Abstract
Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.
Highlights
Calcineurin A was purified by calmodulin-Sepharosewidely distributeCda2+/CaM-dependent phosphoprotein affinity chromatography from Sf9 cells infected with phosphatase
The mechanisms by which the phosphatase activity of CaN A is regulated by CaM and theB subunit are substrat(eDLDVPIPGRFDRRVSVAAEf)oCraN), ambiguous
Stimulation of 6-CaN A Phosphatase Activity by CaN B and CaM-The 6-CaN A, expressed in Sf9 cells and purified on CaM-Sepharose, migrated on SDS-PAGE slightly faster than the A subunit of purified bovine brain CaN (Fig. lA), probably due to the deletion of 10 residues in the 6-CaN A isoform compared to thepurified brain CaN A subunit
Summary
Proteins and Synthetic Peptides-CaM was purified from bovine brain[28], and its concentration was determined by amino acid analysis (Waters Associates PicoTag Systems). Reconstitution of Subunits A and B-Sf9-expressed 6-CaN A (50 nM) was incubated with purified bovine brain CaN B (asindicated in the figure legends) a t 30 "C for 3h in CaN assay buffer (50 mM HEPES, pH 7.5,ml M MnC12, 0.5mM EDTA, 15 mM (3-mercaptoethanol), containing2 mg/ml BSA.
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