Inhibitory domain‐specific antibodies to cytochrome P‐450_scc

Abstract

Highly specific antibodies to cytochrome P-450~ and its F~ and F 2 fragments, representing N-and C-terminal sequences of the hemeprotein respectively, were raised in rabbits. These antibodies were found to be inhibitory (up to 50-90%) for the cholesterol transformation into pregnenolone in the reconstituted system, indicating the involvement of both F 1 and F 2 domains formed by the respective fragments in monooxygenase catalysis. Cytoehrome P-450~¢ in mitoplasts is not accessible for trypsin as revealed by immunological techniques. However, the treatment of submitochondrial particles with trypsin results in two main fragments identified by immunoblotting in the presence of the monospecific antibodies as F~ and F 2 fragments. This indicates that the trypsin sensitive 250-257 region in cytochrome P-450~ molecule connecting both domains is exposed to the matrix side of the inner mitochondrial membrane.

Selective chemical modification of Cys264 with diiodofluorescein iodacetamide as a tool to study the membrane topology of cytochrome P450scc (CYP11A1)

“…tive hinge region (Arg 250-Asn257) is exposed to the mitochondrial matrix space [5,7]. The results obtained in this work suggest that the interdomain hinge in the P45Oscc sequence including cy?…”

“…Immunoblotting was performed in Bio-Rad Mini-Transblot apparatus according to the procedure described previously [4]. P45Oscc concentrations were determined from the absolute spectra or from CO-difference spectra of dithionite-reduced preparations of liposomes using an extinction coefficient of 91 mM_' .…”

“…P45Oscc, AD and AR were purified from bovine adrenocortical mitochondria to electrophoretic homogeneity using specific aBinity adsorbents 171. Antibodies against P45Oscc and its peptide fragments Fl and F2 were raised in rabbits and purified according to the procedure described earlier [4].…”

“…CHL side chain cleavage activity of P45Oscc was determined in the reconstituted system as described earlier [4] followed by treatment of the reaction mixture with CHL oxidase to convert CHL and PG into the 3-one-4-en steroids, i.e. cholestenon and progesterone, respectively [lo].…”

“…We previously used antibodies against intact P45Oscc molecule and its two tryptic fragments, Fl (Ile1-Ar~50) and F2 (Asn257-Ala48'), to study the topology of the hemeprotein in the inner mitochondrial membrane [4,5]. In this paper we studied the functional importance of the central region of the P45Oscc sequence, which includes the trypsin-sensitive, interdomain hinge, and the topology of the region in proteoliposomes by using diio-*Corresponding author.…”