Enzymatic activation of autotaxin by divalent cations without EF-hand loopregion involvement

Citation
J. Lee et al., Enzymatic activation of autotaxin by divalent cations without EF-hand loopregion involvement, BIOCH PHARM, 62(2), 2001, pp. 219-224
Citations number
22
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Pharmacology & Toxicology
Journal title
BIOCHEMICAL PHARMACOLOGY
ISSN journal
0006-2952 → ACNP
Volume
62
Issue
2
Year of publication
2001
Pages
219 - 224
Database
ISI
SICI code
0006-2952(20010715)62:2<219:EAOABD>2.0.ZU;2-B
Abstract
Autotaxin (ATX) is a recently described member of the nucleotide pyrophosph atase/phosphodiesterase (NPP) family of proteins with potent tumor cell mot ility-stimulating activity. Like other NPPs, ATX is a glycoprotein with pep tide sequences homologous to the catalytic site of bovine intestinal alkali ne phosphodiesterase (PDE) and the loop region of an EF-hand motif. The PDE active site of ATX has been associated with the motility-stimulating activ ity of ATX. In this study, we examined the roles of the EF-hand loop region and of divalent cations on the enzymatic activities of ATX. Ca2+ or Mg2+ w as each demonstrated to increase the PDE activity of ATX in a concentration -dependent manner, whereas incubation of ATX with chelating agents abolishe d this activity, indicating a requirement for divalent cations. Non-linear regression analysis of enzyme kinetic data indicated that addition of these divalent cations increases reaction velocity predominantly through an effe ct on V-max. Three mutant proteins, Ala(740)-, Ala(742)-, and Ala(751)-ATX, in the EF-hand loop region of ATX had enzymatic activity comparable to tha t of the wild-type protein. A deletion mutation of the entire loop region r esulted in slightly reduced PDE activity but normal motility-stimulating ac tivity. However, the PDE activity of this same deletion mutant remained sen sitive to augmentation by cations, strongly implying that cations exert the ir effect by interactions outside of the EF-hand loop region. (C) 2001 Else vier Science Inc. All rights reserved.