Figure 8.

Effect of Ca²⁺ on the Mg²⁺-dependent sGC activity. gca^- cells were assayed GC activity in either Ca²⁺-free buffer or at 1 μM free Ca²⁺. The activity was measured in the lysates (A), or in the pellets (B). Experimental details in addition to the general procedure in Materials and Methods: (A) Cells were resuspended in LB from which EGTA was omitted (see Materials and Methods). Lysis occurred either at 1.5 mM EGTA (-) or at 2 μM added Ca²⁺ (+). GC assays were performed in the absence of calcium (-, with 0.75 mM EGTA in the assay), or in the presence of 1 μM free calcium (+, achieved by the addition of 1 mM Ca²⁺ if EGTA was present during lysis). (B) Lysis was performed in the presence (+) or absence (-) of Ca²⁺ as described above or in the presence of Ca²⁺ followed by addition of EGTA 1 min after lysis (+/-). Two minutes after lysis samples were centrifuged and pellets were resuspended in LB (which contained the normal level of 1.5 mM EGTA); GC assays were performed with additional EGTA (1.5 mM final concentration) so no free calcium was present. The data shown are the averages and SE of the means of two independent experiments with lysis in triplicate.