Chloro(2,2':6',2"-terpyridine) platinum inhibition of the renal Na+,K+-ATPase

Chloro(2,2':6',2"-terpyridine)platinum, a bulky, hydrophilic reagent, inhibited the renal sodium pumpwith a single exponential time course. K⁺ increased therate constant of the reaction by about twofold; the K⁺concentration dependence was monotonic, with a half-maximal effect observed at 1 mM, consistent with K⁺ acting at a transportsite. Na⁺, Mg²⁺, eosin, and vanadate did notsignificantly alter the rate of reaction. The results of proteolysisand mass spectrometer analysis were consistent with terpyridineplatinum labeling of Cys452, Cys456, or Cys457. Because phenylarsineoxide reacts with vicinal cysteines and did not prevent terpyridineplatinum modification, terpyridine platinum most likely modifiesCys452. This modification prevents ADP binding; interestingly, theanalogous residue in sarco(endo)plasmic reticulumCa²⁺-ATPase (SERCA) is on the exterior of thenucleotide-binding pocket. Thus it appears that the terpyridineplatinum residue is more accessible in the presence of K⁺than in its absence and that terpyridine platinum modification preventsnucleotide binding.