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 r2P]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 8 5 % 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 MnZf 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 Mnz+ : 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.

Expression, purification, and characterization of the intra-cellular domain of the ANP receptor

“…However, at higher Mn 2+ concentrations, the activity of both constructs decreases whereas higher Mg 2+ concentrations does not seem to have an effect on activity. Such a negative effect on activity by increased Mn 2+ concentrations had been also noted previously in an intact membrane guanylyl cyclase from mouse parotid [15], GCA [16], and adenylate cyclase [17]. Interestingly, the GCA ID and GCA CC+GC protein constructs seem to have different relative Mn 2+ inhibitory effects (the activity for GCA CC+GC dropped 7.4-fold and for GCA ID only 4.4-fold when the [Mn 2+ ] was increased from 2 to 16mM).…”

Differential Inhibition of Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by Purine and Pyrimidine Nucleotides

“…Additionally, hypoxanthine nucleotides bind to other prototypical GTP-utilizing/GTP-binding proteins such as sGC and G-proteins with lower affinity than the corresponding guanine nucleotides (Fig. 4) (39,43,60,64). Considering that MANT-GTP␥S is a potent competitive AC inhibitor (K i for S49 cyc Ϫ membrane AC, 53 nM) (37), we predicted MANT-ITP␥S to be a potent AC inhibitor with reduced affinity for sGC and G-proteins relative to MANT-GTP␥S.…”

Loss of adenylate cyclase activity in variants of Bordetella pertussis

“…In addition, no cyclic AMP was detected in C-mode cultures of strain 18334 when examined at frequent intervals during the growth cycle. Cyclic AMP levels and [ 12] in any of the samples. Although the role of guanosine 3',5 '-cyclic monophosphate (cyclic GMP) in bacteria is obscure, these observations are of interest in view of the reported reciprocal relationship, in some cases, between the levels of cyclic AMP and cyclic GMP [131.…”