Biosynthesis and turnover of Na,K-ATPase in cultured rat hepatoma cells

Author

Norman J. Karin

Date of Award

6-1983

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

John S. Cook

Committee Members

Steven Kennel, Kai-Lin Lee, Leaf Huang, Darrell Doyle

Abstract

Na,K-ATPase is an integral protein of the plasma membrane of cells in all vertebrates. The details of its biosynthesis and, to a lesser degree, its degradation (turnover) remain unclear after more than 25 years of investigation on this enzyme. I have used antibodies prepared to the catalytic (α) subunit of rat kidney Na,K-ATPase to investigate the biosynthesis and turnover of the α subunit in HTC cells, a cultured rat hepatoma line.

Na,K-ATPase was isolated in a form approximately 50% pure, as indicated by its activity and SDS-PAGE profile, by the detergent extraction of rat kidney microsomes, followed by ultracentrifugation through 50% glycerol. The pellet of purified enzyme was denatured in SDS and the α subunit collected by gel filtration. Anti-α IgG, isolated from the serum of rabbits immunized with this preparation, precipitated a major protein of 93,000 daltons from HTC cells labeled with [³⁵S]methionine. The evolutionarily conserved structure of this subunit was confirmed by the cross-reactivity of anti-rat α subunit with the enzyme from hamster, dog and human cells.

No ³⁵S-labeled glycoprotein (β) subunit was detected in the immunoprecipitates from cells lysed under a variety of nondenaturing conditions, or when chemical cross-linking preceded the detergent solubilization. A protein corresponding in size to the β subunit was observed in immunoprecipitates from cells labeled with either [³H]leucine or a mixture of [³H]fucose and [³H]mannose, suggesting that the β peptide of Na,K-ATPase in HTC cells contains little or no methionine.

The α subunit was turned over, when corrected for cell generation time (T_G), with a half-life of .45 T_G and a turnover coefficient of 1.5 T_G^-1. A 5 hour lag period was observed prior to the onset of turnover which was interpreted as the transit time of the newly-synthesized α proteins from intracellular membrane compartments to the plasma membrane. This is inconsistent with theories that α is translated as a soluble protein and inserts directly into the plasma membrane. Furthermore, immunoprecipitable (93,000 daltons) α subunit was found, after brief pulses of [³⁵S]methionine, only in cell membranes, not in the cytosol. I conclude that the α subunit of Na,K-ATPase follows the classic pathway for membrane protein biogenesis, namely translation on bound ribosomes and transport through intracellular (Golgi apparatus) membranes to the plasma membrane.

Recommended Citation

Karin, Norman J., "Biosynthesis and turnover of Na,K-ATPase in cultured rat hepatoma cells. " PhD diss., University of Tennessee, 1983.
https://trace.tennessee.edu/utk_graddiss/13084