ACTIVATORS and INHIBITORS OF BRAIN GLUTAMINASE

Abstract

Abstract— (1) The glutaminase activity of a guinea pig brain dispersion (a 1500g supernatant solution) was tested at pH 7.5 in the presence of a series of organic acids at 20 mm with or without the further addition of 7.5 mm‐phosphate. (2) In the absence of phosphate, glutaminase activity was strongly enhanced by tricarboxylic acids, less strongly by dicarboxylic acids, and slightly, if at all, by monocarboxylic acids. Acidic amino acids were intermediate between mono‐ and dicarboxylic acids. In the presence of 7.5 mm‐phosphate, the addition of 20 mm organic acids resulted in strong potentiation of the activating effect in many cases. The activating effect of even the most active of the organic acids tested, citrate, was only about half of the effect of an equimolar amount of phosphate. (3) At phosphate concentrations approaching the saturation level for activation, the further addition of citrate was without effect. (4) Glutaminase was strongly activated by ITP which was about three times as active as inorganic phosphate. IMP was less active than inorganic phosphate and creatine phosphate had only slight activity which seemed to be accounted for by its content of inorganic phosphate. (5) Glutaminase was activated by fluoride, in the presence as well as in the absence of added phosphate. Chloride, bromide, and iodide, at 100 mm, produced increasing inhibition of the phosphate‐activated reaction. The inhibiting effect of iodide was qualitatively competitive with phosphate. (6) The effects of various other potential inhibitors and activators, including SH‐reagents, d‐glutamine, several amino acids, and amino acid derivatives were studied. (7) The results have been discussed with particular reference to their significance in elucidating the natural function of brain glutaminase. It has been suggested that glutaminase is an allosteric enzyme and that the secondary active site requires a reaction with three anionic groups for full activation.

Kinetic Comparisons Between Soluble and Membrane‐Bound Glutaminase Preparations from pig Brain

“…An additional factor may be the effects of phosphate in stabilizing the soluble enzyme [12, 151 which might, as discussed previously, have resulted in an underestimation of the initial rates at low phosphate concentrations. Apparently cooperative activation by phosphate has also been observed in studies of glutaminase activity in rat kidney mitochondria [29] and in extracts of guinea pig brain [30], in contrast to the hyperbolic behaviour observed with all preparations used here.…”

Glutamine catabolism by heart muscle: Properties of phosphate-activated glutaminase

“…It is also very similar to the Michaelis constants for the kidney (Haser et al, 1985) and brain (Benjamin, 1981;McGeer & McGeer, 1979;Weil-Malherbe, 1969) enzymes, both of which lie in the range 1.6-6.0 mm. The Vm.ax of 4.6 nmol/min per mg of protein (276 ,tmol/h per g of protein) obtained in this work in the presence of 50 mM-phosphate is, however, considerably larger than the value of 42 ,umol/h per g of protein reported by OttawAy (1969).…”

“…(i) It is stimulated by mono-, di-and tri-carboxylic acids, and the extent of stimulation increases with the number of carboxyl groups (Curthoys et al, 1976;Weil-Malherbe, 1972); (ii) it shows a biphasic response to non-physiological [Ca2+], with enhancement in the 0.2-2 mm range and inhibition at the higher levels (Benjamin, 1981); (iii) it is inhibited by the thiol reagent Nethylmaleimide (Haser et al, 1985;Kvamme & Olsen, 1979;Sayre & Roberts, 1958;Weil-Malherbe, 1969). Its sensitivity to stimulation by phosphate and sulphate is, however, lower than that of the brain glutaminase (Bradford & Ward, 1976;Haser et al, 1985), but similar to that of the kidney protein (Godfrey et al, 1977;Haser et al, 1985).…”

Effects of Branched‐Chain L‐Amino Acids, L‐Phenylalanine, and L‐Methionine on the Transport of L‐Glutamine in Rat Brain Cortex In Vitro. Influence of Cations

“…The labeled glutamate contents of the synaptosomal fraction fell, but proportionally to a much smaller extent than that undergone by glutamine. When a phosphate buffer was used in the incubation medium, L-leucine also brought about 60% inhibition of glutamine uptake, but the glutamate value was not so greatly affected, probably partly because of the well-known enhanced activity of glutaminase in the presence of phosphates (see, e.g., Weil-Malherbe, 1969).…”