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European Journal of Biochemistry Aug 1979
Topics: Affinity Labels; Animals; Azo Compounds; Diazooxonorleucine; Drug Stability; Kidney; Kinetics; Maleates; Molecular Weight; Protein Binding; Rats; Stereoisomerism; Structure-Activity Relationship; gamma-Glutamyltransferase
PubMed: 39760
DOI: 10.1111/j.1432-1033.1979.tb13243.x -
The Journal of Biological Chemistry Apr 1979Inactivation of rat renal phosphate-dependent glutaminase by 6-diazo-5-oxo-L-norleucine occurs only under conditions where the enzyme is catalytically active. The...
Inactivation of rat renal phosphate-dependent glutaminase by 6-diazo-5-oxo-L-norleucine occurs only under conditions where the enzyme is catalytically active. The glutaminase activity and the rate of inactivation by the diazoketone exhibit very similar phosphate concentration-dependent activation profiles. Because of this phosphate dependency, it was not possible to differentiate an apparent protection by glutamine from the strong inhibition of inactivation caused by glutamate. The ability of glutamate to protect the glutaminase against inactivation is reversed by increasing concentrations of phosphate. The observed characteristics of inactivation by 6-diazo-5-oxo-L-norleucine differ considerably from those reported for the inactivation by L-2-amino-4-oxo-5-chloropentanoic acid. In addition, the presence of o-carbamoyl-L-serine was found to stimulate inactivation by 6-diazo-5-oxo-L-norleucine, but to protect the glutaminase against inactivation by the chloroketone. Preinactivation of the glutaminase by the diazoketone only slightly reduced the stoichiometry of binding of [5-14C]chloroketone. These observations suggest that 6-diazo-5-oxo-L-norleucine and L-2-amino-4-oxo-5-chloropentanoic acid interact with different sites on the glutaminase which are specific for binding glutamine and glutamate, respectively.
Topics: Animals; Azo Compounds; Binding Sites; Diazooxonorleucine; Glutaminase; Glutamine; Kidney; Kinetics; Phosphates; Protein Binding; Rats
PubMed: 429321
DOI: No ID Found -
Proceedings of the National Academy of... Oct 1978gamma-Glutamyl transpeptidase, a membrane-bound enzyme, functions in the gamma-glutamyl cycle to catalyze utilization of glutathione. It has been postulated that the...
gamma-Glutamyl transpeptidase, a membrane-bound enzyme, functions in the gamma-glutamyl cycle to catalyze utilization of glutathione. It has been postulated that the amino-acid-stimulated utilization of glutathione by gamma-glutamyl transpeptidase reflects an aspect of amino acid translocation. As one approach to the effective in vivo inhibition of this enzyme, the inhibition of the enzyme by L-serine in the presence of borate buffers [Revel, J.P. & Ball, E.G. (1959) J. Biol. Chem. 234, 577-582] was reinvestigated. Inhibition by L-serine, D-serine, and alpha-methyl-DL-serine in the presence of borate is competitive with respect to gamma-glutamyl substrate and such inhibition is parallel to the activity of transpeptidase toward L-gamma-glutamyl, D-gamma-glutamyl, and L-gamma-(alpha-methyl)glutamyl derivatives. L-Serine and borate effectively protect against inactivation of the enzyme by the gamma-glutamyl analogs, 6-diazo-5-oxonorleucine and azaserine, which bind to the gamma-glutamyl site of the enzyme. These studies, kinetic investigations, equilibrium dialysis experiments, and other data support the view that inhibition is produced by formation of serine-borate complex which binds at the gamma-glutamyl binding site of the light subunit of gamma-glutamyl transpeptidase. The data indicate that serine-borate complex is a transition state inhibitor of gamma-glutamyl transpeptidase.
Topics: Azaserine; Borates; Cell Membrane; Diazooxonorleucine; Kinetics; Serine; gamma-Glutamyltransferase
PubMed: 33382
DOI: 10.1073/pnas.75.10.4806 -
The Journal of Cell Biology Oct 1977A quantitative assay was used to measure the rate of collection of a population of embryonic neural retina cells to the surface of cell aggregates. The rate of...
A quantitative assay was used to measure the rate of collection of a population of embryonic neural retina cells to the surface of cell aggregates. The rate of collection of freshly trysinized cells was limited in the initial stages by the rate of replacement of trypsin-sensitive cell- surface components. When cells were preincubated, or "recovered," and then added to cell aggregates, collection occurred at a linear rate and was independent of protein and glycoprotein synthesis. The adhesion of recovered cells was temperature and energy dependent, and was reversibly inhibited by cytochalasin B. Colchicine had little effect on collection of recovered cells. Antiserum directed against recovered cell membranes was shown to bind to recovered cells by indirect immunofluorescence. The antiserum also was shown to inhibit collection of recovered cells to aggregates, suggesting that at least some of the antigens identified might be involved in the adhesion process. The inhibitory effect of the antiserum was dose dependent . Freshly trypsinized cells absorbed neither the immunofluorescence activity nor the adhesion-inhibiting activity. Recovered cells absorbed away both activities. In specificity studies, dorsal neural retina cells adhered to aggregates of ventral optic tectum in preference to aggregates of dorsal optic tectum. The adhesive specificity of the dorsal retina cells was less sensitive to trypsin than the adhesive specificity of ventral retina cells which adhered preferentially to dorsal tectal aggregates only after a period of recovery.
Topics: Animals; Antigens; Cell Adhesion; Cell Membrane; Cells, Cultured; Chick Embryo; Colchicine; Cyanides; Cycloheximide; Cytochalasin B; Diazooxonorleucine; Dinitrophenols; Retina; Superior Colliculi; Trypsin
PubMed: 562349
DOI: 10.1083/jcb.75.1.56 -
The Journal of Biological Chemistry Aug 1977Alkylation of guanosine 5'-monophosphate (GMP) synthetase with the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid (chloroketon) and 6-diazo-5-oxonorleucine...
Alkylation of guanosine 5'-monophosphate (GMP) synthetase with the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid (chloroketon) and 6-diazo-5-oxonorleucine (DON) inactivated glutamine- and NH3-dependent GMP synthetase. Inactivation exhibited second order kinetics. Complete inactivation was accompanied by covalent attachment of 0.4 to 0.5 equivalent of chloroketon/subunit. Alkylation of GMP synthetase with iodacetamide selectively inactivated glutamine-dependent activity. The NH3-dependent activity was relatively unaffected. Approximately 1 equivalent of carboxamidomethyl group was incorporated per subunit. Carboxymethylcysteine was the only modified amino acid hydrolysis. Prior treatment with chloroketone decreased the capacity for alkylation by iodacetamide, suggesting that both reagents alkylate the same residue. GMP synthetase exhibits glutaminase activity when ATP is replaced by adenosine plus PPi. Iodoacetamide inactivates glutaminase concomitant with glutamine-dependent GMP synthetase. Analysis of pH versus velocity and Km data indicates that the amide of glutamine remains enzyme bound and does not mix with exogenous NH3 in the synthesis of GMP.
Topics: Alkylation; Amino Acids; Ammonia; Carbon-Nitrogen Ligases; Diazooxonorleucine; Escherichia coli; Glutaminase; Glutamine; Guanine Nucleotides; Hydrogen-Ion Concentration; Iodoacetamide; Kinetics; Ligases; Pentanoic Acids; Protein Binding
PubMed: 18463
DOI: No ID Found -
European Journal of Biochemistry Aug 1975Glutamine accelerates the degradation of glutamine synthetase in hepatoma tissue culture cells. Compounds structurally related to glutamine were tested for their ability...
Glutamine accelerates the degradation of glutamine synthetase in hepatoma tissue culture cells. Compounds structurally related to glutamine were tested for their ability to mimic or antagonize this effect of glutamine. 6-Diazo-5-oxo-L-norleucine, like glutamine depressed the activity of glutamine synthetase in hepatoma tissue culture cells. L-Methionine sulfone, albizzine, L-methionine sulfoxide, L-gamma-glutamyl hydrazide and gamma-N-methyl-L-glutamine (listed in order of decreasing potency) were antagonists which prevented the effect of glutamine on glutamine synthetase activity. These antagonists had little effect on glutamine transport or protein synthesis of hepatoma tissue culture cells and their effects were reversible. The effects of compounds on gluatmine synthetase activity in cell-free extracts of the cells were examined. Diazo-oxonorleucine and albizzine inhibited neither the transferase nor the synthetase activity of glutamine synthetase. This observation is interpreted to mean that the glutamine-binding site involved in the regulation of glutamine synthetase activity of hepatoma tissue culture cells is not the active site of the enzyme.
Topics: Biological Transport, Active; Carcinoma, Hepatocellular; Cell Line; Diazooxonorleucine; Enzyme Induction; Glutamate-Ammonia Ligase; Glutamine; Kinetics; Liver Neoplasms; Methionine; Neoplasms, Experimental; Sulfones
PubMed: 240712
DOI: 10.1111/j.1432-1033.1975.tb02254.x -
The Journal of Cell Biology May 1975The involvement of mucopolysaccharide synthesis in cell locomotion was investigated by determining the effects of inhibition of synthesis on ruffling membrane activity...
The involvement of mucopolysaccharide synthesis in cell locomotion was investigated by determining the effects of inhibition of synthesis on ruffling membrane activity and cell movement by embryonic heart fibroblasts. Mucopolysaccharide synthesis was inhibited directly by treatment with a glutamine analog, 6-diazo-5-OXO-L-norleucine (DON), and indirectly with cycloheximide. DON treatment reduced synthesis to 20% of control values, and cycloheximide reduced synthesis to less than 10% of control values, as measured by incorporation of [35S]sulfate into mucopolysaccharides. Nevertheless, ruffling membrane activity and cell locomotion continued under both conditions. Cytochalasin B did not inhibit mucopolysaccharide synthesis, although it did stop ruffling and locomotion. These results suggest that if mucopolysaccharides are required for cell movement, they must have long half-lives or represent only a minute fraction of the normal synthetic load.
Topics: Animals; Cartilage; Cell Membrane; Cell Movement; Cells, Cultured; Chick Embryo; Cycloheximide; Cytochalasin B; Diazooxonorleucine; Fibroblasts; Glucosamine; Glycosaminoglycans; Microscopy, Phase-Contrast; Myocardium; Sternum; Sulfates; Sulfur Radioisotopes
PubMed: 123925
DOI: 10.1083/jcb.65.2.286 -
The Journal of Biological Chemistry Dec 1973
Topics: Amidohydrolases; Binding Sites; Carbon Radioisotopes; Chromatography, DEAE-Cellulose; Diazooxonorleucine; Electrophoresis, Disc; Enzyme Activation; Escherichia coli; Glutamine; Hot Temperature; Hydrogen-Ion Concentration; Kinetics; Macromolecular Substances; Models, Chemical; Molecular Weight; Oxidation-Reduction; Protein Binding; Protein Conformation; Protons; Radiation Effects; Sodium Dodecyl Sulfate
PubMed: 4587127
DOI: No ID Found -
The Journal of Biological Chemistry Dec 1973
Topics: Alkylation; Amidohydrolases; Anti-Bacterial Agents; Azo Compounds; Benzoates; Binding Sites; Carbon Radioisotopes; Catalysis; Chemical Phenomena; Chemistry; Diazomethane; Diazooxonorleucine; Escherichia coli; Esters; Glutamates; Glutamine; Hydrolysis; Isoelectric Focusing; Kinetics; Methanol; Tritium; Water
PubMed: 4587126
DOI: No ID Found -
The Journal of Biological Chemistry Sep 1973
Topics: Amino Acid Oxidoreductases; Ammonia; Bacteriophages; Diazooxonorleucine; Enterobacter; Enzyme Repression; Galactosidases; Genotype; Glucose; Glutamate Dehydrogenase; Glutamate-Ammonia Ligase; Glutamates; Glutamine; Histidine; Histidine Ammonia-Lyase; Ketoglutaric Acids; Mutation; Phenotype; Species Specificity; Transaminases
PubMed: 4146916
DOI: No ID Found