STUDIES OF CALCINEURIN-CALMODULIN INTERACTION - PROBING THE ROLE OF ARGININE RESIDUES USING PEPTIDYLARGININE DEIMINASE

Citation
Jm. Imparl et al., STUDIES OF CALCINEURIN-CALMODULIN INTERACTION - PROBING THE ROLE OF ARGININE RESIDUES USING PEPTIDYLARGININE DEIMINASE, Archives of biochemistry and biophysics, 318(2), 1995, pp. 370-377
Citations number
58
Categorie Soggetti
Biology,Biophysics
ISSN journal
00039861
Volume
318
Issue
2
Year of publication
1995
Pages
370 - 377
Database
ISI
SICI code
0003-9861(1995)318:2<370:SOCI-P>2.0.ZU;2-V
Abstract
We have used an enzyme, peptidylarginine deiminase, to convert certain arginyl groups in calcineurin to citrulline. Amino acid analysis show s that only 3 of 34 arginines in calcineurin were deiminated; citrulli ne seems to be localized only in the calcineurin A (CaN A) subunit, Up on incubation with deiminase, the Mn2+/calmodulin-stimulated phosphata se activity decreases to 20-40% of the original activity within 1 h. H owever, the reduction in enzyme activity is fully protected by additio n of calmodulin to the deimination reaction, and only 1.5 mol citrulli ne/mol calcineurin is found in this case. Removal of the calmodulin bi nding domain of the deiminated CaN A by limited proteolysis results in the reactivation of the phosphatase to the same level as digested nat ive calcineurin and also results in the loss of all citrulline residue s. The calmodulin activation curve of the deiminated enzyme is signifi cantly shifted; the calculated apparent K-act using native calmodulin is 15-fold higher than that of native calcineurin while the apparent K -act using a fluorescent derivative of calmdulin, dansyl-calmodulin, i s 10-fold higher. However, the V-m of deiminated calcineurin is simila r to that of native if highly elevated levels of calmodulin are used t o activate the modified calcineurin. To determine directly if the bind ing of calmodulin to calcineurin is affected upon deimination, fluores cence titrations using dansyl-calmodulin were performed. The K-d of de iminated calcineurin determined from these titrations is 10-fold highe r than that of unmodified calcineurin, indicating that calmodulin bind ing is indeed affected. These data indicate that at least one arginine is important for calmodulin binding and is likely located at the calm odulin binding site of the CaN A subunit. (C) 1995 Academic Press, Inc .