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STIMULATION BY PHOSPHATE OF THE ACTIVATION OF GLUTAMATE APODECARBOXYLASE BY PYRIDOXYL‐5′‐PHOSPHATE AND ITS IMPLICATIONS FOR THE CONTROL OF GABA SYNTHESIS
Author(s) -
Martin Sandra Bloom,
Martin David L.
Publication year - 1979
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1979.tb05274.x
Subject(s) - pyridoxal , pyridoxal phosphate , enzyme , chemistry , cofactor , stimulation , glutamate receptor , glutamate decarboxylase , biochemistry , enzyme activator , phosphate , endocrinology , biology , receptor
— Previous studies have shown that inorganic phosphate relieves the inhibition of brain glutamate decarboxylase by ATP. Since the evidence suggested that inhibition by ATP resulted in formation of the inactive apoenzyme, it was possible that P i might relieve this inhibition by promoting activation of the apoenzyme by its cofactor, pyridoxal‐5′‐phosphate. We have investigated this possibility using apoenzyme from rat brain. In most experiments, apoenzyme was prepared by incubating glutamate decarboxylase with 20 μM‐aminooxyacetate followed by exhaustive dialysis. Activation was studied by incubating the enzyme with pyridoxal‐P under various conditions after which the amount of holoenzyme formed was measured by a 5 min enzyme assay. In the absence of P i there was an initially rapid but incomplete activation by pyridoxal‐P which stopped after 15‐20 min. The amount of holoenzyme formed after 20 min increased without saturating as the concentration of pyridoxal‐P was raised from 0.03 to 250 μ m Addition of 1‐10m m ‐P i increased the initial rate of activation and the final degree of activation. P i stimulated activation whether present initially or added after 15 min, indicating that incomplete activation in the absence of P i was not attributable to destruction of pyridoxal‐P or irreversible inactivation of the enzyme. P i reduced the concentration of pyridoxal‐P, giving half maximal activation from about 10 μ m to about 0.07 μ m . Pi also stimulated the residual enzyme activity in the apoenzyme preparation in the absence of added pyridoxal‐P, suggesting that P i may convert the holoenzyme to a more active form. P i had very similar effects on glutamate apodecarboxylase from vitamin B 6 ‐deficient rats and also stimulated the activation of apoenzyme which had been prepared by dissociation of the cofactor by treatment with glutamate, indicating that stimulation by P i is unrelated to the method of preparing apoenzyme. Activation was also strongly stimulated by methylphosphonate and arsenate and weakly stimulated by sulfate. Trichloromethylphosphonate, cacodylate, pyrophosphate and AMP had little or no effect. The results suggest that P i relieves the inhibition by ATP, at least in part, by promoting the activation of glutamate apodecarboxylase, and that P i may be an important factor in the regulation of glutamate decarboxylase in vivo .