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Biomedical subjects

D B Zilversmit

Publications and source records attributed to D B Zilversmit.

At least 55 records · Page 3Linked to original sources
PubMed1982

Heterogeneity of rabbit intestine brush border plasma membrane cholesterol.

Nonspecific lipid transfer protein accelerated cholesterol exchange from brush border vesicles according to a biphasic time course, but sonicated vesicles made from brush border phospholipids and glycosphingolipids showed a single phase exchange. Removal of surface protein with papain or opening brush border vesicles with deoxycholate did not abolish the biphasic exchange pattern. In brush border vesicles treated with cholesterol oxidase, 21 +/- 10% of the free cholesterol was oxidized rapidly, and the remaining cholesterol was oxidized at a slower rate. Opening vesicles with sodium deoxycholate or treatment with phospholipase C, which degraded 55% of the phospholipids, did not increase the size of the rapidly oxidizable cholesterol pool. The rapidly exchangeable and the rapidly oxidizable cholesterol pools appear to represent the same fraction. In double-labeled brush border vesicles 27 +/- 9% of the cholesterol is present in a readily accessible pool, which slowly equilibrates with the remaining membrane cholesterol. The fractional turnover rate of cholesterol in the readily accessible pool equals 0.07 +/- 0.04 h-1 and is increased to 3.35 h-2 by 12 micrograms/ml of nonspecific lipid transfer protein. The heterogeneous distribution of cholesterol in the intact brush border vesicles may not reflect an inside-outside distribution or interaction of cholesterol with membrane lipids but rather an association of more than two-thirds of the membrane cholesterol with a membrane protein fraction.

Animals↗
PubMed1982

Purification and characterization of lipid transfer protein(s) from human lipoprotein-deficient plasma.

Lipid transfer activities from human plasma have been characterized to determine whether triglyceride and cholesteryl ester transfer proteins are identical. After sequential purification by phenyl-Sepharose, CM-cellulose, chromatofocusing, and gel filtration, both triglyceride and cholesteryl ester transfer activities were purified approximately 15,000-fold compared to lipoprotein-deficient plasma, with a 14% recovery of both transfer activities. The gel filtration fraction showed two bands, Mr 58,300 and 66,400, as determined by electrophoresis in sodium dodecyl sulfate. Two samples, each containing predominately one of the two bands, were obtained by selectively combining the eluates from the gel filtration column. The specific activities of triglyceride and cholesteryl ester transfer promoted by the larger protein were within 10% of those for the smaller protein. The relative rates of transfer for cholesteryl ester, triglyceride, retinyl ester, and cholesteryl ether for each fraction were the same. The transfer of triglyceride by either the large or small molecular weight component was almost completely inhibited by mercurial compounds, whereas cholesteryl ester transfer was relatively unaffected. We conclude that triglyceride and cholesteryl ester are transferred by the same plasma protein(s).

Carrier Proteins↗
PubMed1981

A plasma inhibitor of triglyceride and cholesteryl ester transfer activities.

An inhibitor of triglyceride and cholesteryl ester transfer activities has been identified in the plasmas of several animal species. The inhibitor in human plasma has been separated from lipid transfer protein by chromatography on phenyl-Sepharose and CM-cellulose. The inhibitor is characterized as a protein with Mr approximately 35,000 and an isoelectric point of 4 or less. The inhibitor protein decreased the transfer of both triglyceride and cholesteryl ester between all pairs of very low, low, and high density lipoproteins. Although under most conditions the inhibition of triglyceride and cholesteryl ester transfer was similar, the degree of inhibition of lipid transfer was dependent on the lipoprotein pair studied.

Animals↗
PubMed1981

Lipid transfer proteins in the study of artificial and natural membranes.

Lipid transfer proteins, differing in their specificity for the transfer of lipids and for the surfaces on which they act, have been purified from various mammalian tissues and subsequently characterized. Several of their properties make them useful research tools. They have been used alone or with other techniques to study the distribution and mobility of phospholipids in artificial vesicles and in natural membranes, and have been used to create asymmetric phospholipid vesicles. Lipid transfer proteins are capable of altering the lipid composition of membranes by introducing new lipids or by depletion of existing lipids. Some of the transfer proteins can effect a net transfer of phospholipids, glycosphingolipids and cholesterol from one structure to another, whereas others appear to act primarily in promoting exchange. Some lipid transfer proteins are capable of introducing spin labeled and fluorescent lipid analogs into the outer surface of membranes. Because of lipid transfer proteins do not seem to alter membrane lipid asymmetry or permeability of membranes, they are useful tools for studying the effect of lipid substitution on membrane-mediated transport processes and on various membrane-bound enzyme systems.

Animals↗
PubMed1981

Accelerated transfer of neutral glycosphingolipids and ganglioside GM1 by a purified lipid transfer protein.

The transfer of labeled neutral glycosphingolipids from sonicated phosphatidylcholine vesicles to erythrocyte ghosts is greatly stimulated by a nonspecific lipid transfer protein purified from beef liver. Globo-tetraglycosylceramide is transferred at a rate 40% of that for dipalmitoylphosphatidylcholine. II3-alpha-N-Acetylneuraminosyl-gangliotetraglycosylceramide is also transferred by the transfer protein, either from sonicated phosphatidylcholine vesicles or from ganglioside micelles to erythrocyte ghosts. The nonspecific lipid transfer protein catalyzes the net transfer of glycosphingolipids from brush border membrane vesicles (from rabbit intestine) to sonicated phosphatidylcholine/cholesterol vesicles.

Animals↗
PubMed1981

In vivo influx, tissue esterification and hydrolysis of free and esterified plasma cholesterol in the cholesterol-fed rabbit.

The influx of free and esterified cholesterol into various tissues of cholesterol-fed rabbits is calculated from the tissue [3H] cholesterol and [14C] cholesterol content - corrected for radioactivity in contaminating plasma - after a 3--6 h exposure to in vivo-labeled plasma. The plasma free cholesterol was labeled primarily with 3H and the esterified cholesterol with 14C or vice versa. The influx calculation is based on total 3H and 14C in tissues and two linear equations that take into account esterification and hydrolysis of sterol fractions by the tissues. The influx of esterified cholesterol into tissue samples from aorta, heart, small intestine and lung was 10--80 nmol-g--1-h--1, whereas the influx into adrenal, spleen and liver was from 400--2500 nmol--g-1--h-1. The influx of free cholesterol was considerably higher than expected if free and esterified cholesterol had entered the tissues together as part of plasma lipoproteins. This excess of free cholesterol influx can be ascribed to cholesterol exchange between plasma lipoproteins and tissues, which in several tissues amounted to more than 80% of the total free cholesterol influx. From tissue free and esterified cholesterol radioactivity, one can calculate that 20--70% of the newly entered esterified cholesterol was hydrolyzed by various tissues and that most tissues esterified less than 10% of newly entered cholesterol during the experimental period. However, esterification of plasma cholesterol by adrenals averaged 50% of that taken up during a 3-6 h period.

Animals↗
PubMed1981

The separation of apolipoprotein D from cholesteryl ester transfer protein.

This study addresses the question of whether cholesteryl ester transfer protein and apolipoprotein D are identical. The data presented show that these two proteins do not co-purify during hydrophobic and cationic exchange chromatography and are readily separated by molecular sieve chromatography or electrophoresis. Furthermore, the precipitation of apolipoprotein D by specific antisera did not diminish the transfer activity of lipoprotein-deficient plasma. We conclude that apolipoprotein D and cholesteryl ester transfer protein have significantly different physicochemical properties.

Animals↗
PubMed1981

Arterial influx of esterified cholesterol from two plasma lipoprotein fractions and its hydrolysis in vivo in hypercholesterolemic rabbits.

Arterial influx of esterified cholesterol from 2 plasma lipoprotein fractions, d less than 1.019 and d greater than 1.019, and influx of plasma free cholesterol were determined in each of 15 hypercholesterolemic rabbits with approximately the same plasma cholesterol concentrations but with different extents of arterial lesions. The procedure consisted of injecting intravenously into recipient rabbits [14C]- or [3H]cholesterol-labeled lipoproteins prepared from donor rabbits. The esterified cholesterol of one lipoprotein fraction was labeled primarily with one isotope and that of the other lipoprotein fraction was labeled with the other isotope. Thoracic aortas were removed 4-6 h after lipoprotein injections. The arterial influx of esterified cholesterol was up to 50 times higher in rabbits with maximal lesions than in those with minimal cholesterol deposits. the arterial influx of cholesteryl ester derived from d less than 1.019 lipoproteins was about equal to that derived from the d greater than 1.019 fraction. The amount of cholesteryl ester in plasma d less than 1.019 was approximately 3 times that in d greater than 1.019. Thus, per unit of cholesteryl ester concentration the d less than 1.019 lipoproteins delivered about 1/3 as much cholesteryl ester to the artery as the lipoproteins in the higher density fractions. some 5-40% of plasma esterified cholesterol which had entered the artery was hydrolyzed in the artery during the experimental period. The influx of free cholesterol that could not be accounted for by the influx of intact plasma lipoproteins was 5-80% of the free cholesterol influx. This excess probably represents free cholesterol influx by an exchange between the plasma lipoproteins and the intimal surface of the artery.

Animals↗
PubMed1980

Net transfer of phospholipid by the nonspecific phospholipid transfer proteins from bovine liver.

The nonspecific phospholipid transfer proteins from bovine liver catalyze net transfer of phosphatidylcholine and phosphatidylinositol from phosphatidylcholine/phosphatidylinositol multilamellar vesicles (9 : 1; mol/mol) to either intact or totally delipidated human high density lipoprotein. Under indentical conditions, the phosphatidylcholine-specific exchange protein from bovine liver and the phosphatidylinositol/phosphatidylcholine exchange protein from beef heart do not enhance the net transfer of labeled phosphatidylcholine or phosphatidylinositol from multilamellar vesicles to delipidated high density lipoprotein, but do catalyze phospholipid exchange between multilamellar vesicles and intact high density lipoprotein consistent with their reported specificities. In the presence of nonspecific transfer proteins net transfer of mass in also observed from phosphatidylcholine unilamellar vesicles to rat liver inner mitochondrial membranes plus matrix. These are the first exchange proteins demonstrated to have net mass transfer capability.

Animals↗
PubMed1980

Role of phosphatidylinositol in attachment of alkaline phosphatase to membranes.

The mechanism of release of alkaline phosphatase from membranes by phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was studied. Alkaline phosphatase was readily released from pig kidney microsomes by phospholipase C but not by a variety of other treatments, e.g., high ionic strength, extremes of pH, divalent cations, chelating agents, or analogues of the polar head group of phosphatidylinositol. Alkaline phosphatase released from microsomes by phospholipase C did not bind to phospholipid vesicles containing phosphatidylinositol. Alkaline phosphatase solubilized from microsomes by butanol extraction, however, was able to bind phospholipid vesicles even when they contained no phosphatidylinositol. The ability of butanol-extracted alkaline phosphatase to bind to phospholipid vesicles was destroyed by added phosphatidylinositol-specific phospholipase C. Hydrolysis of added phosphatidylinositol by endogenous phospholipase activity in butanol extracts was also accompanied by loss of binding ability. Loss of binding ability was paralleled by a decrease in the apparent molecular weight of alkaline phosphatase. These results indicate that alkaline phosphatase is attached to membranes by a strong interaction with phosphatidylinositol.

Alkaline Phosphatase↗
PubMed1980

Phosphatidylinositol distribution and translocation in sonicated vesicles. A study with exchange protein and phospholipase C.

The distribution of phosphatidylinositol and phosphatidylcholine in sonicated phospholipid vesicles (phosphatidylcholine : diphosphatidylglycerol : phosphatidylinositol, 90 : 5 : 5 mol%) has been determined by the use of exchange protein from beef heart and phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. Approximately 70% of the phosphatidylinositol in the sonicated vesicles was accessible to the exchange protein and 70--75% was accessible to the phospholipase C. A similar proportion (65%) of the phosphatidylcholine was accessible to the exchange protein suggesting that phosphatidylinositol was not preferentially located in either surface of the phospholipid bilayer. The rate of translocation of both phospholipids was very slow but the rate for phosphatidylcholine (t 1/2 = 4--7 days) appeared to be greater than that for phosphatidylinositol (t 1/2 = 8--60 days). Production of asymmetric vesicles by removing phosphatidylinositol from the outer surface with either exchange protein or phospholipase C did not induce rapid phospholipid translocation.

Animals↗
PubMed1979

Purification and characterization of two phospholipid exchange proteins from bovine heart.

Two phospholipid exchange proteins from bovine heart have been purified approximately 2000-fold and judged greater than 90% pure. The proteins are similar in molecular weight (both 33,400 by polyacrylamide gel electrophoresis and 23,500 by gel filtration), in amino acid composition, and in specificity, although they differ in isoelectric points, 5.3 and 5.6. The transfer of phospholipids between artificial membranes is catalyzed by these proteins at the following relative rates: 100 for phosphatidylinositol, 35 for phosphatidylcholine, 5 for sphingomyelin, and 0.1 for phosphatidylethanolamine. The use of these exchange proteins in the study of mixed phospholipid vesicle structure is demonstrated. The purified proteins catalyze the substitution of one membrane phospholipid species for another at a rate comparable to true exchange. The phospholipid exchange activity is inhibited by the presence of sphingomyelin, and also by reagents which react with sulfhydryl groups. Evidence is presented for two sites of N-ethylmaleimide binding on these exchange proteins. Reaction with one site has little effect on activity and occurs in the absence of membranes. Reaction with the second site occurs in the presence of phospholipid vesicles and leads to complete, irreversible inhibition of exchange activity.

Amino Acids↗
PubMed1979

Exchangeability and rate of flip-flop of phosphatidylcholine in large unilamellar vesicles, cholate dialysis vesicles, and cytochrome oxidase vesicles.

Three model membrane systems have been characterized in terms of their interaction with phospholipid exchange proteins. Large unilamellar vesicles of phosphatidylcholine prepared by ether vaporization are shown to be homogeneous by gel filtration. Phospholipid exchange proteins from three sources are capable of catalyzing the rapid exchange of approximately half of the phospholipid from these vesicles. The remaining pool of radioactive phospholipid is virtually nonexchangeable (t1/2 of several days). Small unilamellar vesicles of phosphatidylcholine prepared by cholate dialysis also exhibit two pools of phospholipid (65% rapidly exchangable, 35% very slowly exchangeable) when incubated with beef liver phospholipid exchange protein. Cytochrome oxidase vesicles prepared both by a cholate dialysis method and by a direct incorporation method have been fractionated on a Ficoll discontinuous gradient, and tested for interaction with beef heart exchange protein. Two pools of phospholipid are once again observed (70% rapidly exchangable, 30% nonexchangeable), even for vesicles which have incorporated the transmembranous enzyme at a phospholipid to protein weight ratio of 2. The size of the rapidly exchangeable pool of phosphatidylcholine for each of the vesicle systems is consistent with the calculated fraction of phospholipid in the outer monolayer. The extremely slow rate of exchange of the second pool of the second pool of phospholipid reflects the virtual nonexistence of phospholipid flip-flop in any of these model membranes.

Biological Transport↗