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Amino‐terminal sequence homology of two chick kidney mitochondrial proteins immunoisolated with monoclonal antibodies to the cytochrome P 450 of 25‐hydroxyvitamin D 3 ‐1α‐hydroxylase
Author(s) -
Moorthy Bhagavatula,
Mandel Mark L.,
Ghazarian Jacob G.
Publication year - 1991
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1002/jbmr.5650060214
Subject(s) - cyanogen bromide , sepharose , microbiology and biotechnology , biochemistry , antigen , peptide sequence , monoclonal antibody , chemistry , edman degradation , cytochrome , cytochrome p450 , amino acid , biology , antibody , enzyme , gene , genetics , immunology
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 D 3 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 P 450 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 P 450 heme, which was determined spectrally, was found to be 1.3 nmol cytochrome P 450 per g MAb‐ coupled Sepharose. At this ratio the total binding capacity of the gel was 1 nmol cytochrome P 450 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 P 450 cytochrome and may represent a putative 24‐hydroxylase antigen.