Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform

• Published in: The Journal of biological chemistry 1992-08, Vol.267 (22), p.15965-15969
• Main Authors: PERRINO, B. A, YIU-LIAN FANG, BRICKEY, D. A, SAITH, Y, USHIO, Y, FUKUNAGA, K, MIYAMOTO, E, SODERLING, T. R
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Baculoviridae - genetics; Biological and medical sciences; brain; Brain - enzymology; Calcineurin; calcineurin A; Calmodulin - isolation & purification; Calmodulin - metabolism; Calmodulin-Binding Proteins - genetics; Calmodulin-Binding Proteins - isolation & purification; Calmodulin-Binding Proteins - metabolism; Cattle; Cell Line; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; enzymatic activity; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Insecta; Isoenzymes - genetics; Isoenzymes - isolation & purification; Isoenzymes - metabolism; isoforms; Kinetics; Macromolecular Substances; Molecular Sequence Data; Peptides - chemical synthesis; Peptides - metabolism; Peptides - pharmacology; Phosphoprotein Phosphatases - genetics; Phosphoprotein Phosphatases - isolation & purification; Phosphoprotein Phosphatases - metabolism; Rats; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Substrate Specificity; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)49628-2

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.