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Biomedical subjects

R L Lester

Publications and source records attributed to R L Lester.

At least 73 records · Page 4Linked to original sources
PubMed1977

Changes in phospholipids of Saccharomyces cerevisiae associated with inositol-less death.

Two inositol-requiring strains of Saccharomyces cerevisiae were examined for changes in levels of phospholipids occurring after inositol deprivation. Lack of inositol results in loss of cell viability (inositol-less death) and in very large increases in two phospholipid precursors, phosphatidic acid and CDP-diacylglycerol; the accumulation of other glycerophospholipids continues for a considerable time at normal rates. Phosphatidylinositol accumulation does not occur in the absence of inositol; however, the further metabolism of this lipid continues, with 80 to 90% of this lipid disappearing. This disappearance is matched by increases in the phosphoinositol containing sphingolipids and extracellular glycerophosphoinositol. These changes are not observed when growth is blocked by cycloheximide or by omission of lysine from a lysine auxotroph, most lipids continuing to accumulate long after growth stops. There appears to be no close coordination in the synthesis of the major yeast phospholipids or between protein synthesis and phospholipid synthesis. However, despite very large changes in the composition of yeast phospholipids that can be achieved by altering culture conditions, it appears that the average charge per phospholipid molecule remains fairly constant.

Cell Survival↗
PubMed1976

Rapid breakdown of diphosphoinositide and triphosphoinositide in erythrocyte membranes.

Incubation of rabbit erythrocytes with 32Pi resulted in labeling of membrane diphosphoinositide, triphosphoinositide, and phosphatidic acid. Hypotonic lysis at 37 degress C resulted in an extremely rapid breakdown of the labeled polyphosphoinositides. This breakdown could be retarded by lysis in the presence of EDTA and by lowering the temperature to 0 degrees thus allowing preparation of membranes with minimum breakdown of the labeled lipids. Rapid breakdown of di- and triphosphoinositide in isolated membranes could be initiated by Ca++ or to a lesser extent by Mg++ and prevented by detergents and by heating to 75 degrees C. Assay of radiolabeled lipid was carried out by a method which bypassed prior lipid extraction and which enabled sequential sampling of reactions at 10-second intervals. This method was more convenient than standard procedures and gave yields of di- and triphosphoinositide equivalent to that obtained by the method of Folch.

Animals↗
PubMed1975

The purification and properties of formate dehydrogenase and nitrate reductase from Escherichia coli.

The membrane-bound formate dehydrogenase of Escherichia coli grown anaerobically in the presence of nitrate was solubilized with deoxycholate and purified to near homogeneity. The purification procedure included ammonium sulfate fractionation and chromatography on Bio-Gel A-1.5m and DEAE Bio-Gel A in the presence of the nonionic detergent, Triton X-100. This detergent caused a significant decrease in the molecular weight of the soluble formate dehydrogenase complex and allowed the enzyme then to be resolved from other membrane components. Anaerobic conditions were required throughout due to the sensitivity of the enzyme to oxygen inactivation. Formate dehydrogenase was judged to be at least 93 to 99% pure by the following procedures: polyacrylamide gel electrophoresis in the presence of Triton X-100 and sodium dodecyl sulfate, gel filtration, and sedimentation velocity studies. The purified enzyme exists as a detergent-protein complex (0.20 +/- 0.03 g of Triton X-100/g of protein) which has an S20,w of 18.1 S and a Stokes radius of 76 A. This corresponds to a molecular weight of 590,000 +/- 59,000. The enzyme had an absorbance spectrum of a b-type cytochrome which could be completely reduced by formate. The heme content corresponds to an equivalent weight of 154,000 which suggests a tetrameric structure for the enzyme. Formate dehydrogenase was found to contain (in relative molar amounts): 1.0 heme, 0.95 molybdenum, 0.96 selenium, 14 non-heme iron, and 13 acid-labile sulfide. Neither FAD nor FMN could be detected. The enzyme contains three polypeptides, designated alpha, beta, and gamma, whose molecular weights were estimated by gel electrophoresis in the presence of sodium dodecyl sulfate to be 110,000, 32,000, and 20,000, respectively. After separation of the polypeptides by gel filtration in the presence of sodium dodecyl sulfate alpha, beta, and gamma were found in 1:1.2:0.55 molar ratios. A study of the enzyme obtained from cells grown with [75Se]selenite showed that only the alpha polypeptide contained significant amounts of selenium. The enzyme will catalyze the formate-dependent reduction of phenazine methosulfate, dichlorophenolindophenol, methylene blue, nitroblue tetrazolium, benzyl viologen, methyl viologen, ferricyanide, and coenzyme Q6. Cyanide, azide, p-hydroxymercuribenzoate, iodoacetamide, and oxygen inhibit the enzyme. The procedure which was designed for the purification of formate dehydrogenase also yields a highly purified preparation of nitrate reductase. This nitrate reductase has been shown to contain significant amounts of heme (Enoch, H. G., and Lester, R. L. (1974) Biochem. Biophys. Res Commun. 61,1234-1241). The enzyme contains three polypeptides with molecular weights of 155,000, 63,000, and 19,000. When measured in the presence of Trition X-100 the Stokes radius of nitrate reductase is 75 A and the S20,w is 16 S which corresponds to a molecular weight of 498,000.

Aldehyde Oxidoreductases↗
PubMed1975

The regulated catabolism of endogenous and exogenous phosphatidylinositol by Saccharomyces cerevisiae leading to extracellular glycerophosphorylinositol and inositol.

It was previously shown that phosphatidylinositol catabolism leads to the accumulation of glycerophosphorylinositol in the culture medium of Saccharomyces cerevisiae. We now find that lack of an energy source (glucose) reduces the formation of glycerophosphorylinositol and increases extra-cellular inositol. This situation is reversed by refeeding glucose. [3H]Phosphatidylinositol is the precursor of extra-cellular [3H]inositol with energy-starved cells. Extracellular glycerophosphorylcholine and glycerophosphorylethanolamine accumulate more slowly than glycerophosphorylinositol in the growth medium and do not appear to be a strongly affected by energy starvation. Phosphatidylinositol deacylation appears to occur at the cell surface in a regulated manner. Exogenously added phosphatidylinositol apparently does not mix randomly with the endogenous pool since it is not converted to either inositol-containing sphingolipid or to diphosphoinositide, both previously shown to be derived in part from cellular phosphatidylinositol. Labeled exogenous phosphatidylinositol is, however, quantitatively converted to glycerophosphorylinositol with the probable intermediat formation of monoacyl-glycerophosphorylinositol. Breakdown of exogenous phosphatidylinositol requires an energy source and does not lead to free inositol. Deacylation of exogenously added 1-acyl-glycerophosphorylinositol occurs much faster than deacylation of phosphatidylinositol and does not require an energy source. Glycerophosphorylethanolamine formation from exogenous phosphatidylethanolamine occurs about as fast as the breakdown of phosphatidylinositol and is also inhibited in the absence of energy source. The much slower deacylation of exogenous phosphatidylcholine was also affected by an energy source. Glycerophosphorylinosiyolaccumulates in the culture medium of Kloeckera apiculata, Saccharomyces carlsbergenis, and Neurospora crassa.

Biological Transport↗
PubMed1975

Characterization of Inositol-containing Phosphosphingolipids from Tobacco Leaves: Isolation and Identification of Two Novel, Major Lipids: N-Acetylglucosamidoglucuronidoinositol Phosphorylceramide and Glucosamidoglucuronidoinositol Phosphorylceramide.

A method for a large scale extraction of phosphoglycosphingolipids from the leaves of Nicotiana tabacum L. has been developed. The phosphosphingolipid concentrate consists of a dozen or more polar lipids as judged by thin layer chromatography. Two of these lipids were purified by chromatography on porous silica beads and partially characterized. These lipids are formulated as: N-acetylglucosamidoglucuronidoinositol phosphorylceramide and glucosamidoglucuronidoinositol phosphorylceramide. Although not fully characterized, the other lipids in the concentrate are inositol-containing phosphosphingolipids with a higher carbohydrate content.

Journal Article↗
PubMed1973

Occurrence of phosphonosphingolipids in Bdellovibrio bacteriovorus strain UKi2.

The major phospholipids of two strains of Bdellovibrio bacteriovorus were characterized. Both strain UKi1, which is obligately saprophytic, and strain UKi2, which is facultatively parasitic, contained phosphatidylethanolamine and phosphatidylglycerol as their major glycerophosphatides. A branched, 15-carbon fatty acid is the major component of these alkali-labile lipids. Absent from UKi1 but present in UKi2 were three alkali-stable lipids (compounds 8, 9, and 11) which appear to be phosphosphingolipids. After acid hydrolysis, both compound 8 and 9 yield the identical phosphorus-containing substance that is water soluble, dipolar ionic, and ninhydrin positive. This substance appears to contain a C-P bond since P(i) could not be released from this substance by treatment with alkaline phosphatase or by very harsh mineral acid treatment. Based on chromatographic comparisons, this phosphonate appears to be a novel lipid constituent. Upon degradation, compound 8 yields 1 mol of dihydroxy long-chain base and compound 9 yields 1 mol of a trihydroxy long-chain base. These bases appear to have a 17-carbon, possibly branched, structure based on gas-liquid chromatography retention times. Degradation of both sphingolipids yields a mixture of hydroxy fatty acids, the major component being a branched, 15-carbon hydroxy acid.

Alkaline Phosphatase↗
PubMed1972

Studies on the diversity of inositol-containing yeast phospholipids: incorporation of 2-deoxyglucose into lipid.

Seven inositol-containing phospholipids in Saccharomyces cerevisiae can be resolved by two-dimensional chromatography on silicic acid-impregnated paper. Four of these lipids are stable to mild alkaline methanolysis; one of these has already been characterized as a mannosyl di(inositolphosphoryl) ceramide. Addition of labeled 2-deoxy-d-glucose to a growing culture results in label appearing in five lipids, some of which are alkali-stable and have the same R(F) values as the alkali-stable inositol-containing phospholipids. These lipids are labeled rapidly. The deoxyglucose is incorporated intact, probably in a glycosidic link, since the deoxyglucose is released by mild acid treatment.

Autoradiography↗
PubMed1972

Effects of molybdate, tungstate, and selenium compounds on formate dehydrogenase and other enzyme systems in Escherichia coli.

The role of selenium and molybdenum in the metabolism of Escherichia coli was explored by growing cells in a simple salts medium and examining the metabolic consequences of altering the concentration of molybdenum and selenium compounds in the medium. The addition of tungstate increased the molybdate deficiency of this medium, as reflected by lowered levels of enzyme systems previously recognized to require compounds of molybdenum and selenium for their formation [formate-dependent oxygen reduction, formate dehydrogenase (FDH) (EC 1.2.2.1), and nitrate reductase (EC 1.9.6.1)]. The requirement for selenium and molybdenum appears to be unique to the enzymes of formate and nitrate metabolism since molybdate- and selenite-deficient medium had no effect on the level of several dehydrogenase and oxidase systems, for which the electron donors were reduced nicotinamide adenine dinucleotide, succinate, d- or l-lactate, and glycerol. In addition, no effect was observed on the growth rate or cell yield with any carbon source tested (glucose, glycerol, dl-lactate, acetate, succinate, and l-malate) when the medium was deficient in molybdenum and selenium. dl-Selenocystine was about as effective as selenite in stimulating the formation of formate dehydrogenase, whereas dl-selenomethionine was only 1% as effective. In aerobic cells, an amount of FDH was formed such that 3,200 or 3,800 moles of formate were oxidized per min per mole of added selenium (added as dl-selenocystine or selenite, respectively).

Aerobiosis↗