Enzymatic synthesis and characterizations of cyclic GDP-ribose. A procedure for distinguishing enzymes with ADP-ribosyl cyclase activity

J Biol Chem. 1994 Dec 2;269(48):30260-7.

Abstract

Cyclic nucleotides such as cAMP and cGMP are second messengers subserving various signaling pathways. Cyclic ADP-ribose (cADPR), a recently discovered member of the family, is derived from NAD+ and is a mediator of Ca2+ mobilization in various cellular systems. The synthesis and degradation of cADPR are, respectively, catalyzed by ADP-ribosyl cyclase and cADPR hydrolase. CD38, a differentiation antigen of B lymphocytes, has recently been shown to be a bifunctional enzyme catalyzing both the formation and hydrolysis of cADPR. The overall reaction catalyzed by CD38 is the formation of ADP-ribose and nicotinamide from NAD+, identical to that catalyzed by NADase. The difficulties in detecting the formation of cADPR have led to frequent identification of CD38 as a classical NADase. In this study, we show that both ADP-ribosyl cyclase and CD38, but not NADase, can cyclize nicotinamide guanine dinucleotide (NGD+) producing a new nucleotide. Analyses by high performance liquid chromatography and mass spectroscopy indicate the product is cyclic GDP-ribose (cGDPR) with a structure similar to cADPR except with guanine replacing adenine. Compared to cADPR, cGDPR is a more stable compound showing 2.8 times more resistance to heat-induced hydrolysis. These results are consistent with a catalytic scheme for CD38 where the cyclization of the substrate precedes the hydrolytic reaction. Spectroscopic analyses show that cGDPR is fluorescent and has an absorption spectrum different from both NGD+ and GDPR, providing a very convenient way for monitoring its enzymatic formation. The use of NGD+ as substrate for assaying the cyclization reaction was found to be applicable to pure enzymes as well as crude tissue extracts making it a useful diagnostic tool for distinguishing CD38-like enzymes from degradative NADases.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • ADP-ribosyl Cyclase
  • ADP-ribosyl Cyclase 1
  • Animals
  • Antigens, CD / metabolism
  • Antigens, Differentiation / metabolism
  • Aplysia / enzymology
  • B-Lymphocytes / enzymology
  • Brain / enzymology
  • Calcium / metabolism
  • Cell Membrane / enzymology
  • Chromatography, High Pressure Liquid
  • Cloning, Molecular
  • Dogs
  • Guanosine Diphosphate Sugars / biosynthesis*
  • Humans
  • Membrane Glycoproteins
  • Myocardium / enzymology
  • N-Glycosyl Hydrolases / isolation & purification
  • N-Glycosyl Hydrolases / metabolism*
  • NAD+ Nucleosidase / isolation & purification
  • NAD+ Nucleosidase / metabolism
  • Neurospora crassa / enzymology
  • Ovum / metabolism
  • Pyrophosphatases / isolation & purification
  • Pyrophosphatases / metabolism
  • Recombinant Proteins / metabolism
  • Sea Urchins

Substances

  • Antigens, CD
  • Antigens, Differentiation
  • Guanosine Diphosphate Sugars
  • Membrane Glycoproteins
  • Recombinant Proteins
  • cyclic guanosine diphosphate-ribose
  • N-Glycosyl Hydrolases
  • ADP-ribosyl Cyclase
  • CD38 protein, human
  • NAD+ Nucleosidase
  • ADP-ribosyl Cyclase 1
  • Pyrophosphatases
  • nucleotide pyrophosphatase
  • Calcium