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Biomedical subjects

R Bittman

Publications and source records attributed to R Bittman.

At least 109 records · Page 6Linked to original sources
PubMed1984

Decreases in rates of lipid exchange between Mycoplasma gallisepticum cells and unilamellar vesicles by incorporation of sphingomyelin.

The kinetics of exchange of radiolabeled cholesterol and phospholipids between Mycoplasma gallisepticum cells and an excess of small unilamellar phospholipid/cholesterol vesicles were studied using cells enriched with different phospholipid classes but the same fatty acyl content. The rates of the rapidly exchanging cholesterol and phospholipid pools were markedly slower in sphingomyelin-containing cells than in phosphatidylcholine-containing cells. The decreased rates of spontaneous cholesterol and protein-mediated phospholipid exchange from the outer leaflet of the mycoplasma membrane are attributed to stronger interactions between sphingomyelin and cholesterol than between phosphatidylcholine and cholesterol. On the other hand, an increase in cholesterol exchange rate was found on incorporation of a cyclopentano-1,3-diacyl-2-phosphatidylglycerol, which interacts less with cholesterol than does the analogous glycero-1,2-diacyl-sn-3-phosphatidylglycerol.

Cell Membrane↗
PubMed1984

Isolation of protein(s) containing chloride ion transport activity from thylakoid membranes.

Extracts of detergent-treated thylakoids, when reconstituted into azolectin/cholesterol/dicetyl phosphate vesicles, stimulate chloride ion efflux as measured with a Cl--sensitive electrode. This stimulation is inhibited by piretanide, an active chloride ion transport inhibitor in fish intestinal epithelia. This stimulation is also abolished by pretreatment of extracted proteins with trypsin. Antiserum raised to efflux-active proteins inhibits a cation-driven Cl- influx in nonenergized thylakoids, as measured by a flow dialysis technique.

Biological Transport, Active↗
PubMed1984

Influence of cholesterol on bilayers of ester- and ether-linked phospholipids. Permeability and 13C-nuclear magnetic resonance measurements.

13C-NMR and permeability studies are described for sonicated vesicles of phosphatidylcholines bearing two 16-carbon saturated hydrocarbon chains with (a) one ether linkage at carbon 1 (3) or 2 of glycerol and one ester linkage at carbon 2 or 1 (3) of glycerol; (b) two ether linkages and (c) two ester linkages at carbons 1 (3) and 2 of glycerol. The results of 13C-NMR relaxation enhancement measurements using cholesterol enriched with 13C at the 4 position indicate that no significant relocation of the cholesterol molecules takes place in the bilayer when a methylene group is substituted for a carbonyl group in phosphatidylcholine. The 4-13C atom of cholesterol undergoes similar fast anisotropic motions in diester- and diether -phosphatidylcholine bilayers, as judged by spin-lattice relaxation time measurements in the liquid-crystalline phase; although the fast motions are unaltered, linewidth and spin-spin relaxation time measurements suggested some restriction of the slow motions of cholesterol molecules in bilayers from phosphatidylcholines containing an O-alkyl linkage at the sn-2 position instead of an acyl linkage. At temperatures above the gel to liquid-crystal phase transition, the kinetics of ionophore A23187-mediated 45Ca2+ efflux from vesicles prepared from each type of phosphatidylcholine molecule were the same; the kinetics of spontaneous carboxyfluorescein diffusion from diester- and diether -phosphatidylcholine vesicles were the same, whereas mixed ether/ester phosphatidylcholine molecules gave bilayers which are less permeable. The rate constants were reduced on cholesterol incorporation into the bilayers of each type of phosphatidylcholine molecule. The reductions were not statistically significant for 45Ca2+ release. The rate constants for carboxyfluorescein release were also reduced by cholesterol to the same extent in vesicles from diester-, diether -, and 1-ether, and 1-ether-2-ester-phosphatidylcholines; however, a smaller reduction was noted in bilayers from the 1-ester-2-ether analog. The results provide further evidence that there are no highly specific requirements for ester or ether linkages in phosphatidylcholine for cholesterol to reduce bilayer permeability. This is a reflection of the fact that in both diester- and diether -phosphatidylcholine bilayers, the 4-13C atom of cholesterol is located in the region of the acyl carboxyl group or the glyceryl ether oxygen atom.

Cholesterol↗
PubMed1984

Dissociation kinetics and equilibrium binding properties of polyene antibiotic complexes with phosphatidylcholine/sterol vesicles.

The interactions of sonicated vesicles with the polyene antibiotics amphotericin B, candicidin, mediocidin , and a water-soluble, guanidine derivative of amphotericin B were examined by UV-visible spectroscopy at concentrations below which the polyenes become self-associated. The association constants, Kapp, and the numbers of binding sites per sterol or phospholipid molecule (n) were determined at 30 degrees C and pH 7.4. A single class of binding sites was found, with no evidence of cooperativity. For the binding of mediocidin , amphotericin B, and the guanidine derivative with phosphatidylcholine (PC), PC/cholesterol, and PC/ergosterol vesicles, Kapp was in the range of (1.0-3.0) X 10(6) M-1; Kapp was higher for candicidin-vesicle interaction, reaching 9.0 X 10(6) M-1 with PC/ergosterol vesicles. Binding of the guanidine derivative of amphotericin B to PC vesicles lacking sterol was extensive (n = 0.46); since the other polyenes, which have low aqueous solubilities, had n less than 0.05, positive charges in the mycosamine moiety appear to enhance the extent of polyene antibiotic interaction with the glycerophospholipid head group. Higher values of n (and, therefore, of nKapp ) were found with sterol-containing than with sterol-free vesicles, suggestive of penetration of the polyenes toward the interior of the bilayer when sterol is present. For binding to PC/sterol vesicles, nKapp followed the order of candicidin greater than guanidine derivative of amphotericin B greater than amphotericin B much greater than mediocidin . The values of n and nKapp were appreciably higher for amphotericin B-ergosterol than for amphotericin B-cholesterol interaction in vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Amphotericin B↗
PubMed1984

Spin-label studies of the oligomeric structure of band 3 protein in erythrocyte membranes and in reconstituted systems.

A spin-labeled fatty acid (16-doxylstearic acid), linked by an ester bond to a maleimide or a nitrene residue, was covalently attached to band 3 of erythrocyte membranes. The electron spin resonance spectrum of the spin-labeled protein was examined at different temperatures in: (a) whole erythrocyte ghosts; (b) ghosts depleted of spectrin and actin; (c) alkaline-treated ghosts; (d) vesicles made with purified band 3 reassociated with dimyristoylphosphatidylcholine. Most spectra are composite with a major component corresponding to a large overall splitting. The determination of the percentage of the immobilized component was carried out by pairwise subtraction. At low temperatures (1-7 degrees C), the highest fraction of immobilized component was found in dimyristoylphosphatidylcholine vesicles (approx. 100%); alkaline-treated membranes had approx. 75% of the immobilized component at the same temperature; whole erythrocyte, spectrin/actin-depleted and spectrin/actin/ankyrin-depleted ghosts gave identical results (approx. 60% of immobilized component). The immobilized fraction decreased in all samples with increasing temperature or addition of a nonsolubilizing concentration of dodecyl octaethylene glycol monoether. In dimyristoylphosphatidylcholine vesicles, however, the modification in the ration of the two components was obtained only above the lipid transition temperature (23 degrees C). The strong immobilization of the spin-labeled lipid chain at all temperatures suggested trapping of the lipid chain between proteins. At low temperature, in dimyristoylphosphatidylcholine vesicles or in alkaline-treated ghosts, lipid-protein segregation is likely to take place. In whole erythrocyte ghosts, on the other hand, the large contribution of the motionally restricted component at physiological temperature indicates the oligomeric nature of band 3. Partial dissociation of the oligomers occurs as the temperature is increased, but the presence or absence of cytoskeletal proteins has no influence on the state of oligomerization of band 3.

Actins↗
PubMed1984

Kinetics of cholesterol and phospholipid exchange between Mycoplasma gallisepticum cells and lipid vesicles. Alterations in membrane cholesterol and protein content.

The kinetics of exchange of radiolabeled cholesterol and phospholipids between intact Mycoplasma gallisepticum cells and unilamellar lipid vesicles were investigated over a wide range of cholesterol/phospholipid molar ratio. The change in cholesterol/phospholipid molar ratio was achieved by adapting the sterol-requiring M. gallisepticum to grow in cholesterol-poor media, providing cells with decreased unesterified cholesterol content. At least 90% of the cholesterol molecules in unsealed M. gallisepticum membranes underwent exchange at 37 degrees C as a single kinetic pool in the presence of albumin (2%, w/v). However, we observed biphasic exchange kinetics with intact cells, indicating that cholesterol translocation from the inner to outer monolayers was rate-limiting in the exchange process. Approximately 50% of the cholesterol molecules were localized in each kinetic pool, independent of the cholesterol/phospholipid molar ratio in the cells and vesicles. A striking change in the kinetic parameters for cholesterol exchange occurred between 20 and 26 mol % cholesterol; for example, when the cholesterol/phospholipid molar ratio was decreased from 0.36 to 0.25, the half-time for equilibration of the two cholesterol pools at 37 degrees C decreased from 4.6 +/- 0.5 to 2.5 +/- 0.1 h. Phospholipid exchange rates were also enhanced on decreasing the membrane cholesterol content. The ability of cholesterol to modulate its own exchange rate, as well as that of phospholipids, is suggested to arise from the sterol's ability to regulate membrane lipid order. Extensive chemical modification of the membrane surface by cross-linking of some of the protein constituents with 1,4-phenylenedimaleimide decreased the cholesterol exchange rate. Depletion of membrane proteins by treatment of growing cultures with chloramphenicol increased the cholesterol exchange rate, possibly because of removal of some of the protein mass that may impede lipid translocation. The observations that phospholipid exchange was one order of magnitude slower than cholesterol exchange and that dimethyl sulfoxide, potassium thiocyanate, and potassium salicylate enhanced the cholesterol exchange rate are consistent with a mechanism involving lipid exchange by diffusion through the aqueous phase.

Cholesterol↗
PubMed1984

Distribution and movement of sterols with different side chain structures between the two leaflets of the membrane bilayer of mycoplasma cells.

Mycoplasma gallisepticum was adapted to grow with delta 5-sterols modified in the aliphatic side chain, and stopped-flow kinetic measurements of filipin association were made to estimate the sterol distribution between the two leaflets of the membrane. Cholesterol derivatives with unsaturated side chains (desmosterol, cis- and trans-22-dehydrocholesterol, and cholesta-5,22E,24-trien-3 beta-ol) or an alkyl substituent (beta-sitosterol) were predominantly (86-94%) localized in the outer leaflet of the bilayer. However, cholesterol, 20-isocholesterol, and sterols with side chains of varying lengths (in the 20(R)-n-alkylpregn-5-en-3 beta-ol series where the alkyl group ranged from ethyl to undecyl) were distributed nearly symmetrically between the two halves of the bilayer. Kinetic measurements of beta-[14C]sitosterol and [14C]desmosterol exchange between M. gallisepticum cells and an excess of sonicated sterol/phosphatidylcholine vesicles confirmed the filipin-binding studies. More than 90% of these radiolabeled sterols underwent exchange at 37 degrees C with unlabeled sterols in vesicles over a period of 12-14 h in the presence of 2% (w/v) albumin. beta-[14C]Sitosterol exchange was characterized by biphasic exchange kinetics, indicative of two pools of sitosterol molecules in the cell membrane. Only a single kinetic pool was detected for [14C]desmosterol exchange. Stopped flow measurements of filipin binding to beta-sitosterol and stigmasterol also revealed an asymmetrical localization of these sterols in membranes of growing Mycoplasma. capricolum cells. When an early exponential culture of beta-sitosterol- or stigmasterol-adapted M. capricolum was transferred to a sterol-rich medium at 37 degrees C, approximately three-quarters of the beta-sitosterol or stigmasterol was localized in the outer leaflet after growth was continued for 6 h; in contrast, cholesterol was distributed symmetrically after about 1 h. The asymmetric localization of sterols with alkylated or unsaturated side chains suggests that growth-supporting sterols need not be translocated extensively into the inner leaflet of the bilayers of M. gallisepticum and M. capricolum.

Desmosterol↗
PubMed1982

Organization of unesterified cholesterol in high density lipoproteins probed by filipin.

The initial rate of filipin association with unesterified cholesterol in high density lipoproteins (HDL) was measured by stopped-flow spectrophotometry to assess the roles played by apolipoproteins and phospholipids in modulating the surface exposure of cholesterol. The initial rate of filipin-unesterified cholesterol association was enhanced upon hydrolysis of the glycerophospholipids of human HDL3 by phospholipase A2. Rate enhancements were also observed following trypsin-catalyzed hydrolysis of apolipoprotein A-I in canine HDL and of apolipoproteins A-I and A-II in human HDL3. However, the initial rate of filipin-unesterified cholesterol association was not altered upon incubation of HDL3 with polymorphonuclear cells, which causes hydrolysis of apolipoprotein A-II but leaves apolipoprotein A-I intact. These results are consistent with the general structural model of HDL in which unesterified cholesterol, apolipoproteins and glycerophospholipids are presumed to be localized at the surface of the HDL particle. From these studies and from results indicating that the initial rate of filipin-unesterified association was enhanced in canine HDL hybrids in which 50% of the apolipoprotein A-I had been replaced by apolipoprotein A-II, we also conclude that apolipoprotein A-I in HDL is in closer proximity to unesterified cholesterol than apolipoprotein A-II. Thus, it appears that rapid kinetic measurements of filipin-cholesterol association may be useful in assessing the organization of unesterified cholesterol in serum lipoproteins.

Animals↗
PubMed1982

Equilibrium binding of amphotericin B and its methyl ester and borate complex to sterols.

Scatchard analysis of the binding of amphotericin B to sterols in egg phosphatidylcholine/sterol vesicles revealed that amphotericin B was bound to ergosterol by approximately one order of magnitude more tightly than to cholesterol. Amphotericin B methyl ester formed a tighter complex with each of the sterols investigated than did amphotericin B, but the difference in the apparent binding constants towards ergosterol and cholesterol was not as large as with amphotericin B. Amphotericin B borate formed weaker complexes with vesicle-bound sterols than amphotericin B and amphotericin B methyl ester.

Amphotericin B↗
PubMed1982

Phospholipid interconversions in Mycoplasma capricolum.

Mycoplasma capricolum cells increase their phospholipid content by incorporating exogenous phospholipids from the growth medium. Growing the cells in media with increasing serum concentrations resulted in a massive incorporation of phosphatidylcholine and sphingomyelin (up to about 50% of total phospholipids) into the cell membrane. The incorporation of the exogenous phospholipids had essentially no effect on the rate of cell growth and did not decrease the overall phospholipid biosynthesis of the cells. Thus, the ratio of phospholipid to protein in membranes from cells grown with 5% horse serum was 0.5 (mumol/mg) compared to 0.3 (mumol/mg) in cells grown without serum, and the relative content of charged polar lipids was apparently decreased. The consequence of the incorporation of exogenous phosphatidylcholine was an alteration in the relative amount of the major end-products of the de novo phospholipid biosynthesis; a marked increase in the ratio of diphosphatidylglycerol to phosphatidylglycerol was observed. The possibility that the increase in the ratio of diphosphatidylglycerol to phosphatidylglycerol is part of a control mechanism to maintain a mixture of bilayer and non-bilayer lipids is discussed.

Animals↗
PubMed1982

Isolation of mycoplasma membranes by dicyclohexylcarbodiimide-induced lysis.

A simple procedure was devised to prepared membranes from Mycoplasma gallisepticum cells. The cells were lysed in an isosmotic NaCl solution by dicyclohexylcarbodiimide, which blocks ATPase activity and interferes with the regulation of cell volume. The procedure can be used to isolate membranes of other osmotically resistant mycoplasmas.

Bacteriolysis↗
PubMed1981

Symmetrical distribution and rapid transbilayer movement of cholesterol in Mycoplasma gallisepticum membranes.

The exchange of cholesterol between [14C]cholesterol-labeled Mycoplasma gallisepticum cells and an excess of sonicated egg phosphatidylcholine/cholesterol vesicles (molar ratio of 0.9) was measured. More than 90% of the radioactive cholesterol underwent transfer from intact cells to the vesicles. The kinetics of the transfer was biphasic. About 50% of the radioactive cholesterol was exchanged with a half-time of about 4 h. The residual was exchanged at a slower rate with a half-time of about 9 h at 37 degrees C. Bovine serum albumin had a pronounced effect in enhancing both the fast and slow rates of cholesterol exchange, but did not affect the pool sizes significantly. The half-time for equilibration of the two pools in the presence of 2% albumin, calculated using a reversible two-pool method of analysis, was 6.2 h. The effect of albumin was also obtained with isolated membrane preparations and with cells treated with growth inhibitors, suggesting that this effect is independent of albumin preservation of cell viability. The rate enhancement of albumin was concentration dependent with maximal effects observed with greater than or equal to 2%, where the rates of exchange of both the rapidly and slowly exchanging pools were twice as fast. The mechanism by which albumin may affect the exchange rates is discussed.

Cell Membrane↗
PubMed1981

Determination of cholesterol asymmetry by rapid kinetics of filipin-cholesterol association: effect of modification in lipids and proteins.

The rapid kinetic behavior of filipin association with cholesterol was unaffected by binding of water-soluble proteins to vesicle and mycoplasma membranes and by proteolytic digestion of mycoplasma membrane proteins. The kinetic properties were, however, dependent on the membrane phospholipids, in that the initial rate of filipin association with cholesterol was enhanced by phospholipase A2 treatment by the incorporation of lysophosphatidylcholine, and by increasing the degree of unsaturation in phospholipid vesicles and mycoplasma membranes. The second-order rate constant was also dependent on th mol % of cholesterol in small unilamellar vesicles but not in large unilamellar vesicles. The ratio of rate constants in intact mycoplasma cells relative to isolated membranes provides an estimate of cholesterol distribution in membranes [Bittman, R., & Rottem, S. (1076) Biochem. Biophys. Res. Commun. 71, 318; Clejan, S., Bittman, R., & Rottem, S. (1978) Biochemistry 17, 4579]. This ratio was unaffected by proteolytic digestion of intact cells and by the incorporation of exogenous phospholipids into the Mycoplasma capricolum cell membrane. However, on cross-linking of surface proteins of M. capricolum by dimethylsuberimidate, cholesterol was localized predominantly in the outer half of the bilayer. On aging of mycoplasma cultures, the cholesterol distribution remained constant in membranes of M. capricolum cells but was enriched in the outer leaflet of the Mycoplasma gallisepticum cell membrane. The results of these experiments are discussed in relation to the use of the rapid kinetics of filipin binding as a probe of cholesterol distribution.

Cell Membrane↗
PubMed1981

Effects of sterol structure and exogenous lipids on the transbilayer distribution of sterols in the membrane of Mycoplasma capricolum.

Stopped-flow kinetic measurements of the association of filipin with sterols in intact cells and isolated membranes of Mycoplasma capricolum were used to study the effects of varying the phospholipid in the membrane. The phospholipid composition and content of the membrane were varied by growing cells in an albumin-containing medium with cholesterol, palmitic and oleic acids, and various concentrations of exogenous phospholipids. The exogenous phospholipids (phosphatidylcholine, sphingomyelin, and phosphatidic acid) were incorporated up to levels of approximately 50% of the total membrane phospholipids but had no effect on the distribution of cholesterol between the two halves of the membrane bilayer. The sterol structure was varied by growing the cells with 10/micrograms/mL of either cholesterol, beta-cholestanol, 4,6-cholestadien-3 beta-ol, ergosterol, beta-sitosterol, or stigmasterol. With cholesterol, beta-cholestanol, and 4,6-cholestadien-3 beta-ol, approximately 65% of the sterol was found to be present in the outer half of the lipid bilayer. With ergosterol, beta-sitosterol, and stigmasterol, about 89% of the sterol is localized in the outer half of the membrane bilayer. Thus, the behavior of the alkyl-substituted sterols differs from that of cholesterol. The extent to which a sterol is distributed asymmetrically between the two halves of the bilayer is not related to the extent to which maximum growth is produced. These results suggest that growth-supporting sterols need not be translocated extensively.

Cell Membrane↗
PubMed1978

Cholesterol distribution between the two halves of the lipid bilayer of human erythrocyte ghost membranes.

The binding of filipin with cholesterol in sealed and unsealed ghosts prepared from human erythrocytes and in right-side-out and inside-out vesicles prepared from ghosts follows second order kinetics (first order in each reactant). The second order rate constant of interaction of filipin with cholesterol, determined by stopped flow measurements of the initial rate, is slower in sealed ghosts than in unsealed ghosts by a factor of 2.0, whereas identical rate constants were obtained with right-side-out and inside-out vesicles. These results suggest that the cholesterol accessible to rapid reaction with filipin is distributed symmetrically between the inner and outer halves of the lipid bilayer of erythrocyte ghost membranes.

Cholesterol↗