The Influence of Salts on Pyruvate Kinase from Tissues of Higher Plants

Abstract

Experiments have been conducted to determine the properties and specifically the cation requirements of pyruvate kinase from certain species of higher plants. The concentration of both ADP and PEPA required for saturation of the enzyme from a dialyzed extract of pea seed were determined and from these data the appropriate Michaelis constants were estimated. After the cation requirements were established, evidence was obtained indicating that enzyme activity was proportional to both enzyme concentration and to reaction time under the specified conditions. Detailed studies with the enzyme from pea seed demonstrated an absolute divalent cation requirement that was satisfied by the salts of Mg++ or Mn++. The optimum concentration of these cations was 5 x 10-3 [image]. Co++ at this concentration was approximately 80% as effective as the other two cations. The chloride salts of Ni++ or Ca++ exhibited no activating capacity and Be++ was only slightly stimulatory. The enzyme from pea seed also required a univalent cation salt for maximum activity and this requirement was satisfied by the chloride salts of K+, Rb+ or NH4+. A concentration of 0.05 [image] resulted in maximum activity. NaCl at a concentration of 0.05 [image], which was optimum, resulted in approximately 20% of the activity obtained.with a comparable concentration of K+. The enzyme activity was essentially independent of the anion present with the exception of Br- and I- which were slightly inhibitory at concentrations greater than 0.05 [image]. An absolute requirement of univalent cations for the enzymes from pea seed was not established, however, the low activity of the preparation in absence of added univalent cations was accounted for by the content of K+ and Na+ in the extracts. The Michaelis constants (KA) for the various univalent cation activators were approximately one-fifth the value reported for pyruvate kinase from rabbit muscle. Pyruvate kinase activity was demonstrated in extracts of the seeds of wheat, cotton, oat, corn and beet; in the leaves of tobacco and peas and in the petioles of celery. The enzyme activity of extracts from all these sources was strikingly stimulated by KC1. An absolute requirement for univalent cations was demonstrated with extracts of cotton, oat and corn seed. The pyruvate kinase in those extracts, that did not show an absolute dependence on univalent cations, whould not withstand dialysis periods of sufficient length to remove all endogenous univalent cations.