Inhibition of Na+, K+ Activated ATPase: By Macrocyclic Polyethers

A NADH coupled assay was used to evaluate the effect of crown ethers on the kinetics of Na+, K+ activated ATPase. The coupled assay reaction mixture contained lactic acid dehydrogenase, pyruvate kinase (0.06 mg protein/ml), phosphoenolpyruvate (0.8 mM), NADH (1.41 miu), ATPase (0.079 mg protein/ml), ATP (variable) in a 50mM Tris HC1 buffer, pH 7.8, containing 100 mM NaCl, 10 mM KC1, 5 mM MgCl2 and 3 mM EDTA. Two crown ethers and two cryptand polyethers were used, the dicyclohexano-18-crown-6 (18-C-6) inhibited over the range from 18 mM (9%) to 100 mM (66%), dicyclo-hexano-21-crown-7 (21-C-7) from 9 mM (13%) to 110 mM (93%). The cryptand 4,7,13,16,21-pentaoxa-l,10-diazabicyclo-(8,8,5)-tricosane (k221) inhibited over the range from 2 mM (9%) to 20 mM (82%), while its analogue, 4,7,13,16,21,24-hexaoxa-l,10-diazabicyclo-(8,8,8)-tricosane (k222) inhibited from 0.3 mM (11%) to 18 mM (65%). The kinetic plots exhibits uncompetitive inhibition for 18-C-6, and suggest the same for 21-C-7, k221 and k222, although noncompetitive inhibition cannot be excluded from the latter