Histochemistry of guanylate cyclase activity.

Abstract

Guanylate cyclase liberates pyrophosphate from guanosine triphosphate (GTP). In studies published previously, this phosphate is trapped by lead ions even though it is known that free lead ions inactivate a considerable proportion of this enzymatic activity. To overcome the damaging effects of fixation, this study used fresh cryostat sections stabilized with a sufficient concentration of a collagen-derived polypeptide to ensure no measurable loss of guanylate cyclase activity. To avoid the damaging influence of free lead ions, we used a hidden metal capture reagent, i.e., a complex of lead ammonium citrate/acetate that does not react with GTP but which rapidly forms a precipitate with the pyrophosphate liberated by the enzyme. The lead precipitate is then converted into the colored sulfide which is measured in individual cells by microdensitometry. This system was used to measure guanylate cyclase activity in individual cells in unfixed sections of rat liver.

An improved histochemical method for measuring nitric oxide synthase activity

“…0, with 30 per cent Polypep 5155 in an atmosphere of air, as described previously. 1,7 The results (Table 1) show that, under these conditions, the LACA made in our laboratory was at least as eective as the commercial brand.…”

“…7 For the former the medium contained guanosine triphosphate (10 mM), sodium azide (2 . 5 mM), sodium acetate (0 .…”

“…1 The GTP lithium salt (10 mM) was then added and the pH adjusted to pH 7 . 0 (see 7 ). Finally, for measuring NOS activity, arginine (5 mM) and NADPH (1 mM) were added and the pH was again adjusted to the required pH.…”

The natural substrate for nitric oxide synthase activity

“…As in an earlier microdensitometric study 9 the NOS activity was measured by the increase in soluble guanylate cyclase (sGC) activity induced by the presence of NADPH with either carnosine or arginine.…”

Measurement of nitric oxide synthase activity in sections of rat liver.

“…In the preceding report we described a histochemical method for measuring both membrane-bound and soluble (cytosolic) guanylate cyclase (sGC) activity in hepatocytes (Mehdizadeh et al, 1995). It is known that the cytosolic component is activated by nitric oxide (NO) generated by nitric oxide synthase (NOS) (Waldman and Murad, 1987).…”