Cloning and characterization of the ecto-nucleotidase NTPDase3 from rat brain: Predicted secondary structure and relation to other members of the E-NTPDase family and actin

被引：44

作者：

Vorhoff T. ^{[1
]}

Zimmermann H. ^{[1
]}

Pelletier J. ^{[2
]}

Sévigny J. ^{[2
]}

Braun N. ^{[1
]}

机构：

[1] Biocenter, AK Neurochemistry, J.W. Goethe-University, 60439 Frankfurt am Main

[2] Centre de Rechercheen Rhumatologie et Immunologie, Sainte-Foy, Que.

来源：

Purinergic Signalling | 2005年 / 1卷 / 3期

基金：

加拿大健康研究院;

关键词：

Actin; Apyrase; ATP; Ectonucleotidase; NTPDase; PC12; cell;

D O I：

10.1007/s11302-005-6314-x

中图分类号：

学科分类号：

摘要：

The protein family of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDase family) contains multiple members that hydrolyze nucleoside 5′-triphosphates and nucleoside 5′-diphosphates with varying preference for the individual type of nucleotide. We report the cloning and functional expression of rat NTPDase3. The rat brain-derived cDNA has an open reading frame of 1590 bp encoding 529 amino acid residues, a calculated molecular mass of 59.1 kDa and predicted N- and C-terminal hydrophobic sequences. It shares 94.3% and 81.7% amino acid identity with the mouse and human NTPDase3, respectively, and is more closely related to cell surface-located than to the intracellularly located members of the enzyme family. The NTPDase3 gene is allocated to chromosome 8q32 and organized into 11 exons. Rat NTPDase3 expressed in CHO cells hydrolyzed both nucleoside triphosphates and nucleoside diphosphates with hydrolysis ratios of ATP:ADP of 5:1 and UTP:UDP of 8:1. After addition of ATP, ADP is formed as an intermediate product that is further hydrolyzed to AMP. The enzyme is preferentially activated by Ca2+ over Mg2+ and reveals an alkaline pH optimum. Immunocytochemistry confirmed expression of heterologously expressed NTPDase3 to the surface of CHO cells. PC12 cells express endogenous surface-located NTPDase3. An immunoblot analysis detects NTPDase3 in all rat brain regions investigated. An alignment of the secondary structure domains of actin conserved within the actin/HSP70/sugar kinase superfamily to those of all members of the NTPDase family reveals apparent similarity. It infers that NTPDases share the two-domain structure with members of this enzyme superfamily. © Springer 2005.

引用

页码：259 / 270

页数：11

共 48 条

[41]

Braun N., Sevigny J., Mishra S., Et al., Expression of the ecto-ATPase NTPDase2 in the germinal zones of the developing and adult rat brain, Eur. J. Neurosci., 17, pp. 1355-1364, (2003)

[42]

Vollmayer P., Koch M., Braun N., Et al., Multiple ecto-nucleotidases in PC12 cells: Identification and cellular distribution after heterologous expression, J. Neurochem., 78, pp. 1019-1028, (2001)

[43]

Grinthal A., Guidotti G., Transmembrane domains confer different substrate specificities and adenosine diphosphate hydrolysis mechanisms on CD39, CD39L1, and chimeras, Biochemistry, 41, pp. 1947-1956, (2002)

[44]

Grinthal A., Guidotti G., Dynamic motions of CD39 transmembrane domains regulate and are regulated by the enzymatic active site, Biochemistry, 43, pp. 13849-13858, (2004)

[45]

Smith T.M., Kirley T.L., Expression, characterization, and site-directed mutagenesis of a human E-type ATPase, Ecto-ATPases and Related Ectonucleotidases, pp. 90-105, (2000)

[46]

Hicks-Berger C.A., Yang F., Smith T.M., Kirley T.L., The importance of histidine residues in human ecto-nucleoside triphosphate diphosphohydrolase-3 as determined by site-directed mutagenesis, BBA Protein Struct. Mol. Enzym., 1547, pp. 72-81, (2001)

[47]

Yang F., Hicks-Berger C.A., Smith T.M., Kirley T.L., Site-directed mutagenesis of human nucleoside triphosphate diphosphohydrolase 3: The importance of residues in the apyrase conserved regions, Biochemistry, 40, pp. 3943-3950, (2001)

[48]

Basu S., Murphy-Piedmonte D.M., Kirley T.L., Conserved lysine residue 79 is important for activity of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), Purinergic Signalling, 1, pp. 51-58, (2004)

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