Characterization of alpha alpha, beta beta, gamma gamma and alpha gamma human enolase isozymes, and preparation of hybrid enolases (alpha gamma, beta gamma and alpha beta) from homodimeric forms

Abstract

Four forms (alpha alpha, beta beta, gamma gamma and alpha gamma) of enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) were purified from human brain and skeletal muscle. Each purified preparation showed a single band on SDS-polyacrylamide gel electrophoresis, with a relative mobility corresponding to a molecular weight of about 46 000 for alpha and gamma subunits, or 44 000 for the beta subunit. The four enzymes showed similar values of specific activity (about 80 units/mg), optimal pH (pH 6.8-6.9), and Km for 2-phosphoglycerate (about 3 X 10(-5) M). Amino acid analysis, peptide mapping analysis with a limited proteolysis, and immunochemical studies of the purified enzymes revealed that the three subunits, alpha, beta and gamma, are distinct proteins. Three hybrid enolases (alpha gamma, beta gamma and alpha beta) could be prepared from a mixture of two homodimeric enolases by dissociation and reassociation procedures, followed by isolation with ion-exchange column chromatography.