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  • Title: The stability of chicken nucleoside triphosphate diphosphohydrolase 8 requires both of its transmembrane domains.
    Author: Li CS, Lee Y, Knowles AF.
    Journal: Biochemistry; 2010 Jan 12; 49(1):134-46. PubMed ID: 20000380.
    Abstract:
    Chicken nucleoside triphosphate diphosphohydrolase 8 (NTPDase8) is a cell surface ectonucleotidase with a large extracellular domain (ECD) containing the active site and is anchored to the membrane by two transmembrane domains (TMDs) at the N- and C-termini. Unlike other cell surface NTPDases that have been characterized, the chicken NTPDase8 is not susceptible to substrate inactivation or agents that cause membrane perturbation. To determine if the stability of the enzyme is inherent in its ECD, the cDNA construct of the soluble chicken NTPDase8 was expressed and the protein purified. The ATPase activity of the purified soluble chicken NTPDase8 was less than 15% of that of the purified full-length enzyme. Strikingly, in contrast to the membrane-bound enzyme, the activity of the soluble chicken NTPDase8 decreased with time in a temperature-dependent manner as a result of inactivation by ATP, ADP, and P(i). Truncated mutants in which the ECD is anchored by a single TMD at either the N- or the C-terminus by the native chicken NTPDase TMDs or a TMD from a different NTPDase, human NTPDase2, also displayed a nonlinear time course of ATP hydrolysis. While removal of the N- or C-terminal TMD affected protein expression differently, the truncated mutants were generally similar to the soluble chicken NTPDase8 with respect to ATP, ADP, and P(i) inactivation. Other biochemical characteristics, e.g., ATPase/ADPase ratios, inhibition by azide, and affinity for ATP, were also altered when one or both of the TMDs were removed from the chicken NTPDase8. These results indicate that (1) both TMDs of the chicken NTPDase8 are required to maintain stability of the enzyme and maximal catalytic activity and (2) the conformations of the ectodomain in the soluble enzyme and the truncated mutants differ from that of the full-length chicken NTPDase8.
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