AutoriMeneghelli S.; Luoni L.; De Michelis M.I.
AbstractWe 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.
RivistaJournal Of Biochemistry (tokyo)
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Pagina inizio253
Pagina fine259
Linee di Ricerca IBFMD.P01.005.001