title: Nicotinic acid adenine dinucleotide phosphate (NAADP) degradation by alkaline phosphatase.
abstract: Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous second messenger providing a Ca(2+) trigger in a wide range of cell types. However, its metabolism is not well understood. Here, we demonstrate the presence of endogenous NAADP in HeLa cells. CD38, a promiscuous enzyme described to be involved in NAADP metabolism, was not detectable in HeLa cells. In cell-free extracts of HeLa cells, NAADP was degraded to nicotinic acid adenine dinucleotide (NAAD). The enzyme was enriched in membranes (10,000 × g pellet) and displayed characteristics typical of alkaline phosphatase (AP), e.g. pH optimum at 8-9 and sensitivity to the inhibitors L-homoarginine and L-leucine. Importantly, NAADP at physiological concentrations (50-100 nM) was degraded to NAAD. Expression of AP isoenzymes was analyzed in HeLa cells. Based on the results together with inhibitor studies, the placental AP isoform emerged as the best candidate for NAADP degradation in HeLa cells. In contrast to HeLa cells, Jurkat T cells or HEK293 cells did not express any AP isoenzymes and did not display any NAADP 2'-phosphatase activity. Finally, the placental AP isoform was expressed heterologously in HEK293 cells, resulting in reconstitution of NAADP 2'-phosphatase activity in cell-free extracts. On the basis of the results, we provide evidence for AP as the metabolizing enzyme of NAADP in cells that do not express CD38.
authors.affiliation: The Calcium Signalling Group, Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
authors.forename: Frederike
authors.initials: F
authors.lastname: Schmid
authors.forename: Ralf
authors.initials: R
authors.lastname: Fliegert
authors.forename: Tim
authors.initials: T
authors.lastname: Westphal
authors.forename: Andreas
authors.initials: A
authors.lastname: Bauche
authors.forename: Andreas H
authors.initials: AH
authors.lastname: Guse
mesh_terms: D051997:ADP-ribosyl Cyclase 1 / Q000235:genetics / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D019169:Jurkat Cells; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D008562:Membrane Glycoproteins; D009249:NADP; C024376:NAADP; D000469:Alkaline Phosphatase; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1