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- ArticleHashimoto S, Drevon CA, Weinstein DB, Bernett JS, Dayton S, Steinberg D.Biochim Biophys Acta. 1983 Nov 29;754(2):126-33.The influence of membrane cholesterol on the activities of acyl-CoA: cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl-CoA reductase was examined in three microsomal subfractions (RNA-rich, RNA-poor, and smooth) that had been enriched with cholesterol by incubation with mixed lipoproteins from hypercholesterolemic rabbit serum. Acyl-CoA: cholesterol acyltransferase activity was significantly stimulated in the three subfractions, particularly in the RNA-rich microsomal component. 3-Hydroxy-3-methylglutaryl-CoA reductase, on the other hand, was suppressed (30%) in only one (RNA-poor) of the three microsomal subfractions, despite a 1.4-fold increase in the concentration of membrane cholesterol. An attempt was made to distinguish between an effect based exclusively on an increase in available cholesterol substrate and an activation of acyl-CoA: cholesterol acyltransferase in RNA-rich microsomes enriched with cholesterol. An experimental design was devised so that substrate cholesterol was provided in the form of heated smooth microsomes and acyl-CoA: cholesterol acyltransferase was provided as a separate preparation in the form of RNA-rich microsomes. Appropriate controls were carried out to test for transfer of cholesteryl ester between the two sets of particles. The results suggested that cholesterol enhanced acyl-CoA: cholesterol acyltransferase activity by serving both as a substrate and as a non-substrate modulator.
- ArticleNg VL, Herndon VL, Mendelson CR, Snyder JM.Biochim Biophys Acta. 1983 Nov 29;754(2):218-26.In the present study, the apolipoproteins associated with a purified surfactant fraction isolated from lung lavage of adult rabbits were characterized. Surfactant purity was assessed by the glycerophospholipid composition and by electron microscopic examination. The purified surfactant was delipidated and the apolipoproteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. By use of this technique at least eight proteins or families of proteins were found to be associated with surfactant. Four of these apolipoproteins were families of proteins of 55-70, 29-36, 26-28 and 22-23 kilodaltons (kDa). All of these apolipoprotein families had acidic isoelectric points (pI less than or equal to 5.6), and were specifically bound to a Con A-Sepharose matrix, indicative that these apolipoprotein families are modified by oligosaccharide side-chains. The finding that neuraminidase treatment degraded the 29-36 kDa family is suggestive that this apolipoprotein family contains sialic acid residues. Three major proteins of 66, 43-45 and 35 kDa and a minor protein of 86 kDa were also observed. These proteins had isoelectric points in the more neutral range (pI 6.0-6.5). The 66 kDa protein (pI 6.4) had the same apparent molecular weight and isoelectric point as the major protein of delipidated rabbit serum and as purified rabbit albumin, which suggests that this protein is albumin. These findings are indicative that the apolipoproteins of surfactant are more numerous and complex than previously reported.
- ArticleKenagy RD, Bierman EL.Biochim Biophys Acta. 1983 Nov 29;754(2):174-80.We tested the effects of fibroblast cell density and proliferation on the activities of acid cholesterol esterase and cathepsins, the lysosomal enzymes which degrade low-density lipoprotein. Rates of cell proliferation were increased by: (1) fibroblast conditioned medium, (2) increasing the time since subculture from 3 to 7 days, and (3) decreasing the plating density of cells. Cathepsin activity was consistently decreased as cellular proliferation was increased by these various methods. Changes in acid cholesterol esterase activity were more variable. For example, acid cholesterol esterase activity was consistently a positive function of cell density only at densities under 3 micrograms protein/cm2, while cathepsin activity increased up to densities of 16 micrograms protein/cm2. However, the activities of both enzymes were lower at cell densities of under 3 micrograms cell protein/cm2 compared to confluent cultures. Sparse fibroblast cultures may provide a unique model system to study low-density lipoprotein metabolism since, at low cell density, LDL receptor activity is high while lysosomal activity is low, making it possible that lysosomal degradation could become the rate-limiting step in the process of LDL degradation rather than receptor-mediated internalization of the lipoprotein. This might then allow an accumulation of lipoprotein-derived cholesteryl esters in the cell. Such a model could be relevant to the propensity of arterial cells to become foam cells during atherogenesis.
- ArticleStern N, Tietz A, Gaton E, Wolman M.Biochim Biophys Acta. 1983 Nov 29;754(2):166-73.Pulmonary lipidosis was induced in rats by including 0.36 and 0.54% chlorocyclizine in their diet. Chemical analyses of the lung tissue revealed a very marked increase in phosphatidylcholine concentration. Phosphatidylglycerol and phosphatidylinositol concentrations were also markedly increased. An increase in the phosphatidylcholine content was also observed in lavage fluid and macrophages. Microscopic examination of the cell fraction showed that almost all the cells of the lavage fluid were macrophages and that histochemically demonstrable acid esterase activity was mostly inversely related to storage of lipids in the cells. Sonication of macrophages isolated from normal or chlorocyclizine-treated rats yielded a soluble acid phospholipase (pH optimum, 4.0) and a neutral (pH optimum, 8.2) membrane-bound, CaCl2-dependent enzyme. An inhibitory effect of chlorocyclizine in vitro on the activity of the soluble phospholipase was shown.
- ArticleKrebs KE, Phillips MC.Biochim Biophys Acta. 1983 Nov 29;754(2):227-30.The mean helical hydrophobic moments (muH) have been used to compare the amphipathic helices of several apolipoprotein classes with the helices in membrane proteins, water-soluble globular proteins and surface-active peptides. The amphipathic helices in serum apolipoproteins have similar muH and mean hydrophobicities to helices in water-soluble globular proteins. The intrinsic surface activities of proteins and peptides, as determined by surface pressure at the air/water interface, correlate with the product (muH . F) where muH is the average value of muH for all helices in the molecule, and F is the fraction of alpha-helix structure in the protein.
- ArticleGonen B, Cole T, Hahm KS.Biochim Biophys Acta. 1983 Nov 29;754(2):201-7.We determined the effects of various degrees of chemical modification of low-density lipoprotein (LDL) on its interaction with receptors present on human fibroblasts, human monocyte-derived macrophages and rat peritoneal macrophages. We isolated LDL (d = 1.019-1.063 g/ml) and carbamylated different numbers of lysine residues and tested its cell-interactive properties, including binding, degradation, and stimulation of [3H]oleate incorporation into cholesteryl oleate. Small carbamylation of LDL (approximately 1-2% of lysine residues) resulted in a reduced ability (70-80% of control) to displace 125I-labeled LDL from fibroblast receptors. Modification of 12.5-25% of lysine residues resulted in a marked increase in the ability of LDL to interact with scavenger receptors and an almost total loss in the ability to interact with apolipoprotein B-E receptors. Acetylated LDL and malondialdehyde-modified LDL inhibited competitively the degradation of 125I-carbamylated LDL by human macrophages. Thus, the extent of modification plays an important role in recognition of modified LDL by scavenger receptors. There also seems to be a range of modification over which LDL is not yet recognized by the scavenger receptor, but its interaction with the apolipoprotein B-E receptor is markedly reduced. This perhaps explains how a small in vivo modification of LDL can result in an increase in residence time of LDL in the subendothelial tissue which can lead to further local interactions, ultimately increasing the atherogenicity of the LDL particle.
- ArticleTocher DR, Boyd GS.Biochim Biophys Acta. 1983 Nov 29;754(2):159-65.The lipid droplet fractions from rat adrenal and bovine adrenocortical tissue were isolated by density ultracentrifugation. The droplet fractions were delipidated and the protein components investigated by SDS-polyacrylamide slab gel electrophoresis. The adrenal lipid droplets from both species displayed a qualitatively similar protein profile, and both contained a major apolipoprotein subunit of Mr 40 000. Incubation of intact, non-delipidated lipid droplets with [gamma-32P]ATP in vitro resulted in the phosphorylation of the Mr 40 000 apolipoprotein subunit in the case of rat lipid droplets, but not in the case of bovine lipid droplets. However, following delipidation of the droplets with diethyl ether/ethanol, the Mr 40 000 apolipoprotein subunit was phosphorylated in both cases upon incubation of the delipidated protein fractions with [gamma-32P]ATP in vitro. Labelling with [gamma-32P]ATP and [3H]diisopropyl phosphorofluoridate indicated that the cholesterol ester hydrolase enzyme protein was not a major constituent of the adrenal lipid droplet protein fraction.
- ArticleNoël SP, Dupras R.Biochim Biophys Acta. 1983 Nov 29;754(2):117-25.The aim of this study was to determine the kinetic parameters of the hepatic uptake of VLDL remnant cholesteryl esters. Rat livers were perfused in situ with a broad range of remnant [3H]cholesteryl ester concentrations of known specific radioactivity. Following exactly 3 min of perfusion, hepatic lipids were extracted and labelled cholesteryl esters were separated by thin-layer chromatography and counted. The rate of cholesteryl ester uptake was a saturable process and the apparent kinetic parameters were determined from the Lineweaver-Burk plot of the data. Km and Vmax were calculated to be 72 microM and 35 nmol cholesteryl ester/min per g liver, respectively. For the purpose of comparison, we have expressed our kinetic parameters in terms of number of particles (Vmax = 0.022 nmol particles/min per g liver and Km = 45 nM) and compared our values with those obtained with chylomicron remnants by another group of investigators (Sherrill, B.C., Innerarity, T.L. and Mahley, R.W. (1980) J. Biol. Chem. 255, 1804-1807). We found that the maximal capacity for the removal of VLDL particles was similar to what was observed with rat chylomicron remnants. In contrast, the Km for the uptake process of VLDL remnant particles was approximately four times higher than that of rat chylomicron remnant particles. Our results are consistent with the hypothesis that hepatic removal of both chylomicron and VLDL remnants is mediated by the same receptor, but suggest that the affinity of VLDL remnants for the hepatic removal process is substantially lower, possibly due to structural differences between the two remnant particles.
- ArticleWang CS, Kloer HU.Biochim Biophys Acta. 1983 Nov 29;754(2):142-9.Fractionation of pancreatic juice by heparin-Sepharose and cholate-Sepharose affinity chromatography indicated that pancreatic carboxylesterase can be separated from pancreatic lipase with the former retained and the latter unretained by both columns. The chromatographic behavior of pancreatic carboxylesterase was found to be similar to that of human milk bile salt-activated lipase. The partially purified pancreatic carboxylesterase had a specific activity of 30 mumol/min per mg protein when assayed with p-nitrophenyl acetate. The reaction mechanism of human pancreatic carboxylesterase was studied using p-nitrophenyl acetate as substrate and taurocholate as activator. The reaction of the enzyme was found to follow a rapid-equilibrium random mechanism. Because of the presence of basal activity, the role of taurocholate can be considered as a non-essential activator and the dissociation constant for the enzyme-taurocholate binary complex was determined to be 0.20 mM. The activation effect of taurocholate consists in increasing the affinity of the enzyme to the substrate (5.6-fold) and in increasing the Vmax (2.3-fold). Based on the kinetic property of human pancreatic carboxylesterase and human milk bile salt-activated lipase with p-nitrophenyl acetate, cholesterol oleate and triolein as substrate, we conclude that they share common substrate specificity but show minor differences in kinetic parameters. Fluorescence studies indicated that both enzymes showed a decreased intrinsic tryptophanyl fluorescence upon incubation with taurocholate. This indicates that bile salt caused a conformational change of the enzymes, with a resultant decreased hydrophobicity in the microenvironment of tryptophan residues.
- ArticleChao YS, Kroon PA, Yamin TT, Thompson GM, Alberts AW.Biochim Biophys Acta. 1983 Nov 29;754(2):134-41.Rabbits fed a wheat starch-casein diet develop a marked hypercholesterolemia and have a slower rate of removal of rabbit 125I-labeled low density lipoproteins (LDL) from plasma. Treating rabbits with mevinolin, a highly potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, at a daily dose of 20 mg per animal prevents the increase in plasma and LDL cholesterol. The mevinolin effect is mediated through an increased rate of removal of rabbit 125I-labeled LDL from plasma. To study the role of mevinolin on the regulation of the hepatic LDL receptor in rabbits, the binding of 125I-labeled LDL and 125I-labeled beta-VLDL (beta-migrating very-low-density lipoproteins) to liver membranes prepared from rabbits fed the wheat starch-casein diet with or without mevinolin was investigated. Liver membranes from wheat starch-casein-fed rabbits have no demonstrable EDTA-sensitive binding activity of 125I-labeled LDL and low (37 ng/mg protein) binding activity of 125I-labeled beta-VLDL. Treatment of the wheat starch-casein fed rabbits with mevinolin results in high levels of specific EDTA-sensitive binding of 125I-labeled LDL (28.7 ng/mg protein) and 125I-labeled beta-VLDL (120 ng/mg protein). To assess the functional role of the hepatic LDL receptor in response to mevinolin, the catabolism of 125I-labeled LDL by perfused rabbit livers was studied. Perfused livers from mevinolin-treated rabbits show a 3.3-fold increase in the rate of receptor-dependent catabolism of 125I-labeled LDL (4.6% X h-1) when compared with that of livers from rabbits not treated with mevinolin (1.4% X h-1). Thus, these studies demonstrate that mevinolin prevents the increase of plasma LDL cholesterol level in rabbits fed a wheat starch-casein diet by regulating the levels of hepatic LDL-binding sites and the rate of receptor-dependent catabolism of LDL by the liver.
- ArticleMougin-Schutz A, Vacher D, Girard-Globa A.Biochim Biophys Acta. 1983 Nov 29;754(2):208-17.Pig duodeno-jejunal mucosa was maintained in organ culture for up to 24 h in Eagle's minimum essential medium containing 10% foal serum. Viability was controlled by determination of alkaline phosphatase and sucrase activity in the tissue. [14C]Leucine incorporation into proteins decreased 3-fold between 2 and 24 h. Newly synthesized secreted proteins were analyzed by SDS-polyacrylamide gel electrophoresis of the whole culture medium. Apolipoprotein A-I specifically measured by immunoelectrophoresis represented 10-20% of newly secreted proteins. Only 10% of apolipoprotein A-I secreted was recovered with the lipoprotein fraction (d less than 1.21). Recombination of the medium with porcine lipoproteins or DMPC vesicles prior to ultracentrifugation allowed, respectively, the recovery of 40 and 80% of apolipoprotein A-I secreted. The lipoprotein fractions also contained some apolipoproteins B and C and, after DMPC recombination, an apolipoprotein of Mr 45 000, most likely apolipoprotein A-IV, representing about 3.5% of newly secreted proteins. The d greater than 1.21 fractions all contained a high Mr protein, identified as IgA, and an unidentified protein of Mr approximately 45 000. The addition of colchicine (125 microM) to the culture medium did not significantly modify either tissue enzyme activities or [14C]leucine incorporation. It reduced total secretion by about 40% between 2 and 8 h of incubation, without interfering with apolipoprotein A-I secretion, which then represented up to 35% of secretion products. This raises the question of the mode of secretion of apolipoprotein A-I, which may be related to the high proportion of its which is secreted free.
- ArticleRobinson C, Hoult JR.Biochim Biophys Acta. 1983 Nov 29;754(2):190-200.The effect of four thromboxane A2-like analogues as inhibitors of thromboxane B2 uptake and metabolism to 13,14-dihydro-15-keto-thromboxane B2 was studied in the perfused guinea-pig lung. We used 5-min infusions containing 1 muCi [3H]thromboxane B2 (10 ng/ml) and measured uptake/accumulation (as tissue to medium ratio) and metabolism to 13,14-dihydro-15-ketothromboxane B2 by radio-TLC. The results showed that thromboxane B2 metabolism is saturable and exhibits substantial dose-dependent inhibition of both processes by U46619 and U44069 endoperoxide analogues (50% inhibition, ID50, in the range 0.5-0.9 microM), pinane thromboxane A2 (a thromboxane A2 partial agonist, ID50 against metabolism, 0.7 microM) and the thromboxane A2 mimetic EPO11 (ID50 against metabolism, 2.6 microM). These agents affected uptake and enzyme transformation steps differentially, thus strengthening the evidence that thromboxane B2 metabolism is a multi-step, uptake-dependent process in this tissue. U46619 did not affect prostaglandin F2 alpha metabolism, nor did prostaglandin F2 alpha inhibit thromboxane B2 metabolism, confirming that thromboxane B2 uptake/metabolism is distinct from the process which handles prostaglandins. Of the four analogues, only pinane thromboxane was a significant substrate for 15-hydroxyprostaglandin dehydrogenase and it was also the best inhibitor of 15-hydroxyprostaglandin dehydrogenase in purified enzyme preparations. These results advance our understanding of the inactivation in lung of thromboxane B2 and invite study of thromboxane A2 itself.