Amino-terminal sequence homology of two chick kidney mitochondrial proteins immunoisolated with monoclonal antibodies to the cytochrome P450 of 25-hydroxyvitamin D3-1 alpha-hydroxylase.
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 P450(1) alpha, which inhibit equally the 25-hydroxyvitamin D3 1 alpha- 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 1 alpha-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 1 alpha-hydroxylase P450 cytochrome and may represent a putative 24-hydroxylase antigen.
Moorthy
B
B
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Mandel
M L
ML
Ghazarian
J G
JG
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000276:immunology*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D002645:Chickens; D002846:Chromatography, Affinity; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007668:Kidney / Q000737:chemistry*; D008928:Mitochondria / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry* / Q000276:immunology; D012689:Sequence Homology, Nucleic Acid
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D011506:Proteins; D003577:Cytochrome P-450 Enzyme System; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase