José Carlos da Costa Maia

A CALCIUM-DEPENDENT PROTEIN ACTIVATOR OF MAMMALIAN CYCLIC NUCLEOTIDE PHOSPHODIESTERASE FROM BLASTOCLADIELLA EMERSONII

1. Introduction A heat-stable, acidic calcium-binding protein (PI 4.3), with mol. wt 18 920 and originally described as an activator of cyclic nucleotide phosphodiesterase [I], has been purified and characterized from a variety of mammalian tissues [2-51, as well as from electro- plax of the electric eel [6]. This protein has been shown to exhibit multiple calcium-dependent regula- tory activities, including activation of brain adenylate cyclase [7], human erythrocyte membrane (Mg” t Ca”)dependent ATPase [8], protein kinase [9] and other calcium-sensitive reactions [ 10,111. More recently, crude extracts of > 10 representative invertebrate species were examined and all were found to have the activator protein [ 121, referred to as calmodulin [ 11. Thus, calmodulin appears to be ubiquitous in the animal kingdom. Moreover, it lacks tissue and species specificity, suggesting that it may be a very primitive protein. However, the occurrence of such a protein in bacteria or lower eukaryotes has not been described. Here we report the occurrence, in cells of the phycomycete

Transient cyclic AMP accumulation in germinating zoospores of Blastocladiella emersonii

Blastocladiella emersonii, a unicellular water mold, represents a useful organism with considerable potential for developmental studies [ 1 ] In its cell cycle, the growth period is both preceded (germination) and followed (sporulation) by periods of non growth, which are characterized by dramatic changes in cell structure and function. During germination of B. emersonii zoospores, a sequence of biochemical and morphological changes occurs. Most of this sequence does not appear to require either concommitant transcription or concommitant translation [2-71. Thus, intracellular reorganization and reactivation of metabolism, during this rapid cellular transition in the cell cycle, presumably involve control mechanisms not directly related to gene expression. These conditions led us to speculate that cyclic adenosine 3’,5’mono. phosphate (CAMP) could play a role in regulating the germinatiop process in B. emersonii. We have demonstrated that zoospores contain independent specific enzymes involved in the hydrolysis of cyclic adenosine 3’,5’-monophosphate and cyclic guanosine 3’,5’-monophosphate [8]. CAMP phosphodiesterase activity is at its highest level in zoospores and drops precipituously during the first 20 min of germination [7]. The present paper shows that this drop in activity of CAMP

Calcium efflux during germination ofBlastocladiella emersonii

Abstract Preloaded zoospores ofBlastocladiella emersonii were found to release large quantities of45Ca2+ in the course of germination, whereas no calcium is released from nongerminating zoospores. Studies with inhibitors of germination and of calcium movement seem to support the existence of two temporally as well as developmentally distinct effluxes during this differentiative transition. The early efflux seems to be associated with the triggering of the process and begins prior to other known structural and physiological responses of the zoospores; the late efflux starts just prior to the appearance of the first germling cells and is probably related to the morphological progress of germination. The early efflux of calcium per se is not sufficient to elicit the process, since the calcium ionophore A23187 produces a comparable efflux, but induces germination only in the presence of noneffective cyclic AMP concentrations.

Independent cAMP and cGMP phosphodiesterases in Blastocladiella emersonii

CAMP’ and cGMP’ are now recognized as key intracellular regulators in several biological functions, including growth and morphogenesis [ 1,2] . CAMP levels are low during logarithmic growth but rise when contact inhibited cells reach confluency and stop growing [3,4]. The other naturally occurring cyclic nucleotide, cGMP, might also be involved in cell growth regulation, and evidence for an opposing influence of this nucleotide to CAMP, in at least certain stages of growth, has recently been uncovered [5,6]. The intracellular levels of cyclic nucleotides are controlled by synthesis via adenylate or guanylate cyclases, and by degradation via cyclic nucleotide phosphodiesterases. In many cell types there are at least two and perhaps more forms of phosphodiesterases [7]. Chicken embryonic fibroblasts possess CAMP and cGMP phosphodiesterase activities under separate genetic control [8]. The presence of a CAMP phosphodiesterase activity exhibiting a cyclic fluctuation throughout the life cycle has been demonstrated in Blastocladiella emersonii [9]. Variations in the size of the intracellular pools of CAMP and cGMP during the life cycle of this fungus have been reported; however, a cGMP phosphodiesterase activity was not found [lo] . The present

Serine/threonine protein phosphatases in Dictyostelium discoideum: No evidence for type I activity

Extracts from Dictyostelium discoideum contain type 2A and 2C serine/threonine-specific protein phosphatases with properties very similar to those from mammals according to their sensitivity to okadaic acid and to their dependence for divalent cations. In contrast, no type 1 protein phosphatase is found at any time of development, neither in the cytosolic nor in the particulate fraction, using glycogen phosphorylase a, casein, histone or the non-proteinous 4-Methylumbelliferyl phosphate as substrates. Both type 2A and 2C protein phosphatase activities remain constant throughout the development cycle.

Changes in cGMP phosphodiesterase levels during growth and differentiation in Blastocladiella EmersonII

Blastocladiella emersonii, a unicellular phycomycete represents a useful organism for experimental studies on the regulatory role of cyclic nucleotides (see ref [ 1 ] for a recent review of the cell cycle). Thus, the levels of CAMP** and cGMP** as well as enzyme activities concerning their metabolism were found to change markedly during the life cycle [2-51. In all living systems so far studied, cGMP is synthesized via guanylate cyclase and hydrolysed via cyclic nucleotide phosphodiesterases. In principle, any alteration in the amount or activity of either of these two enzymes would affect the intracellular cGMP concentration. Thus, it is important to find out if these two enzyme activities are altered as a function of the cell cycle. cGMP levels in B. emersonii are low during the growth stage rising about SO1 OO-fold at a defined stage in its life cycle, during sporulation [3]. We have demonstrated that B. emersonii contains independent specific enzymes involved in the hydrolysis of CAMP and cGMP [6]. The present communication shows that the specific activity of cGMP phosphodiesterase dramatically changes throughout the life cycle of B. emersonii, particularly during germination and sporulation. The results indicate that the observed variation in cGMP levels when cells sporulate [3] reflects the changes in enzymatic activities, mainly in cGMP phosphodiesterase.