Purification of two enolases from human brain using a chromatofocusing column

When a purified preparation of rat alpha gamma enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) was applied to a chromatofocusing column, the enolase was almost completely dissociated and recombined to form alpha alpha and gamma gamma enolases, which were eluted at different fractions from the column. Using these phenomena, two homo-dimer forms (alpha alpha and gamma gamma) of human brain enolase were purified from a crude preparation of the hybrid alpha gamma enolase by the chromatofocusing, and subsequent chromatography on a QAE-Sephadex column (alpha alpha) or a DEAE-Sephadex column (gamma gamma). Each purified preparation showed a single band on SDS-gel electrophoresis with a relative mobility corresponding to a molecular weight of about 50 000. Amino acid analysis, peptide mapping analysis with a limited proteolysis and immunochemical studies of the purified alpha alpha and gamma gamma enolases revealed that the two subunits, alpha and gamma, are distinct proteins. The antisera to human alpha alpha or gamma gamma enolase cross-related with the respective form of rat enolase.