STUDIES OF THE SODIUM BINDING CENTERS OF THE PROTEOLYSIS PRODUCTS OF MEMBRANE-BOUND NA-ATP-ASE BY NA-23-NMR( K+)

Changes in the Na-23(+) relaxation times during the <<exhaustive>> try ptic or chymotryptic cleavage of membrane-bound pig kidney Na+, K+-ATR -ase were studied in TES-TMA buffer containing NaCl. The effect of NaC l concentration on the relaxation times of Na-23(+) in the products of proteolytic digestion of Na+, K+-ATR-ase was studied. These products included the water-soluble and membrane-bound fractions of the enzyme as well as the membrane-bound fractions obtained during the two-step t ryptic digestion of Na+, K+-ATR-ase in the presence of 0,1 M NH4HCO3. The data obtained show that upon the <<ehaustive>> tryptic proteolysis , the Na+-binding sites of Na+, K+-ATR-ase remained membrane-bound, wh ereas after the <<exhaustive>>, chymotryptic proteolysis some of the N a+-binding sites are transferred to water-soluble state (K-d = 13,7 mM ). Centrifugation, resuspendition, and removal of Na+ cations by dialy sis altered the characteristics of the Na+-binding sites in the mebran e-bound proteolytic products. The high affinity binding sites of the m embrane-bound product obtained after the 1-st step of two-step tryptic hydrolysis of Na+, K+-ATR-ase displayed decreased affinity for Na The low affinity Na+-binding sites of the membrane-bound products of the second step of tryptic diggestion were altered to a greater extent tha n of the first step.