Biliverdin-IXa Reductase and Biliverdin-IXP Reductase from Human Liver

This report describes for the first time the identification of four forms of biliverdin reductase including two biliverdin-MP reductases and two biliverdin-Ma reductases, designated isozymes I and I1 and isozymes I11 and IV, respectively, in human liver cytosolic fractions. The four forms of biliverdin reductase were purified to homogeneity. There was a 7,80&15,000-fold increase in specific activity when compared with the crude preparation, and the recovery was 8-26%. The purified enzymes were monomers with a molecular weight of about 21,000 (isozymes I and 11) and 34,000 (isozymes I11 and IV). The enzymes were strictly specific for biliverdin, and no other oxidoreductase activities were detected in the purified preparations. The purified enzymes used NADPH and NADH as electron donors for the reduction of biliverdin. The apparent K , values of isozymes I, 11,111, and IV for NADPH were 35.9, 13.1, 10.9, and 34.1 JIM, respectively, whereas those for NADH were 5.6, 8.2, 7.9, and 23.4 m ~ , respectively. It was assumed that NADPH rather than NADH was the physiological electron donor in the intracellular reduction of biliverdin. "he apparent K,,, value of isozymes I and I1 for biliverdin-MP in the NADPH system was 0.3 p~ whereas those of isozymes I11 and IV for biliverdin-Ma were 1.0 and 0.8 JIM, respectively. Isozymes I and I1 used biliverdin-MP, -My, and M S as substrates but not biliverdinMa, and isozymes I11 and IV preferred biliverdin-Ma as the most effective substrate among the four biliverdin isomers. The NADPH-dependent enzyme activities were inhibited by substrate concentrations in excess of 3 4 p ~ . The NADPH-dependent enzyme activities, especially isozymes I11 and IV, were sensitive to SH reagents including iodoacetamide, p-chloromercuribenzoic acid, and N-ethylmaleimide. The optimum pH of the reaction with NADPH for isozymes I and I1 was 8.2 whereas that for isozymes I11 and IV was 7.4. The proportion of the total activity of isozymes I and 11 to that of isozymes I11 and IV was considerably higher in the fetal than in the adult liver.