Alterations in 32P-labelled intermediates during flux activation of human platelet glycolysis

Using a newly developed isotopic tracer technique for the measurement of 32P-labelled intermediates in glycolysis and nucleotide metabolism in platelets, we studied the variations in 32P-labelled intermediates during activation of the glycolytic flux by cyanide and platelet-activating agents. The major variations occurred in [32P]Fru-1,6-P2, dihydroxy acetone phosphate, ATP and Pi. There was a quantitative covariance between the increase in lactate production and the rise in [32P]Fru-1,6-P2 induced by different platelet-activating agents. In contrast, cyanide induced weaker activation of the flux and greater accumulation of [32P]Fru-1,6-P2. Variations in 32P-labelled intermediates were apparent 5 s after flux activation, but the major changes in [32P]Fru-1,6-P2 occurred much later and fell in periods in which a constant lactate formation was maintained. The cyanide-induced changes in 32P-labelled intermediates depended on the extracellular level of glucose, showing a predominant ATP----Pi conversion in glucose-depleted medium that shifted to an ATP----Fru-1,6-P2 conversion at excess glucose. At about 50 microM glucose, flux activation occurred without major changes in [32P]Fru-1,6-P2, dihydroxy acetone phosphate and Pi, with only a small fall in [32P]ATP. The data provide evidence for a role of the aldolase reaction in flux control and demonstrate rapid changes in Fru-1,6-P2 and ATP during flux activation with an additional role for Fru-1,6-P2 as an energy buffer during post-activation periods.