Specific involvement of calmodulin and non‐specific effect of tropomyosin in the sensitivity to ouabain of Na+, K+‐ATPase in murine plasmocytoma cells

Abstract

The Kd for ouabain for inhibition of Na+,K+-ATPase isolated from murine plasmocytoma MOPC 173 cells is 120 microM, but when isolated in the presence of EDTA, it is 100-fold lower (1.2 microM). Simultaneous addition of muscle tropomyosin and calcium to sensitive membranes restored the original insensitivity (tropomyosin bound to the membranes in an irreversible and saturable manner). For comparison 86Rb influx into intact cells, mediated by the Na+,K+-pump, is half-maximally inhibited at 50 microM ouabain. Calcium converts the enzyme to an insensitive form. This appeared to involve calmodulin because after extraction of calmodulin with EDTA and EGTA from sensitive membranes, they could not be made insensitive by the addition of tropomyosin and Ca2+. Addition of exogenous calmodulin to these calmodulin-depleted membranes was required, in addition to tropomyosin and Ca2+, to decrease the ouabain sensitivity. The involvement of calmodulin was further assessed by measuring the range of Ca2+ concentrations required to convert to the insensitive form. At saturating concentrations of tropomyosin, increasing free [Ca2+] up to 3 microM led to an heterogeneous population of Na+,K+-ATPase forms. The calcium dependency was a saturable process. The shift to the insensitive form was half maximal at 0.65 + 0.11 microM free Ca2+ and was abolished by the addition of troponin I or trifluoroperazine (0.1 mM). These results suggest that, in murine plasmocytoma cells, the intrinsic sensitivity of Na+,K+-ATPase to ouabain might be regulated by a calmodulin-dependent process within a submembrane contractile-like environment.