NUCLEOTIDE-BINDING IS INSUFFICIENT TO INDUCE COLD INACTIVATION OF THEVACUOLAR-TYPE ATPASE FROM MAIZE ROOTS

Although it has been established that the stability of V-type ATPase a t 0 to 4 degrees C is diminished by the presence of substrate, the mec hanism of inactivation is poorly understood. The ability of various nu cleotide analogs that have been identified as either competitive or no n-competitive inhibitors of ATP-dependent proton transport to induce c old inactivation of the maize (Zen mays L.) vacuolar-type H+-ATPase at 0 degrees C was compared to that of substrate ATP. There was little i f any loss in activity over 30 min incubation at 0 degrees C in the pr esence of 10 mM KNO3 and 2 mM MgSO4. Addition of 2 mM ATP promoted sig nificant inhibition over this time period. The noncompetitive inhibito rs, 5'-adenylylimidodiphosphate and 2'-,3'-O-(4-benzoylbenzoyl)-adenos ine triphosphate, did not accelerate the loss of activity. Therefore, binding of nucleotides was not sufficient for cold inactivation. The c ompetitive inhibitor: dialdehyde derivative of AMP, did accelerate col d inactivation of the H+-ATPase but to a much lesser extent than ATP. During cold incubation, ATP and the dialdehyde derivative of AMP were hydrolyzed, whereas the two non-competitive inhibitors were not. Hydro lysis of nucleotides at 0 degrees C was associated with the degree of cold inactivation. These results suggest that hydrolysis of nucleotide s at 0 degrees C rather than binding caused instability in the H+-ATPa se complex that led to cold inactivation.