Further purification, characterization and salt activation of acyl-CoA synthetase from Escherichia coli.
Acyl-CoA synthetase was further purified from Escherichia coli in good yield and fold purification by affinity chromatography on CoA-Sepharose 4B. The molecular weight of the active form of the purified enzyme was estimated as 45 000 by Sephadex G-100 and 47 000 by Sephadex G-200. Sedimentation equilibrium ultracentrifugation analysis revealed a molecular weight of 50 000. The sedimentation coefficient was calculated as 4.4 S. An absorption maximum at 276 nm was observed in the ultraviolet light absorption spectrum. The molar extinction coefficient was 9.2 X 10(4). Kinetic constants were determined for trans fatty acids. All ions tested, including chaotropic and lyotropic ions, stimulated or inhibited acyl-CoA synthetase activity depending on their concentrations in the assay system. In a series of chaotropes, the lower concentration required to maximally activate acyl-CoA synthetase in increasing order of potency of chaotropic ions. The inhibitory effect of chaotrope on the enzyme activity was reversible. These data suggest that salts have a common mode of action and influence acyl-CoA synthetase activity primarily through their effect on the solution structure.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D003066:Coenzyme A Ligases / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D009994:Osmolar Concentration; D010743:Phospholipids / Q000494:pharmacology; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012492:Salts / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014461:Ultracentrifugation; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D000596:Amino Acids; D010743:Phospholipids; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012492:Salts; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase