Endocrinology, Vol 123, 2491-2498, Copyright © 1988 by Endocrine Society

ARTICLES

Characterization of monoclonal antibodies specific to bovine renal vitamin D hydroxylases

RE Bort and JF Crivello
Department of Physiology and Neurobiology, University of Connecticut, Storrs 06268.

Monoclonal antibodies (MAbs) have been produced which recognize specific epitopes on bovine renal mitochondrial vitamin D3 1 alpha- and 24-hydroxylases. Renal mitochondria cytochrome P-450s were partially purified to 0.5-2 nmol/mg by Emulgen 911 and cholate solubilization, followed by chromatography on a 2-(4,6-dichloro-O- biphenyloxy)ethylamine HBR affinity column. Reduced carbon monoxide difference spectra determined that this preparation contained 0.5-2 nmol P-450/mg protein. This preparation contained both 1 alpha- and 24- hydroxylase activities, and Eadie-Hofstee plots of product formation as a function of substrate concentrations have maximum velocities of 1.4 and 4 pmol product/30 min.mg protein and Km values of 690 and 1300 nM, respectively. Bovine renal hydroxylases were isolated by immunoprecipitation from this partially purified P-450 preparation with a polyclonal antibody specific for rat liver microsomal cytochrome P- 450 RLM5. This polyclonal antibody immunoprecipitated both 1 alpha- and 24-hydroxylase activities as well as renal mitochondrial cytochrome P- 450, as determined by reduced CO spectra. Bovine renal mitochondrial components were immunoisolated and used to immunize BALB/c mouse spleen cells in vitro. MAbs then produced were screened for 1) immunoisolation of renal mitochondrial hydroxylase activity from a partially purified preparation, 2) immunohistochemical detection of antigen in renal proximal tubule cells, and 3) immunoquantitation of renal hydroxylases in a solid phase sandwich (enzyme-linked immunosorbent assay) and by 4) Western blot analysis. MAbs were isolated with specifically immunoprecipitated 1 alpha-hydroxylase activity, 24-hydroxylase activity, or both. In 10 micron sections of bovine kidney, antibodies detected antigen only in proximal tubule cells on the basal surface, which is rich in mitochondria. No antigen was detected in sections of pancreas or liver. In the solid phase sandwich enzyme-linked immunosorbent assay, MAbs detected 1 alpha and 24-hydroxylases only in renal mitochondria and not in liver microsomes or adrenal gland mitochondria. In a Western blot, MAbs specific for epitopes expressed on both hydroxylases detected a single band(s) at 52,000-53,000 daltons. Apparently it is not possible to discriminate between hydroxylases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blots. By these criteria, MAbs have been generated which are specific to epitopes expressed on bovine renal mitochondrial 1 alpha- and 24-hydroxylases.

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