Interaction of Triton X-100 with particulate cardiac guanylate cyclase: Comparison between Mg 2+- and Mn 2+-supported enzyme activities in particulate, detergent-solubilized and detergent-insoluble fractions

Abstract

Guanylate cyclase activity was determined in a 1000 g particulate fraction derived from rabbit heart homogenates using Mg 2+ or Mn 2+ as sole cation in the presence and absence of Triton X-100. With Mg 2+, very little guanylate cyclase activity could be detected in the original particulate fraction assayed with or without Triton, or in the particulate fraction treated with varying concentrations of Triton (detergent-treated mixture) prior to enzyme assay. However, the detergent-solubilized supernatants as well as the detergent-insoluble residues (pellets) derived from detergent-treated mixtures possessed appreciable Mg 2+-supported enzyme activity. With Mn 2+, significant enzyme activity was detectable in the original particulate fraction assayed without Triton. Much higher activity was seen in particulate fraction assayed with Triton and in detergent-treated mixtures; the supernatants but not the pellets derived from detergent-treated mixtures possessed even greater activity. The sum of enzyme activity in pellet and supernatant fractions greatly exceeded that of the mixture. When the pellets and supernatants derived from detergenttreated mixtures were recombined, measured enzyme activities were similar to those of the original mixture. With Mg 2+ or Mn 2+, the specific activity of guanylate cyclase in pellet and supernatant fractions varied considerably depending on the concentration of Triton used for treatment of the particulate fraction; treatment with low concentrations of Triton (0.2–0.7 μmol/mg protein) gave supernatants showing high activity whereas treatment with relatively greater concentrations of the detergent (>0.7 μmol/mg protein) gave pellets showing high activity. The relative distribution of guanylate cyclase in pellet and supernatant fractions expressed as a function of Triton concentration during treatment (of the particulate fraction) showed that 50 to 80% of the recovered enzyme activity remained in supernatants at low detergent concentrations whereas 50 to 80% of the recovered activity resided in the pellets at higher detergent concentrations. Inclusion of excess Triton in the enzyme assay medium did not alter the specific activity profiles and the relative distribution patterns of the cyclase in pellet versus supernatant fractions. The results demonstrate the inherent potential of cardiac particulate guanylate cyclase to utilize Mg 2+ in catalyzing the synthesis of cyclic GMP. However, it appears that some factor(s) endogenous to the cardiac particulate fraction severely impairs the expression of Mg 2+-dependent activity; Mn 2+-dependent activity is also affected by such factor(s) but apparently less severely. Further, the results suggest that previously reported activities of cardiac particulate guanylate cyclase, despite being assayed with Mn 2+ and in the presence of Triton X-100, represent underestimation of what otherwise appears to be a highly active enzyme system capable of utilizing physiologically relevant divalent cation such as Mg 2+.