NADH-cytochrome b5 reductase from the insect Ceratitis capitata. Enzyme properties and membrane binding capacity

Abstract

Abstract 1. 1. NADH-cytochrome b 5 reductase was purified from microsomes of Ceratitis capitata larvae by a procedure involving its solubilization with Triton X-100, DEAE-cellulose anion exchange and hydroxylapatite adsorption chromatography, and affinity chromatography on 5′-ADP-agarose. The overall purification from isolated microsomes was 172-fold and the yield was 13%. 2. 2. The purified enzyme was free from cytochrome b 5 , cytochrome P450 and NADPH-cytochrome c(P450) reductase. The preparation showed a major protein band with an apparent molecular weight of 31,000 D by SDS-polyacrylamide slab gel electrophoresis, whereas the estimated molecular weight ranged between 240,000 D and 260,000 D by chromatography on Sepharose-6B gels in the presence of 0.2% Triton X-100. 3. 3. The enzyme was capable to reduce potassium ferricyanide and 2,6-dichlorophenolindophenol with NADH as electron donor. NADPH was ineffective as electron donor. Reduction of cytochrome c required the presence of NADH and cytochrome b 5 which has been purified from larval microsomes. 4. 4. Influence of ionic strength, pH, temperature and thiol reagents on the enzyme activity is reported. 5. 5. The purified flavoprotein exhibited membrane-binding capacity confirming its amphypathic nature. Reductase bound to microsomal membrane preparations appears to be functionally similar to the endogenous proteins.