Proton-dependent inhibition of yeast and brain hexokinases by aluminum in ATP preparations.

Abstract

The aluminum present as a contaminant in ATP preparations can cause strong inhibition of yeast hexokinase P-II activity at pH 7.0 or below but has little or no inhibitory effect at a pH of 7.5 or greater. The inhibition is reversed by citrate, 3-phosphoglycerate, malate, phosphate, and catecholamines, all of which have previously been described as activators of hexokinase at low pH. We suggest that these agents activate the enzyme only by virtue of their ability to coordinate with aluminum present in the assay system. The presence of aluminum is also responsible for the "negative cooperativity" observed at low pH with respect to Mg'ATP concentration-i.e., the inhibition by aluminum is uncompetitive at low Mg'ATP concentrations but becomes competitive at high Mg'ATP concentrations. The inhibition is thought to be due to formation of a complex of AI-ATP with the enzyme, with a dissociation constant (K;) of 0.1 tM. Yeast hexokinase P-I is somewhat less sensitive to Al than is hexokinase P-II, and yeast glucokinase is not detectably affected. The hexokinase in rat brain (type I) shows a p11-dependent inhibition by Al similar to that observed with the yeast hexokinases, whereas the rat muscle (type II) enzyme is less sensitive, suggesting a possible relationship to aluminum encephalopathy in man. Kosow and Rose (1) showed that, when yeast hexokinase P-I1 was inhibited by lowering the pH from the optimum of 8.0 to values around 7.0 or below, it could be activated either by raising the Mg-ATP concentration ("substrate activation" or "negative cooperativity") or by adding certain metabolitese.g., citrate, 3-phosphoglycerate, malate, or phosphate. We reported (see refs. 2 and 3, compare with ref. 4) that the inhibition by protons was not seen with certain buffers at pH 6.9, but that inhibition was then obtained by adding low concentration of ADP or GDP, and that this inhibition was reversed by any of the activators listed above.We now report that the degree of inhibition of yeast hexokinase by protons is dependent on the aluminum content of the ATP (or ADP) used, and that the activation by excess Mg-ATP or by the above-mentioned metabolites is due to their ability to reverse inhibition by aluminum. We also report a similar sensitivity of brain hexokinase to aluminum and suggest a possible relation to cases of encephalopathy associated with aluminum toxicity in man.MATERIALS AND METHODS Yeast hexokinase isozymes P-I and P-I were prepared as described (5). They were dialyzed vs. 50 mM potassium phosphate, pH 7/0.1 mM EDTA and stored frozen as concentrated solutions of approximately 20 mg of protein per ml. These frozen stocks were diluted 1:1000 each day in 50 mM Tris-HCI, pH 6.9, containing 0.1 mM EDTA. From 0.005 to 0.03 units was added to reaction mixtures in 2-to 4-,gl volumes. The specific activity of the stock P-II was 650 units/mg and that of the stock P-I was 160 units/mg.Glucose-6-phosphate dehydrogenase, purity grade I, was purchased from Boehringer Mannheim. It was dialyzed vs. 50 mM Tris-HCl, pH 6.9/...

Effects of aluminium on electrical and mechanical properties of frog atrial muscle

“…The secondary increase in contractile force was most probies to ATP molecules 107 more strongly than Mg2+, ably not due to mechanisms depending on the sodium grang inactive ATP-A1, which disrupts the activity of yeast dient, since aluminium had the same effects in the absence of )rain hexokinases (Womack & Colowick, 1979). We may any significant gradient.…”

Biological Water Dynamics and Entropy: A Biophysical Origin of Cancer and Other Diseases

“…In 1979, Womack and Colowick demonstrated proton-dependent inhibition of yeast and brain hexokinases by Al in ATP preparations [255]. In 1984, Lai and Blass demonstrated inhibition of brain glycolysis by Al in rat brain with IC50 values between 4 and 9 microM for cytosolic and mitochondrial hexokinase inhibition [256].…”

Isolation and functional characterization of a novel plastidic hexokinase from Nicotiana tabacum

“…Since cooperativity may be artificial due to several experimental flaws [18], a more thorough investigation of the phenomenon is obligatory. Influence of Al 3+ ions as contaminants in commercial ATP preparations could be ruled out by measuring activity at pH 8.4 and by addition of 0.5 mM NaCitrate to the buffer [19]. Since activity was measured in crude extracts, parameters may differ from those of purified enzymes.…”