An unexpected effect of an ouabain-sensitive ATPase activity on the amount of antigen-antibody complexes formed in situ.
A classical method of indirect immunofluorescence was applied on various kinds of lightly fixed and permeabilized cells to analyze the formation of the complexes between a nuclear antigen and its antibody (AAC). The amount of AAC decreased dramatically when the incubation with the first antibody was realized in the presence of ATP in a sodium-rich medium with 0.5 mM KCl. Addition of sodium vanadate, a general inhibitor of ATPases, ouabain or tetrabutylammonium ion, specific inhibitors of the Na+,K+-ATPase, prevented this effect. The established role of this enzyme is to increase free-K+ concentration and decrease free Na+ concentration in the cell. It is not surprising to find an ATPase still active since light fixation and permeabilization do not destroy phosphatases. But it is rather surprising to find something looking like Na+,K+-ATPase activity in permeabilized cells. The importance of potassium in this puzzling result is suggested by the fact that appearance of ACC was equally suppressed if the incubation was made in the absence of ATP in a potassium-rich medium without sodium. Results are discussed, taking into account the properties of cell-associated water and recently found interrelation between Na+,K+-ATPase and tubulin. In any case, the results seem interesting in the field of immunocytochemistry.
Mentré
P
P
Station INRA (806), Institut de Biologie Physico-Chimique, Paris, France. mentre@ibpc.fr
Debey
P
P
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D004622:Embryo, Mammalian; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008815:Mice, Inbred Strains; D009687:Nuclear Proteins / Q000276:immunology; D009865:Oocytes; D010042:Ouabain / Q000494:pharmacology*; D011188:Potassium / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000936:Antigen-Antibody Complex; D009687:Nuclear Proteins; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium