Search

The possibility that carrier proteins are involved in bile acid synthesis was investigated using rat liver homogenates. The 105 000 X g supernatant fraction was found to contain heat stable proteins that bound the bile acid precursor, 7alpha-hydroxy-4-cholesten-3-one, and increased the amount of 7alpha, 12alpha-dihydroxy-4-cholesten-3-one formed by the microsomal enzyme, 12alpha-hydroxylase. Subsequent studies were carried out to determine if squalene and sterol carrier protein or albumin, two lipid binding proteins present in the 105 00 X g supernatant fraction of rat liver homogenates, may be responsible for the effects seen with this fraction. Squalene and sterol carrier protein bound several water insoluble bile acid precursors, including 7alpha-hydroxy-4-cholesten-3-one, and increased the apparent activity of 12alpha-hydroxylase. Squalene and sterol carrier protein, however, did not bind either cholic acid or chenodeoxycholic acid. Rat serum albumin also bound 7alpha-hydroxy-4-cholesten-3-one and increased the apparent activity of 12alpha-hydroxylase. Kinetic analysis indicated that the apparent stimulation of 12alpha-hydroxylase by squalene and sterol carrier protein and albumin was due to increased solubilization of the substrate, 7alpha-hydroxy-4-cholesten-3-one. Thus, these studies indicate that bile acid precursor carrier proteins are present in the 105 000 Xg supernatant fraction of rat liver homogenates and suggest that squalene and sterol carrier protein or albumin may participate as carrier proteins in bile acid synthesis.

Rat liver sterol carrier protein (SCP), a major regulator of lipid metabolism and transport, undergoes a rapid turnover and dramatic circadian variation in amount. The level of SCP was quantitated by a specific immunochemical assay using an antibody to homogeneous liver SCP. During a 12-h dark, 12-h light cycle, liver exhibits a biphasic pattern in SCP level. A 7-fold increase in SCP (i.e. from 1 to 7 mg/g of liver) occurs in the dark period, peaking at the midpoint and returning to basal levels by the beginning of the light period. A similar but smaller pattern of variation in SCP amount occurs in the light cycle. To elucidate the basic mechanism responsible for these changes in SCP level, the relative synthetic rate of SCP and mRNA functional activity for SCP were measured during the dark-light cycle. Alterations in the rate of SCP synthesis can account for the variations in SCP concentration. Although large changes occur in relative synthetic rate, no significant changes were found in the level of mRNA for SCP. Therefore, the circadian rhythm in SCP synthesis and amount does not reflect variations in the concentration of mRNA for SCP, but instead is caused by some mechanism controlling the efficiency of translation of SCP mRNA.

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)

The striking changes in amount of rat liver SCP (sterol carrier protein) during a 24-h dark-light cycle are due to alterations in the relative synthetic rate of SCP. However, functional SCP mRNA, measured by a cell-free translational assay, does not fluctuate in the dark-light cycle. Since cell-free translational assays do not always reflect the actual abundance of an mRNA molecule, a specific cDNA hybridization assay was used to directly quantitate SCP mRNA sequences. The cDNA probe was selected from a rat liver library by hybridization to a mixture of synthetic oligonucleotides containing a portion of the sequence of SCP mRNA. The relative amount and size distribution of the SCP mRNA species (approximately 700-800 nucleotides) does not change during the diurnal cycle. To explore possible mechanisms of this translational control, the polysomal distribution of SCP mRNA was compared at the maximum and minimum points of SCP synthesis. No significant amounts of SCP mRNA were present in nonpolysomal ribonucleoprotein particles. Furthermore, no alteration in the relative level of SCP mRNA associated with polysomes or in polysome size occurs at the maximum and minimum points of SCP synthesis. Thus, changes in total SCP mRNA levels or its polysomal distribution cannot account for the diurnal variation in SCP synthesis.

The rat liver enzyme system, which catalyzes reduction of delta 5,7,24-cholestatrien-3 beta-ol to cholesterol (delta 5-cholesten-3 beta-ol), converted radiolabeled delta 5,7,22-cholestatrien-3 beta-ol to delta 5,22-cholestadien-3 beta-ol, but not to cholesterol. This enzyme system thus contains membrane-bound delta 7- and delta 24-reductase and no delta 22-reductase. Kinetic and competition studies showed that the enzyme system contains a single delta 5,7-sterol delta 7-reductase, which is not influenced by unsaturation at the delta 22-position of the sterol side chain. The identity of delta 5,22-cholestadienol was established by chromatographic, spectral and chemical analyses. Use of the enzyme system and readily available delta 5,7,22-cholestatrienol provides a facile procedure for specific production of delta 5,22-cholestadien-3 beta-ol in quantity.