JoséCarlos Da Costa Maia

Adenylate cyclase activity and cyclic AMP metabolism during cytodifferentiation of blastocladiella emersonii

Abstract Particulate cell fractions of zoospores of Blastocladiella emersonii contain an adenylate cyclase activity which can be solubilized by 1% Triton X-100. The enzyme requires Mn 2+ and is not inhibited or stimulated by NaF or cathecolamines. The enzyme specific activity fluctuates according to the various phases of the life cycle of the organism. It is barely detectable at the vegetative, growth phase. During formation and release of zoospores it reaches levels 70-fold higher. The zoospores keep a high level of adenylate cyclase, which, however, decreases during germination. At the round cell stage of germination it is 40% of the initial level and decreases further in the following phase, the germling formation. Intracellular level of cyclic AMP was similarly measured at germination. Some of its fluctuation is tentatively explained by unparalleled variations in the activities of adenylate cyclase and cyclic AMP phosphodiesterase. There was also found a certain excretion of cyclic AMP during this phase which certainly contributes for the drop in the intracellular concentration of the nucleotide during the transition of round cell to germling.

Developmental changes in translatable RNA species and protein synthesis during sporulation in the aquatic fungus Blastocladiella emersonii

Protein synthesis during sporulation in Blastocladiella emersonii is developmentally regulated as revealed using [35S]methionine pulse labeling and two-dimensional gel electrophoresis. A large increase in the synthesis of several proteins is associated with particular stages. A large number of basic proteins are synthesized exclusively during late sporulation. Changes in translatable mRNA species were also detected by two-dimensional gel electrophoresis of the polypeptides produced in a cell-free rabbit reticulocyte lysate primed with RNA prepared at different stages of sporulation. The synthesis of several proteins during sporulation seems to be transcriptionally controlled. Most of the sporulation-specific messages are not present in the mature zoospores.

Induction of blastocladiella emersonii germination by cyclic adenosine-3′, 5′-monophosphate

Abstract The role of adenosine 3′,5′-monophosphate (cyclic AMP) in the control of Blastocladiella emersonii germination was studied. This differentiative transition may be induced by replacing K+, a classical inducer, by cyclic AMP or by competitive inhibitors of cyclic AMP phosphodiesterase activity. When zoospores are treated simultaneously with two inducers at non-effective concentrations, a synergistic effect is observed between cyclic AMP and either KCl or adenine. The calcium ionophore A23187 per se is not able to elicit germination, but the association of A23187 and sub-optimal concentrations of cyclic AMP is effective. These results suggest that germination may depend on a correlation between the intracellular mobilization of calcium and cyclic AMP levels.

Studies on the adenosine 3',5'-monophosphate-dependent protein kinase of Blastocladiella emersonii.

Abstract Using a combination of Chromatographic and sucrose density gradient techniques under carefully controlled conditions of pH and protease inhibitors, we demonstrate that there is only one form of adenosine 3′,5′-monophosphate-dependent protein kinase in the cytosol fraction of the Blastocladiella emersonii zoospore. If any of these conditions are omitted during extract preparation, one obtains what are apparently multiple forms of the enzyme, which are in reality artifacts due to extensive endogenous proteolytic activity. This endogenous protease is stimulated by alkaline pH and inhibited by antipain. The zoospore protein kinase is similar to type II protein kinase from mammalian cells in several aspects including Chromatographic behavior on DEAE-cellulose column, conditions for subunit dissociation and reassociation, as well as the molecular weight value of the regulatory subunit.

Changes in cyclic AMP binding and protein kinase activities during growth and differentiation of Blastocladiella emersonii

Multiple protein kinases in the water mould Blastocladiella emersonii are described. A cyclic AMP-independent protein kinase which prefentially phosphorylates casein remains unchanged during vegetative growth of the cells and in the two phases of differentiation: germination and sporulation. In contrast, cyclic AMP-dependent protein kinase activity and cyclic AMP binding components are induced during the sporulation.