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Journal of Biochemistry Advance Access originally published online on November 15, 2007
Journal of Biochemistry 2008 143(2):253-259; doi:10.1093/jb/mvm218
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© 2007 The Japanese Biochemical Society.

Heparin Stimulates a Plasma Membrane Ca2+-ATPase of Arabidopsis thaliana

Silvia Meneghelli{dagger}, Laura Luoni{dagger} and Maria Ida De Michelis*

Dipartimento di Biologia ‘L. Gorini’, Università di Milano, CNR Istituto di Biofisica - Sezione di Milano, via G. Celoria 26, 20133 Milano, Italy

*To whom correspondence should be addressed. Tel: 39 02 50314822, Fax: 39 02 50314815, E-mail: mariaida.demichelis{at}unimi.it

Received August 3, 2007; Accepted November 1, 2007


  Abstract

We have studied the effect of heparin, a glycosaminoglycan widely used in releasing tags from fusion proteins, on isoform 8 of Arabidopsis thaliana PM Ca2+-ATPase (ACA8) expressed in Saccharomyces cerevisiae strain K616. Heparin stimulates hydrolytic activity of ACA8 with an estimated K0.5 value for the complex of 15 ± 1 µg ml–1, which is unaffected by free [Ca2+]. Heparin increases Vmax up to 3-fold while it does not significantly affect the apparent Km for free Ca2+ and for the nucleoside triphosphate substrate. The heparin effect is not additive with that of exogenous calmodulin and heparin is ineffective on a mutant devoid of the N-terminal auto-inhibitory domain ({Delta}74–ACA8). Altogether, these results indicate that heparin activation is due to partial suppression of the auto-inhibitory function of ACA8 N-terminus. Pull-down assays using heparin–agarose gel show that heparin directly interacts with ACA8. Binding to the heparin–agarose gel occurs also with a peptide reproducing ACA8 sequence 1M-I116. Several single-point mutations within ACA8 sequence A56–T63 significantly alter the enzyme response to heparin, suggesting that heparin interaction with this site may be involved in ACA8 activation. These results highlight a new difference between the plant PM Ca2+-ATPase and its animal counterpart, which is inhibited by heparin.

Key Words: Arabidopsis thaliana, Ca2+-ATPase, calmodulin, heparin, plasma membrane

Abbreviations: ACA8, auto inhibited Ca2+-ATPase isoform 8; Brij 58, polyoxyethilene 20 cethyl ether; BTP, bis tris propane (1,3-bis[tris(hydroxymethyl)methylamino]propane); CaM, calmodulin; ER, endoplasmic reticulum; GST, glutathione S-transferase; NTA, nitrilotriacetic acid; PM, plasma membrane; PMCA, animal plasma membrane Ca2+-ATPase

{dagger}Equal contributors to this work.


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