The Functional Role of Thiol Groups in Protease‐Solubilized NADPH—Cytochrome c Reductase from Pork‐Liver Microsomes

• Published in: European journal of biochemistry Vol. 76; no. 2; pp. 365 - 371
• Main Authors: LAZAR, Tibor, EHRIG, Helga, LUMPER, Ludwig
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 15.06.1977
• Subjects: Animals; Cytochrome Reductases - metabolism; Kinetics; Microsomes, Liver - enzymology; NADPH-Ferrihemoprotein Reductase - isolation & purification; NADPH-Ferrihemoprotein Reductase - metabolism; Oxidation-Reduction; Peptide Hydrolases; Solubility; Sulfhydryl Compounds - analysis; Swine
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1977.tb11604.x

The total —SH content of protease‐solubilized NADPH—cytochrome c reductase (EC 1.6.2.4) from pork liver microsomes was determined to be 5.9 ± 0.3 mol thiol groups per mol of protein. Only three —SH groups of the protease‐solubilized NADPH – cytochrome c reductase could be modified by 0.34 or 1.0 mM 5,5′‐dithio‐bis(2‐nitrobenzoate) at pH 7.5 and + 4°C. More than 95% of the original enzymatic activity was lost during this treatment. In the presence of 1 mM NADP+ only two —SH groups of the NADPH – cytochrome c reductase reacted with 5,5′‐dithio‐bis(2‐nitrobenzoate). After removal of the competitive inhibitor NADP+, an enzyme derivative with the specific activity of the unmodified enzyme was obtained, containing two cysteinyl residues as mixed disulfides with 2‐nitro‐5‐thiobenzoate. Renewed treatment of the modified reductase with 0.34 or 1.0 mM 5,5′‐dithio‐bis(2‐nitrobenzoate) resulted in the modification of one additional thiol group under complete inactivation (k for modification = 0.027 min−1, k for inactivation = 0.039 min−1 at 0.34 mM Nbs2, pH 7.5, + 4°C). Kinetic analysis confirmed the suggestion that a single thiol group of the NADPH—cytochrome c reductase was protected by NADP+ against the reaction with 5,5′‐dithio‐bis(2‐nitrobenzoate). Cytochrome c (58 μM) apparently enhanced the effect of NADP+. The modification of the accessible thiol groups by 1 mM 5,5′‐dithio‐bis(2‐nitrobenzoate) did not affect the half‐reduced state of the NADPH–cytochrome c reductase.