Effect of divalent cations on NADH-dependent and NADPH-dependent cytochrome b5 reduction by hepatic microsomes

Abstract

The effect of Ca 2+ or Mg 2+ on cytochrome b 5 reduction by porcine liver microsomes was examined using trypsin-solubilized cytochrome b 5 as a substrate. The reduction of exogenous cytochrome b 5 by microsomes was low at 1.2 μ m cytochrome b 5 (3.9 or 2.7 nmol/min/mg protein, respectively, with NADH or NADPH). The addition of CaCl 2 greatly enhanced either NADH-dependent or NADPH-dependent cytochrome b 5 reduction. At 2 m m CaCl 2, the reduction rate was increased to 23-or 18-fold of control, respectively with NADH or NADPH. The concentration for half-maximal effect (EC 50) was 0.5 or 0.6 m m in the NADH or NADPH systems, respectively. MgCl 2 also stimulated cytochrome b 5 reduction with a EC 50 value of 1.0 m m in the NADH system or 0.6 m m in the NADPH system. The comparison with the result with KCl indicated that the activation by CaCl 2 or MgCl 2 is caused mainly by their divalent cation moiety. The K m value for cytochrome b 5 was decreased and the V max was increased by calcium with either the NADH- or the NADPH-dependent system. NADH—ferricyanide reductase activity was not affected by calcium, but NADPH—ferricyanide reductase activity was stimulated as well as NADPH—cytochrome c reductase activity. In the presence of Triton X-100, divalent cations were inhibitory in NADH-dependent cytochrome b 5 reduction, and in contrast, stimulative in NADPH-dependent reaction. These findings suggest that the activation of cytochrome b 5 reduction by divalent cations in the NADH system is mainly due to an increasing accessibility of the substrate, and in the NADPH system, in addition to this, a direct effect of divalent cations on NADPH—cytochrome P450 reductase is also involved.