Manganese and magnesium dependent properties and inner plasma membrane surface localization of guanylate cyclase from murine plasmocytoma cells.

The particulate fraction from murine plasmocytoma cells contained 90 per cent of the total guanylate cyclase activity. Triton X-100 produced a 6 fold stimulation of guanylate cyclase activity in plasma membrane enriched fractions obtained by zonal centrifugation. Isolated inside out (10) vesicles contained 9 times more activity than rightside out (RSO) vesicles. This difference was abolished by Triton X-100 treatment of the vesicles indicating that the catalytic site of guanylate cyclase is located on the inner face of the plasma membrane. Kinetic studies of membranous guanylate cyclase showed that optimal activity was found with manganese. Only 20 per cent of this activity was obtained with magnesium. The Km for GTP with magnesium (1.4 mM) was about 7 fold greater than with manganese (0.2 mM). Positive cooperativity was obtained in both cases and the Hill coefficients were 1.8 for manganese and 1.6 for magnesium. Physiological concentrations of ATP were found to inhibit both manganese and magnesium supported activities indicating a possible regulatory mechanism for this nucleotide in vivo.