ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 252, No. 1, January, pp. 136-144,1987 Calcium Effects on Calmodulin Lysine Reactivities’ DAVID P. GIEDROC: DAVID PUETT, SUDHIR K. SINHA, AND KEITH BREW4 Department of Biochenzist?y and the Reprcductive Sciences and Endoknology Laboratories, University of Miami School of Medicine, Miami, Florida 33101 Received July 28,1986, and in revised form September 23,1986 The differential reactivities of individual lysines on porcine testicular calmodulin were determined by trace labeling with high specific activity [3H]acetic anhydride as a function of the molar ratio of Ca2+ to calmodulin. In progressing from the Ca2+-depleted form of the protein to a Ca2+:calmodulin molar ratio of 51, six of the seven lysyl residues exhibited a modest 1.5 to 3.0-fold increase in reactivity. Lys 75, in contrast, was enhanced in reactivity greater than 20-fold. When the change in reactivity of each lysine was normalized as a percentage of the maximum change, most of the residues were found to fall into two distinct classes. One class, comprising lysines 94 and 148 from the two carboxy terminal Ca2+-binding domains 3 and 4, respectively, exhibited about 90% of their reactivity change when the Ca2+:calmodulin molar ratio was 2:1, and these residues were perturbed very little upon further addition of Ca ‘+ The other class, encompassing . lysines 13,21, and 30 from the amino terminal domain 1 and Lys 75 from the extended helix connecting the two globular lobes of calmodulin, underwent most of their overall reactivity change (55-‘70%) between 2 and 5 equivalents of Ca2+per mol of calmodulin. Lys 77 was distinct in its pattern of change, undergoing approximately equal changes with each Ca2+ increment. These results are consistent with a model where Ca2+first binds to the two carboxy terminal sites of calmodulin with no apparent preference, concomitant with minor alterations in the microenvironments of lysines in the unoc- cupied amino terminal domains. The third and fourth Ca2+ ions then bind to these latter two domains, again with no evidence of preference, with little change in the lysine reactivities at the carboxy terminus of the molecule. The environments of groups in the central helix appear to undergo changes in a manner that reflects their proximity to the amino and carboxy terminal domains. In the course of this work, it was found that Lys 94 in apocalmodulin is specifically perturbed by the addition of EGTA, suggesting that the chelating agent may interact with calmodulin at or near the third Ca2+-binding domain. 0 1987 Academic Press, Inc. Calmodulin is a ubiquitous Ca2+-binding l-10 PM). The specific coordination of Ca2+ protein that plays a pivotal role in the po- by these proteins occurs through multiple tentiation of cellular responses to Ca2+in- conserved EF-hand structures, a domain flux (see Ref. (1) for review). This protein is a member of a group of homologous pro- teins which bind Ca2+ with high affinity (Kd a Present address: Department of Molecular Bio- physics and Biochemistry, Yale University, New ’ This work was supported by National Institutes Haven, CT 06510. of Health Research Grants GM35415, GM21363, and 3 Present address: IMREG, Inc., New Orleans, LA AM33973. This research was presented by D. P. Gied- 70112. rot in partial fulfillment of the requirements for the ‘To whom correspondence should be addressed: Ph.D. degree from Vanderbilt University, Nashville, Department of Biochemistry, University of Miami Tennessee, where he was supported in part by NIH School of Medicine (R-629), P.O. Box 016129, Miami, Training Grant HD07043. FL 33101. 0003-9861/87 $3.00 136 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.