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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 µg ml−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 (Δ74–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. [ABSTRACT FROM PUBLISHER]

Type IIB Ca2+-ATPases have a terminal auto-inhibitory, domain the action of which is suppressed by calmodulin (CaM) binding. Here, we show that a peptide (6His-1M-I116) corresponding to the first 116 aminoacids (aa) of At-ACA8, the first cloned isoform of Arabidopsis thaliana plasma membrane Ca2+-ATPase, inhibits the activity of the enzyme deprived of the N-terminus by controlled trypsin treatment 10-fold more efficiently than a peptide (41I-T63) corresponding only to the CaM-binding site. A peptide (268E-W348) corresponding to 81 aa of the small cytoplasmic loop of At-ACA8 binds peptide 6His-1M-I116 immobilized on Ni–NTA agarose. Peptide 268E-W348 stimulates Ca2+-ATPase activity. Its effect is not additive with that of CaM and is suppressed by tryptic cleavage of the N-terminus. These results provide the first functional identification of a site of intramolecular interaction with the terminal auto-inhibitory domain of type IIB Ca2+-ATPases. [Copyright &y& Elsevier]