Mechanism of 25-hydroxyvitamin D<sub>3</sub> 24-hydroxylation: incorporation of oxygen-18 into the 24 position of 25-hydroxyvitamin D<sub>3</sub>

Madhok, Thelma C.; Schnoes, Heinrich K.; DeLuca, Hector F.

doi:10.1021/bi00629a015

Cited by 44 publications

(9 citation statements)

References 32 publications

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“…Previous studies using solubilized cytochrome P-450 preparations from normal mammalian kidney have failed to demonstrate 24-hydroxylase activity [5,6]. However, other studies support the involvement of cytochrome P-450 in 24-hydroxylase activity [28][29][30]. The present studies also support this concept, as demonstrated by the presence of reconstituted 24-hydroxylase activity in a cytochrome P-450 fraction isolated by DEAE-Sepharose chromatography.…”

Section: Discussionsupporting

confidence: 80%

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

Gray

Ghazarian

1989

Biochemical Journal

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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.

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Section: Discussionsupporting

confidence: 80%

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

Gray

Ghazarian

1989

Biochemical Journal

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“…Under normal physiological conditions the synthesis of la,25(OH)2D3 is minimal, but another kidney mitochondrial enzyme, designated '25(OH)D3 24-hydroxylase', catalyses the transformation of 25(OH)D3 to 24,25(OH)2D3 [4,5]. The 24-hydroxylase is also a cytochrome P-450-catalysed mono-oxygenase complex [6,7]. We had previously demonstrated [6] that exposure of normal chicks to aminophylline increases the 24-hydroxylase activity dramatically.…”

Section: Introductionmentioning

confidence: 99%

Reciprocal post-translational regulation of renal 1α- and 24-hydroxylases of 25-hydroxyvitamin D3 by phosphorylation of ferredoxin. mRNA-directed cell-free synthesis and immunoisolation of ferredoxin

Mandel

Moorthy

Ghazarian

1990

Biochemical Journal

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We have used a cell-free rabbit reticulocyte translational system programmed with polyadenylated [poly(A)+] RNA prepared from chick kidney tissue to study the synthesis of nascent ferredoxin, a class of iron-sulphur-containing proteins functional in the renal mitochondrial 1 alpha- and 24-hydroxylases of 25-hydroxyvitamin D3. The synthesis of ferredoxin was monitored by determining [35S]methionine incorporation into ferredoxin and quantified by SDS/PAGE and autoradiography after immunoprecipitation from the total translation products. Compared with normal controls, vitamin D deprivation caused a significant increase in the net synthesis of nascent ferredoxin with an Mr of 12,000-13,000. [3H]Orotate incorporation as uridine into kidney poly(A)+ RNA was stimulated by aminophylline, a potent inducer of 25-hydroxyvitamin D3 24-hydroxylase; however, the amount of nascent ferredoxin synthesis was the same as in normal controls. Also, partially purified chick kidney mitochondrial cyclic AMP-stimulated protein kinase catalysed the phosphorylation of ferredoxin in vitro. The catalytic activity of the ferredoxin in 1 alpha- and 24-hydroxylations of 25-hydroxyvitamin D3 in reconstituted systems consisting of cytochrome P-450 and ferredoxin reductase was altered with ferredoxin phosphorylation. The phosphorylation caused inhibition of the 1 alpha-hydroxylase activity while at the same time it stimulated the 24-hydroxylase. Authentic 1 alpha,25- and 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 were used as standards to monitor the separation of the enzymic products by h.p.l.c. using methanol/water (4:1, v/v) as solvent. These results indicate that, in the absence of vitamin D or its metabolites in the deficient state, the synthesis of ferredoxin necessary for the 1 alpha-hydroxylase is accentuated, whereas the stimulation of the 24-hydroxylase requires the phosphorylation of existing ferredoxin without a net gain in its synthesis. This would suggest a post-translational regulation of the 1 alpha- and 24-hydroxylases. A model delineating the various aspects of this study is presented.

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“…Following on from vitamin D synthesis, FAD is required as a cofactor for vitamin D conversions to its active metabolic form of 25 (OH) vitamin D (44), which was the form of vitamin D assayed in this project. Furthermore, vitamin D levels and its ROCs indicating lower levels of vitamin D in cases than controls, correlated most closely with biomarker domains for impaired visual dysfunction and auditory dysfunction in psychosis (Table 4), both of which also held high correlates for disability and severity measures in the psychosis condition (Table 2).…”

Section: Methodsmentioning

confidence: 99%

Fundamental Role of Methylenetetrahydrofolate Reductase 677 C → T Genotype and Flavin Compounds in Biochemical Phenotypes for Schizophrenia and Schizoaffective Psychosis

Fryar-Williams

2016

Front. Psychiatry

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The Mental Health Biomarker Project (2010–2016) explored variables for psychosis in schizophrenia and schizoaffective disorder. Blood samples from 67, highly characterized symptomatic cases and 67 gender and age matched control participants were analyzed for methyl tetrahydrofolate reductase (MTHFR) 677C → T gene variants and for vitamin B6, B12 and D, folate, unbound copper, zinc cofactors for enzymes in the methylation cycle, and related catecholamine pathways. Urine samples were analyzed for indole-catecholamines, their metabolites, and oxidative-stress marker, hydroxylpyrolline-2-one (HPL). Rating scales were Brief Psychiatric Rating Scale, Positive and Negative Syndrome Scale, Global Assessment of Function scale, Clinical Global Impression (CGI) score, and Social and Occupational Functioning Assessment Scale (SOFAS). Analysis used Spearman’s correlates, receiver operating characteristics and structural equation modeling (SEM). The correlative pattern of variables in the overall participant sample strongly implicated monoamine oxidase (MAO) enzyme inactivity so the significant role of MAO’s cofactor flavin adenine nucleotide and its precursor flavin adenine mononucleotide (FMN) within the biochemical pathways was investigated and confirmed as 71% on SEM of the total sample. Splitting the data sets for MTHFR 677C → T polymorphism variants coding for the MTHFR enzyme, discovered that biochemistry variables relating to the wild-type enzyme differed markedly in pattern from those coded by the homozygous variant and that the hereozygous-variant pattern resembled the wild-type-coded pattern. The MTHFR 677C → T-wild and -heterozygous gene variants have a pattern of depleted vitamin cofactors characteristic of flavin insufficiency with under-methylation and severe oxidative stress. The second homozygous MTHFR 677TT pattern related to elevated copper:zinc ratio and a vitamin pattern related to flavin sufficiency and risk of over-methylation. The two gene variants and their different biochemical phenotypes govern findings in relationship to case-identification, illness severity, duration of illness, and functional disability in schizophrenia and schizoaffective psychosis, and establish a basis for trials of gene-guided precision treatment for the management of psychosis.

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Mechanism of 25-hydroxyvitamin D₃ 24-hydroxylation: incorporation of oxygen-18 into the 24 position of 25-hydroxyvitamin D₃

Cited by 44 publications

References 32 publications

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

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

Reciprocal post-translational regulation of renal 1α- and 24-hydroxylases of 25-hydroxyvitamin D3 by phosphorylation of ferredoxin. mRNA-directed cell-free synthesis and immunoisolation of ferredoxin

Fundamental Role of Methylenetetrahydrofolate Reductase 677 C → T Genotype and Flavin Compounds in Biochemical Phenotypes for Schizophrenia and Schizoaffective Psychosis

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Mechanism of 25-hydroxyvitamin D3 24-hydroxylation: incorporation of oxygen-18 into the 24 position of 25-hydroxyvitamin D3

Cited by 44 publications

References 32 publications

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

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

Reciprocal post-translational regulation of renal 1α- and 24-hydroxylases of 25-hydroxyvitamin D3 by phosphorylation of ferredoxin. mRNA-directed cell-free synthesis and immunoisolation of ferredoxin

Fundamental Role of Methylenetetrahydrofolate Reductase 677 C → T Genotype and Flavin Compounds in Biochemical Phenotypes for Schizophrenia and Schizoaffective Psychosis

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Mechanism of 25-hydroxyvitamin D₃ 24-hydroxylation: incorporation of oxygen-18 into the 24 position of 25-hydroxyvitamin D₃