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Journal of Biochemistry Advance Access published online on November 15, 2007
Journal of Biochemistry, 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 Meneghelli1, Laura Luoni1 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.

Corresponding author: 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., mariaida.demichelis{at}unimi.it, TEL 39-02-50314822 FAX 39-02-50314815

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 A. thaliana PM Ca2+-ATPase (ACA8) expressed in S. 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 three 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 ({triangleup}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

1Silvia Meneghelli and Laura Luoni equally contributed to this work.


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