Partial Purification of NADH-Cytochrome b₅ Reductase from Rabbit Liver Microsomes with Detergents and Its Properties

抄録

NADH-Cytochrome b₅ reductase [EC 1. 6. 2. 2] (fp₁) was solubilized from rabbit liver microsomes with Triton X-100 and sodium deoxycholate and purified 30-fold in the presence of Triton X-100. This preparation was called “detergent-solubilized fp1” or simply “d-fp₁” to distinguish it from the fp₁ preparation purified after solubilization by lysosomal digestion (“lysosome-solubilized fp₁” or “1-fp₁”). The d-fp₁ preparation still contained phospholipids and was contaminated by cytochromes P-450 and P-420, but was free from cytochrome b₅ and NADPH-cytochrome c reductase system. A considerable amount of non-heme iron, showing an electron spin resonance (ESR) signal at g=4.3, was also present in the preparation, but this iron did not seem to be functional in the catalysis.
When the preparation was subjected to Sephadex gel filtration in the presence of 1.0% deoxycholate, the reductase activity was eluted at a position corresponding to a molecular weight of 45, 000. After treatment with lysosomes or partially puri-fied cathepsin D, the elution volume of the reductase activity corresponded to a smaller molecular weight of about 28, 000, which was similar to the value reported for 1-fp₁.
d-fp₁ could react with cytochrome b₅ purified using detergent (“d-b₅”) as well as with that solubilized by trypsin (“t-b₅”) to reconstitute the NADH-cytochrome c reductase activity. However, it interacted with d-b₅ much better than with t-b₅. The highly efficient interaction with d-b₅ was abolished when d-fp₁ was digested with lysosomes or cathepsin D, though the reactivity of d-fp₁ with t-b₅ was unaffected by these treatments. It is suggested that the fp₁ preparations obtained previously (1-fp₁) are functionally incomplete.