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 mgml 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 ("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.

Heparin Stimulates a Plasma Membrane Ca2+-ATPase of Arabidopsis thaliana / S. Meneghelli, L. Luoni, M. I. De Michelis. - In: JOURNAL OF BIOCHEMISTRY. - ISSN 0021-924X. - 143:2(2008), pp. 253-259.

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

S. Meneghelli
Primo
;
L. Luoni
Secondo
;
M. I. De Michelis
Ultimo
2008

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 mgml 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 ("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.
Arabidopsis thaliana; Ca2+-ATPase; Calmodulin; Heparin; Plasma membrane
Settore BIO/04 - Fisiologia Vegetale
2008
Article (author)
File in questo prodotto:
Non ci sono file associati a questo prodotto.
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/49029
Citazioni
  • ???jsp.display-item.citation.pmc??? 2
  • Scopus 8
  • ???jsp.display-item.citation.isi??? 7
social impact