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S Rottem

Publications and source records attributed to S Rottem.

At least 145 records · Page 8Linked to original sources
PubMed1978

Cholesterol distribution and movement in the Mycoplasma gallisepticum cell membrane.

The time course and extent of transfer of [14C]-cholesterol from resting Mycoplasma gallisepticum cells or membrane preparations to high-density lipoproteins were studied. More than 90% of the total cholesterol in isolated, unsealed membrane preparations was exchanged in a single kinetic process. In intact cells, however, cholesterol exists in two different environments. Cholesterol in one environment, representing approximately 50% of the total unesterified cholesterol, is readily exchanged with the cholesterol of high-density lipoproteins, with a half-time of about 4 h at 37 degrees C. The rate of exchange of [14C]cholesterol from the other environment was exceedingly slow, with a half-time of about 18 days. The fraction of the total cholesterol in the readily exchangeable cholesterol pool in intact cells increased somewhat upon aging of the culture. Electron spin resonance spectra of nitroxide-labeled stearic acids incorporated into membranes of M. gallisepticum cells indicated increased rigidity at the late exponential phase of growth. These results suggest that cholesterol is present in approximately equal concentrations on both surfaces of the M. gallisepticum membrane and that in resting cells the rate of movement of cholesterol molecules from the inner to outer halves of the lipid bilayer is exceedingly slow or nonexistent.

Cell Membrane↗
PubMed1978

Thermal regulation of the fatty acid composition of lipopolysaccharides and phospholipids of Proteus mirabilis.

The fatty acid composition of the lipid A moiety of the lipopolysaccharide and phospholipid fractions of Proteus mirabilis changed significantly on varying the growth temperature. A decrease in the growth temperature from 43 degrees C to 15 degrees C resulted in a decrease in the palmitic acid content of the lipopolysaccharide from 19.4% of total fatty acids at 43 degrees C to 1.4% at 15 degrees C, and by the appearance of an unsaturated fatty acid residue, hexadecenoic acid. Changes in the 3-hydroxy-myristic acid content of the lipid A were minimal. The decrease in the growth temperature also resulted in a decrease in the saturated fatty acid content of the phospholipid fraction, which was accompanied by an increase in their fluidity, as measured by the freedom of motion of spin-labeled fatty acids incorporated into dispersions made of the phospholipids. Nevertheless, the fluidity obtained with membrane phospholipids extracted from the cells grown at various temperatures were essentially the same when fluidity was determined at the growth temperature, supporting the hypothesis that variations in the fatty acid composition of membrane phospholipids serve to produce membranes having a constant fluidity at different temperatures of growth.

Chromatography, Gas↗
PubMed1978

Cerulenin-induced changes in the lipopolysaccharide content and phospholipid composition of Proteus mirabilis.

Inhibition of Proteus mirabilis growth by cerulenin, a specific inhibitor of fatty acid biosynthesis, was reversed by exogenously supplied fatty acid mixtures containing oleic acid and palmitic or pentadecanoic acids. The growth rate of the cells treated with cerulenin in the presence of the fatty acid mixtures was slower, however, than that of untreated cells, and their lipopolysaccharide content was decreased by 30-50%, resulting in an increased sensitivity of the organisms to rifamycin and vancomycin. Polyacrylamide gel electrophoresis of the lipopolysaccharide fraction from cerulenin-treated cells revealed that of the two P. mirabilis lipopolysaccharide types, the relative amount of the higher molecular weight lipopolysaccharide was reduced from 50% to 30% of the total lipopolysaccharide. Fatty acid analysis of the phospholipid and lipopolysaccharide fractions from cells grown with cerulenin, pentadecanoate, and oleate revealed that over 60% of the native even-numbered fatty acids of the phospholipid fraction was substituted by the odd-numbered fatty acid, while no incorporation of either the pentadecanoate or oleate could be demonstrated in the lipid A moiety of the lipopolysaccharide. The only change in the lipid A observed was an increase in the content of 3-hydroxymyristic acid accompanied by a decrease in the nonhydroxylated fatty acids, supporting the highly conserved nature of this molecule.

Antifungal Agents↗
PubMed1978

Evidence linking penicillinase formation and secretion to lipid metabolism in Bacillus licheniformis.

The formation of penicillinase by cultures of Bacillus licheniformis was preferentially suppressed by cerulenin, an antibiotic known to specifically inhibit fatty acid synthesis in microorganisms. The effect was studied at cerulenin concentrations that had almost no effect on the rate of cell growth and overall protein synthesis, but that reduced the rate of [14C]acetate incorporation (by 50 to 70%), indicating partial inhibition of lipid synthesis. The levels of both the released enzyme (exopenicillinase) and its cell-bound precursor were reduced to the same extent (70% to 80%). Enzyme formation was gradually resumed after the removal of cerulenin or the addition of a mixture of fatty acids prepared from lipids extracted from B. licheniformis. Reversal was less effective as the time interval between treatment with cerulenin and addition of fatty acids increased. We conclude that de novo synthesis of fatty acids is required for the formation of both the membrane-bound and extracellular penicillinase. Suppression of the membrane-bound enzyme is a likely consequence of the altered membrane (decreased lipid-to-lipid ratio and increased density) seen in cerulenin-treated preparations. The corresponding suppression of exopenicillinase is consistent with the view that it is derived from the membrane-bound form. A mechanism linking the general class of exportable proteins to specific aspects of lipid synthesis is discussed.

Acetates↗
PubMed1977

Effect of variations in lipopolysaccharide on the fluidity of the outer membrane of Escherichia coli.

The lipid hydrocarbon chains in the outer membrane of gram-negative bacteria appear from previous experiments to be less mobile than in the cytoplasmic membrane. To determine whether lipopolysaccharide, a unique outer membrane component, is a cause of this restricted mobility, outer membranes differing in the amount of lipopolysaccharide, and the length of the polysaccharide side chain, were prepared from Escherichia coli J5. Cytoplasmic membranes were prepared for comparison. The probes, 5- and 12-doxylstearate, were introduced into these membranes, electron spin resonance spectra were analyzed, and the order parameter (S) and empirical motion parameter (tau0) were calculated. Outer membrane preparations containing long chain lipopolysaccharide were much less fluid by these criteria than were preparations containing short chain lipopolysaccharide. Removing about 40% of the lipopolysaccharide from the former preparations greatly increased their fluidity. The lipid in the cytoplasmic membrane preparations was more fluid than in the outer membrane and cytoplasmic membranes were similar to each other regardless of the composition of the outer membrane. These results indicate that lipopolysaccharide, and especially the polysaccharide portion, directly or indirectly causes the restricted mobility of the lipid hydrocarbon chains observed in the outer membrane.

Cell Membrane↗
PubMed1977

Membrane-bound thioesterase activity in mycoplasmas.

Thioesterase activity was found in all mycoplasmas tested. Activity was highest in Acholeplasma species, whereas most of the sterol-requiring Mycoplasma species showed little activity. The thioesterase activity of Acholoplasma laidlawii is confined to the cell membrane. The enzyme could not be released from the membrane by either low- or high-ionic-strength solutions, with or without ethylenediaminetetraacetic acid, nor solubilized by detergents. The enzyme has a general specificity for long-chain saturated and unsaturated fatty acid thioesters. The preferred substrates among the saturated fatty acyl derivatives are the myristyl and palmityl derivatives. Arrhenius plots of thioesterase activities in A. laidlawii membranes enriched with elaidic or palmitic acids showed discontinuities at 12 and 18 degrees C, respectively. The possible regulatory significance of the thioesterase activity for the fatty acid synthetase and the possibllity that the activity of the enzyme is controlled by the physical state of membrane lipids are discussed.

Acholeplasma↗
PubMed1976

Interaction between tetanolysin and Mycoplasma cell membrane.

1. A partially purified tetanolysin preparation lysed the sterol-requiring Mycoplasma capricolum cells but had no effect on M. capricolum cells adapted to grow with no or very little cholesterol. The sterol-non-requiring Acholeplasma laidlawii cells grown either in a cholesterol-rich or a cholesterol-poor medium were unaffected by the tetanolysin preparation. 2. The lysis of M. capricolum cells by the tetanolysin preparation was temperature dependent, inhibited by cholesterol, sublytic concentrations of lucensomycin, and Mg2+. The sensitivity to lysis was greatly affected by the age of the culture, being highest in cells from the early logarithmic phase of growth and declining sharply thereafter. 3. Isolated M. capricolum membranes were capable of binding large amounts of the tetanolysin activity (up to 30 hemolytic units per mug membrane protein), 20 times as much as membranes of the adapted strain. The binding of tetanolysin activity to membranes was almost the same at 4,22, or 37 degrees C, and was very little affected by the age of the culture. The binding capacity of the membranes was not affected by the removal of 60-70% of membrane proteins by pronase digestion but markedly decreased with the removal of membrane lipids. 4. Of the five polypeptide bands detected in electrophorograms of the partially purified tetanolysin preparation, two bands (mol. wt. 44 000 and 42 000) were found to bind to the cholesterol-containing mycoplasma membrane preparation. EPR spectrometry revealed that the freedom of motion of fatty acid spin labels in the tetanolysin-treated membranes was markedly higher than that in untreated membranes. 5. The concept that tetanolysin interacts specifically with membrane cholesterol resulting in the shielding of cholesterol from its interaction with membrane phospholipids is discussed.

Acholeplasma laidlawii↗
PubMed1976

Motion of spin-labeled fatty acids in murine macrophages. Relation to cellular phagocytic activity.

Macrophage membrane fluidity was investigated with respect to cellular phagocytic activity through the use of fatty acid spin labels. Spin-labeled fatty acid derivatives were incorporated into intact mouse peritoneal macrophages by exchange from bovine serum albumin. The electron spin resonance (ESR) spectra of the spin-labeled fatty acids in the macrophages showed a pronounced temperature dependence and a decrease in the hyperfine splittings (2 T11) of the spectra as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives. Spin-labeled macrophages underwent a time- and temperature-dependent decay, which was inhibited by preincubating the cells with mercuric chloride, heating at 56 degrees C, or by fixing them with 0.25% glutaraldehyde. No correlation between the phagocytic activity of macrophages and membrane freedom of motion could be demonstrated. Treatment of macrophages with anti-macrophage serum or extended in vitro cultivation inhibited cellular phagocytic activity but exerted no effect on the motional freedom of the macrophage membrane. Enrichment of the fatty acid composition of the macrophage membrane with cis- or trans-unsaturated fatty acids had striking effects on cellular phagocytic activity, while no significant changes could be detected in the freedom of motion of incorporated fatty acid spin labels at the degree of specific enrichment achieved here. Thus no correlation between cellular phagocytic activity and lipid motion could be detected.

Animals↗
PubMed1976

Characterization of the mycoplasma membrane proteins. VI. Composition and disposition of proteins in membranes from aging Mycoplasma hominis cultures.

Membranes of Mycoplasma hominis cells from cultures progressing from the mid to the end of the logarithmic phase of growth became richer in protein, poorer in phospholipids and cholesterol, heavier in density, and more viscous as determined by EPR. The membrane-bound ATPase activity declined steeply. Electrophoretic analysis failed to show marked changes in membrane protein composition on aging, apart from an increase in the staining intensity of one protein band (Mr approximately 130 000) concomitant with a decrease in the staining intensity of several minor protein bands of high molecular weight. To test for possible changes in the disposition of the various membrane proteins on aging of cultures, a comparison was made of the susceptibility of membrane proteins of intact cells and isolated membranes to trypsinization and lactoperoxidase-mediated iodination. The iodination values and the percent of membrane protein released by trypsinization of intact cells were similar in cells from cultures of different ages, indicating no significant changes in the organization of the proteins on the outer surface. On the other hand, trypsinization and iodination of isolated membranes were found to be most markedly affected by the culture age, indicating significant changes in the organization of the proteins on the inner membrane surface. Thus, the iodination values of isolated membranes decreased by almost two fold, while the percentage of protein released from the membrane by trypsin increased from 28% to 50% during the experimental period. It is suggested that aging in M. hominis cultures is accompanied by a continuous increase in the packing density of the protein molecules on the inner surface of the cell membrane.

Bacterial Proteins↗
PubMed1976

Effect of cerulenin on growth and lipid metabolism of mycoplasmas.

Cerulenin markedly inhibited the growth of Acholeplasma laidlawii. A. axanthum and A. granularum were less susceptible, whereas the sterol-requiring Mycoplasma species examined showed very little susceptibility. The inhibition was not reversed by the addition of long-chain fatty acids to the medium. At a concentration of 20 mug/ml, cerulenin inhibited the incorporation of [(14)C]acetate into A. laidlawii membrane lipids, but it had no effect on either protein or nucleic acid biosynthesis. Cerulenin inhibited both the de novo synthesis of long-chain fatty acids and the elongation of medium-chain fatty acids. As a result, carotenoid biosynthesis was stimulated, and increased amounts of oleic and elaidic acids were incorporated into membrane polar lipids. Our studies support the concept that cerulenin can serve as a useful tool for obtaining better control of fatty acid composition of A. laidlawii membranes.

Acholeplasma↗
PubMed1976

Biosynthesis of saturated and unsaturated fatty acids by a T-strain mycoplasma (Ureaplasma).

A human T mycoplasma (Ureaplasma urealyticum) incorporated radioactivity into its lipids from [1-14C]acetate in the growth medium. Methanolysis of the lipids showed the label to be confined almost entirely to the methyl esters of the fatty acids. About 80% of the label was associated with the methyl esters of the saturated fatty acids, and the rest was found in the unsaturated methyl ester fraction. Gas-liquid chromatography of the saturated methyl esters showed the label to be present in the peaks of palmitate, myristate, and stearate, whereas in the unsaturated methyl ester fraction most of the radioactivity emerged in the peak of palmitoleate. The addition of either oleic or palmitic acid to the growth medium markedly decreased the organisms' incorporation of radioactivity from acetate. It is concluded that the T mycoplasma strain is capable of de novo synthesis of both saturated and unsaturated fatty acids, in this respect differing from all of the Mycoplasma and Acholeplasma strains investigated to date.

Acetates↗
PubMed1976

Cholesterol requirement of mycoplasmas as determined by microtiter test using polyene antibiotics.

A microtiter metabolic inhibition test was used to determine the effect of filipin and lucensomycin on the growth of representative species of Mycoplasma and Acholeplasma. The cholesterol-requiring species tested were found to be very susceptible to the two antibiotics, whereas the cholesterol nonrequiring species were not. The utilization of this method for differentiation between the Mycoplasma and Acholeplasma species is suggested and its advantages are discussed.

Acholeplasma↗
PubMed1975

The outer membrane of Proteus mirabilis. I. Isolation and characterization of the outer and cytoplasmic membrane fractions.

1. The crude envelope preparation obtained by sonication of Proteus mirabilis cells in the presence of lysozyme was separated into outer and cytoplasmic membrane fractions by sucrose density gradient centrifugation. The outer membrane fraction accounted for about two thirds of the dry weight of the envelope preparation. 2. In thin sections, the outer and cytoplasmic membrane fractions were shown to consist of vesicles bounded by a single trilaminar membrane, but those of the outer membrane were considerably smaller and were frequently open, forming C-shaped structures. The cytoplasmic membrane vesicles were cleaved by freeze fracturing to expose fracture faces studded with particles, while the outer membrane fragments resisted cleavage. 3. The outer membrane fraction consisted of protein (similar to 40%), lipopolysaccharide (similar to 36%) and lipid (similar to 18%) and had a density of about 1.22 g/cm3. The cytoplasmic membrane fraction consisted mostly of protein (similar to 56%) and lipid (similar to 38%), had a density of about 1.16 g/cm3, and contained almost all the NADH oxidase, succinate and D-lactate dehydrogenase activities of the crude envelope preparation. 4. Electrophoresis in polyacrylamide gels containing sodium dodecylsulfate revealed over 20 polypeptide bands in the cytoplasmic membrane fraction and only 6-7 in the outer membrane fraction. The outer membrane electrophorogram was dominated by a major band (mol. wt 40 000) which was resolved into two bands when electrophoresed in an acidic gel system. Amino acid analysis revealed a higher content of polar amino acids in the protein moiety of the outer membrane.

Amino Acids↗