Biosynthesis of the Na,K-ATPase in Madin-Darby canine kidney cells. Activation and cell surface delivery.
Madin-Darby canine kidney cells were used to study events in the postsynthetic processing and cell surface delivery of Na,K-ATPase. The photoactivable 2-nitro-5-azidobenzoyl (NAB) derivative of ouabain and an anti-ouabain antibody were employed in experiments designed to determine the time intervals required for newly synthesized Na,K-ATPase to achieve the capacity to bind ouabain and to arrive at the cell surface. Ouabain-binding capacity was assessed in Madin Darby canine kidney cells which were pulse-labeled with [35S]methionine. At various chase intervals cells were disrupted by probe sonication and the resultant vesicles were permeabilized. Vesicles were incubated with NAB-ouabain and, following UV photolysis, solubilized and subjected to immunoprecipitation with an anti-ouabain antibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitates revealed that newly synthesized Na,K-ATPase can carry out type II (Mg2+ and Pi supported) ouabain binding throughout the course of its postsynthetic processing. In contrast, the ability to carry out type I (Na+, Mg2+, and ATP-supported) ouabain binding is not attained until 10 min after the completion of the sodium pump's synthesis. Experiments in which intact pulse-labeled cells were incubated with NAB-ouabain revealed that the Na,K-ATPase arrives at the cell surface as soon as 50 min after its synthesis. These results suggest that postsynthetic processing is required before the newly synthesized Na,K-ATPase can display its full repertoire of catalytic functions. This processing seems to be complete prior to the newly synthesized sodium pump's arrival at the cell surface.
Caplan
M J
MJ
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Forbush
B
B
Palade
G E
GE
Jamieson
J D
JD
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D002463:Cell Membrane Permeability; D004285:Dogs; D004789:Enzyme Activation; D015151:Immunoblotting; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D010042:Ouabain / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D000936:Antigen-Antibody Complex; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase