Abstract
Stabilization and inhibition of hepatic microsomal glucose-6-P phosphohydrolase (EC 3.1.3.9) by F- requires the presence of Al3+ ions. At millimolar concentrations, reagent grade NaF inhibited glucose-6-P hydrolysis and protected the enzyme against inactivation induced by heat in the presence of 0.025% (w/v) Triton X-100 or by reaction of the catalytic site with the histidine-specific reagent, diethyl pyrocarbonate. The presence of millimolar EDTA in all test systems abolished the effectiveness of NaF, yet EDTA by itself was without significant influence on the kinetics of phosphohydrolase reaction, the thermal stability of the enzyme or its reactivity with diethyl pyrocarbonate. Although ultrapure NaF was ineffectual in all test systems, its potency as a competitive inhibitor or protective agent was markedly increased by micromolar AlCl3 or when assays were carried out in flint glass test tubes. The latter response is explained by the well documented ability of fluoride solutions to extract Al3+ from glass at neutral pH. Our analysis indicates that the effectiveness of fluoride in all test systems derives from the formation of a specific complex with Al3+, most likely Al(F)4-. The apparent dissociation constant for interaction of the enzyme and Al(F)4- is 0.1 microM. The combination of NaF and AlCl3 holds promise as an unusually effective and versatile means to stabilize this notoriously labile enzyme during efforts to purify it.
Highlights
From the Division of Nutritional Sciencesand Section of Biochemistry, Molecularand Cellular Biology of the Division of Biological Sciences,Savage Hall, Cornell University, Ithaca, New York 14853
Stabilization and inhibition of hepatic microsomal idly equilibrates across the membrane, presumably via a pasglucose-6-P phosphohydrolase (EC 3.1.3.9) by F- re- sive, nonmediated process [2]
At millimolar concen- Despite its important position at the terminus of the glutrations, reagent gradeNaF inhibited glucose-6-P hy- coneogenic and glycogenolytic pathways in liver and kidney, drolysisand protected the enzyme against inactivatiowne know relatively little aboutthe components of the glucoseinduced by heat in the presence0.o0f26%(w/v)Triton 6-phosphatase system compared with other key enzymes of
Summary
From the Division of Nutritional Sciencesand Section of Biochemistry, Molecularand Cellular Biology of the Division of Biological Sciences,Savage Hall, Cornell University, Ithaca, New York 14853. X-100 orby reactionofthe catalytic site with the the glucogenic pathways. This situation has arisen in part histidine-specificreagent, diethyl pyrocarbonate. The presence of millimolarEDTA in all test systems abolished theeffectiveness of NaF, yet EDTA byitself was without significant influence on the kinetics of phosphohydrolase reaction, the thermasltability of theenzyme or its reactivity with diethyl pyrocarbonate. Burchell and Burchell [5] were able to make significant progress in the purification following their discovery that sodium fluoride was an unusually effective stabilizer of the “solubilized”. Thelatter response is explained by thwe ell mechanism by which sodium fluoride stabilizes glucose-6-P documentedability of fluoride solutiontso extract Ais+ phosphohydrolase. The apparent dissociation constant inactivation of the enzyme by the histidine reagent, DEPC,’
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