Comparison of the immunochemical properties of human placental and bovine adrenal cholesterol side-chain cleavage enzyme complex.
The immunochemical relatedness between human and bovine proteins catalyzing the cholesterol side-chain cleavage reaction was investigated. In dot-immunobinding analysis, antibodies against bovine adrenocortical cytochrome P-450SCC, adrenodoxin, and adrenodoxin reductase recognized the corresponding proteins in a dose-dependent manner in mitochondrial preparations from human placenta. Limited proteolysis with trypsin cleaved bovine P-450SCC into fragments F1 and F2, which represent the NH2- and C-terminal parts of P-450SCC, respectively. Identical trypsin treatment yielded similar-size fragments from human placental P-450SCC. In Western immunoblots, anti-F1 and anti-F2 antibodies recognized the corresponding fragments in both trypsin-digested bovine and human P-450SCC. Antibodies against bovine P-450SCC, fragments F1 and F2, adrenodoxin and adrenodoxin reductase inhibited cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria by 24-51%, but failed to affect the activity in human placental mitochondria. These data indicate that human and bovine P-450SCC share common antigenic determinants located outside the enzyme active site. The immunological similarity between bovine adrenodoxin and human ferredoxin allowed for a simple purification protocol of human placental P-450SCC by adrenodoxin affinity chromatography. The P-450SCC obtained by this method was electrophoretically homogeneous and showed characteristics typical to P-450SCC.
Usanov
S A
SA
Institute of Bioorganic Chemistry, BSSR Academy of Sciences, Minsk USSR.
Honkakoski
P
P
Lang
M A
MA
Pasanen
M
M
Pelkonen
O
O
Raunio
H
H
D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000276:immunology* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D005287:Ferredoxin-NADP Reductase / Q000276:immunology; D005288:Ferredoxins / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000276:immunology; D008928:Mitochondria / Q000201:enzymology / Q000276:immunology; D010446:Peptide Fragments / Q000032:analysis; D010920:Placenta / Q000201:enzymology*; D013057:Spectrum Analysis
D005288:Ferredoxins; D007527:Isoenzymes; D010446:Peptide Fragments; D002786:Cholesterol Side-Chain Cleavage Enzyme; D005287:Ferredoxin-NADP Reductase