PMID:1650254

Citation	Hellmich, MR and Strumwasser, F (1991) Purification and characterization of a molluscan egg-specific NADase, a second-messenger enzyme. Cell Regul. 2:193-202
Abstract	An egg-specific NADase has been purified to homogeneity from the ovotestis of the opisthobranch mollusk Aplysia californica. Unlike other NADases, the Aplysia enzyme generates primarily cyclic-ADP-ribose (cADPR) rather than ADP-ribose from NAD. cADPR has been shown to stimulate the release of Ca2+ from microsomes prepared from sea urchin egg and, when injected into intact eggs, to activate the cortical reaction, multiple nuclear cycles, and DNA synthesis. The Aplysia enzyme was initially identified as an inhibitor of cholera and pertussis toxin-catalyzed ADP-ribosylation. By the use of an NADase assay, it was purified from the aqueous-soluble fraction of ovotestis by sequential column chromatography. The enzyme has an apparent molecular mass of 29 kDa, a Km for NAD of 0.7 mM, and a turnover rate of approximately 27,000 mol NAD.min-1.mol enzyme-1 at 30 degrees C. Monoclonal antibodies were generated to the NADase. Immunoblots of two-dimensional gels revealed multiple isoforms of the enzyme, with pls ranging from 8.1 to 9.8. The multiple isoforms were resolved with a cation exchange high-pressure liquid chromatography column and shown to generate cADPR. Immunohistochemical analysis of cryostat sections of Aplysia ovotestis shows that the enzyme is specific to the eggs and restricted to large 5- to 10-microns granules or vesicles. To date the cADPR-generating enzyme activity has been identified in various organisms, including mammals. The Aplysia enzyme is the first example in which the enzyme that generates cADPR has been purified. All of the available evidence indicates that this NADase is a second-messenger enzyme, implying that other NADases may serve a similar function.
Links	PubMed PMC361751
Keywords	Adenosine Diphosphate Ribose/metabolism; Animals; Aplysia/enzymology; Blotting, Western; Calcium/metabolism; Cell Fractionation; Chromatography, High Pressure Liquid; Isoenzymes; Microscopy, Fluorescence; Microsomes/metabolism; NAD+ Nucleosidase/chemistry; NAD+ Nucleosidase/immunology; NAD+ Nucleosidase/isolation & purification; NAD+ Nucleosidase/metabolism; Ovum/enzymology; Second Messenger Systems
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
APLCA:NADA		GO:0003953: NAD+ nucleosidase activity	ECO:0000314:		F		Figure 2 shows the direct assay of the extracted and purified protein and its NADase activity when nicotinamide is present. Figure 3 shows the enzymatic kinetics of this enzyme and its product formation.	complete CACAO 4899

References

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