Distribution of PMCA V_max among normal RBCs. One experiment with protocol 2 is shown (see “Experimental protocols”) representative of 5 with similar results. (A) Duplicate measurement of the PMCA V_max by the load-extrusion protocol. The indicated mean V_max values were obtained by linear regression through the first 6 time-points after cobalt addition. The mean rate of Ca²⁺ extrusion fell progressively when [Ca_T]_i dropped below about 100 μmol (340 g Hb)^-1. (B) RBCs from the same batch as those used for the V_max measurements reported in panel A underwent the load-extrusion protocol without ⁴⁵Ca tracer and were sampled for hemolysis curves at the indicated times after ionophore addition (the number of curves exceeded the repertoire of available symbols in the software used leading to cyclic repetition; however, the bottom-up sequence can be followed unambiguously). All samples were postincubated in LK medium. There was a monotonic upward progression of the curves with time from the fully dehydrated pattern (1-min point) to the fully recovered pattern (7-min point). (C) Percent cells with emptied Ca²⁺ loads as a function of time after cobalt addition (Δt). The percent of Ca²⁺-emptied RBCs was computed from the ordinate readings shown in panel B at a relative tonicity of 0.3, taking the distance between lowest and highest reading as 100% (see “Materials and methods”). The equation y = axⁿ/(bⁿ + xⁿ) was found to give a good empirical fit of the experimental points in the 5 experiments of this series, slightly better than that obtained with other sigmoid saturation functions, as judged by eye and by least means squares analysis. The parameter values obtained by least mean squares fit to the equation in this experiment were as follows: a = 100, b = 2.2, and n = 5.5. (D) Derivative of the curve fit through the experimental points in panel C (continuous line) compared with a Gaussian curve fit to the rising branch of the derivative curve (dotted line). Both curves are plotted as a function of V_max estimated from (Ca²⁺ load)/Δt. The Ca²⁺ load in the suspension used for hemolysis curve sampling (B) was 390 μmol (340 g Hb)^-1. Compared with the Gaussian curve, the derivative curve reporting the actual V_max distribution among RBCs shows a marked right-shifted skew, observed in all the experiments of this series (Table 3). The parameter values of the actual V_max distribution are shown in the figure and were calculated as follows: mean = Σ(y_ix_i)/Σy_i;SD = ((Σ(y_i(x_i-mean))²)/Σy_i)^1/2; Skew = (Σ((y_i(x_i-mean))/SD)³)/Σy_i.