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J Biol Chem. 1990 Feb 25;265(6):3528-34.

Biosynthesis of the Na,K-ATPase in Madin-Darby canine kidney cells. Activation and cell surface delivery.

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Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.


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.

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