Mechanism of inhibition of glycolysis by vanadate.
Vanadate is known to inhibit several phosphatases including Na+, K+-ATPase, alkaline phosphatase, and glyceraldehyde-3-P dehydrogenase. Inhibition presumably results because vanadium adopts a stable structure which resembles the transition state of phosphate during the reactions involving these enzymes. We performed experiments to further examine the effects of vanadate (VO3-4) on erythrocyte (red blood cells (RBC] glycolytic intermediates. RBC obtained from human subjects were centrifuged and washed with lactated Ringer's 5% dextrose. 31P nuclear magnetic resonance analysis of the RBC revealed the characteristic peaks for the 3-phosphate and 2-phosphate of 2,3-diphosphoglycerate (DPG), inorganic phosphate (Pi), and ATP. Incubation of RBC with 10(-6) M VO3-4 led to a disappearance of ATP and 2,3-DPG while the peak for Pi increased. By the end of 4 h over 90% of the VO3-4 had been reduced to VO2+ (vanadyl) in the RBC. The effects of 10(-4) M iodoacetamide and 10(-5) M ethacrynic acid, known inhibitors of glyceraldehyde-3-P dehydrogenase that act by interactions with sulfhydryl groups (-SH) of the enzyme, were similar to those of VO3-4. Incubation with vanadyl did not affect the peaks for Pi, 2-DPG, or 3-DPG. Furthermore, using electron spin resonance we demonstrated that in the presence of glyceraldehyde-3-P dehydrogenase, VO3-4 is reduced to VO2+. The findings demonstrate that VO3-4 inhibits glycolysis at micromolar concentrations and that the ion is reduced to VO2+ in the cell. The similarity of the effect of VO3-4 to those of iodoacetamide and ethacrynic acid suggests that interactions with -SH groups is its mechanism of inhibition. Since under physiological conditions intracellular VO3-4 concentrations are in the micromolar range and may exist in oxidized and/or reduced forms, VO3-4 could regulate the activity of glyceraldehyde-3-P dehydrogenase through changes in the redox state of the enzyme rather than by substituting for the PO3-4 ion.
D004229:Dithiothreitol / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D004976:Ethacrynic Acid / Q000494:pharmacology; D006019:Glycolysis / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology*
D014639:Vanadium; D014638:Vanadates; D004976:Ethacrynic Acid; D004229:Dithiothreitol