Immunochemical specificity of placental NADPH cytochrome c ( P-450) reductase in neoplastic and non-neoplastic human tissue

Abstract

NADPH cytochrome c ( P-450) reductase was purified from human placental microsomes using a combination of affinity and gel filtration chromatography. Affinity chromatography using agarose-hexane-adenosine 2′5 diphosphate resulted in two protein bands being detected by SDS-PAGE of approximate M Ws 68 and 75 kDa. Fractions containing the two proteins were pooled, and then resolved using Sephacryl S-200. Both of the purified proteins displayed enzyme activity, measured by their ability to reduce cytochrome c. The 75 kDa protein obtained was used to immunize three female New Zealand white rabbits. The IgG fraction was partly purified from rabbit sera which suppressed placental microsomal NADPH cytochrome c reductase activity by >80% using 33% ammonium sulphate. The procured antibody suppressed androstenedione aromatase activity in microsomal preparations of human placental and breast adipose tissue, and NADPH cytochrome c reductase activity in prostate (benign and malignant), MDA-MB-231 breast cancer cells, breast adipose, Hep G2 hepatoma cells and placental microsomal preparations. The extent of NADPH cytochrome c reductase inhibition varied in the order of malignant prostate < benign prostate<MDA<breast adipose<Hep G2<placenta. The results suggest that human placental NADPH cytochrome c ( P-450) reductase shares common antigenic epitopes pertinent to its capability of reducing cytochrome c in all of the above-mentioned tissues. In attempting to associate possible changes in NADPH cytochrome c reductase activity imposed by neoplasia to the obtained immunochemical cross reactivity and enzyme activity results, it was noted that microsomes obtained from MDA cells exhibited enzyme activity significantly less than that of breast adipose microsomes (1.6 and 8.1 nmol/min/mg protein, respectively) and by comparison showed 6% less homology towards the placental antibody. The results obtained for benign and malignant prostate showed no significant difference between the neoplastic states as adjudged by enzyme activity and immunochemical assays.