Catalytic properties of the retinal rod outer segment disk ADP-ribosyl cyclase

Abstract

AbstractCyclic 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 estimatedKmfor NGD+of 12.5 ± 0.3μM, aVmaxof 26.50 ± 0.70 pmol cyclic GDP-ribose synthesized/min/mg, and optimal pH of 6.5. The effect of divalent cations (Zn2+, Cu2+, and Ca2+) was also tested. Micromolar Zn2+and Cu2+inhibited the disk ADPR-cyclase activity (half maximal inhibitory concentration, IC50= 1.1 and 3.6μM, respectively). By contrast, Ca2+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 Ca2+release from the disks at various rates in response to change in free Ca2+concentrations, caused by lightversusdark 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 Zn2+and may shed light to the role of Cu2+and Zn2+deficiency in retina.