Skip Navigation


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
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
143/2/253    most recent
mvm218v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Meneghelli, S.
Right arrow Articles by De Michelis, M. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Meneghelli, S.
Right arrow Articles by De Michelis, M. I.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 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.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.