Figure 1

Detection of mono- and diphosphate nucleotides generated by LALP70 or LALP70v apyrase activity. Crude membrane preparations containing expressed LALP70 were incubated with ³H-UTP/UTP in the presence of 5 mM Ca²⁺ and 500 μM Mg²⁺. After indicated time points, samples were separated by thin layer chromatography and tritiated nucleotides were detected with a thin layer radioactivity scanner (A). Peak 1 contains ³H-UTP, peak 2 ³H-UDP and peak 3 ³H-UMP, and the integrated amount of counts per peak represents the amount of product generated by the enzyme in a certain time intervall. The peak positions were calibrated using unlabeled nucleotides, which were separated onto fluorescence covered HPTLC plates and detected by UV-light (λ = 260 nm; (B)). When a crude membrane preparation from cells transfected with the expression vector only were used in the assay described in (A), less than 5% of the ³H-UTP/UTP was cleaved during a 30 minute incubation (C).