Abstract
Goat antibody IgG produced against bovine corpus luteum mitochondrial cytochrome P-450 (P-450scc) associated with cholesterol side chain cleavage (CSCC) was used to compare immunological characteristics of mitochondrial cytochrome P-450s from the bovine adrenal cortex (BAM), bovine corpus luteum (BCLM), and human placenta (HPM). In Ouchterlony double diffusion, anti-P450scc produced a single band with BAM and BCLM P-450scc, but not with HPM P-450scc or BAM P-450 11 beta. Appropriate concentrations of this anti-P-450scc IgG inhibited the conversion of cholesterol to pregnenolone in BCLM and BAM preparations equivalently, but inhibition of placental P-450scc was considerably less. The addition of BCLM iron sulfur protein and iron sulfur protein reductase to HPM P-450scc increased CSCC approximately 5-fold. Under these conditions, anti-P-450scc inhibited CSCC in HPM. Solubilized and sonicated BCLM preparations were inhibited equivalently but more than whole mitochondria. Addition of anti-P-450scc IgG to BAM increased 11 beta-hydroxylation activity in concentration-dependent fashion. It appears that the cytochrome P-450sccs from BAM and BCLM are very similar if not identical, but immunologically different from HPM P-450scc. The BAM P-450 11 beta is immunochemically distinct from BCLM P-450scc. The CSCC and 11 beta-hydroxylation systems of the adrenal are intimately linked because inhibition of P-450scc markedly stimulated 11 beta-hydroxylation. Finally, the inhibition of CSCC activity of BAM, BCLM, and HPM P-450 indicates that the antigenic effect is directed toward the active site.
Highlights
Goat antibody IgG produced against bovine corpus luteum mitochondrial cytochrome P-450 (P-45OS,) associated with cholesterol side chain cleavage (CSCC) was used to compare immunological characteristics of mitochondrial cytochrome P-450s from the bovine adrenal cortex(BAM), bovine corpus luteum(BCLM), and human placenta (HPM)
The ferredoxin associated with CSCC in placental mitochondria was not inhibited by the anti-adrenodoxin antibodlyeading to the interpretation that the human placentaflerredoxin may be different from that of the bovine corpus luteum and adrenaclortex
Preparation of ISP Reductase and ISP-Both ISP and ISP reductase were partially purified from bovine corpus luteum mitochondria as previously described (1); both preparations were free from cytochrome P-450,by spectrophotometric analysis and assay for CSCC activity
Summary
Materials-[26-’4C]Cholesterol ( 5 4 mCi/mmol), and [3H]H20 (100 pCi/nmol) were purchased from New England Nuclear. For studies of inhibition with IgG fractions, specified amounts of immune or preimmune IgG fractions were added to the P-450 preparation and an identical volume of 0.01 M borate buffer was added to the control incubation mixture. Iron sulfur protein and ISP reductase isolated from bovinecorpora lutea were added to theplacental mitochondria These preparations were shown to be devoid of P-450,, by spectroscopy and devoid of enzymatic activity. Inhibition of CSCC activity by the IgG was determined by comparison with the control flask after incubation for 60 min. One nmol of P-450 in Buffer A was incubated with 0.1 ml of 2 nm deoxycorticosterone in propylene glycol, 4 mM glucose 6-phosphate, 1 unit of glucose-6phosphate dehydrogenase, 1.1 mMMgC12, 11 mMCaC12, and the specified volume of the preimmune or immune IgG fraction in a total volume of 2 ml. Effect of anti-P-4503, on 11/3-hydroxylationby sonicated bovine adrenal corticalmitochondria
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