phase high performance liquid chromatography (HPLC), ion-exchange HPLC, and SDS-urea gel electrophoresis. The enzyme subunit contains 308 amino acid residues and has a molecular weight of 33,920 daltons. The coenzyme-binding site was determined by treatment of the enzyme, containing bound pyridoxal 5-phosphate, with tritiated sodium borohydride prior to trypsin digestion. Monitoring radioactivity incorporation and peptide map comparisons with an apoenzyme tryptic digest, allowed identification of the pyridoxylated-peptide which was isolated by reverse-phase HPLC and sequenced. The coenzyme-binding site is a lysyl residue at position 159. Some peptides were further characterized by fast atom bombardment mass spectrometry.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Fri Jan 01 00:00:00 EST 1988},
month = {Fri Jan 01 00:00:00 EST 1988}
}

Thesis/Dissertation:
Other availability
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