James J. Keirns

Adenosine 3',5' cyclic monophosphate in Euglena gracilis.

Abstract Euglena gracilis contains in high concentration the enzymes for the synthesis and degradation of cyclic AMP. The synthetic enzyme, adenyl cyclase is mainly associated with a particulate fraction which sediments at 7,000–30,000xg whereas the degradative enzyme, 3′5′ nucleotide phosphodiesterase, is soluble (does not sediment at 78,000xg). The adenyl cyclase activity is stimulated somewhat by prostaglandins and by catecholamines, agents which markedly stimulate cyclase in appropriate mammalian tissues. There is no detectable activity of guanyl cyclase, the enzyme which synthesizes cyclic GMP. Euglena also contains a cyclic AMP stimulated protein kinase which is associated with a particulate fraction sedimenting at 30,000xg.

Isolation of cyclic AMP and cyclic GMP by thin-layer chromatography. Application to assay of adenylate cyclase, guanylate cyclase, and cyclic nucleotide phosphodiesterase

Abstract A procedure is described for isolation of cAMP and cGMP by thin-layer chromatography on polyethylenimine cellulose. Chromatographs are developed (descending) twice in the same direction with two different solvents. This procedure separates cAMP and cGMP from other radioactive metatolites of [3H] or [14C] ATP or GTP. Application of this isolation method to assay of adenylate cyclase, (EC 4.6.1.1), guanylate cyclase (EC 4.6.1.2), and cyclic nucleotide phosphodiesterase (EC 3.1.4.17) has proven convenient and provides results of unusual quality.

Adenosine 3′, 5′-cyclic monophosphate in Schistosoma mansoni

Abstract Homogenates of adult Schistosoma mansoni (blood flukes), isolated from the porto-mesenteric veins of infected mice, contain substantial activities of adenylyl cyclase, cyclic AMP phosphodiesterase, and a cyclic AMP stimulated protein kinase. The adenylyl cyclase, which is largely sedimentable at 10,000xg, is stimulated 20-fold by 10mM sodium fluoride and 1.4 to 2-fold by serotonin, glucagon, prostaglandins E1, E2 or B1. The phosphodiesterase, which is largely sedimentable at 10,000xg, is inhibited by both aminophylline and papaverine but is not influenced by 10mM sodium fluoride. The protein kinase, which is present in the 10,000xg supernatant is stimulated 4 to 8-fold by either cyclic AMP or cyclic GMP. There is a preference for cyclic AMP ( K 1 2 = 1.1×10 −7 M ) over cyclic GMP ( K 1 2 = 4.5×10 −6 M ). If intact worms are incubated in a glucose free medium there is a mobilization of glycogen stores which is preceded by a rise in cyclic AMP concentration. In a medium with 5mM glucose there is neither a rise in cyclic AMP nor mobilization of glycogen.

The role of cyclic nucleotides in visual excitation

Abstract Early studies of vertebrate rod outer segment cyclic nucleotide metabolism indicated that illumination of photoreceptor membranes causes a marked reduction in apparent cyclic AMP production. Current studies confirm these observations and explain light effects in terms of an unanticipated, ATP-dependent activation of phosphodiesterase. Here we present recent progress in our understanding of the components and mechanisms of this light dependent activation. We also describe the characteristics and substrate preference (cyclic GMP) of the photoreceptor phosphodiesterase, and a suggested model for the role of cyclic nucleotides in photoreceptor physiology.

Light regulation of adenosine 3′, 5′ cyclic monophosphate levels in vertebrate photoreceptors

Abstract The regulation of adenylyl cyclase activity (measured as the production of labeled cyclic-AMP (cAMP) from labeled ATP) in frog rod outer segment photoreceptor membranes has been studied. The activity was found to be more than 20 times higher in fully dark-adapted than in bleached material. A number of experimental parameters must be carefully regulated to obtain this large ratio of dark to light activities. The disrupted rod outer segments must not be washed, since a soluble factor is essential for inhibition of activity by light. The photoreceptor membranes should be assayed for adenylyl cyclase immediately after preparation since the specific activity and light-dark differences are substantially reduced on storage at 4°C. Mechanical trauma (homogenization or sonication) causes some decrease in dark activity and a lessening of sensitivity to illumination. Preparation and assay of the dark material must be carried out using infrared light since there is a marked disproportionality between photic bleaching of rhodopsin and loss of cyclase activity. This disproportionality was further studied by mixing dark and totally bleached material in various proportions. With as little as 2% of the material bleached the cyclase activity is the same as in fully bleached material. The inhibitory action of the bleached material is destroyed by heating but is not affected by dialysis or treatment with tryspin. If the photoreceptor material is fractionated by sedimentation at 60 000 × g and 200 000 × g, the rhodopsin and adenylyl cyclase are primarily in the 60 000 × g pellet. The inhibitor is present in both pellets but not in the high speed supernatant. However, the high speed supernatant is essential for action of the inhibitor on unbleached material. Bleached (but not dark) photoreceptor material was found to inhibit cyclase in brain, liver and melanoma. Recent work carried out after presentation of the above findings has indicated that although the activity of cAMP phosphodiesterase is not affected by light under the usual assay conditions, in the presence of ATP light produces a marked stimulation of this activity. This stimulation of phosphodiesterase exhibits many of the characteristics previously attributed to the inhibition of cyclase and accounts completely or in large part for the apparent light dark differences in cyclase activity. At present the evidence points to phosphodiesterase as the main locus of light regulation of cAMP concentrations in photoreceptors, though a role for the light regulation of cyclase activity is not excluded.

THE ROLE OF CYCLIC NUCLEOTIDES IN VISUAL EXCITATION

Early studies of vertebrate rod outer segment cyclic nucleotide metabolism indicated that illumination of photoreceptor membranes causes a marked reduction in apparent cyclic AMP production. Current studies confirm these observations and explain light effects in terms of an unanticipated, ATP-dependent activation of phosphodiesterase. Here we present recent progress in our understanding of the components and mechanisms of this light dependent activation. We also describe the characteristics and substrate preference (cyclic GMP) of the photoreceptor phosphodiesterase, and a suggested model for the role of cyclic nucleotides in photoreceptor physiology.