Aluminum ions are required for stabilization and inhibition of hepatic microsomal glucose-6-phosphatase by sodium fluoride.
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.
D000535:Aluminum / Q000378:metabolism* / Q000494:pharmacology; D000077410:Aluminum Chloride; D017607:Aluminum Compounds; D000818:Animals; D002712:Chlorides; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005952:Glucose-6-Phosphatase / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D012969:Sodium Fluoride / Q000494:pharmacology*
D017607:Aluminum Compounds; D002712:Chlorides; D000077410:Aluminum Chloride; D011092:Polyethylene Glycols; D012969:Sodium Fluoride; D017830:Octoxynol; D004492:Edetic Acid; D000535:Aluminum; D005952:Glucose-6-Phosphatase; D004047:Diethyl Pyrocarbonate