Guanylate Cyclase : Assay and Properties of the Particulate and Supernatant Enzymes in Mouse Parotid

Abstract

A new, very sensitive, rapid and reliable assay for guanylate cyclase has been established based on conversion of [³²P]GTP to [³²P]guanosine 3′: 5′-monophosphate and its separation on Dowex 50 and aluminium oxide columns. The optimum conditions for the assay of mouse parotid guanylate cyclase have been established and using this procedure the properties of the enzyme have been investigated. The enzyme was found in both the particulate and supernatant fractions. The particulate enzyme was activated 12-fold by Triton X-100 and the supernatant enzyme activity increased 2-fold. In the presence of detergent guanylate cyclase activity was distributed 85% in the particulate and 15% in the supernatant fractions, respectively. The particulate activity was localised in a plasma membrane fraction. Guanylate cyclase activity was also assayed in a wide variety of other tissues. In all cases enzymatic activity was found in both the particulate and supernatant fractions. The distribution varied with the tissue but only the intestinal mucosa had a greater proportion of total guanylate cyclase activity in the particulate fraction than the parotid. The two enzymes showed some similar properties. Their pH optima were pH 7.4, both enzymes were inhibited by ATP, dATP, dGTP and ITP, required Mn²⁺ for activity and plots of activity versus Mn²⁺ concentration were sigmoidal. However, in many properties the enzymes were dissimilar. The ratios of Mn²⁺ to GTP for optimum activity were 4 and 1.5 for the supernatant and plasma-bound enzymes, respectively. The slope of Hill plots for the supernatant enzyme with varying Mn²⁺ was 2. The particulate enzyme plots also had a slope of 2 at low Mn²⁺ concentration but at higher concentrations (above 0.7 mM) the Hill coefficient shifted abruptly to 4. Calcium ions reduced sigmoidicity of the kinetics lowering the Hill coefficient, activated the enzyme at all Mn²⁺ concentrations but had no effect on the Mn²⁺: GTP ratio with the supernatant enzyme while with the plasma membrane enzyme Ca²⁺ had no effect on the sigmoid form of the kinetics at low Mn²⁺ but prevented the shift to a greater Hill coefficient at higher Mn²⁺, inhibited the activity at low Mn²⁺ and shifted the Mn²⁺ : GTP optimum ratio to 4. For the particulate enzyme plots of activity versus GTP concentration were sigmoid (n = 1.3), while the supernatant enzyme exhibited hyperbolic kinetics.