INTERACTIONS OF CS-DEPENDENT PYRUVATE-KINASE AND MALATE-DEHYDROGENASEFROM THE YEASTS RHODOTORULA-RUBRA AND SACCHAROMYCES-CEREVISIAE( AND OTHER MONOVALENT CATIONS (LI+,NA+,K+,RB+,NH4+) WITH K+)

The activities of K+-dependent pyruvate kinase and malate dehydrogenas e in cell-free extracts from Saccharomyces cerevisiae X2180-1B and R. rubra NCYC 797 were examined in the presence of Cs+ and other monovale nt cations (Li+, Na+, K+, Rb+, NH4+) in order to assess their ability to substitute for K+ in activation of these enzymes and therefore thei r suitability as probes for studying the importance and function of K. Varying the monovalent cation concentration (up to 100 mM) in S. cer evisiae did not inhibit activity of either of the enzymes assayed. In comparison, NADPH-dependent malate dehydrogenase from R. rubra was inh ibited approximately 90+/-1% in the presence of K+, Cs+, Na+, Rb+ and Li+ at a concentration of 100 mM. In contrast, NADH-dependent malate d ehydrogenase from R. rubra showed constant enzyme activity irrespectiv e of the monovalent cation present indicating that NADH-dependent mala te dehydrogenase may be of low monovalent cation specificity in both y easts or possesses no binding sites for monovalent cations apart from K+. Similarly Cs+ and the other monovalent cations tested did not affe ct the activity of pyruvate kinase from S. cerevisiae. In contrast pyr uvate kinase from R. rubra was less inhibited by cations with ionic ra dii approximating more closely to that of K+ (133 pm). NH4+ (143 pm) a nd 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 io ns such as Cs+ (166 pm) inhibited activity. Kinetic analysis of pyruva te kinase from R. rubra revealed an increased V-max of 14.4 X 10(-2) m u mol lactate produced min(-1) (mg protein)(-1) at high [K+] (50-200 m M) compared to a V-max of 7.5 x 10(-2) mu mol lactate produced min(-1) (mg protein)(-1) at low [K+] (1-25 mM) which indicated the presence o f additional K+-binding sites on the enzyme; K-m values of 42.5 and 4. 4 mM were obtained for high (50-200 mM) and low (1-25 mM) K+ concentra tion ranges respectively. Inhibition of K+ activation of pyruvate kina se by Cs+ was examined over the K+ concentration range 1-25 mM K+ and Cs+ concentrations of 25-100 mM. Kinetic analysis revealed that Cs+ in hibition was predominantly uncompetitive over these concentration rang es indicating that the Cs+ combines with the enzyme-activator (K+) com plex and not with the free enzyme to form an inactive enzymatic struct ure. 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.