Interactions of Cs + and other monovalent cations (Li +, Na +, K +, Rb +, NH 4+) with K +-dependent pyruvate kinase and malate dehydrogenase from the yeasts Rhodotorula rubra and Saccharomyces cerevisiae

Abstract

The activities of K + -dependent pyruvate kinase and malate dehydrogenase in cell-free extracts from Saccharomyces cerevisiae X2180-1B and R. rubra NCYC 797 were examined in the presence of Cs + and other monovalent cations (Li + , Na + , K + , Rb + , NH 4 + ) in order to assess their ability to substitute for K + in activation of these enzymes and therefore their suitability as probes for studying the importance and function of K + . Varying the monovalent cation concentration (up to 100 nw) in S. cerevisiae did not inhibit activity of either of the enzymes assayed. In comparison, NADPH-dependent malate dehydrogenase from R. rubra was inhibited approximately 90 ±1 % in the presence of K + , Cs + Na + , Rb + and Li + at a concentration of 100 m m . In contrast, NADH-dependent malate dehydrogenase from R. rubra showed constant enzyme activity irrespective of the monovalent cation present indicating that NADH-dependent malate dehydrogenase may be of low monovalent cation specificity in both yeasts or possesses no binding sites for monovalent cations apart from K + . Similarly, Cs + and the other monovalent cations tested did not affect the activity of pyruvate kinase from S. cerevisiae . In contrast, pyruvate kinase from R. rubra was less inhibited by cations with ionic radii approximating more closely to that of K + (133 pm). NH 4 + (143 pm) and Rb + (147 pm) could partially substitute for K + in enzyme activation, whereas smaller ions such as Na + (97 pm) or Li + (78 pm) or larger ions such as Cs + (166 pm) inhibited activity. Kinetic analysis of pyruvate kinase from R. rubra revealed an increased V max of 14·4 × 10 −2 mol lactate produced min −1 (mg protein) −1 at high [K + ] (50–200 m m ) compared to a V max of 7·5 × 10 −2 umol lactate produced min −1 (mg protein) −1 at low [K + ] (1–25 m m ) which indicated the presence of additional K + -binding sites on the enzyme; K m values of 42·5 and 4·4 m m were obtained for high (50–200 m m ) and low (1–25 m m ) K + concentration ranges respectively. Inhibition of K + activation of pyruvate kinase by Cs + was examined over the K + concentration range 1–25 m m K + and Cs + concentrations of 25–100 m m . Kinetic analysis revealed that Cs + inhibition was predominantly uncompetitive over these concentration ranges indicating that the Cs + combines with the enzyme-activator (K + ) complex and not with the free enzyme to form an inactive enzymatic structure. It is concluded that Cs + -sensitive K + -dependent enzymes, such as pyruvate kinase from R. rubra , may represent an intracellular site for toxicity of inessential monovalent cations.