glutaminase and maleic-acid

A key evolutionary development facilitating land colonization in terrestrial isopods (Isopoda: Oniscidea) is the intermittent liberation of waste nitrogen as volatile ammonia. Intermittent ammonia release exploits glutamine (Gln) as an intermediary nitrogen store. Here, we explore the relationship between temporal patterns of ammonia release and Gln accumulation in three littoral oniscideans from Southern California. Results are interpreted in terms of water availability, habitat, activity patterns, and ancestry. A two-way experimental design was used to test whether ammonia excretion and Gln accumulation follow a tidal or diel periodicity. Ammonia excretion was studied in the laboratory using chambers with or without available seawater and using an acid trap to collect volatile ammonia. Ligia occidentalis releases ammonia directly into seawater and accumulates Gln during low tide (48.9 ± 6.5 μmol g⁻¹ at low tide, 24.1 ± 3.0 μmol g⁻¹ at high tide), indicating that excretion is tidally constrained. Alloniscus perconvexus and Tylos punctatus can excrete ammonia directly into seawater or utilize volatilization. Both species burrow in sand by day and show a diel excretory pattern, accumulating Gln nocturnally (31.8 ± 2.7 μmol g⁻¹ at dawn and 21.8 ± 2.3 μmol g⁻¹ at dusk for A. perconvexus; 85.7 ± 15.1 μmol g⁻¹ at dawn and 25.4 ± 2.9 μmol g⁻¹ at dusk for T. punctatus) and liberating ammonia diurnally. Glutaminase shows higher activity in terrestrial (0.54-0.86 U g⁻¹) compared to intertidal (0.25-0.31 U g⁻¹) species, consistent with the need to generate high PNH₃ for volatilization. The predominant isoform in Armadillidium vulgare is phosphate dependent and maleate independent; phosphate is a plausible regulator in vivo.

Topics: Ammonia; Animals; Arthropod Proteins; Behavior, Animal; California; Circadian Rhythm; Ecosystem; Enzyme Activation; Glutaminase; Glutamine; Isopoda; Kinetics; Maleates; Models, Biological; Pacific Ocean; Phosphoric Acids; Phylogeny; Species Specificity; Tidal Waves; Trees

The activity of gamma-glutamyl transpeptidase was inhibited and stimulated by hippurate with and without acceptor molecules, respectively. Substrate activation was observed when acceptor molecules was not present but the activation was disappeared by the addition of hippurate. When glutamine was absent the inhibition degree of hippurate at pH 8.0 was greater than at pH 7.0, whereas in the presence of both hippurate and glutamine the results were opposed. These show a possibility that glutaminase is activated by hippurate under the condition of physiological pH. The activation of glutaminase by hippurate was also confirmed directly. The inhibition degree of transfer activity by glutamine was increased along the increase of acceptor concentration only in the presence of hippurate. Gamma-Glu-Phe-Gly satisfying prerequisites for donors doesn't act as a substrate, which shows that L-gamma-glutamyl-p-nitroanilide itself hardly acts as an acceptor. This contradicts to an idea that substrate activation caused by autotranspeptidation. The change of apparent Km and Vmax according to the change of pH in the presence of hippurate was different from that in the presence of acceptors. This shows a possibility that the active site on small subunit is inhibited and the latent active site in large subunit is exposed by the interaction of hippurate to the enzyme.

Topics: Animals; Cattle; Enzyme Activation; gamma-Glutamyltransferase; Glutamates; Glutamic Acid; Glutaminase; Glutamine; Hippurates; Hydrogen-Ion Concentration; Hydrolysis; Kidney; Kinetics; Maleates; Substrate Specificity

Owing to increased enzymatic hydrolysis of glutamine, additional ammonia is formed in blood and plasma specimens with increased gamma-glutamyltransferase activity (gamma-GT, EC 2.3.2.2.). The close correlation between gamma-GT and glutaminase (EC 3.5.1.2) activities (r = 0.97) in plasma, the inhibition with 6-diazo-5-oxo-L-norleucin or borate plus serine, and the activation with maleate clearly show that gamma-GT itself is the glutamine-deamidating enzyme in plasma. Under pathological conditions, increased gamma-GT activity can increase the rate of ammonia formation in plasma more than 30-fold the mean values of healthy subjects. Increased in vitro formation of ammonia caused by increased gamma-GT activity is an important source of false-positive findings in ammonia determination. Because of the high prevalence of pathological gamma-GT activities in clinical populations, blood specimens for ammonia analysis should be preserved by addition of borate plus serine.

Topics: Ammonia; Borates; Chromatography, Gel; Diazooxonorleucine; False Positive Reactions; gamma-Glutamyltransferase; Glutamates; Glutamic Acid; Glutaminase; Glutamine; Humans; Maleates; Serine; Specimen Handling

Phosphate-activated glutaminase (EC 3.5.1.2) in synaptosomal preparations is inhibited 40-60% by the sulphydryl group reagent N-ethylmaleimide (NEM), forming the basis for distinction between NEM-sensitive and NEM-insensitive glutaminases. The NEM effect cannot be explained by differential effects on distinct glutaminases because other glutaminases have not been detected, and the synaptosomal glutaminase activity can be fully accounted for by the activity of phosphate-activated glutaminase. By fractionation of mitochondria isolated from synaptosomal preparations, which are preincubated with and without NEM, both NEM-sensitive and NEM-insensitive glutaminases are found to be localized to the inner mitochondrial membrane. Variations in pH (7.0-7.6) and the phosphate concentration (5-10 mM) affect chiefly NEM-sensitive glutaminase, demonstrating that this glutaminase may be subject to regulation by compounds in the cytosol having restricted permeability to the inner mitochondrial membrane. Since p-hydroxymercuribenzoate, which is known to be impermeable to the inner mitochondrial membrane, inhibits glutaminase similarly to NEM, phosphate-activated glutaminase is assumed to be compartmentalized within the inner mitochondrial membrane. Thus, NEM-sensitive glutaminase is localized to the outer face and NEM-insensitive glutaminase to the inner region of this membrane and probably also to the matrix region.

Topics: Animals; Enzyme Activation; Ethylmaleimide; gamma-Glutamyltransferase; Glutaminase; Hot Temperature; Hydrogen-Ion Concentration; Hydroxymercuribenzoates; Intracellular Membranes; Maleates; Phosphoric Acids; Rats; Synaptosomes