The present investigation identifies bovine liver mitochondrial NADase (NAD+ glycohydrolase) as a member of the class of bifunctional ADP-ribosyl cyclases/cyclic ADP-ribose hydrolases, known to be potential second messenger enzymes. These enzymes catalyse the synthesis and degradation of cyclic ADP-ribose, a potent intracellular calcium-mobilizing agent. The mitochondrial enzyme utilized the NAD+ analogues nicotinamide guanine dinucleotide (NGD+) and nicotinamide hypoxanthine dinucleotide (NHD+) to form fluorescent cyclic purine nucleoside diphosphoriboses. ADP-ribosyl cyclase activity was also demonstrated using 32P-labelled NAD+ as substrate. The identity of NADase and ADP-ribosyl cyclase was supported by their co-migration in SDS/polyacrylamide gels. Cyclase activity was visualized directly within the gel by detecting the formation of fluorescent cyclic IDP-ribose from NHD+. The enzyme catalysed the hydrolysis of cyclic ADP-ribose to ADP-ribose. Moreover, in the presence of nicotinamide and cyclic ADP-ribose the enzyme synthesized NAD+. Both the ADP-ribosyl cyclase and NADase activities of the enzyme were strongly inhibited by reducing agents. Treatment of the NADase with dithiothreitol caused the apparent inactivation of the enzyme. Subsequent removal of the reducing agent and addition of oxidized glutathione led to a partial recovery of enzymic activity. The results support a model for pro-oxidant-induced calcium release from mitochondria involving cyclic ADP-ribose as a specific messenger, rather than the non-enzymic modification of proteins by ADP-ribose.
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September 1997
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Research Article|
September 01 1997
Identification of bovine liver mitochondrial NAD+ glycohydrolase as ADP-ribosyl cyclase
Mathias ZIEGLER;
Mathias ZIEGLER
1
1Institut für Biochemie, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany
1To whom correspondence should be addressed.
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Dierk JORCKE;
Dierk JORCKE
1Institut für Biochemie, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany
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Manfred SCHWEIGER
Manfred SCHWEIGER
1Institut für Biochemie, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany
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Publisher: Portland Press Ltd
Received:
December 13 1996
Revision Received:
April 22 1997
Accepted:
April 23 1997
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1997
1997
Biochem J (1997) 326 (2): 401–405.
Article history
Received:
December 13 1996
Revision Received:
April 22 1997
Accepted:
April 23 1997
Citation
Mathias ZIEGLER, Dierk JORCKE, Manfred SCHWEIGER; Identification of bovine liver mitochondrial NAD+ glycohydrolase as ADP-ribosyl cyclase. Biochem J 1 September 1997; 326 (2): 401–405. doi: https://doi.org/10.1042/bj3260401
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