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 Ca2+-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 mu g ml(-1), which is unaffected by free [Ca2+]. Heparin increases V-max up to 3-fold while it does not significantly affect the apparent K-m 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 M-1-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 Ca2+- ATPase and its animal counterpart, which is inhibited by heparin.