Monoclonal antibodies to chick renal calcium regulating hemeproteins. Biochemical and immunohistochemical interactions with mitochondrial 25-hydroxyvitamin D3 hydroxylases
| Autor: | M L, Mandel, B, Moorthy, S J, Swartz, J C, Garancis, J G, Ghazarian |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
Hybridomas
Molecular Sequence Data Antibodies Monoclonal Kidney Mitochondria Ferredoxin-NADP Reductase Immunoenzyme Techniques Isoenzymes Kidney Tubules Proximal Epitopes Mice Cytochrome P-450 Enzyme System Immunoglobulin M Multienzyme Complexes Organ Specificity Adrenal Glands Steroid Hydroxylases Microsomes Liver Animals Cholestanetriol 26-Monooxygenase Calcium Amino Acid Sequence Vitamin D Vitamin D3 24-Hydroxylase Chickens |
| Zdroj: | Journal of clinicallaboratory immunology. 31(1) |
| ISSN: | 0141-2760 |
| Popis: | The kidney mitochondrial monooxygenases known as 25-hydroxyvitamin D3 1 alpha- and 24R-hydroxylases are two analogous enzymes which utilize the vitamin as a common substrate for the catalytic production of 1 alpha,25-dihydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3. These two enzymes are complexes of NADPH-ferredoxin reductase, and an (Fe-S)-cluster containing ferredoxin with a redox potential that allows the ultimate transfer of reducing equivalents to the terminal oxidases distinctly known as cytochromes P-450(1) alpha and P-450(24). We have used in vitro immunizations of splenocytes obtained from mice sensitized with the purified cytochrome P-450(1) alpha to generate three hybridoma clones from fusion with p3 x 63.Ag8.653 myeloma ATCC cells which selectively secrete monoclonal antibodies (MAbs) of the IgM class. The MAbs have been partially purified by ammonium sulfate fractionation followed by size separation chromatography on Sephacryl S-200-HR. We have compared the structural similarities and differences between the two kidney enzymes in Western Blot analyses using horseradish peroxidase conjugated goat anti-mouse Igs specific for the heavy and light chains of mouse IgA, IgG and IgM. The MAbs from all three clones recognized and interacted with apparent common epitopes of the two hydroxylases but selectively discriminated against liver microsomal P-450LM2 type and adrenal mitochondrial P-450SCC cytochromes. The cytochromes P-450(1) alpha and P-450(24) were detected as two separate bands with approximate molecular weights of 57 and 55 KDa, respectively. In reconstitution of hydroxylase activities in vitro, the MAbs were equally effective in inhibiting the 1 alpha-hydroxylation and 24R-hydroxylation reactions. The ratio of micrograms of Igs to pmol cytochrome P-450 for a 50% inhibition of either activity was approximately 25. These results, collectively, seem to suggest the existence of a precursor-product relationship between the kidney mitochondrial 1 alpha- and the 24R-hydroxylases, or perhaps, a common ancestral origin. Immunochemical peroxidase anti-peroxidase staining of kidney tissue first exposed to the MAbs revealed that only the proximal tubular segment of the nephron was specifically enriched with the cytochromes. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|