Evidence for carrier proteins in bile acid synthesis The effect of squalene and sterol carrier protein and albumin on the activity of 12α-hydroxylase

Grabowski, Gregory A.; McCoy, Kim E.; Williams, Gale C.; Dempsey, Mary E.; Hanson, Russell F.

doi:10.1016/0005-2760(76)90235-6

Cited by 21 publications

(5 citation statements)

References 23 publications

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“…Previous investigations using radiolabeled cholesterol did not demonstrate the presence of cholesterol in purified SCP (Dempsey et al, 1981) because, in in vitro assays for binding, cholesterol caused aggregation of SCP to higher molecular weight oligomers (Grabowski et al, 1976;Daum & Dempsey, 1979). This phenomenon in combination with sterol micellization and nonspecific binding (Kandutsch & Shown, 1981;Ishibashi & Bloch, 1981) precluded the determination of cholesterol binding by gel filtration assays.…”

Section: Discussionmentioning

confidence: 96%

Fluorescence of .DELTA.5,7,9(11),22-ergostatetraen-3.beta.-ol in micelles, sterol carrier protein complexes, and plasma membranes

Fischer¹,

Cowlen²,

Dempsey³

et al. 1985

Biochemistry

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The fluorescent sterol analogue delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was synthesized and purified by reverse-phase high-performance liquid chromatography. Dehydroergosterol in aqueous solution had a critical micelle concentration of 25 nM and a maximum solubility of 1.3 microM as ascertained from fluorescence polarization and light scattering properties, respectively. Several lines of evidence indicated a close molecular interaction of dehydroergosterol with purified rat liver squalene and sterol carrier protein (SCP). SCP increased the maximal solubility of dehydroergosterol in aqueous buffer. The fluorescence emission spectrum of dehydroergosterol was blue shifted upon addition of SCP. The fluorescence lifetime of dehydroergosterol in aqueous buffer was 2.3 ns; addition of SCP resulted in the appearance of a second lifetime component near 12.4 ns. The SCP increased the fluorescence polarization of monomeric dehydroergosterol in aqueous buffer from 0.033 to 0.086. Scatchard analysis of the binding data indicated that dehydroergosterol interacted with purified rat liver SCP with an apparent KD = 0.88 microM and Bmax = 4.8 microM. At maximal binding, 1.0 mol of dehydroergosterol was specifically bound per mole of SCP. The close molecular interaction of dehydroergosterol with SCP was also demonstrated by energy-transfer experiments. The intermolecular distance between SCP and bound dehydroergosterol was evaluated by fluorescence energy transfer from tyrosine residues of SCP to the conjugated triene series of double bonds in dehydroergosterol. The transfer efficiency was 36%, and R, the apparent distance between the tyrosine energy donor and the dehydroergosterol energy acceptor, was 19 A. The significance of these data obtained in vitro for dehydroergosterol interaction with SCP was also tested in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 96%

Fluorescence of .DELTA.5,7,9(11),22-ergostatetraen-3.beta.-ol in micelles, sterol carrier protein complexes, and plasma membranes

Fischer¹,

Cowlen²,

Dempsey³

et al. 1985

Biochemistry

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“…No explanation for the inhibitory effects can be offered at present. Grabowski et al (1976) reported that squalene and sterol carrier protein stimulated 12a-hydroxylation in microsomes. The preparation of sterol carrier protein (fatty-acid-binding protein) isolated as described by Dempsey et al (1981) used in the present study did not stimulate the reaction.…”

Section: Discussionmentioning

confidence: 99%

“…The reason for this discrepancy is not known. It is possible that the preparation used by Grabowski et al (1976) contained some sterol carrier protein2, which stimulates 12a-hydroxylation in microsomes.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, Seltman et al (1985) reported that sterol carrier protein2 stimulates microsomal 7a-hydroxylation of cholesterol and suggested that it might play a regulatory role in bile-acid biosynthesis. Dempsey and associates have reported that a sterol carrier protein prepared by their procedures, as well as serum albumin, stimulates microsomal 12a-hydroxylation (Grabowski et al, 1976;Dempsey et al, 1981). The two sterol carrier proteins are not identical .…”

Section: Introductionmentioning

confidence: 99%

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The role of sterol carrier protein2 and other hepatic lipid-binding proteins in bile-acid biosynthesis

Lidström-Olsson¹,

Wikvall²

1986

Biochemical Journal

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The ability of different lipid-binding proteins in liver cytosol to affect enzyme activities in bile-acid biosynthesis was studied in whole microsomes (microsomal fractions) and mitochondria and in purified enzyme systems. Sterol carrier protein2 stimulated the 7a-hydroxylation of cholesterol and the 12a-hydroxylation of 5,-cholestane-3a,7a-diol in microsomes and the 26-hydroxylation of cholesterol in mitochondria 2-3-fold. It also stimulated the oxidation of 5-cholestene-3,/,7a-diol into 7a-hydroxy-4-cholesten-3-one in microsomes. The stimulatory effect of sterol carrier protein2 was much less with purified cholesterol 7a-and 26-hydroxylase systems than with microsomes and mitochondria. No stimulatory effect of sterol carrier protein2 was observed with purified 12a-hydroxylase and 3,#-hydroxy-A5-C27-steroid

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“…It was later reported (Bhattacharyya & DeLuca, 1974) that the enzyme is predominantly found in the microsomal fraction of rat liver homogenates and requires the soluble fraction of the cell for maximal activity. The cytosol contains a heat-labile factor that both protects the substrate and stimulates conversion of cholecalciferol into 25(OH)D3 and may well be analogous to the system described for cholesterol biosynthesis and for bile acid biosynthesis (Ritter & Dempsey, 1971;Scallen et al, 1971;Grabowski et al, 1976). In addition, some 25-hydroxylation was observed in the mitochondrial fraction, especially at high substrate concentration (Bhattacharyya & DeLuca, 1973), as well as production of a more-polar metabolite not identical with 25(OH)D3.…”

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confidence: 99%

Characteristics of the rat liver microsomal enzyme system converting cholecalciferol into 25-hydroxycholecalciferol. Evidence for the participation of cytochrome p-450

Madhok¹,

DeLuca²

1979

Biochemical Journal

112

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Properties of the rat hepatic cholecalciferol 25-hydroxylase have been studied. An assay system has been developed in which 25-hydroxycholecalciferol production is linear for at least 2h in both homogenates and microsomal fraction. Furthermore, the initial reaction velocity is linearly related to the amount of liver tissue or microsomal fraction. This enzyme system also metabolizes an analogue of cholecalciferol, namely dihydrotachysterol 3, into 25-hydroxydihydrotachysterol 3. The 25-hydroxylase is in the microsomal fraction and not in mitochondria. It has a Km of 44 nM for cholecalciferol and 360 nM for dihydrotachysterol 3. Its activity is not altered by dietary concentrations of calcium and phosphorus. Vitamin D-deficient rats have higher activities of the hepatic 25-hydroxylase than those receiving 25 ng of cholecalciferol daily. The 25-hydroxylase is inhibited by metyrapone. An atmosphere of CO/O2 (9:1, v/v) inhibits the reaction by 87%. This inhibition is partially reversed by white light. Additionally, cholecalciferol and 25-hydroxycholecalciferol competitively inhibit aminopyrine demethylase. These results support the idea that the cholecalciferol 25-hydroxylase is a cytochrome P-450-dependent mono-oxygenase.

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Evidence for carrier proteins in bile acid synthesis The effect of squalene and sterol carrier protein and albumin on the activity of 12α-hydroxylase

Cited by 21 publications

References 23 publications

Fluorescence of .DELTA.5,7,9(11),22-ergostatetraen-3.beta.-ol in micelles, sterol carrier protein complexes, and plasma membranes

Fluorescence of .DELTA.5,7,9(11),22-ergostatetraen-3.beta.-ol in micelles, sterol carrier protein complexes, and plasma membranes

The role of sterol carrier protein2 and other hepatic lipid-binding proteins in bile-acid biosynthesis

Characteristics of the rat liver microsomal enzyme system converting cholecalciferol into 25-hydroxycholecalciferol. Evidence for the participation of cytochrome p-450

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