Either the Carboxyl- or the Amino-Terminal Region of the Human Ecto-ATPase (E-NTPDase 2) Confers Detergent and Temperature Sensitivity to the Chicken Ecto-ATP-diphosphohydrolase (E-NTPDase 8)

Abstract

Human ecto-ATPase (E-NTPDase 2) and chicken ecto-ATP-diphosphohydrolase (E-NTPDase 8) are cell surface nucleotidases with two transmembranous domains, one each at the N- and C-termini. Hydrolysis of substrates occurs in active sites residing in their extracellular domains. Human ecto-ATPase activity is decreased by NP-40 and at temperatures higher than 37 degrees C. Reduction of activity is abolished by prior cross-linking of the ecto-ATPase by lectin and chemical cross-linking agents [Knowles, A. F., and Chiang, W.-C. (2003) Arch. Biochem. Biophys. 418, 217-227]. In contrast, the chicken ecto-ATP-diphosphohydrolase is not inhibited by NP-40, and activity is approximately 2-fold higher at 55 degrees C. To determine if the transmembranous domains of the two E-NTPDases mediate their respective responses to detergents and high temperature, we first constructed a chimera (ck-hu ACR5) in which the C-terminus of the chicken ecto-ATP-diphosphohydrolase is substituted by the corresponding region of the human ecto-ATPase. While this chimera displays many similar enzymatic characteristics as the parental chicken ecto-ATP-diphosphohydrolase, its inhibition by NP-40, high temperature, and substrate resemble that of the human ecto-ATPase, which donates the C-terminus including the C-terminal transmembranous domain. Additionally, comparison of the effects of ConA, disuccinimidyl suberate, and glutaraldehyde on the parental enzymes and the chimera indicated that catalysis which occurs in the extracellular domains of the two E-NTPDases responds differently to conformational constraints. Enzyme activity of a second chimera (ck-hu ACR1) in which the N-terminus of the chicken ecto-ATP-diphosphohydrolase is substituted by the corresponding region of the human ecto-ATPase is also inhibited by NP-40 and is less active at 55 degrees C; however, its temperature dependence differs from that of ck-hu ACR5. These results indicate that (1) the C- and N-termini of the two E-NTPDases encompassing the two transmembranous domains are important elements in determining the sensitivity of the human ecto-ATPase to NP-40 and high temperatures; (2) incorporation of either the C- or N-terminus of the human ecto-ATPase alone in the chicken ecto-ATP-diphosphohydrolase is sufficient to impart negative regulation on ATP hydrolysis due to membrane perturbation; and (3) interactions of the two sets of heterologous transmembranous domains are not equivalent, which are most likely related to their different amino acid sequences.

Molecular Cloning and Characterization of Expressed Human Ecto-Nucleoside Triphosphate Diphosphohydrolase 8 (E-NTPDase 8) and Its Soluble Extracellular Domain

“…The biochemical properties of the soluble human E-NTPDase 8 differ markedly from the membrane-bound enzyme with respect to resistance to detergent inhibition, substrate utilization, effects of pH and temperature, and inhibition by azide. These findings, together with previously published results from our laboratory (20), demonstrate the importance of the transmembranous domains in determining the structure and regulating the activity of membrane-bound E-NTPDases.…”

Cellular function and molecular structure of ecto-nucleotidases

“…Considerable evidence has been accumulated demonstrating that the two TMDs play a role in the function and regulation of the enzymes in addition to anchoring the protein in the membrane. However, individual paralogues differ in their TMD sequences resulting in variable outcome of membrane perturbing experiments [124,125].…”

The GDA1_CD39 superfamily: NTPDases with diverse functions

“…Chicken NTPDase8 is unique in its resistance to detergent inactivation. It shows a linear time course of ATP hydrolysis and its activity increases with temperature [180]. These properties were expected to be retained by the ECD, but the soluble chicken NTPDase8 displayed a temperature dependent loss of activity in the presence of ATP, ADP, and Pi [179], i.e., the ECD became susceptible to inactivation by the substrates and product.…”