Dimethyl sulfoxide-induced conformational state of Na(+)/K(+)-ATPase studied by proteolytic cleavage.
Effects of dimethyl sulfoxide (Me(2)SO) on substrate affinity for phosphorylation by inorganic phosphate, on phosphorylation by ATP in the absence of Na(+), and on ouabain binding to the free form of the Na(+)/K(+)-ATPase have been attributed to changes in solvation of the active site or Me(2)SO-induced changes in the structure of the enzyme. Here we used selective trypsin cleavage as a procedure to determine the conformations that the Na(+)/K(+)-ATPase acquires in Me(2)SO medium. In water or in Me(2)SO medium, Na(+)/K(+)-ATPase exhibited after partial proteolysis two distinct groups of fragments: (1) in the presence of 0.1 M Na(+) or 0.1 M Na(+) + 3 mM ADP (enzyme in the E1 state) cleavage produced a main fragment of about 76 kDa; and (2) in the presence of 20 mM K(+) (E2 state) a 58-kDa fragment plus two or three fragments of 39-41 kDa were obtained. Cleavage in Me(2)SO medium in the absence of Na(+) and K(+) exhibited the same breakdown pattern as that obtained in the presence of K(+), but a 43-kDa fragment was also observed. An increase in the K(+) concentration to 0.5 mM eliminated the 43-kDa fragment, while a 39- to 41-kDa doublet was accumulated. Both in water and in Me(2)SO medium, a strong enhancement of the 43-kDa band was observed in the presence of either P(i) + ouabain or vanadate, suggesting that the 43-kDa fragment is closely related to the conformation of the phosphorylated enzyme. These results indicate that Me(2)SO acts not only by promoting the release of water from the ATP site, but also by inducing a conformation closely related to the phosphorylated state, even when the enzyme is not phosphorylated.
Villas-Boas Tribuzy
Adriano
A
Departmento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil.
Fontes
Carlos F L
CF
Nørby
Jens G
JG
Barrabin
Hector
H
D000818:Animals; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000502:physiology; D011487:Protein Conformation / Q000187:drug effects; D012964:Sodium / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D012964:Sodium; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium; D004121:Dimethyl Sulfoxide