Conformational states of the (Na+ + K+)-transporting ATPase. Formation of 240 000-Mr and 116 000-Mr polypeptides in the presence of a bifunctional thiol probe.
Interpeptide cross-linking of alpha-subunits with concomitant loss of Na+ + K+-transporting ATPase (Na+, K+-ATPase) activity was found when the purified lamb kidney enzyme was treated with the bifunctional thiol reagent 4,4'-difluoro-3,3'-dinitrodiphenyl sulphone (F2DNS). Several forms of the enzyme could be clearly distinguished: one binding ATP (non-phosphorylated enzyme, E1 X ATP), a phosphorylated form (E2-P) and a phosphoenzyme-ouabain complex (E2P X ouabain). A polypeptide of approx. Mr 240 000 and probable alpha 2 composition comprised up to 5-20% of the total polypeptides after reaction of the lamb kidney Na+, K+-ATPase with F2DNS. The amount of this polypeptide formed was related to the conformational state of the enzyme. The presence of adenine nucleotide greatly diminished the amount of 240 000-Mr polypeptide formed and provides evidence for an enzyme-adenine-nucleotide complex under conditions where the enzyme is not phosphorylated. F2DNS reacted with the enzyme in the presence of Mg2+, Pi and ouabain to form a new polypeptide with an approx. Mr of 116 000, and comprised 23% of the total, whereas the 240 000-Mr polypeptide comprised 9% of the total. This suggests that the 116 000-Mr polypeptide is a characteristic marker of the E2P X ouabain complex. By using specific antibodies it was established that both the 240 000- and 116 000-Mr polypeptides contained alpha-, but not beta-, subunits of the Na+, K+-ATPase.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003432:Cross-Linking Reagents; D004139:Dinitrofluorobenzene / Q000031:analogs & derivatives; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000201:enzymology; D010455:Peptides / Q000032:analysis*; D011487:Protein Conformation; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000032:analysis
D003432:Cross-Linking Reagents; D010455:Peptides; D013438:Sulfhydryl Compounds; C013936:difluorodinitrobenzene sulfone; D000255:Adenosine Triphosphate; D004139:Dinitrofluorobenzene; D000254:Sodium-Potassium-Exchanging ATPase