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Characterization of an Arabidopsis calmodulin-like domain protein kinase purified from Escherichia coli using an affinity sandwich technique.

Abstract：

:A full-length cDNA encoding a calcium-dependent protein kinase with a calmodulin-like domain from Arabidopsis thaliana (AK-1 for Arabidopsis kinase-1) has been expressed as a fusion protein (called AK-1-6H) in Escherichia coli and purified to near homogeneity with high specific activity (typically 2000 nmol min-1 mg-1) using an "affinity sandwich" technique. AK-1-6H protein phosphorylation activity using histone as substrate was stimulated up to 50-fold by the addition of calcium alone or up to 5-fold by the addition of specific phospholipids alone; together calcium and these lipids acted synergistically to give up to 100-fold stimulation. We earlier reported that, of a wide array of lipids tested, only phosphatidylinositol and lysophosphatidylcholine stimulated histone phosphorylation by AK-1 [Harper, J. F., Binder, B. M., & Sussman M. R. (1993) Biochemistry 32, 3282-3290]. The properties of lipid stimulation were further explored by testing the effects of lipids on autophosphorylation and on other catalytic properties of the kinase. Although phosphatidylinositol stimulated autophosphorylation up to 11-fold, lysophosphatidylcholine was inactive. Basic peptides such as polylysine (average M(r) approximately 37,100) were potent, mixed-type inhibitors of AK-1-6H with an IC50 of 2 nM. In the presence of phosphatidylinositol, the inhibition was reduced and the IC50 for polylysine was increased to 341 nM. As with autophosphorylation, lysophosphatidylcholine was inactive in alleviating the basic peptide inhibition, which suggests that this lipid's stimulatory effects using exogenous substrate are distinct from those of phosphatidylinositol. These results are consistent with a model in which phosphoinositides directly interact with the kinase protein and alleviate a catalytic block caused by basic charges.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Binder BM,Harper JF,Sussman MR

doi

10.1021/bi00174a008

subject

Has Abstract

pub_date

1994-03-01 00:00:00

pages

2033-41

issue

eissn

0006-2960

issn

1520-4995

journal_volume

pub_type

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Characterization of an Arabidopsis calmodulin-like domain protein kinase purified from Escherichia coli using an affinity sandwich technique.