Regulation by turnover of Na,K-ATPase in HeLa cells.

As in most if not all animal cells, HeLa Na,K-ATPase is an essential enzyme of the cell surface. The three ways, referred to in the Introduction, in which it is regulated may be summarized as follows: 1) The activity of the enzyme under normal conditions responds almost linearly to small perturbations in internal (Na+); this is short-term regulation. 2) In the normal steady state, the enzyme is one of the rapidly turning over components of the cell surface; this is part of long-term regulation. The functional importance of turnover to cell homeostasis depends on the reversibility of any event that may inactive the enzyme. Thus turnover is an important, but not the only, means of recovery from ouabain intoxication, and for an inactivating ligand that dissociates more readily than ouabain turnover may be relatively unimportant. Conversely, turnover may be the only means of recovery from thermal inactivation at physiological temperatures. 3) Under conditions of prolonged stress, turnover itself may be modulated so as to enhance the number of active enzymes on the cell surface. Regulation by turnover has the advantage of permitting a prompt response to a changing cell environment, whereas regulation by synthesis would introduce a delay in response at least equal to the transit time. Broadman et al [1974] have suggested that the signal for the increase in Na,K-ATPase is elevated cellular (Na+), but how this is translated into a mechanism for altering the specific clearance of this enzyme from the membrane is not known.

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

HeLa cells in log growth have 10(6) surface Na-K-ATPase molecules as estimated by the specific binding of [3H]-ouabain. Studies utilizing ouabain as a label show that the ligand is internalized at a rate corresponding to the turnover of three sets of Na-K-ATPase enzymes per generation. The label is taken up exclusively into a particulate cell compartment where it is codistributed with beta-hexosaminidase, identifying the internal compartment as lysosomal. Turnover is an important parameter in the recovery of the cells from glycoside intoxication. The unmetabolized glycoside is subsequently released by exocytosis. 13C-density-labeled Na-K-ATPase has been identified by specific phosphorylation of its catalytic subunit with [32P]ATP or [33P]ATP, and the rate of turnover of the density label is shown to be the same as the internalization of the ouabain-labeled site. There is a transit time of about 4 h from the onset of synthesis of the catalytic subunit to its insertion in the surface membrane; 2,800 catalytic subunits are synthesized per minute per cell, and 2,100 are turned over K+-starved cells respond to the stress in 24-30 h with modulation of the surface density of Na-K-ATPase the synthetic rate remains constant; the number of functional enzymes per cell is controlled by change in the rate constant for turnover.

Na+, K+-ATPase in HeLa cells after prolonged growth in low K+ or ouabain.

Effects of long-term, subtotal inhibition of Na+-K+ transport, either by growth of cells in sublethal concentrations of ouabain or in low-K+ medium, are described for HeLa cells. After prolonged growth in 2 X 10(-8) M ouabain, the total number of ouabain molecules bound per cell increases by as much as a factor of three, mostly due to internalization of the drug. There is only about a 20% increase in ouabain-binding sites on the plasma membrane, representing a modest induction of Na+, K+-ATPase. In contrast, after long-term growth in low K+ there can be a twofold or greater increase in ouabain binding per cell, and in this case the additional sites are located in the plasma membrane. The increase is reversible. To assess the corresponding transport changes, we have separately estimated the contributions of increased intracellular [Na+] and of transport capacity (number of transport sites) to transport regulation. During both induction and reversal, short-term regulation is achieved primarily by changes in [Na+]i. More slowly, long-term regulation is achieved by changes in the number of functional transporters in the plasma membrane as assessed by ouabain binding Vmax for transport, and specific phosphorylation. Parallel exposure of cryptic Na+,K+-ATPase activity with sodium dodecyl sulfate in the plasma membranes of both induced and control cells showed that the induction cannot be accounted for by an exposure of preexisting Na+,K+-ATPase in the plasma membrane. Analysis of the kinetics of reversal indicates that it may be due to a post-translational event.