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Biomedical subjects

D Haas

Publications and source records attributed to D Haas.

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

Chromosome mobilization by the R plasmid R68.45: a tool in Pseudomonas genetics.

The conjugative plasmid R68.45 mobilizes the chromosome of Pseudomonas aeruginosa strain PAO from multiple sites located in different chromosome regions. In interrupted matings on the plate, selection for any single marker tested resulted in entry times of 3-5 min. When selection was imposed for two markers linked in R68.45-mediated conjugation, double recombinants appeared after a delay which corresponded approximately to the map distance between the two markers as measured by the sex factor FP2. Thus, R68.45 and FP2 appear to promote chromosome transfer at similar rates, but R68.45, unlike FP2, seems to give non-polarized transfer. R68.45 may be used to estimate map distances between linked markers located in those chromosome regions where other sex factors do not produce enough recombinants to permit accurate measurement of entry times. In R68.45 matings on the plate, most recombinants inherited short donor chromosome fragments (usually less than 10 min long) and lost the R plasmid during purification. Used like a "large" generalized transducing phage, R68.45 has proved valuable in construction of PAO strains with desired genotypes.

Chromosomes, Bacterial↗
PubMed1978

Enhancement of the mitochondrial Ca2+ uptake rate by phenethylbiguanide and other organic cations with hypoglycemic activity.

At concentrations below 50 micronM, phenethylbiguanide enhanced the initial rate of enertized Ca2+ uptake into energized guinea pig liver mitochondria by as much as 45 per cent; Ca2+-stimulated O2 uptake increased in parallel. The biguanide concentration that enhanced Ca2+ uptake maximally was at least 15 times lower than that required for 50 per cent inhibition of respiration. Kinetic studies indicated that the enhanced rate of Ca2+ transport resulted from an increase in Vmax, while Km for Ca2+ was unaffected by the biguanide. Several other organic cations known to lower blood sugar in intact animals or to block the hepatic gluconeogenic response to glucagon also enhanced the mitochondrial Ca2+ uptake rate; three of these compounds did not inhibit respiration even at high concentrations. One organic cation, triethyltin, which is a potent respiratory inhibitor that does not affect blood sugar, had no effect on Ca2+ uptake. We concluded that enhancement of the mitochondrial Ca2+ uptake rate is related to the mechanism of therapeutic blood sugar lowering by these drugs, probably by impairing the gluconeogenic response of the liver to glucagon.

Animals↗
PubMed1977

The genetic organization of arginine biosynthesis in Pseudomonas aeruginosa.

Six loci coding for arginine biosynthetic enzymes in Pseudomonas aeruginosa strain PAO were identified by enzyme assay: argA (N-acetylglutamate synthase), argB (N-acetylglutamate 5-phosphotransferase), argC (N-acetylglutamate 5-semialdehyde dehydrogenase), argF (anabolic ornithine carbamoyl-transferase), argG (argininosuccinate synthetase), and argH (argininosuccinase). One-step mutants which had a requirement for arginine and uracil were defective in carbamoylphosphate synthase, specified by a locus designated car. To map these mutations we used the sex factor FP2 in an improved interrupted mating technique as well as the generalized transducing phages F116L and G101. We confirmed earlier studies, and found no clustering of arg and car loci. However, argA, argH, and argB were mapped on a short chromosome segment (approx. 3 min long), and argF and argG were cotransducible, but not contiguous. N-Acetylglutamate synthase, the enzyme which replenishes the cycle of acetylated intermediates in ornithine synthesis of Pseudomonas, appears to be essential for arginine synthesis since argA mutants showed no growth on unsupplemented minimal medium.

Aldehyde Oxidoreductases↗
PubMed1977

Mutants of Escherichia coli "cryptic" for certain periplasmic enzymes: evidence for an alteration of the outer membrane.

Mutants in which the expression of periplasmic enzymes by whole cells is reduced (termed "cryptic") are also found to show greatly reduced uptake of labeled adenosine 5'-monophosphate (5'-AMP), providing a rapid assay for crypticity. The crypticity of 3'- and 5'-nucleotidase has been examined as a function of substrate concentration. The Km for 3'- or 5'-AMP increases in the cryptic mutants when whole cells are used as the enzyme source. The Vmax is not altered. Electrophoretic analysis of protein prepared from cell envelopes showed that three cryptic mutants have a polypeptide absent from the outer membrane and a relatively high proportion of a polypeptide in the inner membrane. Analysis of the molar ratios of constituent sugars of the lipopolysaccharides showed no differences between three cryptic mutants and the parent strain. One cryptic mutant (3--41), however, has altered sensitivity to phage T4. By selection for phage resistance, derivatives of the cryptic mutants that are deoxycholate sensitive have been obtained. These mutants are no longer cryptic. We suggest that cryptic mutants have an altered outer membrane, with decreased permeability to 3'- and 5'-AMP, as a result of an altered polypeptide.

Adenosine Monophosphate↗
PubMed1976

(+)-alpha-(N-1-phenethyl) urea stereospecifically inhibits Ca2+ - but not ADP-stimulated mitochondrial respiration.

The (+) isomer of alpha-(N-1-phenethyl) urea is a moderately potent inhibitor of Ca2+ -stimulated mitochondrial respiration of 45Ca2+ uptake (50 percent inhibition at 0.18 mM) while having no effect on adenosine diphosphate-stimulated respiration; the (-) isomer is without effect up to 4 mM. beta-Phenethylurea does not inhibit with either stimulus. The data support the involvement of a Ca2+ -specific protein in energized mitochondrial Ca2+ uptake.

Adenosine Diphosphate↗
PubMed1976

R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa.

The R factor R68 readily promotes chromosome transfer in Pseudomonas aeruginosa strain PAT, but shows little such sex factor activity in strain PAO. A variant of this plasmid, R68.45, has been isolated which produces recombinants in PAO plate matings at frequencies of 10(-3)--10(-5) per donor cell for markers in the 0-60 min region of the chromosome. Little or nor chromosome transfer was shown in liquid media. The kinetics of chromosome transfer were studied by interrupting matings on solid media with nalidixic acid. Five chromosomal markers, mapping in widely spaced regions of the chromosome all entered 3-5 min after initiation of mating. These results, combined with linkage studies, indicated that R68.45, unlike the Pseudomonas sex factors FP2 and FP39, promotes chromosome transfer from a range of origin sites and can thus be used for mapping the region of the P. aeruginosa chromosome later than 40 min. R68.45 and other similar variants were isolated from rare chromosomal recombinants appearing in crosses between PAO(R68) donors and PAO recipients in which selection for ARGB+ was made. Selection for other chromosomal markers did not result in such variants suggesting that plasmides of the R68.45 type arise by recombination of genetic material between the R68 plasmid and certain regions of the bacterial chromosome.

Chromosome Mapping↗
PubMed1975

N-acetylglutamate 5-phosphotransferase of Pseudomonas aeruginosa. Purification and ligand-directed association-dissociation.

N-Acetylglutamate 5-phosphotransferase (ATP: N-acetyl-L-glutamate 5-phosphotransferase EC 2.7.2.8), the second enzyme of arginine biosynthesis, was purified over 2000-fold from Pseudomonas aeruginosa. The purification procedure involved a heat treatment, ammonium sulfate precipitation, and chromatography on DEAE-cellulose, Sephadex G-150, and hydroxyapatite. The purified enzyme was greater than 90% pure as judged by analytical polyacrylamide gel electrophoresis. A molecular weight of approximately 230000 was obtained by gel filtration. Electrophoresis in sodium dodecyl sulfate gels gave a single band corresponding to a molecular weight of 29000. Due to the capacity for self-association, the enzyme can exist in different states of aggregation depending on the nature of ligands and the concentrations of phosphate buffer. As estimated by gel filtration, the molecular weight was about 230000 in the presence of N-acetyl-L-glutamate. With L-arginine, the feedback inhibitor, and MgATP forms of smaller molecular weight (minimum of approximately 65000) were found. A concurrent change in the sedimentation coefficient as a function of ligands was demonstrated by sucrose gradient centrifugation. The synthesis of N-acetylglutamate 5-phosphotransferase was not repressed by exogenous L-arginine or its precursors.

Drug Stability↗
PubMed1975

N-acetylglutamate 5-phosphotransferase of Pseudomonas aeruginosa. Catalytic and regulatory properties.

Some kinetic properties of N-acetylglutamate 5-phosphotransferase (ATP: N-acetyl-L-glutamate 5-phosphotransferase EC 2.7.2.8) purified approx. 2000-fold from Pseudomonas aeruginosa have been studied. The enzyme required Mg2+ for activity. Mn2+, Zn2+, Co2+, and Ca2+, in this order, could replace Mg2+ partially. The substrate specificity was narrow: N-carbamoyl-L-glutamate and N-formyl-L-glutamate were phosphorylated, but at a lower rate than N-acetyl-L-glutamate; N-propionyl-L-glutamate was almost inactive as a substrate. dATP, but neither GTP nor ITP, could be used instead of ATP. The enzyme had a broad pH optimum from pH 6.5 to 9. Feedback inhibition by L-arginine was markedly dependent on pH. Above pH 9 no inhibition was observed. L-Citrulline was three times less potent an inhibitor than L-arginine. The enzyme showed Michaelis-Menten kinetics, even at low concentration of the second substrate. The apparent Km was 2 mM for N-acetyl-L-glutamate (at 10 mM ATP) and approx. 3 mM for ATP (at 40 mM N-acetyl-L-glutamate). In the presence of L-arginine the rate-concentration curves for N-acetyl-L-glutamate became signoidal, while no cooperativity was detected for ATP. A method was developed allowing the determination of N-acetyl-L-glutamate in the nanomolar range by means of purified enzyme.

Cations, Divalent↗
PubMed1975

N-Acetylglutamate synthase of Escherichia coli regulation of synthesis and activity by arginine.

N-Acetylglutamate synthase, the first enzyme of arginine biosynthesis, was stabilized in crude extracts from Escherichia coli. At 4 degrees the enzyme lost less than 5% of activity per day. L-Arginine repressed the formation of N-acetylglutamate synthase. Under conditions of genetic or physiological derepression, a specific activity of approximately 50 nmol per min per mg of protein was measured. No activity (i.e. less than 0.2 nmol per min per mg of protein) could be detected in extracts from cells grown under conditions of repression, whereas an intermediate level was found in cell cultivated on minimal medium. In a 6-fold purified preparation L-arginine inhibited the enzyme. Of 11 precursors and analogues of arginine tested only O-[L-norvalyl-5]-isourea inhibited N-acetylglutamate synthase as strongly as L-agrinine.

Acetyl Coenzyme A↗