Enzymes of diadenosine tetraphosphate (Ap4A) catabolism in the green alga Scenedesmus obliquus

Abstract

The nucleotide diadenosine 5',5"-P1 ,p-tetraphosphate ( A p d )is found in all cells in concentrations ranging from 0.05 to >lo0 pM. It been implicated in the regulation of the cellular response to metabolic stresses, including heat shock, oxidative stress and DNA damage [I]. Other evidence has supported a role for Ap4A in DNA replication in eukaryotes [2] while extracellular signalling and neurotransmitter functions for Ap4A and its homologues ADA, ApsA and ApbA have also been reported 131.Regulation of intracellular Ap4A levels must depend on both the rates of synthesis and degradation. With regard to the enzymes of Ap4A catabolism, all higher eukaryotes studied so far have an asymmetrically cleaving A@ pyrophosphohydrolase (Ap4A f AMP + ATP) [4] while lower eukaryotes can have any one of (1) an asymmetrical hydrolase, e.g Schizosaccharomyces pombe [ 5 ] ; (ii) a symmetrical hydrolase (like prokaryotes, Ap4A -2ADP), e.g. Physarum jwlycephalum; or (iii) a reversible Ap4A phosphorylase ( A p d + pi -ATP + ADP), e.g. Saccharomyces cerevisiae and Euglenu gracilis [6]. The reasons for this variation are unknown. We have now found that the green alga Scenedesmus obliquus CCAP 27616A and related organisms possess both asymmetricallycleaving Ap& hydrolase a d A@ phosphorylase activities.S. obliquus was grown heterotrophically at 30'C in 1 litre batches to late log phase as previously described 171. Cells (1 g)were broken in a Braun Mikrodismembrator in 3 ml 30 mM benzamidine, 5 pM E-64, a high speed supernatant prepared, and a sample (0.5 ml) analysed by high performance gel filtration (Fig. I). Surprisingly, both Ap& phosphorylase and asymmetrical Ap4A hydrolase activities were readily detected with specific activities of 0.65 and 0.28 Ulmg protein and molecular masses of 46 and 26 kDa respectively. Cultures were judged free of contamination by several criteria; furthermore, both activities were also detected in extracts of S. basiliensis, S. quadricauda and Chlorellu vulguris. Therefore, both types of A p~c a t a b o l i c enzyme must be present within the same cell. Both enzymes were therefore purified to homogeneity from S.

Cloning and expression of diadenosine 5′,5‴-P1,P4-tetraphosphate hydrolase from Lupinus angustifolius L

“…saccharomyces pombe [14], the green alga Scenedesmus obliquus [15], humans and other mammals [16][17][18][19], and plants [20,21]. Seeds of the grain legumes Lupinus luteus and Lupinus angustifolius are a rich source of this enzyme [20], although, apart from suggestions that link Ap % A hydrolase to stress responses in tomatoes [21], there is no other information on the role that it might play in plants.…”