Abstract

Adenosine cyclic 3',5'-monophosphate (cAMP)-dependent protein kinase activity has been found in both the 27,000 x g particulate and supernatant fractions of homogenates from the liver fluke, Fasciola hepatica. The enzyme activity was 2-5 times greater in the anterior end ("head") of the fluke than in the posterior end. Half-maximal activation of the protein kinase in both fractions was obtained at 0.1-0.4 µM cAMP. When the enzyme was assayed in the presence of cyclic nucleotide, the apparent K_m for protamine was 0.1 mg/ml and the apparent K_m for MgATP was 20-50 µM in the two homogenate fractions. Incubation of fluke heads for 5 min with 1 mM 5-hydroxytryptamine resulted in an increase in protein kinase activity of both homogenate fractions when assayed in the absence of added cAMP. The protein kinase activity assayed in the presence of 5 µM cAMP was increased in the particulate fraction and decreased in the supernatant fraction. The degree of activation of the enzyme by the cyclic nucleotide was markedly reduced in both homogenate fractions from heads that had been incubated with 5-hydroxytryptamine. The time course of accumulation of endogenous cAMP correlated with activation of protein kinase, except during the first minute of incubation with 5-hydroxytryptamine. The degree of activation of protein kinase by added cAMP was inversely correlated with endogenous cyclic nucleotide during incubation with 5-hydroxytryptamine. Incubation of fluke heads with both 5-hydroxytryptamine and D-lysergic acid diethylamide together reduced the activation of protein kinase by 5-hydroxytryptamine. Incubation of fluke heads with only D-lysergic acid diethylamide had no effect on protein kinase activity, cAMP increased phosphorylation of fluke protein in both fractions of the homogenate. This phosphorylation was less sensitive to activation by the cyclic nucleotide when the fluke heads had been incubated with 5-hydroxytryptamine. Thus 5-hydroxytryptamine can stimulate protein kinase activity toward endogenous proteins. The results indicate that the physiological effects of 5-hydroxytryptamine are mediated by cAMP activation of protein kinase.