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Biomedical subjects

R D Simoni

Publications and source records attributed to R D Simoni.

At least 91 records · Page 5Linked to original sources
PubMed1980

Genetic and biomedical studies demonstrating a second gene coding for asparagine synthetase in Escherichia coli.

Genetic experiments have indicated that asparagine auxotrophs of Escherichias coli K-12 can be made asparagine prototrophs at either of two sites on the chromosome and that wild-type strains require both sites to be mutated to produce asparagine auxotrophy. The former asn locus is now called asnA, and the new gene is designated asnB. The asnB gene is located near gal.AsnA+ asnB and asnA asnB+ strains were constructed, and the asparagine synthetic reaction was characterized in extracts. These studies revealed that the asnA gene codes for the enzyme previously described (H. Cedar and J.H. Schwartz, J. Biol. Chem. 244: 4112-4121, 1969), whereas the asnB gene is involved in the production of an enzyme which differs from the one previously described in its specific activity in extracts, its stability at low and high temperatures, and its apparent ability to use either glutamine or ammonia as amide nitrogen donor. Physiological studies showed that either enzyme alone is sufficient to allow a maximal growth rate under conditions of asparagine limitation.

Asparagine↗
PubMed1979

Human serum albumin. Spectroscopic studies of binding and proximity relationships for fatty acids and bilirubin.

Binding and proximity relationships of hydrophobic ligands on human serum albumin have been studied using absorption, fluorescence, circular dichroism, and electron paramagnetic resonance spectroscopy. The ligands studied were bilirubin, two conjugated linear polyene fatty acids, cis-parinaric acid and cis-eleostearic acid, and three nitroxide derivatives of stearic acid with doxyl groups at positions 5, 10, and 12, respectively. Binding of polyene fatty acids was monitored by absorption peak shifts, induced circular dichroism, enhancement of fluorescence, and energy transfer between albumin's single tryptophanyl residue and the polyene chromophore. Induced circular dichroism studies indicate excitonic ligand-ligand interaction between bound fatty acids. Fluorescence enhancement of cis-parinaric acid was analyzed using a stepwise multiple equilibrium model, and six binding constants in the range 10(8) to 10(6) M-1 were obtained, in agreement with previous measurements for other fatty acids. The temperature dependence of the equilibrium constants indicates that the binding enthalpy is nearly zero. Fluorescence energy transfer was similarly used to quantitate bilirubin binding to albumin. Energy transfer, nitroxide quenching of fluorescence, and electron paramagnetic resonance spectroscopy were used to elucidate binding geometries which support and extend proposed structural models for albumin. It is suggested that the first two fatty acids bind side-by-side in an antiparallel fashion in domain III of human serum albumin.

Bilirubin↗
PubMed1979

Lactose carrier protein of Escherichia coli. Transport and binding of 2'-(N-dansyl)aminoethyl beta-D-thiogalactopyranoside and p-nitrophenyl alpha-d-galactopyranoside.

The elevated level of lactose carrier protein present in cytoplasmic membranes derived from Escherichia coli strain T31RT, which carries the Y gene of the lac operon on a plasmid vector (Teather, R. M., et al. (1978) Mol. Gen. Genet. 159, 239--248), has allowed the detection of a complex between the carrier and the fluorescent substrate 2'-(N-dansyl)-aminoethyl beta-D-thiogalactopyranoside (Dns2-S-Gal). Binding is accompanied by a 50-nm blue shift in the emission maximum of the dansyl residue. The complex (dissociation constant, KD = 30 micron) rapidly dissociates upon addition of competing substrates such as beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside or upon reaction with the thiol reagent p-chloromercuribenzenesulfonate. Binding of both Dns2-S-Gal and p-nitrophenyl alpha-D-galactopyranoside (alpha-NPG) occurs spontaneously in the absence of an electrochemical potential gradient across the membrane. Comparison of equilibrium binding experiments using Dns2-S-Gal or alpha-NPG and differential labeling of the carrier with radioactive amino acids shows that the carrier binds 1 mol of substrate per mol of polypeptide (molecular weight 30 000). In addition to specific binding to the lactose carrier, Dns2-S-gal binds unspecifically to lipid vesicles or membranes, as described by a partition coefficient, K = 60, resulting in a 25-nm blue shift in the emission maximum of the dansyl group. Both Dns2-S-Gal and alpha-NPG are not only bound by the lactose carrier but also transported across the membrane by this transport protein in cells and membrane vesicles. The fluorescence changes observed with dansylated galactosides in membrane vesicles in the presence of an electrochemical gradient (Schuldiner et al. (1975) J. Biol. Chem. 250, 1361--1370)) are interpreted as an increase in unspecific binding after translocation.

Biological Transport, Active↗
PubMed1978

Isolation and preliminary characterization of rat liver cells resistant to 7-ketocholesterol.

Two clones of the rat liver cell line, GAI, were selected and propagated in 4 microgram/ml of the cholesterol analogue, 7-ketocholesterol. One of the variants was also found to be resistant to another toxic analogue, 25-hydroxycholesterol. 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) activity was elevated in both variant clones, but was not increased substantially upon incubation of the cells in lipid-depleted medium. The reductase activity was increased 3-fold in the wild type cells under the same conditions. Incubation of the cells in lipid-depleted medium resulted in a 3-fold increase in the rate of sterol synthesis from [14C]acetate in all three types. In the presence of 2 microgram/ml 7-ketocholesterol, the reductase activity in all three cell lines decreased at an equal rate, but activity of the enzyme was still measurable after 48 h in the presence of the analogue. Sterol synthesis was reduced almost to zero in the wild type cells under these conditions, while the variant cells retained 12--15% of their capacity for sterol synthesis. These results are interpreted to indicate that 7-ketocholesterol, besides affecting sterol synthesis at the level to 3-hydroxy-3-methylglutaryl coenzyme A reductase, must also affect sterol synthesis at another step further along the synthetic pathway.

Acetates↗
PubMed1977

Incorporation of a naturally occurring fluorescent fatty acid into lipids of cultured mammalian cells.

A fluorescent fatty acid, cis-parinaric acid (cis-PnA), has been biosynthetically incorporated by cultured mammalian cells. The fatty acid probe is predominantly incorporated into phospholipids, with a small amount of incorporation into the neutral lipid fraction. Both phosphatidylethanolamine and phosphatidylcholine are labeled with the fluorescent probe. The temperature dependence of the fluorescence intensity of cis-PnA, either biosynthetically incorporated or added as the free fatty acid, suggests thermally induced structural reorganizations in phospholipids isolated from cultured cells enriched in palmitic acid. Results are essentially identical with both forms of the probe.

Cells, Cultured↗
PubMed1977

Activation of purified 3-hydroxy-3-methylglutaryl-CoA reductase by phospholipids.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), the enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis, has been purified by two previously reported procedures. Enzyme purified by the method of Heller, R. and Shrewsbury, M. (1976) J. Biol. Chem. 251, 3815-3822) shows up to 3-fold enhancement of activity by various types of lipid dispersions while the enzyme purified by the procedure of Tormanen et al. ((1976) Biochem. Biophys. Res. Commun. 68, 754-762) shows no activation. These results suggest that interaction with microsomal membrane lipids may be important in determining the activity of this enzyme. Analysis of bound lipid showed that enzyme prepared by the procedure of Tormanen contained at last 50 times as much phospholipid on a weight basis as enzyme prepared by Heller and Shrewsbury. Analysis of both preparations by gel-electrophoresis indicates that enzyme activities of the two comigrate, but in neither case does activity coincide with the major protein species.

Alcohol Oxidoreductases↗
PubMed1977

Conjugated polyene fatty acids as fluorescent probes: synthetic phospholipid membrane studies.

The preparation of polyene fatty acid membrane probes cis- and trans-parinaric acid and parinaroylphosphatidylcholines and their use in studies of several one- and two- component lipid systems are described. The fluorescence quantum yield of trans-parinaric acid in dipalmitoylphosphatidylcholine at 20 degrees C is approximately 0.3; the quantum yield in aqueous solution is negligibly small. Thermal-phase transitions in single-component phospholipid dispersions are monitored with absorption and fluorescence excitation peak position, fluorescence intensity, lifetime, and polarization. The transition temperatures observed are consistent with previous determinations. Shifts in the absorption peak position are related to the bilayer expansion as it undergoes the gel to liquid-crystalline transition, while fluorescence depolarization provides semiquantitative information concerning molecular motion of the probe in the bilayer. A long fluorescence lifetime component is observed for parinaric acid in the solid phase (up to 50 ns), and a short lifetime component is observed (ca. 5 ns) in the fluid phase of dipalmitoylphosphatidylcholine; both lifetime components are observed in the transition region. In most phospholipids, cis-parinaric acid detects the melting transition at about 1 degree C lower than trans-parinaric acid. Partitioning experiments involving mixed populations of phospholipid vesicles show that trans-parinaric acid preferentially associates with solid-phase lipids, while cis-parinaric acid shows a more equal distribution between solid and fluid lipids. The binding of cis-parinaric acid to dipalmitoylphosphatidylcholine at 25 degrees C is described as partitioning of parinaric acid between lipid vesicles and the aqueous phase with a partition coefficient of 5 X 10(5). Several rates are observed in the binding process which are interpreted as rapid outer monolayer uptake and a much slower process of interlamellar exchange. The phase diagram of the binary lipid mixture dipalmitoylphosphatidylcholine-dipalmitoylphosphatidylethanolamine has also been examined and found to be essentially identical to the one constructed using a nitroxide probe.

Fatty Acids, Unsaturated↗
PubMed1977

Conjugated polyene fatty acids as fluorescent probes: biosynthetic incorporation of parinaric acid by Escherichia coli and studies of phase transitions.

The use of the fluorescent fatty acid, parinaric acid (9, 11, 13, 15-octadecatetraenoic acid) (PnA), was studied in cells of an unsaturated fatty acid auxotroph of Escherichia coli. Growth conditions were found that permitted biosynthetic incorporation of PnA (up to 3%) into membrane phospholipids during growth on oleic or elaidic acid. Fluorescence measurements of incorporated PnA revealed phase transitions in cells, membranes, and phospholipids at temperatures that reflected the fatty acid composition of the sample. Transitions had a well-defined onset from high temperature, while the lower and end point was less well defined. cis- and trans-PnA (cis, trnas, trans, cis, and all trans, respectively) gave comparable results. Similar phase transitions were detected with PnA, which was not biosynthetically incorporated. Fluorescence of tryptophan was measured in E. coli membranes as a function of concentration of PnA. Significant quenching of tryptophan fluorescence by PnA was observed.

Cell Membrane↗
PubMed1976

Sugar transport. Properties of mutant bacteria defective in proteins of the phosphoenolpyruvate: sugar phosphotransferase system.

The bacterial phosphotransferase system (PTS) catalyzes the transfer of the phosphoryl group from phosphoenolpyruvate to its sugar substrates, PTS sugars, concomitant with the translocation of these sugars across the bacterial membrane. The phosphorylation of a given sugar requires four proteins, two general proteins, Enzyme I, and the histidine-containing phosphocarrier protein of the PTS (HPr), used for all sugars, and a pair of proteins specific for that sugar, designated an Enzyme II complex. The phosphotransferase system has been implicated in regulating the induction of synthesis of some catabolic enzyme systems required for the utilization of sugars that are not substrates of the phosphotransferase system, and this and the accompanying reports are concerned with this phenomenon in Salmonell typhimurium and Escherichia coli. Mutants defective in Enzyme I (ptsI), HPr (ptsH), and certain Enzymes II were isolated, and their abilities to ferment and grow on a wide range of sugars and other compounds were determined. The mutants showed the expected properties on PTS sugars, but in addition, ptsH and tight ptsI mutants were unable to utilize certain non-PTS sugars, including maltose, melibiose, glycerol, glycerol-P, mannose-6-P, and, in E. coli, lactose. Leaky Enzyme I mutants could utilize these carbohydrates, but were unable to use them in the presence of a PTS sugar such as methyl alpha-D-glucopyranoside. In accord with the results reported by other laboratories, the inability of the mutants to utilize the non-PTS sugars was explained by the fact that these cells could not be normally induced to synthesize the corresponding catabolic enzyme systmes. This phenomenon is designated PTS-mediated repression. PTS-mediated repression was also observed in wild type cells, but by comparing wild type and leaky pts mutants it was shown that the sensitivity to repression by PTS sugars was greatest in mutants containing the lowest levels of Enzyme I or HPr. Furthermore, ptsI mutants containing a second site mutation in a gene for an Enzyme II were not repressed by the sugar substrate of that Enzyme II, although repression by other PTS sugars was not affected. Transport and other studies further indicated that neither appreciable uptake nor metabolism of the PTS sugars was required for these compounds to effect repression. The ptsH mutants showed the same phenotypic properties as the ptsI mutants with some important exceptions. First, they could ferment and grow on a PTS sugar, fructose. Second, after growth on fructose, (and to a lesser extent on glucose or mannose), such mutants were capable of utilizing other PTS sugars for a few generations. Third, growth of the ptsH mutants on fructose relieved PTS-mediated repression; after growth on fructose, but not on lactate, the mutants could grow for several generations on non-PTS sugars. Preliminary experiments indicated that growth on fructose resulted in the formation of one or more proteins that could substitute for HPr in the utilization of both PTS and non-PTS sugars.

Acetylglucosamine↗
PubMed1976

Regulation of carbohydrate uptake in gram-positive bacteria.

Uptake of glycerol and other carbohydrates by Staphylococcus aureus cells is sensitive to regulation by sugar substrates of the phosphoenolpyruvate:sugar phosphotransferase system. Inhibition requires an intact phosphotransferase system. In contrast to results obtained with Gram-negative bacteria, it appears that intracellular sugar phosphate is the inhibiting species.

Binding, Competitive↗
PubMed1975

Energy transduction in Escherichia coli. Genetic alteration of a membrane polypeptide of the (Ca2+,Mg2+)-ATPase.

Recent genetic analyses of the membrane components involved in energy transduction in Escherichia coli have concentrated on the (Ca2+, Mg2+)-ATPase complex (EC 3.6.1.3). Many mutants have been described with altered biochemical properties and defects in energy-requiring processes such as oxidative phosphorylation, transhydrogenase activity, and active transport of several solutes. This report describes the isolation of a mutant strain of E. coli that is defective in several energy-requiring processes. The strain BG-31 was obtained by "localized mutagenesis" using phage P1c1. The mutation maps at approximately 73.5 min on the E. coli chromosome. Reversion and suppression analyses indicate that the defect is the result of a single amber mutation. This strain is unable to utilize succinate, D-lactate, or malate for growth. Mutant cells are unable to couple the energy derived from the hydrolysis of ATP to the active transport of proline, although coupling of energy derived from electron transport to solute transport appears normal when examined in both cells and isolated membrane vesicles. Isolated membranes of the mutant are unable to couple the energy derived from the hydrolysis of ATP to transhydrogenase activity while they can utilize the energy generated from electron transport to drive transhydrogenase activity. Extracts of strain BG-31 have normal levels of (Ca2+, Mg2+)-ATPase activity. The ATPase portion of the complex, bacterial F1 (BF1), is poorly attached to the membrane portion of the complex. In vitro reconstitution of transhydrogenase activity with stripped membrane fractions and crude preparations of BF1 localize the defect in strain BG-31 to the membrane portion of the complex. Analysis of membranes of the strain BG-31 by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate demonstrate the absence of a single polypeptide of molecular weight about 54,000 and the appearance of a new polypeptide of lower molecular weight, about 25,000. Analysis of a spontaneous revertant of BG-31 shows complete restoration of the parental phenotype including the gel patterns. The characterization of this mutant provides the first demonstration of the consequences of a structural gene mutation on a polypeptide in the membrane portion of the complex and represents the initial stages in what we hope will be the biochemical definition and functional characterization of this important energy-transducing system.

2,6-Dichloroindophenol↗
PubMed1975

Conjugated polyene fatty acids as membrane probes: preliminary characterization.

The use of fluorescent conjugated polyenoic fatty acids as probes of membrane structure is introduced. alpha- and beta-parinaric acid (cis, trans, trans, cis-, and all trans-9,11,13,15-octadecatetraenoic acid) and synthetic lecithins containing an alpha-parinaric acid chain in position 2 are prepared and their absorption and fluorescence properties are determined. Phase transitions are detected as fluorescence changes at characteristic temperatures when either the free fatty acid probes or the labeled phospholipid probe are included in sonicated aqueous dispersions of L-alpha-dimyristoyl lecithin, L-alpha dipalmitoyl lecithin, or L-alpha-distearoyl lecithin. The phase transitions are detected at about 23 degrees C (dimyristoyl), 44 degrees C (dipalmitoyl), and 53 degrees C (distearoyl lecithin). Binding of alpha-parinaric acid to bovine serum albumin is measured by shifts in the absorption spectrum and enhanced quantum yield of the fatty acid upon binding and by energy transfer between 2 tryptophyl residues in bovine serum albumin and alpha-parinaric acid. Approximately six binding sites are detected. Other applications of these probe molecules, including phase transitions of phospholipid/cholesterol dispersions, induced circular dichroism of parinaric acid bound to albumin, and biosynthetic incorporation of parinaric acid into biological membranes, are discussed.

Fatty Acids, Unsaturated↗