Purification of carnosine synthetase from avian muscle by affinity chromatography and determination of its subunit structure.
An extract of chick pectoral muscle was prepared in which the level of carnosine synthetase (L-histidine; beta-alanine ligase (AMP-forming), EC 6.3.2.11) activity was approx. 10-times that of previous preparations. In affinity chromatography studies, this material was applied to a Cibracon blue-agarose column, and elution of carnosine synthetase by carnosine was attempted. Results indicated that the elution was not specific as the eluate contained large amounts of myosin. An (NH4)2SO4 fraction (21--30% satn.) of the crude extract was prepared which, in comparison to the crude extract, had a higher specific activity, was more stable on storage at 4 degrees C and had much lower myosin content. On affinity chromatography of this fraction, apparently homogeneous carnosine synthetase was eluted with carnosine, and the specific activity of the preparation was 1700-times that of the fresh crude extract. Amino acid analysis of the preparation indicated that it had a very high histidine content (141 per 1000 residues). On analysis of the preparation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), a polypeptide of Mr 119 000 was observed, whereas gel permeation chromatography of the native enzyme indicated an Mr of 250 000, suggesting that the native enzyme is a dimer.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002645:Chickens; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010453:Peptide Synthases / Q000302:isolation & purification*
D000596:Amino Acids; D046911:Macromolecular Substances; D010453:Peptide Synthases; C019844:carnosine synthetase