Assay of 25-Hydroxyvitamin D31α-Hydroxylase in Rat Kidney Mitochondria

Eto, Takeshi; Nakamura, Yoshito; Taniguchi, Tomotaka; Miyamoto, Koji; Nagatomo, Junji; Maeda, Yorio; Higashi, Shinji; Okuda, Kyuichiro; Setoguchi, Toshiaki

doi:10.1006/abio.1998.2567

Cited by 5 publications

(6 citation statements)

References 20 publications

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“…The relatively high K m of mouse CYP27B1 for adrenodoxin and the low concentrations of adrenodoxin in kidney mitochondria [51] suggest that adrenodoxin concentration may limit CYP27B1 activity in vivo. In support of this, Eto et al [52] have shown that the activity of CYP27B1 in rat kidney mitochondria is greatly increased when the mitochondria are solubilized with detergent and supplemented with exogenous adrenodoxin reductase and adrenodoxin.…”

Section: Discussionmentioning

confidence: 85%

Metabolism of substrates incorporated into phospholipid vesicles by mouse 25-hydroxyvitamin D3 1α-hydroxylase (CYP27B1)

Tang

Voo

Nguyen

et al. 2010

The Journal of Steroid Biochemistry and Molecular Biology

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Section: Discussionmentioning

confidence: 85%

Metabolism of substrates incorporated into phospholipid vesicles by mouse 25-hydroxyvitamin D3 1α-hydroxylase (CYP27B1)

Tang

Voo

Nguyen

et al. 2010

The Journal of Steroid Biochemistry and Molecular Biology

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“…However, based on mathematical fitting of our in vitro experimental data and an assumed CYP27B1 rate of 0.1 µM/hr, we estimated the basal amount of CYP27B1 to be 0.1 nM. K m of CYP27B1 was set at 1 µM based on reports measuring the K m between 0.38–2.7 µM [29], [47], [48], [49]. We also assumed the potential for cooperativity in enzymatic conversion of 25OHD to 1,25(OH) 2 D but cooperativity in 25OHD binding to CYP27B1 then also requires 25OHD/VDR affinity to VDRE to be greater than that of 1,25(OH) 2 D/VDR to compensate and fit the in vitro data.…”

Section: Methodsmentioning

confidence: 99%

Vitamin D Binding Protein and Monocyte Response to 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D: Analysis by Mathematical Modeling

et al. 2012

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Vitamin D binding protein (DBP) plays a key role in the bioavailability of active 1,25-dihydroxyvitamin D (1,25(OH)2D) and its precursor 25-hydroxyvitamin D (25OHD), but accurate analysis of DBP-bound and free 25OHD and 1,25(OH)2D is difficult. To address this, two new mathematical models were developed to estimate: 1) serum levels of free 25OHD/1,25(OH)2D based on DBP concentration and genotype; 2) the impact of DBP on the biological activity of 25OHD/1,25(OH)2D in vivo. The initial extracellular steady state (eSS) model predicted that 50 nM 25OHD and 100 pM 1,25(OH)2D), <0.1% 25OHD and <1.5% 1,25(OH)2D are ‘free’ in vivo. However, for any given concentration of total 25OHD, levels of free 25OHD are higher for low affinity versus high affinity forms of DBP. The eSS model was then combined with an intracellular (iSS) model that incorporated conversion of 25OHD to 1,25(OH)2D via the enzyme CYP27B1, as well as binding of 1,25(OH)2D to the vitamin D receptor (VDR). The iSS model was optimized to 25OHD/1,25(OH)2D-mediated in vitro dose-responsive induction of the vitamin D target gene cathelicidin (CAMP) in human monocytes. The iSS model was then used to predict vitamin D activity in vivo (100% serum). The predicted induction of CAMP in vivo was minimal at basal settings but increased with enhanced expression of VDR (5-fold) and CYP27B1 (10-fold). Consistent with the eSS model, the iSS model predicted stronger responses to 25OHD for low affinity forms of DBP. Finally, the iSS model was used to compare the efficiency of endogenously synthesized versus exogenously added 1,25(OH)2D. Data strongly support the endogenous model as the most viable mode for CAMP induction by vitamin D in vivo. These novel mathematical models underline the importance of DBP as a determinant of vitamin D ‘status’ in vivo, with future implications for clinical studies of vitamin D status and supplementation.

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“…To assay mitochondrial P450 accurately, saturation of electron-transferring proteins is necessary (19,20). When inadequate quantities of adrenodoxin and NADPH-adrenodoxin reductase were used, 27-hydroxylase activity could not be measured accurately (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Another method using exogenous and endogenous cholesterol as a substrate was developed (18), which requires an oxidation step to quantify in HPLC. To measure the activity more simply and effectively, we used 7 ␣ -hydroxy-4-cholesten-3-one (HCO) as a substrate and were able to monitor its 27-hydroxylated product at 240 nm in HPLC.In addition, when the mitochondrial suspension was used as an enzyme source, the maximal velocity could not be measured, because electron-transferring proteins were not saturated (19,20). To solve this problem, we solubilized the enzyme from the rat liver mitochondria and fortified it with saturated amounts of adrenodoxin and NADPH-adrenodoxin reductase as the electron transfer system.…”

mentioning

confidence: 99%

“…In addition, when the mitochondrial suspension was used as an enzyme source, the maximal velocity could not be measured, because electron-transferring proteins were not saturated (19,20). To solve this problem, we solubilized the enzyme from the rat liver mitochondria and fortified it with saturated amounts of adrenodoxin and NADPH-adrenodoxin reductase as the electron transfer system.…”

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confidence: 99%

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Assay method for mitochondrial sterol 27-hydroxylase with 7α-hydroxy-4-cholesten-3-one as a substrate in the rat liver

Ota

Eto

Tanaka

et al. 2003

Journal of Lipid Research

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Mitochondrial sterol 27-hydroxylase (EC 1.14.13.15) is an important enzyme, not only in the formation of bile acids from cholesterol intermediates in the liver but also in the removal of cholesterol by side chain hydroxylation in extrahepatic tissues. The enzyme has been assayed by complicated methods using radiolabeled substrates or deuterium-labeled tracers. These methods may be inaccurate for measuring enzyme activity, because the amount of electron-transferring proteins may be insufficient for maximal velocity. To solve this problem, after solubilization of the enzyme from rat liver mitochondria with n -octyl-␤ -D -glucopyranoside (OGP), we measured the enzyme activity by incubating the solubilized enzyme with saturated amounts of electron-transferring proteins. In our assay system, using 7 ␣ -hydroxy-4-cholesten-3-one (HCO) as a substrate, we could easily measure the product, 7 ␣ ,27-dihydroxy-4-cholesten-3-one, with HPLC monitoring absorbance at 240 nm. The product formation was proportionate to the time up to 5 min and the protein concentration up to 0.5 mg of protein/ml. The maximal velocity of the enzyme was 1.1 nmol/min/mg of protein, which was 4-to 16-fold higher than previously reported values. A simple and accurate assay method for sterol 27-hydroxylase in rat liver mitochondria is herein described. It is well known that sterol 27-hydroxylase (CYP27A1) has an important role in the oxidation of the side chain of sterols in the formation of bile acids from cholesterol in the liver (1-4). In addition, the enzyme has another important role in the removal of cholesterol by side chain hydroxylation (5). Furthermore, this enzyme is present not only in the liver but also in extrahepatic tissues, i.e., brain (5), lung alveolar macrophages (6-9), endothelial cells (7, 10-12), ovary (13), intestine, skin, long bone, and calvaria (14).To measure enzyme activity, 5 ␤ -cholestane-3 ␣ ,7 ␣ ,12 ␣ -triol (C-triol) has been used as a substrate. However, this assay system requires radiolabeled substrate (2-4) or deuterium-labeled tracers in the gas chromatography-mass spectrometry with selected ion monitoring (GC-SIM) methods (15-17). These assay methods are complicated and time-consuming. Another method using exogenous and endogenous cholesterol as a substrate was developed (18), which requires an oxidation step to quantify in HPLC. To measure the activity more simply and effectively, we used 7 ␣ -hydroxy-4-cholesten-3-one (HCO) as a substrate and were able to monitor its 27-hydroxylated product at 240 nm in HPLC.In addition, when the mitochondrial suspension was used as an enzyme source, the maximal velocity could not be measured, because electron-transferring proteins were not saturated (19,20). To solve this problem, we solubilized the enzyme from the rat liver mitochondria and fortified it with saturated amounts of adrenodoxin and NADPH-adrenodoxin reductase as the electron transfer system. In this study, we describe a simple and accurate assay method for mitochondrial sterol 27-hydroxylation.

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Assay of 25-Hydroxyvitamin D31α-Hydroxylase in Rat Kidney Mitochondria

Cited by 5 publications

References 20 publications

Metabolism of substrates incorporated into phospholipid vesicles by mouse 25-hydroxyvitamin D3 1α-hydroxylase (CYP27B1)

Metabolism of substrates incorporated into phospholipid vesicles by mouse 25-hydroxyvitamin D3 1α-hydroxylase (CYP27B1)

Vitamin D Binding Protein and Monocyte Response to 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D: Analysis by Mathematical Modeling

Assay method for mitochondrial sterol 27-hydroxylase with 7α-hydroxy-4-cholesten-3-one as a substrate in the rat liver

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