Caged Cyclic ADP-Ribose
SYNTHESIS AND USE (*)
- From the Department of Physiology, University of Minnesota, Minneapolis, Minnesota 55455 and Molecular Probes, Inc., Eugene, Oregon 97402
- ¶ To whom correspondence should be addressed: 6-182 Lyon Lab., Dept. of Physiology, University of Minnesota, Minneapolis, MN 55455. Tel: 612-625-7120; Fax: 612-625-0991.
Abstract
Cyclic ADP-ribose (cADPR) is a recently discovered cyclic nucleotide with Ca
mobilizing activity. Caged cADPR was synthesized by reacting cADPR with 2-nitrophenethyldiazoethane. Elemental analyses,
1H NMR, and extinction coefficient measurements indicate that the product contains only one caging group. Anion exchange high
pressure liquid chromatography separated caged cADPR into two forms, which most likely represent isomers. Both forms could
be uncaged with equal efficiency by UV exposure to regenerate cADPR. Photolysis of caged cADPR was accomplished effectively
with a spectrofluorimeter. The efficiency of uncaging depended on wavelength with UV light shorter than about 320 nm being
the most effective. Caged cADPR was biologically inactive and could induce Ca
release from sea urchin egg homogenates only after photolysis. Specificity of the Ca
release was shown by inhibition by 8-amino-cADPR, a specific antagonist of cADPR. To demonstrate its utility in live cells,
caged cADPR was microinjected into sea urchin eggs. Photolysis using a mercury light source effectively regenerated cADPR
and resulted in Ca
mobilization and activation of cortical exocytosis in the eggs.
Footnotes
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↵* This work was supported by National Institutes of Health Grants HD17484 and HD32040 (to H. C. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- cADPR
-
Cyclic ADP-ribose
- HPLC
-
high pressure liquid chromatography
- GTP
S -
guanosine 5′-3-O-(thio)-triphosphate.
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- Received November 2, 1994.
- Revision received January 31, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
