Evidence for essential carboxyls in the cation-binding domain of the Na,K-ATPase.

Abstract Treatment of isolated canine renal Na,K-ATPase with a stable diazomethane analog, 4-(diazomethyl)-7-(diethylamino)-coumarin (DEAC), results in enzyme inactivation. The inactivation rate was dramatically increased when the enzyme was treated with DEAC in the presence of ATP and Mg2+ (in imidazole buffer) or Pi and Mg2+, conditions which produce enzyme phosphorylation. Inactivation in the presence of Pi and Mg2+ could be partially prevented by Na+ and almost completely prevented by K+. The quantity of DEAC covalently bound to the Na,K-ATPase was determined spectrophotometrically. The extent of inactivation was linearly related to the amount of K-protectable DEAC incorporation. Complete inactivation of ATPase activity occurred with 2.14 +/- 0.18 nmol of DEAC covalently bound/mg of protein. This suggests that only 1 or 2 carboxyl residues/catalytic center (estimated by high affinity ADP binding) are involved in the modification leading to inactivation. The modified enzyme exhibited normal levels of high affinity [3H]ADP (and hence ATP) binding, thus, the nucleotide-binding domain of the enzyme seems unaffected by the modification. In contrast, under conditions where native enzyme was able to occlude 3.82 nmol of K+ ions/mg of protein, DEAC-modified enzyme occluded only 0.33 nmol of K+ ions. Na+ occlusion by the enzyme (in the presence of oligomycin) was also reduced (by 80%) following treatment with DEAC. Phosphorylation by [32P]inorganic phosphate and Na(+)-activated phosphorylation of the modified enzyme with [32P]ATP yielded reduced levels of phosphoenzyme (about 36%) compared to native enzyme. The DEAC-modified [32P]phosphoenzyme formed from [32P]ATP was insensitive to the addition of K+ ions, under conditions which led to the rapid hydrolysis of native phosphoenzyme. Gel electrophoresis of modified protein revealed strong fluorescence labeling of the alpha-subunit, which was substantially reduced if treatment with DEAC was performed in the presence of K+ ions. Partial tryptic digestion and electrophoretic analysis revealed normal degradation patterns in the presence of ADP (E1 form) but the typical patterns, seen with K+ ions (E2K) or Na+ ions (E1Na) in native enzyme, were absent. A typical E2-like tryptic degradation pattern was seen, however, in the presence of vanadate ions and ouabain, suggesting that the modification does not freeze the enzyme in an E1 conformation and that the enzyme is still able to undergo the E1E2 conformational transition after modification. Our results suggest that a small number of carboxyl residues in the sodium pump alpha-subunit (perhaps one) are essential for K+ and Na+ binding and stabilizing the occluded enzyme cation forms. Esterification of the carboxyl groups by DEAC inactivates the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)