scholarly journals Solubilization and reconstitution of kidney 25-hydroxyvitamin D3 1 α- and 24-hydroxylases from vitamin D-replete pigs

1989 ◽  
Vol 259 (2) ◽  
pp. 561-568 ◽  
Author(s):  
R W Gray ◽  
J G Ghazarian
Keyword(s):  
Normal Diet ◽  
Partial Purification ◽  
Hydroxylase Activity ◽  
Dihydroxyvitamin D3 ◽  
Specific Content ◽  
Protein Binding Assay ◽  
Separate Fraction ◽  
Excess Substrate ◽  
Solvent Systems ◽  
Cytochrome P 450

Pig kidney mitochondrial 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylase activities have been solubilized with cholate/Emulgen 911 and reconstituted with NADPH, ferredoxin reductase and ferredoxin. All three of these components are required for full catalytic activity of both enzymes. Both products were identified by co-chromatography with authentic metabolites on both normal and reverse-phase h.p.l.c. using solvent systems which were shown to separate 10-oxo-19-nor-25-hydroxyvitamin D3 from 1,25-dihydroxyvitamin D3 [1,25-(OH)2-D3]. In addition, periodate treatment of the 24,25-(OH)2-D3 product resulted in complete loss of the product as measured by protein-binding assay. Further purification by p-chloroamphetamine-Sepharose chromatography of a solubilized extract from a pig fed a normal diet increased the specific content of the cytochrome P-450 from 0.019 to 0.239 nmol/mg and the 1 alpha-hydroxylase activity from 4.75 to 268 pmol/h per mg. Activity of the 24-hydroxylase in the crude solubilized extract was 6.3 pmol/h per mg, but was undetectable after partial purification by a p-chloroamphetamine-Sepharose column. However, further fractionation of this material by DEAE-Sepharose chromatography resulted in a further increase in 1 alpha-hydroxylase activity to 430 pmol/h per mg and detection of 24-hydroxylase in a separate fraction at a level of 53 pmol/h per mg. Production of 1,25-(OH)2-D3 was linear with time up to 2 h and was dependent upon ferredoxin concentration as well as cytochrome P-450 concentration in the range of 0-40 nM. In the presence of excess ferredoxin and adequate amounts of cytochrome P-450, 1,25-(OH)2-D3 production was also dependent upon substrate concentrations in the range of 0.25 to 2.5 microM yielding an estimated Km of 1 microM. In the presence of excess substrate and ferredoxin, the catalytic-centre activity of the enzyme was estimated to be 1 h-1.

scholarly journals Partial purification of cytochrome P -450 of Candida tropicalis and reconstitution of hydroxylase activity

FEBS Letters ◽  
1979 ◽  
Vol 105 (1) ◽  
pp. 143-146 ◽  
Author(s):  
J.C. Bertrand ◽  
M. Gilewicz ◽  
H. Bazin ◽  
M. Zacek ◽  
E. Azoulay
Keyword(s):  
Candida Tropicalis ◽  
Partial Purification ◽  
Hydroxylase Activity ◽  
Cytochrome P 450

scholarly journals Androgen hydroxylation catalysed by a cell line (SD1) that stably expresses rat hepatic cytochrome P-450 PB-4 (IIB1)

1989 ◽  
Vol 260 (1) ◽  
pp. 81-85 ◽  
Author(s):  
D J Waxman ◽  
D P Lapenson ◽  
J J Morrissey ◽  
S S Park ◽  
H V Gelboin ◽  
...  
Keyword(s):  
Cell Line ◽  
Gene Product ◽  
Rat Liver ◽  
Membrane Fraction ◽  
Liver Microsomes ◽  
Parental Cell Line ◽  
Rat Liver Microsomes ◽  
Hydroxylase Activity ◽  
Specific Content ◽  
Cytochrome P 450

Androgen hydroxylation catalysed by Chinese hamster fibroblast SD1 cells, which stably express cytochrome P-450 form PB-4, the rat P450IIB1 gene product, was assessed and compared to that catalysed by purified cytochrome P-450 PB-4 isolated from rat liver. SD1 cell homogenates catalysed the NADPH-dependent hydroxylation of androstenedione and testosterone with a regioselectivity very similar to that purified by P-450 PB-4 (16 beta-hydroxylation/16 alpha-hydroxylation = 6.0-6.8 for androstenedione; 16 beta/16 alpha = 0.9 for testosterone). Homogenates prepared from the parental cell line V79, which does not express detectable levels of P-450 PB-4 or any other cytochrome P-450, exhibited no androgen 16 beta- or 16 alpha-hydroxylase activity. The hydroxylase activities catalysed by the SD1 cell homogenate were selectively and quantitatively inhibited (greater than 90%) by a monoclonal antibody to P-450 PB-4 at a level of antibody (40 pmol of antibody binding sites/mg of SD1 homogenate) that closely corresponds to the P-450 PB-4 content of the cells (48 pmol of PB-4/mg of SD1 homogenate). Fractionation of cell homogenates into cytosol and microsomes revealed that the P-450 PB-4-mediated activities are associated with the membrane fraction. Although the P-450 PB-4-specific content of the SD1 microsomes was 15% of that present in phenobarbital-induced rat liver microsomes, the P-450 PB-4-dependent androstenedione 16 beta-hydroxylase activity of the SD1 membrane fraction was only 2-3% of that present in the liver microsomes. This activity could be stimulated several-fold, however, by supplementation of SD1 microsomes with purified rat NADPH P-450 reductase. These studies establish that a single P-450 gene product (IIB1) can account for the hydroxylation of androgen substrates at multiple sites, and suggest that SD1 cells can be used to assess the catalytic specificity of P-450 PB-4 with other substrates as well.

Enzymatic Synthesis of 4′-and 3′, 4′ -Hydroxylated Flavanones and Flavones with Flower Extracts of Sinningia cardinalis

1987 ◽  
Vol 42 (11-12) ◽  
pp. 1193-1199 ◽  
Author(s):  
K. Stich ◽  
G. Forkmann
Keyword(s):  
Enzymatic Synthesis ◽  
Microsomal Fraction ◽  
Chalcone Synthase ◽  
Condensation Reaction ◽  
Flavonoid Biosynthesis ◽  
Hydroxylase Activity ◽  
Key Enzyme ◽  
Flavone Synthase ◽  
Flavone Synthase Ii ◽  
Cytochrome P 450

Flowers of Sinningia (syn. Rechsteineria) cardinalis contain glycosides of the flavones apigenin (4′-OH) and luteolin (3′,4′-OH) respectively, and of the related 3-deoxyanthocyanidins apigeninidin and luteolinidin. Studies on substrate specificity of the key enzyme of flavonoid biosynthesis, chalcone synthase, revealed that the 3′,4′-hydroxylated flavonoids are formed by hydroxylation of flavonoid compounds rather than by incorporation of caffeoyl-CoA into the flavonoid skeleton during the condensation reaction. In fact, flavonoid 3′-hydroxylase activity could be demonstrat­ed in the microsomal fraction of the flower extracts. The enzyme catalyses hydroxylation of naringenin and apigenin in the 3′-position to eriodictyol and luteolin, respectively, with NADPH as cofactor. Besides flavanone 3′-hydroxylase a further NADPH-dependent enzyme activity (flavone synthase II) was observed in the microsomal fraction catalysing the oxidation of naringenin to apigenin and of eriodictyol to luteolin. The Cytochrome P-450 inhibitor ancymidol was found to abolish completely flavone synthase II activity, whereas flavonoid 3′-hydroxylase activity was not impaired.

24,25-dihydroxyvitamin D3 in serum: sample purification with Sep-Pak C-18 cartridges and liquid chromatography before protein-binding assay.

1983 ◽  
Vol 29 (10) ◽  
pp. 1806-1807 ◽  
Author(s):  
M L Traba ◽  
M Babé ◽  
C de la Piedra ◽  
A Marín
Keyword(s):  
Liquid Chromatography ◽  
Protein Binding ◽  
Serum Sample ◽  
High Performance ◽  
Binding Assay ◽  
Reversed Phase ◽  
Specific Method ◽  
Dihydroxyvitamin D3 ◽  
Protein Binding Assay ◽  
24,25 Dihydroxyvitamin D3

Abstract We describe a precise, specific method for measuring 24,25-dihydroxyvitamin D3 in human serum. A 2-mL serum sample is extracted with acetonitrile and passed through a Sep-Pak C-18 cartridge. The sample is further purified by "high-performance" liquid chromatography under isocratic conditions on a normal-phase column (Radial-Pak silica-gel cartridge), then subjected to a protein-binding assay. The mean concentration of 24,25-dihydroxyvitamin D3 in serum from 22 normal adults (measured during the spring) was 2.9 micrograms/L (SD 1.9, range 6.3-0.42 microgram/L). The intra-assay CV was 7.7%, the interassay CV 11.2%. Purification of the sample with Sep-Pak C-18 and liquid chromatography on normal plus reversed-phase columns leads to a mean value of 3.4 micrograms/L (SD 1.6 micrograms/L, n = 12), not significantly different from results with our method.

Rat renal 25-hydroxyvitamin D3 1- and 24-hydroxylases: their in vivo regulation

1984 ◽  
Vol 246 (2) ◽  
pp. E168-E173 ◽  
Author(s):  
Y. Tanaka ◽  
H. F. DeLuca
Keyword(s):  
Vitamin D ◽  
Inorganic Phosphorus ◽  
Hydroxylase Activity ◽  
Deficient Diet ◽  
Dihydroxyvitamin D3 ◽  
1,25 Dihydroxyvitamin D3 ◽  
Dietary Phosphorus ◽  
Low Calcium

The effects of thyroparathyroidectomy, parathyroid hormone, 1,25-dihydroxyvitamin D3, dietary calcium, dietary phosphorus, age, and sex on the renal 25-hydroxyvitamin D3 1- and 24-hydroxylases measured in vitro in rats have been studied. Thyroparathyroidectomy of vitamin D-deficient rats abolishes 25-hydroxyvitamin D3 1-hydroxylase activity, and administration of bovine parathyroid extract to the thyroparathyroidectomized rat restores diminished 1-hydroxylase activity. Both suppression and restoration of the enzyme activities require many hours (18-24 h) independent of rapid changes in serum calcium and inorganic phosphorus levels in response to these manipulations. Administration of 1,25-dihydroxyvitamin D3 to vitamin D-deficient rats suppresses 25-hydroxyvitamin D3 1-hydroxylase activity and stimulates 25-hydroxyvitamin D3 24-hydroxylase activity within 48 h. Rats maintained on a low-calcium or a low-phosphorus diet with a daily supplement of 20 IU vitamin D3 show high 25-hydroxyvitamin D3 1-hydroxylase activity and low 24-hydroxylase activity as compared with rats similarly treated but fed a diet containing adequate calcium or adequate phosphorus. When vitamin D-sufficient rats having suppressed renal 25-hydroxyvitamin D3 1-hydroxylase activity are placed on a low-calcium vitamin D-deficient diet for 7 days, the 1-hydroxylase activity is greatly stimulated in 6-wk-old rats but much less so in rats with advancing age.

Inhibition of 25-hydroxyvitamin D3-1-hydroxylase by chronic metabolic acidosis

1982 ◽  
Vol 243 (4) ◽  
pp. E265-E271
Author(s):  
G. S. Reddy ◽  
G. Jones ◽  
S. W. Kooh ◽  
D. Fraser
Keyword(s):  
Vitamin D ◽  
Metabolic Acidosis ◽  
Rat Kidney ◽  
Ion Concentration ◽  
Hydroxylase Activity ◽  
Renal Metabolism ◽  
Dihydroxyvitamin D3 ◽  
Hydrogen Ion Concentration ◽  
Chronic Metabolic Acidosis ◽  
Negative Linear Correlation

Chronic metabolic acidosis had been shown to influence the renal metabolism of 25-hydroxyvitamin D3. Using the isolated perfused rat kidney model, we evaluated the rates of synthesis of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in vitamin D-depleted [D(-)] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] in vitamin D-replete [D(+)] rats. Metabolic acidosis was induced in both groups of rats by feeding aqueous ammonium chloride for 9 days. Kidneys isolated from D(-) acidotic rats (mean pH, 7.11) exhibited a decreased rate of 1,25(OH)2D3 synthesis (0.79 +/- 0.17 pmol produce . h-1 . g kidney-1) when compared with that (1.27 +/- 0.09) of D(-) nonacidotic (mean pH, 7.33) rats. There was a significant negative linear correlation between the rate of synthesis of 1,25(OH)2D3 and the hydrogen ion concentration of the animal (r = 0.79, P less than 0.005). The rate of synthesis of 24,25(OH)2D3 by the kidneys from D(+) acidotic (mean pH, 7.06) and nonacidotic (mean pH, 7.39) rats did not differ (0.81 +/- 0.21 vs. 0.60 +/- 0.12 pmol product . h-1 . g kidney-1). It is concluded that chronic acidosis suppressed 1-hydroxylase activity, but does not suppress 24-hydroxylase activity.

Effects of 1,25-dihydroxyvitamin D3 on colonic calcium transport in vitamin D-deficient and normal rats

1984 ◽  
Vol 246 (3) ◽  
pp. G268-G273
Author(s):  
M. J. Favus ◽  
C. B. Langman
Keyword(s):  
Vitamin D ◽  
Calcium Transport ◽  
Calcium Absorption ◽  
Biological Response ◽  
Vehicle Control ◽  
Normal Diet ◽  
Dihydroxyvitamin D3 ◽  
Vitamin D Intake ◽  
1,25 Dihydroxyvitamin D3

To determine whether prior vitamin D intake influences the intestinal calcium absorptive action of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], we measured in vitro the two unidirectional transepithelial fluxes of calcium across descending colon segments from rats fed either a vitamin D-deficient or normal diet and injected with either 10, 25, or 75 ng of 1,25(OH)2D3 or vehicle alone. Vitamin D deficiency abolished net calcium absorption [J net, -2 +/- 2 vs. 12 +/- 2 (SE) nmol X cm-2 X h-1, P less than 0.001], and 10 ng of 1,25(OH)2D3 raised J net to levels found in normal rats. Larger doses (25 and 75 ng) increased J net above levels in normal rats given the same dose. In normal rats only 75 ng of 1,25(OH)2D3 increased calcium J net above vehicle control values (12 +/- 2 vs. 38 +/- 4 nmol X cm-2 X h-1, P less than 0.001). Circulating 1,25(OH)2D3 measured by radioreceptor assay was well correlated with calcium transport. For each dose of 1,25(OH)2D3 higher serum 1,25(OH)2D3 levels were reached in vitamin D-deficient rats. Only the 75-ng dose increased circulating 1,25(OH)2D3 and colonic calcium transport in normal rats. Intravenous [3H]-1,25(OH)2D3 disappeared more rapidly from the circulation of normal rats, suggesting that accelerated metabolic degradative processes for 1,25(OH)2D3 may be present in normal but not in vitamin D-deficient rats and may account for the lack of a biological response to 1,25(OH)2D3 in normal animals.

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