Low chemical specificity of the nicotinic acetylcholine receptor sterol activation site.
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The nicotinic acetylcholine receptor (nAcChoR) has an absolute requirement for cholesterol if agonist-stimulated channel opening is to occur [Biochemistry 25 (1986) 830]. Certain non-polar analogs could replace cholesterol in vectorial vesicle permeability assays. Using a stopped-flow fluorescence assay to avoid the limitations of permeability assays imposed by vesicle morphology, it was shown that polar conjugates of cholesterol could also satisfy the sterol requirement [Biochim. Biophys. Acta 1370 (1998) 299]. Here this assay is used to explore the chemical specificity of sterols. Affinity-purified nAcChoRs from Torpedo were reconstituted into bilayers at mole ratios of 58:12:30 [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA)/steroid]. When the enantiomer of cholesterol was used, or when the stereochemistry at the 3-hydroxy group was changed from beta to alpha by substituting epicholesterol for cholesterol, activation was still supported. The importance of cholesterol's planar ring structure was tested by comparing planar cholestanol (5alpha-cholestan-3beta-ol) with nonplanar coprostanol (5beta-cholestan-3beta-ol). Both supported activation. Thus, these steroids support activation independent of structural features known to be important for modulation of lipid bilayer properties. This provides indirect support for a steroid binding site possessing very lax structural requirements. (+info)
Transport of plasma membrane-derived cholesterol and the function of Niemann-Pick C1 Protein.
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To visualize the intracellular transport of plasma membrane-derived cholesterol under physiological and pathophysiological conditions, a novel fluorescent cholesterol analog, 6-dansyl cholestanol (DChol), has been synthesized. We present several lines of evidence that DChol mimics cholesterol. The cholesterol probe could be efficiently incorporated into the plasma membrane via cyclodextrin-donor complexes. The itinerary of DChol from the plasma membrane to the cell was studied to determine its dependence on the function of Niemann-Pick C1 (NPC) protein. In all cells, DChol moved from the plasma membrane to the endoplasmic reticulum. Its further transport to the Golgi complex was observed but with marked differences among various cell lines. DChol was finally transported to small (approximately 0.5 microm diameter) lipid droplets, a process that required functional acyl-CoA:cholesterol acyltransferase. In human NPC fibroblasts, NPC-like cells, or in cells mimicking the NPC phenotype, DChol was found in enlarged (>1 microm diameter) droplets. When the NPC-phenotype was corrected by transfection with NPC1, DChol was again found in small-sized droplets. Our data show that NPC1 has an essential role in the distribution of plasma membrane-derived cholesterol by maintaining the small size of cholesterol-containing lipid droplets in the cell. (+info)
Structural features of sterols required to inhibit human sperm capacitation.
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Ejaculated mammalian sperm must undergo a final maturation (capacitation) before they can acrosome-react and fertilize eggs. Loss of cholesterol is an essential step in the capacitation of human sperm. Experimentally maintaining a high level of cholesterol inhibits capacitation, but the mechanism is unknown. The present study investigated the structural features that are required for cholesterol's inhibitory activity. Human sperm also contain much desmosterol, which is lost from sperm during capacitation. Preventing the loss of desmosterol inhibited capacitation (as assessed by acrosomal responsiveness), with an effectiveness approximately equal to cholesterol's inhibitory activity. Other structural analogs were added to the incubation medium to replace sperm cholesterol and desmosterol. Most inhibited capacitation, including those that lacked cholesterol's 3beta-OH group (cholesteryl methyl ether and epicholesterol) and those with modified C17 groups (ergosterol and diosgenin). Two steroids did not inhibit capacitation well. Coprostanol, which has a nonplanar steroid nucleus, had low inhibitory activity that could be explained by an elevated endogenous cholesterol concentration. Epicoprostanol, which has a nonplanar ring structure and a 3alpha-OH group, promoted rather than inhibited capacitation. The inhibitory activity of the analogs was correlated with their ability to promote order of egg phosphatidylcholine as measured by fluorescence anisotropy. In summary, a planar ring structure is required for sterol inhibitory activity, but a 3beta-OH group and a saturated cholesterol-like aliphatic tail on C17 are not required. The present results support the hypothesis that sperm sterols block capacitation by increasing order of phospholipids. (+info)
Liquid-crystalline collapse of pulmonary surfactant monolayers.
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During exhalation, the surfactant film of lipids and proteins that coats the alveoli in the lung is compressed to high surface pressures, and can remain metastable for prolonged periods at pressures approaching 70 mN/m. Monolayers of calf lung surfactant extract (CLSE), however, collapse in vitro, during an initial compression at approximately 45 mN/m. To gain information on the source of this discrepancy, we investigated how monolayers of CLSE collapse from the interface. Observations with fluorescence, Brewster angle, and light scattering microscopies show that monolayers containing CLSE, CLSE-cholesterol (20%), or binary mixtures of dipalmitoyl phosphatidylcholine(DPPC)-dihydrocholesterol all form bilayer disks that reside above the monolayer. Upon compression and expansion, lipids flow continuously from the monolayer into the disks, and vice versa. In several respects, the mode of collapse resembles the behavior of other amphiphiles that form smectic liquid-crystal phases. These findings suggest that components of surfactent films must collapse collectively rather than being squeezed out individually. (+info)
On the substrate specificity of human CYP27A1: implications for bile acid and cholestanol formation.
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The mitochondrial sterol 27-hydroxylase (CYP27A1) is required for degradation of the C27-sterol side chain in bile acid biosynthesis. CYP27A1 seems, however, to have roles beyond this, as illustrated by patients with a deficient sterol 27-hydroxylase due to mutations of the CYP27A1 gene [cerebrotendinous xanthomatosis (CTX)]. These subjects have symptoms ranging from accumulation of bile alcohols and cholestanol to accelerated atherosclerosis and progressive neurologic impairment. The present work describes a detailed investigation on the substrate specificity of recombinant human CYP27A1. In accordance with some previous work with rat liver mitochondria, the activity in general increased with the polarity of the substrate. An obvious example was the finding that cholesterol was 27-hydroxylated more efficiently than cholesterol oleate but less efficiently than cholesterol sulfate. The oxysterols 24S-hydroxycholesterol and 25-hydroxycholesterol were 27-hydroxylated less efficiently than cholesterol, possibly due to steric hindrance. Surprisingly, sterols with a 3-oxo-Delta4 structure were found to be hydroxylated at a much higher rate than the corresponding sterols with a 3beta-hydroxy-Delta5 structure. The rates of hydroxylation of the sterols were: 7alpha-hydroxy-4-cholesten-3-one>4-cholesten-3-one>7alpha-hydroxycholesterol>24-h ydroxy-4-cholesten-3-one> cholesterol>25-hydroxy-4-cholesten-3-one>24-hydroxycholesterol>or=25-hydroxychole sterol. The possibility is discussed that the findings may have implications for oxysterol-mediated regulation of gene expression. The very high activity of CYP27A1 towards the cholestanol precursor 4-cholesten-3-one may be of importance in connection with the accumulation of cholestanol in patients with CTX. (+info)
Electrophysiological studies in cerebrotendinous xanthomatosis.
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Seven patients with cerebrotendinous xanthomatosis (CTX) were studied by electrophysiological techniques. The percentages of abnormalities detected in nerve conduction studies and electroencephalograms were 28.6% (two patients) and 100%, respectively. All patients showed prolonged central conduction times in short latency somatosensory evoked potentials (SSEPs) by tibial nerve stimulation but normal SSEPs by median nerve stimulation. Brain stem auditory evoked potentials and visual evoked potentials were abnormal in three (42.9%) and four patients (57.1%), respectively. These electrophysiological parameters were correlated with the ratio of serum cholestanol to cholesterol concentration. The results of SSEPs suggest that the polyneuropathy in CTX is caused by distal axonopathy affecting longer axons before shorter axons (central-peripheral distal axonopathy). (+info)
Mechanism and stereochemistry in the sequential enzymatic saturation of the two double bonds in cholesta-4,6-dien-3-one.
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The mechanism and stereochemistry in connection with enzymatic conversion of cholesta-4,6-dien-3-one into cholestanol was studied. Rat and mouse liver microsomes are able to catalyze NADPH-dependent sequential saturation of the two double bonds. Evidence was obtained that the saturation of the delta 6-double bond includes transfer of a hydride ion from the B-side of the cofactor to the 7-position of the steroid (mainly 7 beta-position), followed by addition of a proton to the 6 alpha-position (mainly trans addition). The saturation of the delta 4-double bond includes transfer of a hydride ion from the B-side of the cofactor to the 5 alpha-position of the steroid followed by addition of a proton to the 4 beta-position (trans addition). The reduction of the 3-oxo group was found to involve transfer of a hydride ion from the B-side of the cofactor NADPH to the 3 alpha-position of the steroid. The results are in accord with the contention that the enzymatic saturation of the two double bonds involves a polarization of the 3-oxo group making C-7 electrophilic and C-6 nucleophilic in connection with the saturation of the delta 6-double bond and C-5 electrophilic and C-4 nucleophilic in connection with the saturation of the delta 4-double bond. (+info)
Conversion of cholesterol injected into man to cholestanol via a 3-ketonic intermediate.
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Cholesterol-3-(3)H,4-(14)C was injected intravenously in man and its transformation to cholestanol was studied. From the (3)H: (14)C ratios in cholestanol isolated from blood, evidence for the participation of a ketonic intermediate in the conversion was obtained. In a second subject given cholestanol-3-(3)H,4-(14)C the (3)H: (14)C ratios in blood sterols remained unchanged for as long as 1 wk after the injection, which showed that cholestanol did not lose tritium by interconversion with cholestanone. (+info)