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1. An assay, based on the transfer of label from [gamma-32P]ATP to [32P]phosphoenolpyruvate, suitable for a steady-state kinetic analysis of pyruvate kinase in the reverse direction (i.e. phosphoenolpyruvate synthesis), is described. 2. This assay was used in a kinetic investigation of the rabbit muscle enzyme including initial-rate and product-inhibition experiments, at a pH of 7.4 and constant concentrations of total K+ and free Mg2+. 3. These studies indicate that there is a random release of ADP and phosphoenolpyruvate from the enzyme and that there is a competitive substrate inhibition by ATP. Some of the results were suggestive that the rapid-equilibrium assumption, generally used for this enzyme was not valid. 4. Techniques were developed to measure the rate of isotopic exchange between all the substrate-product pairs. 5. By using these techniques the rates of isotopic exchange at chemical equilibrium were measured. The results indicate that this enzyme does not catalyse a truly rapid-equilibrium random mechanism, although in the forward reaction all initial-rate data obtained to date are consistent with this assumption.

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Pyruvatekinase from the hepatopancreas of the common shore crab, Carcinus maenas, was purified to a specific activity of 240 units/mg of protein in the assay conditions described. 2. In one method of purification the enzymic activity could be resolved into two fractions after chromatography on DEAE-cellulose. Fructose 1, 6-diphosphate was able to effect the conversion of one form (peak 1) into the second (peak 2). 3. In the presence of a saturating concentration of fructose 1, 6-diphosphate both forms of the enzyme were kinetically similar. 4. Polyacrylamide-gel electrophoresis of the enzyme 1 day after preparation showed a single protein band. On storage at least three protein bands became visible, all of which were associated with pyruvate kinase activity. 5. Chromatography of the enzyme on Sephadex G-200 indicated a mol.wt. of 247000, but in the presence of fructose 1, 6-diphosphate the elution volume of the enzyme increased corresponding to a mol.wt. of 193000. 6 Dissociation of the enzyme in sodium dodecyl sulphate and 2-mercaptoethanol followed by polyacrylamide-gel electrophoresis produced one major protein band with a mol.wt. of 55000.