Jacob G. Ghazarian

Modulation of 25-hydroxyvitamin D3-24-hydroxylase by aminophylline: A cytochrome P-450 monooxygenase system☆

Abstract The chick kidney mitochondrial 25-hydroxyvitamin D 3 -24-hydroxylase is a cytochrome P-450 catalyzed monooxygenase system. The administration of aminophylline [3,7-Dihydro-1,3-dimethyl-1H-purine-2,6-dione compounded with 1,2-ethanediamine (2:1)], a cyclic nucleotide phosphodiesterase inhibitor, to vitamin D 3 -sufficient chicks at a dose of 78 mg/kg body weight increased the 24-hydroxylase activity by 10-fold without a corresponding increase in the kidney mitochondrial cytochrome P-450 concentration. The product of the reaction comigrated with authentic 24,25-dihydroxyvitamin D 3 in high pressure liquid chromatography and was quantitatively sensitive to periodate oxidation cleavage. The increase in the enzyme activity occurred while serum calcium concentration fell from 14 to 9 mg%. Serum phosphate concentration remained unchanged. These results support the idea that the regulation of kidney 24-hydroxylase activity may be mediated by tissue levels of cyclic nucleotides.

Vitamin d and 25-hydroxyvitamin d in rainbow trout (Salmo gairdneri): cytochrome p-450 and biotransformations of the vitamins

Abstract 1. 1. Rainbow trout tissue microsomal cytochrome P-450 concentrations nmol/mg of protein were as follows: liver, 0.22-2-0.28; kidney, 0.016–0.044; intestinal mucosa, 0.069–0.089. Cytochrome b5 concentrations of the tissues in the same order were 0.120-0.0240, 0.202–0.224, respectively. 2. 2. Gills were able to esterify vitamin D in vivo while the liver and g gall bladder metabolized 25-hydroxyvitamin D to yet unidentified more polar products. 3. 3. Liver microsomes were capable of metabolizing vitamin D and 25-hydroxyvitamin D to more polar products, but the product of vitamin D transformation was not 25-hydroxyvitamin D. Kidney microsomerscould not metabolize either substrate, but kidney mitochondria converted only vitamin D to a more polar product.

NADPH-cytochrome c reductase in outer membrane of kidney mitochondria. Purification and properties.

Abstract NADPH-cytochrome c reductase of vitamin D 3 -deficient chick kidney mitochondria has been purified approximately 1100-fold to a specific activity of 788 nmol cytochrome c reduced/min/mg protein. Analytical gel electrophoresis of the purified enzyme revealed two bands when stained for protein, but only the more anodic band demonstrated NADPH-tetrazolium reductase activity. The relative ease of solubilization of the reductase without the use of lipases, proteases, or detergents was the first line of evidence that suggested a novel mitochondrial localization for this previously unreported NADPH-linked cytochrome c reductase. The apparent properties of the reductase suggest that the enzyme is a component of kidney mitochondrial outer membrane. The kinetic determination of Michaelis constants with respect to NADPH, cytochrome c , and NADH gave the following values: K m NADPH = 1.7 μM , K m cyt c = 3.4 μM , and K m NADH = 20 mM . These constants were different from those of the intact kidney microsomal reductase: K m NADPH = 5.5 μM , K m cyt c = 10.5 μM , and K m NADH = 13.3 μM . The mitochondrial as well as the intact microsomal reductases exhibited a ping-pong kinetic mechanism for NADPH-mediated cytochrome c reduction. Spectrofluorometric measurements demonstrated the presence of equimolar amounts of FAD and FMN. The oxidized enzyme has absorption maxima at 280 and 450 nm with a shoulder at 415 nm. Upon reduction with NADPH a distinct loss in the 450-nm absorption was observed. Ouchterlony immunodiffusion studies with rabbit antiserum to chick renal mitochondrial ferredoxin did not reveal cross-reactivity when the purified reductase was the antigen. This excludes the involvement of a ferredoxin-type iron-sulfur protein in the NADPH-mediated reduction of cytochrome c by the purified reductase.

Chick kidney microsomal cytochrome P-450 involvement in the metabolism of 25-hydroxyvitamin D3.

Abstract A new metabolic activity is described for liver, kidney, and intestinal mucosal microsomes of rachitic chicks that is associated with the metabolism of 25-hydroxyvitamin D3 (25-OH-D3). Cytochrome P-450 was shown to be an integral component of this metabolism, supporting the monooxygenase nature of the microsomal activities. The 25-OH-D3 was the specific substrate inasmuch as the reactions could not be demonstrated to occur when vitamin D3, 1,25-dihydroxyvitamin D3, or cholesterol was the substrate. The microsomal cytochrome P-450 concentrations of rachitic chick liver, kidney, and intestinal mucosa were 0.108, 0.068, and 0.270 nmol/mg of protein, respectively. The cytochrome b5 concentrations of the tissues in the same order were 0.198, 0.123, and 0.306 nmol/mg of protein. Normal chick tissue P-450 and b5 concentrations were the same as those in the rachitic. The kidney enzyme has a pH optimum of 6.6 to 7.5 and an apparent Km of 2.5 μ m . At a substrate concentration of 0.417 μ m , the rate of 25-OH-D3 metabolism was proportional to a microsomal protein concentration of up to 3 mg/ml. The activity was inhibited 100% by 10 μ m n-ethylmaleimide, 1 m m o-phenanthroline, 1 m m metyrapone, or by a carbon monoxide:oxygen mixture of 90:10 ( v v ). A 50% inhibition of activity was produced by 5 m m sodium cyanide, but 10 μ m antimycin A and rotenone were ineffective. Supporting evidence for the participation of a cytochrome P-450 species in the renal microsomal metabolism of 25-OH-D3 included the type I substrate-induced spectral shift at 0.5 μ m 25-OH-D3 with a modified positive absorption at 405 nm and a negative absorption at 422 nm. These findings suggest that a metabolically active cytochrome P-450-substrate complex is involved in the renal microsomal biotransformations of 25-OH-D3 as a component of the regulatory processes of vitamin D3 metabolism.

Induction of 25-OH-vitamin D3 24- and 23-hydroxylase activities m partially purified renal extracts from pigs given exogenous 1,25-(OH)22D3

A renal mitochondrial cytochrome P 450 preparation from pigs treated with exogenous 1,25-(OH)2D3 was reconstituted with an NADPH-generating system, adrenodoxin and adrenodoxin reductase. The reconstituted system catalyzed the conversion of the substrate, 25-OH-D3, to metabolites comigrating with authentic 23,25-(OH)2D3 and 24,25-(OH)2D3 in both straight- and reverse-phase high-performance liquid chromatography systems, which achieve separation of these metabolites from each other as well as from other vitamin D metabolites. The putative 23,25-(OH)2D3 product was resistant to periodate treatment, while the 24,25-(OH)2D3 product was sensitive, providing additional evidence for the identity of the products. Although induction of 24-hydroxylase activity has been studied using renal homogenates from several species, only recently have techniques become available to study the activity of the enzyme in a solubilized and reconstituted form. Using these techniques, the present study shows that production of 24,25-(OH)2D3 was increased more than 80-fold with 1,25-(OH)2D3 treatment compared with untreated controls, an effect much greater than that previously observed with homogenates. In addition, production of both 23,25-(OH)2D3 and 24,25-(OH)2D3 varied with substrate concentration and was consistent with a monooxygenase-linked enzyme reaction.

Avian kidney mitochondrial hemeprotein P-4501α: isolation, characterization and NADPH-ferredoxin reductase-dependent activity

We describe the isolation of cytochrome P-4501 alpha from chick-kidney mitochondria. Although, gel permeation HPLC yielded 41% of the total amount of P-450 present in cholate-solubilized hemeproteins, it produced a highly purified mixture from which the P-4501 alpha could be purified to homogeneity in a final detergent-free state by a single-step application of hydrophobic interaction HPLC using hydroxypropyl silica. The purified P-4501 alpha traveled as a single band in SDS gel electrophoresis with an apparent Mr = 57,000. The absolute spectrum of the P-4501 alpha (Fe3+) form gave a lambda max at 403 nm. This characteristic lends support to the anomalous high-spin heme electron paramagnetic resonance spectrum and the heme structure of P-4501 alpha which we have previously reported (Ghazarian et al. (1980) J. Biol. Chem. 255, 8275-8281; Pedersen et al. (1976) J. Biol. Chem. 251, 3933-3941). In reconstitution experiments with ferredoxin-dependent NADPH-cytochrome c (P-450) reductase complexes, P-4501 alpha catalyzed the hydroxylation of 25-hydroxy-9,10-secocholesta-5,7,10(19)-trien-3 beta-ol at the C-1 position exclusively with a turnover number of 0.03 min-1. This number is identical to that obtained from measurements of the catalytic activity in intact mitochondria, indicating that only one major species of cytochrome P-450 occurs in chick-kidney mitochondria. The complete responsiveness of cytochrome P-450 concentrations in intact mitochondria to the vitamin D status of chicks provided additional evidence that the major cytochrome P-450 species present in renal mitochondria is uniquely associated with vitamin D metabolism.

Inhibition of 25-hydroxyvitamin D 1α-hydroxylase by renal mitochondrial protein kinase-catalyzed phosphorylation

Partially purified chick kidney mitochondrial Type II protein kinase catalyzes the phosphorylation of 1 alpha-hydroxylase cytochrome P-450 without affecting the rate of product formation in vitro when 1 alpha-hydroxylase activity is reconstituted by the addition of [ferredoxin] and [ferredoxin reductase] to the phosphorylated cytochrome. The cytochromes effective concentration, or its general spectral properties did not change upon phosphorylation. However, when the cytochrome and its ferredoxin were present simultaneously during the phosphorylation reaction, reconstitution of 1 alpha-hydroxylase activity by the addition of ferredoxin reductase failed to catalyze product formation. Although a several fold increase in the kinase activity could be demonstrated in the presence of cAMP, the above phosphorylation effects appear to be cAMP-independent.

Studies on vitamin D3 metabolism. Discrete liver cytosolic binding proteins for vitamin D3 and 25-hydroxyvitamin D3

Abstract Two separate liver cytosolic proteins have been partially purified and identified by their selectivity for binding either [1α,2α(n)- 3 H]vitamin D 3 or 25-hydroxy [26(27)-methyl- 3 H]vitamin D 3 . The chromatographic properties of the two proteins were not distinguishable by ion-exchange nor were they dependent upon the vitamin D 3 nutritional status of the birds. However, in molecular exclusion chromatography, the binding proteins can be successfully resolved into two discrete entities. Their binding properties suggest that they are not identical with plasma vitamin D 3 binding protein.

Biotransformations of 25-hydroxyvitamin D3 by kidney microsomes☆

Abstract Vitamin D3-deficient chick kidney microsomes in vitro metabolize 25-hydroxy-[26(27)-methyl-3H]-vitamin D3 to yet structurally unidentified polar metabolites previously designated MIC-I and MIC-II. Kidney microsomes of vitamin D3-repleted chicks could not be demonstrated to produce these metabolites when 3H was the radioactive isotope in positions C-26 and C-27 of the substrate. However, when 25-hydroxy-[26,27-14C]-vitamin D3 was the radioactive substrate, MIC-I and MIC-II production was independent of the vitamin D3 status of the chicks. These results suggest that under conditions of vitamin D3-sufficiency, there is augmented sequential kidney metabolism of 25-hydroxyvitamin D3 to products with modified side-chains involving C-26 and/or C-27. It is possible that this metabolism is responsible for the regulation of kidney cellular concentrations of 25-hydroxyvitamin D3.

Diurnal rhythmicity in plasma 24,25-dihydroxyvitamin D3: correlation with intestinal calcium transport and plasma minerals.

Abstract Plasma 24,25-(OH) 2 D 3 exhibits diurnal rhythmic variations in the adult rat. The decrease in 24,25-(OH) 2 D 3 occurring at the onset of feeding during light-dark transition coincides with a decrease in plasma calcium but is inversely related to plasma phosphate and to increased intestinal calcium transport activity.