Catalytic properties of the retinal rod outer segment disk ADP-ribosyl cyclase.
Cyclic ADP-ribose (cADPR) is a second messenger modulating intracellular calcium levels. We have previously described a cADPR-dependent calcium signaling pathway in bovine rod outer segments (ROS), where calcium ions play a pivotal role. ROS ADP-ribosyl cyclase (ADPR-cyclase) was localized in the membrane fraction. In the present work, we examined the properties of the disk ADPR-cyclase through the production of cyclic GDP-ribose from the NAD(+) analogue NGD(+). The enzyme displayed an estimated K(m) for NGD(+) of 12.5 ± 0.3 μM, a V(max) of 26.50 ± 0.70 pmol cyclic GDP-ribose synthesized/min/mg, and optimal pH of 6.5. The effect of divalent cations (Zn(2+), Cu(2+), and Ca(2+)) was also tested. Micromolar Zn(2+) and Cu(2+) inhibited the disk ADPR-cyclase activity (half maximal inhibitory concentration, IC50=1.1 and 3.6 μM, respectively). By contrast, Ca(2+) ions had no effect. Interestingly, the properties of the intracellular membrane-associated ROS disk ADPR-cyclase are more similar to those of the ADPR-cyclase found in CD38-deficient mouse brain, than to those of CD38 or CD157. The novel intracellular mammalian ADPR-cyclase would elicit Ca(2+) release from the disks at various rates in response to change in free Ca(2+) concentrations, caused by light versus dark adaptation, in fact there was no difference in disk ADPR-cyclase activity in light or dark conditions. Data suggest that disk ADPR-cyclase may be a potential target of retinal toxicity of Zn(2+) and may shed light to the role of Cu(2+) and Zn(2+) deficiency in retina.
Department of Biology, University of Genoa, Genova, Italy.
Andrea
A
Fabiano
Isabella
I
Panfoli
Daniela
D
Calzia
Maurizio
M
Bruschi
Silvia
S
Ravera
Angela
A
Bachi
Angela
A
Cattaneo
Alessandro
A
Morelli
Giovanni
G
Candiano
D036541:ADP-ribosyl Cyclase / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D003300:Copper / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006150:Guanine Nucleotides / Q000378:metabolism; D046529:Microscopy, Electron, Transmission / Q000379:methods; D018482:Muscle, Skeletal / Q000201:enzymology / Q000648:ultrastructure; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009994:Osmolar Concentration; D010775:Photic Stimulation; D012160:Retina / Q000166:cytology*; D017948:Retinal Rod Photoreceptor Cells / Q000166:cytology*; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
D006150:Guanine Nucleotides; D009243:NAD; C102728:nicotinamide guanine dinucleotide; D003300:Copper; D012243:Rhodopsin; D036541:ADP-ribosyl Cyclase; D000254:Sodium-Potassium-Exchanging ATPase; D015032:Zinc; D002118:Calcium