Fig. 6.

Phosphorylation of olfactory particulate guanylyl cyclase by protein kinases. A, In vitrophosphorylation of guanylyl cyclase by various protein kinases. Olfactory particulate guanylyl cyclase was solubilized and purified by a single chromatography step on a GTP–agarose column and incubated with PKA (250 U/20 μg), PKG (300 U/1 μg), Ca²⁺/calmodulin PK (CamPK, 300 U/500 ng), or PKC (300 U/50 ng) for 30 min (Promega) at 30°C. One unit is the amount of each kinase required to incorporate 1 pmol of phosphate into the substrates of each kinase per minute. PKA was incubated in either the absence or presence of 1 mm 8-Br-cAMP. The effect of PKA-specific inhibitor was also tested. PKG was stimulated in the presence of 1 mm 8-Br-cGMP. With CamPK, pGC was incubated in the presence of 10 μm calmodulin and 10 mm Ca²⁺. PKC was stimulated by adding 20 μg of phosphatidylserine and 250 nm 12-O-tetradecanoyl phorbol 13-acetate.B, Effect of PKA on particulate guanylyl cyclase activity. Purified pGC was phosphorylated by the catalytic subunit of protein kinase A for 30 min, after which cGMP levels were determined. Guanylyl cyclase was assayed at high (1 mm) or low (10 μm) GTP concentrations to delineate high and lowK_m activities. The results shown are representative of six independent experiments and presented as percentage of control ± SEM.