Diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorylase and Ap4A pyrophosphohydrolase activities have been purified from extracts of the green alga Scenedesmus obliquus. Both activities were also detected in Scenedesmus brasiliensis, Scenedesmus quadricauda and in Chlorella vulgaris. This is the first time that both types of enzyme have been detected in the same species. The Ap4A phosphorylase has a molecular mass of 46-48 kDa, a broad pH optimum between 7.5 and 9.5, and requires a divalent ion for activity (Mg2+ > Co2+ > Ca2+ = Mn2+ = Cd2+ > Zn2+). It degrades substrates with at least four phosphate groups and always produces a nucleoside 5'-diphosphate product. The Km values for Ap4A and Pi are 5.3 microM and 160 microM, respectively, and kcat. = 1.8 s-1. Arsenate, vanadate, molybdate, chromate and tungstate can substitute for phosphate. The enzyme also catalyses Ap4A synthesis (Keq. = [Ap4A] [Pi]/[ATP][ADP] = 9 x 10(-4)) and ADP arsenolysis. The Ap4A hydrolase has a molecular mass of 26-28 kDa, an alkaline pH optimum of 8.8-9.8, and prefers Zn2+ as the stimulatory ion (Zn2+ > Mg2+ > Mn2+ > Co2+ > Cd2+). It degrades substrates with at least four phosphate groups, having a slight preference for Ap5A, and always produces a nucleoside 5'-triphosphate product. The Km value for Ap4A is 6.6 microM and kcat. = 1.3 s-1. It is inhibited competitively by adenosine 5'-tetraphosphate (Ki = 0.67 microM) and non-competitively by fluoride (Ki = 150 microM). A 50-54 kDa dinucleoside 5',5'''-P1,P3-triphosphate (Ap3A) pyrophosphohydrolase was also detected in S. obliquus, S. quadricauda and C. vulgaris. The corresponding enzyme in S. brasiliensis (> 100 kDa) may be a dimer

The enzyme diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) pyrophosphohydrolase has been purified to homogeneity from firefly lanterns. It is a single polypeptide of M(r) 16,000 with a Km for Ap4A of 1.9 microM and kcat = 3.6 s-1. It is inhibited competitively by adenosine 5'-tetraphosphate (Ki = 7.5 nM) and non-competitively by fluoride ions (Ki = 50 microM). The specific activity of the enzyme in crude extracts of at least 20 milliunits/mg protein is 10-100 times higher than in any other eukaryote so far examined. Interestingly, firefly luciferase is known to synthesize Ap4A and related adenine-containing dinucleoside tetraphosphates in vitro. The high activity of Ap4A hydrolase in lanterns may be related to this ability and could be relevant to the use of the luciferase gene as a reporter gene.

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) phosphorylase, previously only known in lower eukaryotes, has been detected in extracts of the cyanobacteria Anabaena flos-aquae, Anabaena variabilis and Synechococcus sp. The 32 kDa enzyme was partially purified from A. flos-aquae and separated from a 23 kDa hydrolytic activity. It had a pH optimum of 9.5 and required a bivalent cation for activity: Mg2+, Mn2+, Ca2+, Co2+ or Zn2+. Diadenosine tri-, tetra- and penta-phosphates were all effective substrates (relative rates 0.85, 1.00 and 0.27 respectively), while the hexaphosphate was a poor substrate and the diphosphate was inactive. ADP was always one of the products of phosphorolysis. Arsenate and vanadate could substitute for phosphate (relative rates 1.80, 2.25 and 1.00 respectively), but tungstate and sulphate could not. Chromate and molybdate were poor substrates. A search of the GenBank non-redundant database revealed a putative Ap4A phosphorylase gene in the cyanobacterium Synechocystis sp. The gene showed significant blocks of identity/similarity with yeast Ap4A phosphorylases I and II, particularly the latter.

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.