Purification and characterization of a molluscan egg-specific NADase, a second-messenger enzyme.

Hellmich, Mark R.; Strumwasser, Felix

doi:10.1091/mbc.2.3.193

Cited by 137 publications

(79 citation statements)

References 17 publications

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“…A similar enzyme was also isolated from the mollusk, Aplysia californica, where ADP-ribosyl cyclase was characterized as a 29-kDa cytoplasmic protein purified from the ovotestis [53]. In 1991, a cDNA for this cyclase was cloned [54].…”

Section: The Nad ϩ Connectionmentioning

confidence: 99%

The metamorphosis of a molecule: from soluble enzyme to the leukocyte receptor CD38

Ferrero

Malavasi

1999

Journal of Leukocyte Biology

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Human CD38 is a 45-kDa type II membrane glycoprotein with an intricate pattern of expression in leukocytes, although evidence is accumulating of its quite widespread expression in cells of nonvascular origin. CD38 is a member of a nascent eukaryotic gene family encoding cytosolic and membrane-bound enzymes whose substrate is NAD, a coenzyme ubiquitously distributed in nature. Functionally, CD38 is an eclectic molecule with the ability not only to catalyze but also to signal, to mobilize calcium, and to adhere to itself, to hyaluronan, and to other ligands. Interaction with CD38 on various leukocyte subpopulations has profound though diverse consequences on their life-span, but these effects seem to be independent of the enzymatic activity of the molecule. CD38 challenges our expectations of a surface molecule and we must sift through its many guises to unmask its true nature. J. Leukoc. Biol. 65: 151-161; 1999.

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Section: The Nad ϩ Connectionmentioning

confidence: 99%

The metamorphosis of a molecule: from soluble enzyme to the leukocyte receptor CD38

Ferrero

Malavasi

1999

Journal of Leukocyte Biology

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“…An aqueous soluble enzyme, thought to be an NADase, has been purified recently from the ovotestis of Aplysia californica (Hellmich and Strumwasser, 1991). This paper shows that the Aplysia enzyme catalyzes the conversion of NAD+ to cADPR and nicotinamide.…”

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confidence: 99%

“…These results suggest that cADPR could be a general second messenger for mobilizing intracellular Ca2`stores. Although the activity of the enzyme responsible for cyclizing NAD+ to cADPR has been detected in a variety of mammalian tissue extracts as well as in sea urchin eggs (Rusinko and Lee, 1989) (Hellmich and Strumwasser, 1991). Figure 1a shows that a single Spectra/gel CM column could be used effectively for purification.…”

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confidence: 99%

ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite.

1991

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Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The activity of the enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that cADPR may be a general messenger for Ca2+ mobilization in cells. An aqueous soluble enzyme, thought to be an NADase, has been purified recently from the ovotestis of Aplysia californica (Hellmich and Strumwasser, 1991). This paper shows that the Aplysia enzyme catalyzes the conversion of NAD+ to cADPR and nicotinamide. The Aplysia enzyme was purified by fractionating the soluble extract of Aplysia ovotestis on a Spectra/gel CM column. The purified enzyme appeared as a single band of approximately 29,000 Da on SDS-PAGE but could be further separated into multiple peaks by high-resolution, cation-exchange chromatography. All of the protein peaks had enzymatic activity, indicating that the enzyme had multiple forms differing by charge. Analysis of the reaction products of the enzyme by anion-exchange high-pressure liquid chromatography (HPLC) indicated no ADP-ribose was produced; instead, each mole of NAD+ was converted to equimolar of cADPR and nicotinamide. The identification of the product as cADPR was further substantiated by proton NMR and also by its Ca(2+)-mobilizing activity. Addition of the product to sea urchin egg homogenates induced Ca2+ release and desensitized the homogenate to authentic cADPR but not to IP3. Microinjection of the product into sea urchin eggs elicited Ca2+ transients as well as the cortical exocytosis reaction. Therefore, by the criteria of HPLC, NMR, and calcium-mobilizing activity, the product was identical to cADPR. To distinguish the Aplysia enzyme from the conventional NADases that produce ADP-ribose, we propose to name it ADP-ribosyl cyclase.

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“…It is present on the cell surface in some cases but is also localized in various intracellular organelles (15, 18 -20). The Aplysia ADP-ribosyl cyclase, on the other hand, is a soluble protein that shares about 30% sequence identity with CD38 (21) and is found in large quantities in the Aplysia ovotestis (22,23). Both CD38 and the cyclase are novel multifunctional enzymes capable of synthesizing NAADP from NADP by catalyzing the exchange of nicotinamide in NADP with nicotinic acid (10).…”

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confidence: 99%

Acidic Residues at the Active Sites of CD38 and ADP-ribosyl Cyclase Determine Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Synthesis and Hydrolysis Activities

Graeff

Liu

Kriksunov

et al. 2006

Journal of Biological Chemistry

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Nicotinic acid adenine dinucleotide phosphate (NAADP) is a novel metabolite of NADP that has now been established as a Ca 2؉ messenger in many cellular systems. Its synthesis is catalyzed by multifunctional enzymes, CD38 and ADP-ribosyl cyclase (cyclase). The degradation pathway for NAADP is unknown and no enzyme that can specifically hydrolyze it has yet been identified. Here we show that CD38 can, in fact, hydrolyze NAADP to ADP-ribose 2-phosphate. This activity was low at neutrality but greatly increased at acidic pH. This novel pH dependence suggests that the hydrolysis is determined by acidic residues at the active site. X-ray crystallography of the complex of CD38 with one of its substrates, NMN, showed that the nicotinamide moiety was in close contact with and has since been shown in a wide variety of cell types, from plants to human (reviewed in Refs. 3-5). NAADP is endogenously present in cells and its level is modulated by various agonists and stimuli, establishing its role as a second messenger (Refs. 6 and 7 and reviewed in Refs. 8 and 9). In contrast to the widespread functional activity of NAADP in cells, only two homologous proteins, mammalian CD38 and Aplysia ADP-ribosyl cyclase, have so far been identified as enzymes responsible for the synthesis of NAADP (10). CD38 is a membrane-bound protein first thought of as a lymphocyte antigen (reviewed in Refs. 11-13) but has since been found to be widely expressed in virtually all tissues examined (14 -17). It is present on the cell surface in some cases but is also localized in various intracellular organelles (15, 18 -20). The Aplysia ADP-ribosyl cyclase, on the other hand, is a soluble protein that shares about 30% sequence identity with CD38 (21) and is found in large quantities in the Aplysia ovotestis (22, 23). Both CD38 and the cyclase are novel multifunctional enzymes capable of synthesizing NAADP from NADP by catalyzing the exchange of nicotinamide in NADP with nicotinic acid (10). Both can also cyclize NAD to produce cyclic ADP-ribose (cADPR) (10, 24), another novel Ca 2ϩ messenger (reviewed in Refs. 3-5). The crystal structures of both proteins have been solved (25,26) and the catalytic glutamate residues at their enzymatic active sites identified by site-directed mutagenesis (27,28). Expectedly, a large degree of structural homology is observed between them (26).As a second messenger, NAADP levels in cells are expected to respond to physiological stimuli. Indeed, agonists and stimuli are shown to activate rapid and transient changes in NAADP levels (7,29,30), indicating the presence in cells of efficient pathways for not only the synthesis but also the degradation of NAADP. As a nucleotide, NAADP is sensitive to degradative enzymes, such as nucleotide phosphodiesterase or alkaline phosphatase (31,32). Whether these general enzymes are responsible for its degradation in cells is not known. NAADP is insensitive to regular NADase (32), and so far, no enzyme has yet been identified that can specifically hydrolyze NAADP. Here we show, unexpectedl...

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Purification and characterization of a molluscan egg-specific NADase, a second-messenger enzyme.

Cited by 137 publications

References 17 publications

The metamorphosis of a molecule: from soluble enzyme to the leukocyte receptor CD38

The metamorphosis of a molecule: from soluble enzyme to the leukocyte receptor CD38

ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite.

Acidic Residues at the Active Sites of CD38 and ADP-ribosyl Cyclase Determine Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Synthesis and Hydrolysis Activities

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