Heparin stimulates a plasma membrane Ca2+-ATPase of Arabidopsis thaliana.
We have studied the effect of heparin, a glycosaminoglycan widely used in releasing tags from fusion proteins, on isoform 8 of Arabidopsis thaliana PM Ca(2+)-ATPase (ACA8) expressed in Saccharomyces cerevisiae strain K616. Heparin stimulates hydrolytic activity of ACA8 with an estimated K(0.5) value for the complex of 15 +/- 1 microg ml(-1), which is unaffected by free [Ca(2+)]. Heparin increases V(max) up to 3-fold while it does not significantly affect the apparent K(m) for free Ca(2+) 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 (Delta74-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 (1)M-I(116). 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 Ca(2+)-ATPase and its animal counterpart, which is inhibited by heparin.
Meneghelli
Silvia
S
Dipartimento di Biologia L. Gorini, Università di Milano, CNR Istituto di Biofisica-Sezione di Milano, via G. Celoria 26, Milan, Italy.
Luoni
Laura
L
De Michelis
Maria Ida
MI
D017360:Arabidopsis / Q000201:enzymology*; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology*
D006493:Heparin; D000252:Calcium-Transporting ATPases