Amino-terminal sequence homology of two chick kidney mitochondrial proteins immunoisolated with monoclonal antibodies to the cytochrome P450 of 25-hydroxyvitamin D3-1α-hydroxylase

Abstract

We demonstrate the unique capability of monoclonal antibodies for the specific immunodetection and characterization of two antigenic proteins occurring in normal chick kidney mitochondrial extracts. The antigens were adsorbed to cyanogen bromide-activated Sepharose gel coupled to monoclonal antibodies (MAbs) of the IgM class raised against cytochrome P_4501α, which inhibit equally the 25-hydroxyvitamin D₃ 1α- and 24-hydroxylase catalytic activities (Mandel et al. 1990 J Clin Lab Immunol, in press). The two identified antigenic proteins are polypeptides with apparent molecular weights of 57,000 and 55,000 daltons. The 1α-hydroxylase cytochrome P₄₅₀ has been shown to have a molecular weight of 57,000 daltons (Mandel et al. 1990 Biochim Biophys Acta 1034:239–246). The optimal antigen: Gel ratio for maximal antigen binding as cytochrome P₄₅₀ heme, which was determined spectrally, was found to be 1.3 nmol cytochrome P₄₅₀ per g MAb- coupled Sepharose. At this ratio the total binding capacity of the gel was 1 nmol cytochrome P₄₅₀ per g Sepharose. The two polypeptides were desorbed with 0.1% Emulgen 911 in 25% glycerol at pH 3.0 and separated by SDS-gel electrophoresis. The amino-terminal sequences of the two antigens were determined by automated Edman degradation with an on-line analyzer of PTH derivatives. The sequences in both antigens were 100% homologous. Complete amino acid composition analysis also revealed that their amino acid compositions were highly similar. These findings suggest that the smaller protein may be a proteolytic cleavage product of the 1α-hydroxylase P₄₅₀ cytochrome and may represent a putative 24-hydroxylase antigen.