Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.16 (calcineurin)
 17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used an enzyme, peptidylarginine deiminase, to convert certain arginyl groups in calcineurin to citrulline. Amino acid analysis shows that only 3 of 34 arginines in calcineurin were deiminated; citrulline seems to be localized only in the calcineurin A (CaN A) subunit. Upon incubation with deiminase, the Mn2+/calmodulin-stimulated phosphatase activity decreases to 20-40% of the original activity within 1 h. However, the reduction in enzyme activity is fully protected by addition of calmodulin to the deimination reaction, and only 1.5 mol citrulline/mol calcineurin is found in this case. Removal of the calmodulin binding domain of the deiminated CaN A by limited proteolysis results in the reactivation of the phosphatase to the same level as digested native calcineurin and also results in the loss of all citrulline residues. The calmodulin activation curve of the deiminated enzyme is significantly shifted; the calculated apparent Kact using native calmodulin is 15-fold higher than that of native calcineurin while the apparent Kact using a fluorescent derivative of calmodulin, dansyl-calmodulin, is 10-fold higher. However, the Vm of deiminated calcineurin is similar to that of native if highly elevated levels of calmodulin are used to activate the modified calcineurin. To determine directly if the binding of calmodulin to calcineurin is affected upon deimination, fluorescence titrations using dansyl-calmodulin were performed. The Kd of deiminated calcineurin determined from these titrations is 10-fold higher than that of unmodified calcineurin, indicating that calmodulin binding is indeed affected. These data indicate that at least one arginine is important for calmodulin binding and is likely located at the calmodulin binding site of the CaN A subunit.
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PMID:Studies of calcineurin-calmodulin interaction: probing the role of arginine residues using peptidylarginine deiminase. 773 65

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.
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PMID:Response of recombinant calcineurin to metal ions, reduction-oxidation agents, and enzymatic modification. 1240 72