Response of recombinant calcineurin to metal ions, reduction-oxidation agents, and enzymatic modification.
Recombinant calcineurin heterodimer with the full length delta-isoform of the catalytic subunit (CaN(500)) was expressed in insect cells using the baculovirus system and compared to native bovine brain enzyme in its response to divalent metal ions, redox reagents, and enzymatic modification of arginine residues. The response to various metal ions showed essentially the same profile as bovine brain calcineurin, although Co2+ and Zn2+ did not support recombinant activity as well. Kinetic analysis showed that metal ion and substrate binding were not independent, as found for the bovine brain calcineurin. Incubation with DTT or ascorbate alone caused similar effects on the activity of both enzymes, but different responses were observed when incubated with both DTT and ascorbate; only the recombinant enzyme showed activation. Arginine deimination of recombinant calcineurin by peptidylarginine deiminase resulted in the loss of 60-80% of its phosphatase activity with protection observed if calmodulin was present. Recombinant calcineurin was reactivated by treatment with the protease clostripain, suggesting that deimination of an arginine in the carboxyl terminal domain may be responsible for the loss of phosphatase activity and decreased calmodulin binding [Arch. Biochem. Biophys. 318 (1995) 370]. Supporting this conclusion, a truncated variant of the catalytic subunit lacking the carboxyl terminus showed no loss of phosphatase activity compared to full length calcineurin subunit and contained lower amounts of citrulline than the full length subunit after deimination. These different responses of recombinant calcineurin are consistent with conformational differences compared to bovine brain calcineurin and raise questions about its utility for studying the mechanism of calcineurin.
Rhode
David J
DJ
Department of Molecular Sciences, University of Tennessee, Memphis, TN 38163, USA.
Imparl-Radosevich
Jennifer
J
Bartleson
Cheryl
C
Spannaus-Martin
Donna J
DJ
Martin
Bruce L
BL
D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry; D001921:Brain / Q000201:enzymology; D019703:Calcineurin / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D004229:Dithiothreitol / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008670:Metals / Q000737:chemistry*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D008670:Metals; D011994:Recombinant Proteins; D019703:Calcineurin; D001205:Ascorbic Acid; D004229:Dithiothreitol