Enzyme Properties of Aplysia ADP-Ribosyl Cyclase: Comparison with NAD Glycohydrolase of CD38 Antigen.

J-STAGE
  • Inageda Kiyoshi
    Department of Life Science, Tokyo Institute of Technology
  • Takahashi Katsunobu
    Department of Life Science, Tokyo Institute of Technology
  • Tokita Ken-ichi
    Department of Life Science, Tokyo Institute of Technology
  • Nishina Hiroshi
    Department of Life Science, Tokyo Institute of Technology
  • Kanaho Yasunori
    Department of Life Science, Tokyo Institute of Technology
  • Kukimoto Iwao
    Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokyo
  • Kontani Kenji
    Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokyo
  • Hoshino Shin-ichi
    Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokyo
  • Katada Toshiaki
    Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokyo

抄録

An ecto-enzyme of NAD glycohydrolase (NADase) induced by retinoic acid in HL-60 cells is attributed to the molecule of CD38 antigen [Kontani, K., Nishina, H., Ohoka, Y., Takahashi, K., and Katada, T. (1993) J. Biol. Chem. 268, 16895-16898]. CD38 antigen has an amino acid sequence homologous to Aplysia ADP-ribosyl cyclase which generates cyclic adenosine diphosphoribose (cADPR) and nicotinamide (NA) from β-NAD+. On the basis of this sequence homology, we compared enzyme properties between CD38 NADase expressed as a fusion protein in Escherichia coli and ADP-ribosyl cyclase purified from the ovotestis of Aplysia kurodai. 1) β-NAD+ analogs, nicotinamide 1, N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide, did not serve as good substrates for the ADP-ribosyl cyclase, suggesting that the intact adenine ring of β-NAD+ was required for the cyclase-catalyzed reaction. On the other hand, CD38 NADase utilized the NAD analogs to form ADP-ribose and NA. 2) Kinetic analyses of the ADP-ribosyl cyclase reaction revealed that NA was first released from the substrate (β-NAD+)-enzyme complex, followed by the release of another product, cADPR, which was capable of interacting with the free enzyme. 3) The enzyme reaction catalyzed by the ADP-ribosyl cyclase was fully reversible; β-NAD+ could be formed from cADPR and NA with a velocity similar to that observed in the degradation of β-NAD+. However, CD38 NADase did not catalyze the reverse reaction to form β-NAD+ from ADP-ribose and NA. 4) The CD38 NADase activity was, but the ADP-ribosyl cyclase activity was not, inhibited by dithiothreitol. These results indicated that enzyme reactions catalyzed by Aplysia ADP-ribosyl cyclase and CD38 NADase were quite different from each other in terms of their substrate specificities, reversible reactions, and susceptibilities to dithiothreitol, though both enzymes cleaved the N-glycoside bond of β-NAD+ resulting in the liberation of NA.

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