Purification of two enolases from human brain using a chromatofocusing column

• Published in: Biochimica et biophysica acta Vol. 717; no. 2; pp. 348 - 354
• Main Authors: Shimizu, Atsuko, Suzuki, Fujiko, Kato, Kanefusa
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 06.08.1982
• Subjects: Amino Acids - analysis; Brain - enzymology; Chromatofocusing; Chromatography, Ion Exchange - methods; Cross Reactions; Enolase; Human brain; Humans; Immunodiffusion; Isoenzymes - isolation & purification; Macromolecular Substances; Molecular Weight; Peptide Fragments - analysis; Phosphopyruvate Hydratase - isolation & purification; Protein Multimerization; Human brain; Enolase; Chromatofocusing
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(82)90189-1

When a purified preparation of rat αγ 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 αα and γγ enolases, which were eluted at different fractions from the column. Using these phenomena, two homo-dimer forms (αα and γγ) of human brain enolase were purified from a crude preparation of the hybrid αγ enolase by the chromatofocusing, and subsequent chromatography on a QAE-Sephadex column (αα) or a DEAE-Sephadex column (γγ). 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 αα and γγ enolases revealed that the two subunits, α and γ, are distinct proteins. The antisera to human αα or γγ enolase cross-reacted with the respective form of rat enolase.