Subunit separation in reversed micelle system reveals the existence of active centers both on light and heavy γ-glutamyltransferase subunits

• Published in: FEBS letters 1991-12, Vol.295 (1), p.73-76
• Main Authors: Kabanov, Alexander V., Nametkin, Sergey N., Chernov, Nikolai N., Klyachko, Natalya L., Levashov, Andrey V.
• Format: Article
• Language: English
• Subjects: (3-carboxy-4-nitro)aniline; (4-nitro)aniline; active sites; Aerosol OT (sodium bis(2-ethylhexyl)sulfosuccinate); Analytical, structural and metabolic biochemistry; AOT; AT-125; Binding Sites; Biological and medical sciences; CNA; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; gamma -glutamyltransferase; gamma-Glutamyltransferase - antagonists & inhibitors; gamma-Glutamyltransferase - isolation & purification; gamma-Glutamyltransferase - metabolism; GluCNA; GluNA; glycylglycine; GlyGly; Isoxazoles - pharmacology; Kinetics; L-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid; Macromolecular Substances; Mammalia; Micellar enzymology; Micelles; PAGE; polyacrylamide gel electrophoresis; Protein subunit; Reversed micelle; separation; subunits; Supra-macromolecular structure; Transferases; Tumor Cells, Cultured; γ-glutamyl(3-carboxy-4-nitro)anilide; γ-glutamyl(4-nitro)anilide; γ-Glutamyltransferase; γ-GT
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(91)81388-O

Regulation of supra-macromolecular composition and catalytic activity of a heterodimeric enzyme, γ-glutamyltransferase, in the system of Aerosol OT (sodium bis(2-ethylhexyl) sulfosuccinate) reversed micelles in octane were studied. Variation of the surfactant hydration degree (parameter, determining dimensions of the polar inner cavity of the micelle) causes a reversible dissociation of the enzyme to light and heavy subunits. Both enzyme subunits possess catalytic activity. The light and heavy subunits of the enzyme were separated on a preparative scale in a reversed micelle system using ultracentrifugation. The active centers of γ-glutamyltransferase were studied using its irreversible inhibitor — AT-125 (L·(αS.5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). Separation of the γ-glutamyltransferase subunits results in the ‘opening’ of a new active center located at the heavy subunit. In the dimer form of the enzyme this center is masked and it is not accessible to both substrate and inhibitor molecules.