Turnover and regulation of Na, K-ATPase in HeLa cells

When the plasma membranes were isolated from cells which had been grown in ¹³C-substituted amino acids, the Na,K-ATPase, specifically labeled with [γ-³²P]ATP, sedimented in the sodium dodecyl sulfate (SDS)- containing density gradients more rapidly than control enzyme labeled with [γ-³³P]ATP. Turnover was measured by resuspending ¹³C-substituted cells in control medium and following the decrease in density of ¹³C-substituted enzyme as it was replaced by enzyme of control density. A lag period of about 4 to 4.5 h, or 0.16 to 0.2 of the generation time (T_G) was observed, followed by the first-order disappearance of the ¹³C-substituted enzyme. I attribute the lag to the transit time from the cellular locus of synthesis to the surface membrane. Turnover, corrected for dilution of the ¹³C-enriched enzyme by net growth, had a half-time of 5.5 h, or 0.24 T_G, corresponding to a rate of constant of 3.1/T_G.

In parallel experiments I have measured the turnover of [¹⁴C] leucine-labeled protein in both total cell protein and cell- surface protein. Total protein turnover showed no lag and was approximately first-order, with a mean half-time of about 60 h. Plasma membrane protein showed a pronounced lag, followed by a complex turnover of at least two components. The data support a model of plasma membrane turnover in which the individual proteins are turned over at different rates.

Significant isotope effects were observed when HeLa cells were cultured in ²H substituted amino acid-containing medium. Rates of growth and of protein synthesis were significantly lower than controls; eventually, the deuterated cells ceased growing and lysed. These effects could be reduced by labeling cells for less than 24 hours, but could not be eliminated. In contrast, cells grew well in medium containing ¹³C-substituted amino acids; such cells incorporated [¹⁴C] leucine into protein at the control rate.

Density shifts obtained for specifically phosphorylated, ¹³C-labeled Na,K-ATPase were equivalent to or larger than those obtained for ²H-labeled enzyme, indicating that greater incorporation of ¹³C-amino acids compensated for the theoretically smaller density shifts expected with ¹³C-amino acids. The first-order rate constant for removal from the surface (1.5/T_G) obtained using ²H-substituted amino acids gave a half-time for turnover of the Na,K-ATPase (0.45 T_G or 21 h), substantially longer than obtained using ¹³C-substituted amino acids.

Effects of long-term, subtotal inhibition of Na⁺,K⁺ transport, by growth of cells in low-[K⁺] medium, are also described for HeLa cells. After long-term growth in low-[K⁺] there was two-fold or greater increase in ouabain binding per cell. The increase was reversible. To assess the corresponding transport changes for cells grown in low-[K⁺] I have separately estimated the contributions of increased intracellular [Na⁺] and of transport capacity (number of transport sites) to transport regulation. During induction as well as its reversal, More slowly, long-term regulation was achieved by changes in the number of functional transporters in the plasma membrane.

Parallel exposure of cryptic Na⁺ ,K⁺-ATPase activity with SDS in the plasma membranes of both induced and control cells, as assayed by specific phosphorylation, showed that the long-term response cannot be accounted for by an exposure of pre-existing Na⁺ ,K⁺-ATPase in the plasma membrane. Analyses of the kinetics of the induction and its reversal and the steady state turnover of the enzyme following induction show that it is caused by a decrease in the rate of degradation of the enzyme.