Tryptic modification of red-cell sodium pump behaviour.
Inside-out membrane vesicles derived from human red cells were used to probe the effects of controlled tryptic digestion on the sodium pump as it exists in situ. Digestion of the enzyme in its E1 conformation resulted in several alterations which are generally similar to those reported for the purified kidney enzyme, namely (i) greater loss in overall hydrolytic activity compared to level of phosphoenzyme intermediate and (ii) cleavage of the alpha-subunit by trypsin as well as chymotrypsin at the cytoplasmic surface to yield a fragment of approx. 78 kDa. Tryptic digestion effected similar rates of inactivation of pump-mediated Na+-K+(Rb+) exchange, (ATP- plus ADP)-dependent Na+-Na+ exchange and, in the absence extracellular alkali cation, 'uncoupled' Na+ flux (Na+/0 flux). Alteration in the Na+:Rb+(K+) stoichiometry following trypsin cleavage could not be detected. The conformational transitions of phosphoenzyme and dephosphoenzyme are affected similarly by trypsin, as evidenced by similar inactivation rates of reactions through the 'forward' sequence involving the E1P to E2P transition as well as through the 'reserve' sequence involving the E1 to E2 transition.
D009597:4-Nitrophenylphosphatase / Q000032:analysis; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D011487:Protein Conformation; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology*
D000255:Adenosine Triphosphate; D012964:Sodium; D009597:4-Nitrophenylphosphatase; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium