Determination of native oligomeric state and substrate specificity of rat NTPDase1 and NTPDase2 after heterologous expression in Xenopus oocytes.
NTPDase1 and NTPDase2 are two related plasma membrane-located enzymes involved in the extracellular degradation of nucleoside 5'-tri- and -diphosphates. They differ regarding their hydrolysis ratios for ATP and ADP. Both enzymes have a predicted transmembrane domain close to the N- and C-terminus, respectively, connected by an extensive extracellular domain that carries the active site. We expressed the rat-derived enzymes in Xenopus laevis oocytes and analyzed their quarternary structure. As revealed by application of blue native PAGE and a comparison of glutaraldehyde cross-linking, native NTPDase1 and NTPDase2 occur in oligomeric form. Oligomer formation of the cell surface-located pool of the enzymes was verified by surface iodination. The two enzymes differed in oligomeric structure and in oligomer complex stability. NTPDase1 preferentially occurred as a dimer that could be dissociated into monomeric forms in the presence of Coomassie Brilliant blue G-250 and dithiothreitol whereas NTPDase2 revealed higher oligomeric forms up to tetramers, largely resistant to dithiothreitol. Our results further suggest that the enzymes exist in varying oligomeric states. In contrast to NTPDase1, substrate specificity of NTPDase2 was altered with prolonged expression time, resulting in a decrease in the ATPase/ADPase activity ratio from 10 : 1 to 2.5 : 1. This was accompanied by a transition into a higher oligomeric state. Our results suggest that despite close sequence identity, NTPDase1 and NTPDase2 differ in oligomeric structure. Dynamic alterations in oligomeric state may induce changes in substrate preference and thus influence the pattern of extracellular nucleotide degradation in situ.[1]References
- Determination of native oligomeric state and substrate specificity of rat NTPDase1 and NTPDase2 after heterologous expression in Xenopus oocytes. Failer, B.U., Aschrafi, A., Schmalzing, G., Zimmermann, H. Eur. J. Biochem. (2003) [Pubmed]
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