Opposite behavior of two isozymes when refolding in the presence of non-ionic detergents.
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GroEL has a greater affinity for the mitochondrial isozyme (mAAT) of aspartate aminotransferase than for its cytosolic counterpart (cAAT) (Mattingly JR Jr, Iriarte A, Martinez-Carrion M, 1995, J Biol Chem 270:1138-1148), two proteins that share a high degree of sequence similarity and an almost identical spatial structure. The effect of detergents on the refolding of these large, dimeric isozymes parallels this difference in behavior. The presence of non-ionic detergents such as Triton X-100 or lubrol at concentrations above their critical micelle concentration (CMC) interferes with reactivation of mAAT unfolded in guanidinium chloride but increases the yield of cAAT refolding at low temperatures. The inhibitory effect of detergents on the reactivation of mAAT decreases progressively as the addition of detergents is delayed after starting the refolding reaction. The rate of disappearance of the species with affinity for binding detergents coincides with the slowest of the two rate-limiting steps detected in the refolding pathway of mAAT. Limited proteolysis studies indicate that the overall structure of the detergent-bound mAAT resembles that of the protein in a complex with GroEL. The mAAT folding intermediates trapped in the presence of detergents can resume reactivation either upon dilution of the detergent below its CMC or by adding beta-cyclodextrin. Thus, isolation of otherwise transient productive folding intermediates for further characterization is possible through the use of detergents. (+info)
Antilithiasic effect of beta-cyclodextrin in LPN hamster: comparison with cholestyramine.
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Beta-Cyclodextrin (BCD), a cyclic oligosaccharide that binds cholesterol and bile acids in vitro, has been previously shown to be an effective plasma cholesterol lowering agent in hamsters and domestic pigs. This study examined the effects of BCD as compared with cholestyramine on cholesterol and bile acid metabolism in the LPN hamster model model for cholesterol gallstones. The incidence of cholesterol gallstones was 65% in LPN hamsters fed the lithogenic diet, but decreased linearly with increasing amounts of BCD in the diet to be nil at a dose of 10% BCD. In gallbladder bile, cholesterol, phospholipid and chenodeoxycholate concentrations, hydrophobic and lithogenic indices were all significantly decreased by 10% BCD. Increases in bile acid synthesis (+110%), sterol 27-hydroxylase activity (+106%), and biliary cholate secretion (+140%) were also observed, whereas the biliary secretion of chenodeoxycholate decreased (-43%). The fecal output of chenodeoxycholate and cholate (plus derivatives) was increased by +147 and +64%, respectively, suggesting that BCD reduced the chenodeoxycholate intestinal absorption preferentially. Dietary cholestyramine decreased biliary bile acid concentration and secretion, but dramatically increased the fecal excretion of chenodeoxycholate and cholate plus their derivatives (+328 and +1940%, respectively). In contrast to BCD, the resin increased the lithogenic index in bile, induced black gallstones in 34% of hamsters, and stimulated markedly the activities of HMG-CoA reductase (+670%), sterol 27-hydroxylase (+310%), and cholesterol 7alpha-hydroxylase (+390%). Thus, beta-cyclodextrin (BCD) prevented cholesterol gallstone formation by decreasing specifically the reabsorption of chenodeoxycholate, stimulating its biosynthesis and favoring its fecal elimination. BCD had a milder effect on lipid metabolism than cholestyramine and does not predispose animals to black gallstones as cholestyramine does in this animal model. (+info)
Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles.
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The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-beta-cyclodextrin (MbetaCD) to selectively extract cholesterol from the plasma membrane. MbetaCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MbetaCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MbetaCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MbetaCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles. (+info)
Cellular cholesterol regulates expression of the macrophage type B scavenger receptor, CD36.
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CD36, the macrophage type B scavenger receptor, binds and internalizes oxidized low density lipoprotein (OxLDL), and may potentially play a role in the development of atherosclerosis. We reported that the native and modified low density lipoproteins increased CD36 mRNA and protein ( J. Biol. Chem. 272: 21654-21659). In this study, we investigated the effect of alterations of cellular cholesterol content on macrophage expression of CD36. Depletion of cholesterol by treatment with beta-cyclodextrins (beta-cyclodextrin [beta-CD] and methylated beta-cyclodextrin [MebetaCD]) significantly decreased CD36 mRNA and 125I-labeled OxLDL binding. Conversely, loading macrophages with cholesterol or cholesteryl ester (acetate) with MebetaCD:cholesterol complexes increased CD36 mRNA, 125I-labeled OxLDL binding, and CD36 surface expression as determined by fluorescence activated cell sorting. Thus, CD36 expression paralleled cellular cholesterol levels after removal of cholesterol with beta-cyclodextrins or addition of cholesterol with MebetaCD:cholesterol complexes. Neither cholesterol depletion nor loading altered expression of type A scavenger receptor mRNA. Kinetics studies showed that changes in CD36 mRNA occurred after changes of cellular cholesterol. Neither beta-cyclodextrins nor MebetaCD:cholesterol altered CD36 mRNA half-life in the presence of actinomycin D, suggesting that alterations in CD36 expression by cholesterol occur at the transcriptional level. These experiments demonstrate that CD36 expression is enhanced by cholesterol and down-regulated by cholesterol efflux, and imply that macrophage expression of CD36 and foam cell formation in atherosclerotic lesions may be perpetuated by a cycle in which lipids drive expression of CD36 in a self-regulatory manner. (+info)
Critical role for cholesterol in Lyn-mediated tyrosine phosphorylation of FcepsilonRI and their association with detergent-resistant membranes.
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Tyrosine phosphorylation of the high affinity immunoglobulin (Ig)E receptor (FcepsilonRI) by the Src family kinase Lyn is the first known biochemical step that occurs during activation of mast cells and basophils after cross-linking of FcepsilonRI by antigen. The hypothesis that specialized regions in the plasma membrane, enriched in sphingolipids and cholesterol, facilitate the coupling of Lyn and FcepsilonRI was tested by investigating functional and structural effects of cholesterol depletion on Lyn/FcepsilonRI interactions. We find that cholesterol depletion with methyl-beta-cyclodextrin substantially reduces stimulated tyrosine phosphorylation of FcepsilonRI and other proteins while enhancing more downstream events that lead to stimulated exocytosis. In parallel to its inhibition of tyrosine phosphorylation, cholesterol depletion disrupts the interactions of aggregated FcepsilonRI and Lyn on intact cells and also disrupts those interactions with detergent-resistant membranes that are isolated by sucrose gradient ultracentrifugation of lysed cells. Importantly, cholesterol repletion restores receptor phosphorylation together with the structural interactions. These results provide strong evidence that membrane structure, maintained by cholesterol, plays a critical role in the initiation of FcepsilonRI signaling. (+info)
Sweet taste responses of mouse chorda tympani neurons: existence of gurmarin-sensitive and -insensitive receptor components.
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Inhibitory effects of gurmarin (gur) on responses to sucrose and other sweeteners of single fibers of the chorda tympani nerve in C57BL mice were examined. Of 30 single fibers that strongly responded to 0. 5 M sucrose but were not or to lesser extent responsive to 0.1 M NaCl, 0.01 M HCl, and 0.02 M quinine HCl (sucrose-best fibers), 16 fibers showed large suppression of responses to sucrose and other sweeteners by lingual treatment with 4.8 microM (approximately 20 microg/ml) gur (suppressed to 4-52% of control: gur-sensitive fibers), whereas the remaining 14 fibers showed no such gur inhibition (77-106% of control: gur-insensitive fibers). In gur-sensitive fibers, responses to sucrose inhibited by gur recovered to approximately 70% of control responses after rinsing the tongue with 15 mM beta-cyclodextrin and were almost abolished by further treatment with 2% pronase. In gur-insensitive fibers, sucrose responses were not inhibited by gur, but were largely suppressed by pronase. These results suggest existence of two different receptor components for sweeteners with different susceptibilities to gur in mouse taste cells, one gur sensitive and the other gur insensitive. Taste cells possessing each component may be specifically innervated by a particular type of chorda tympani neurons. (+info)
Intracellular phosphotyrosine induction by major histocompatibility complex class II requires co-aggregation with membrane rafts.
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Cross-linking MHC class II molecules human leukocyte antigen (HLA-DR) on the surface of THP-1 cells was found to induce their entry into the glycolipid-enriched membrane fraction of the plasma membrane. At the cellular level, this resulted in the synergistic co-aggregation of class II with cholera toxin, a marker of membrane rafts. The accompanying induction of intracellular protein tyrosine phosphorylation could be inhibited by treating cells with methyl-beta-cyclodextrin, a drug that chelates membrane cholesterol and thereby disperses membrane rafts. Signaling could also be inhibited by treating cells with the Src-family kinase inhibitor PP1. Together, these results show that the induced association of class II molecules with membrane rafts can contribute to their aggregation on the cell surface and mediate an association with intracellular protein-tyrosine kinases. (+info)
Comparison of the capacity of beta-cyclodextrin derivatives and cyclophanes to shuttle cholesterol between cells and serum lipoproteins.
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Previous studies from this laboratory have demonstrated that low concentrations of cyclodextrins (<1.0 mm), when added to serum, act catalytically as cholesterol shuttles to accelerate the exchange of free cholesterol between cells and serum lipoproteins. As cholesterol shuttles, cyclodextrins have the potential to serve as pharmacological agents for modifying cholesterol metabolism. In the present study, we have quantitated the cholesterol-shuttling capacity of a series of newly synthesized beta-cyclodextrin derivatives (betaCDs), with varying structure, and two double-decker cyclophanes. The general protocol is as follows. [(3)H]cholesterol-labeled CHOK1 cells are incubated for 2 h with the test compounds alone or together with 5% human serum, and efflux of the cellular [(3)H]cholesterol is measured. As methyl beta-cyclodextrin (MbetaCD) served as the basis for comparison, initial experiments were conducted that demonstrated there was a dose-dependent stimulation of cell cholesterol efflux as the concentration of MbetaCD increased, with an EC(50) that was calculated to be 0.05 mm. To determine the cholesterol-shuttling capacity of the newly synthesized compounds, cell cholesterol efflux is measured when the compounds are present alone, at a concentration of 0.05 mm, or together with 5% human serum. Our results demonstrate that the double-decker cyclophanes are the most efficient cholesterol shuttles. Under our experimental conditions, methyl beta-cyclodextrin (MbetaCD) approximately doubles the efflux of cell cholesterol to serum, whereas one of the double-decker cyclophanes produces a 4-fold stimulation in efflux. Four of the beta-cyclodextrin derivatives (betaCDs) display shuttling ability similar to that of MbetaCD. Furthermore, there does not appear to be a structural pattern among the other betaCDs which could explain their shuttling capacity. (+info)