Abstract
The Ca2+-dependent association of beta-endorphin and trifluoperazine with porcine testis calmodulin, as well as the effects of removing Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) treatment, were investigated by the procedure of differential kinetic labeling. This technique permitted determination of the relative rates of acylation of each of the epsilon-amino groups of the seven lysyl residues on calmodulin by [3H]acetic anhydride under the different conditions. In all cases, less than 0.52 mol of lysyl residue/mol of calmodulin was modified, thus ensuring that the labeling pattern reflects the microenvironments of these groups in the native protein. Lysines 75 and 94 were found to be the most reactive amino groups in Ca2+-saturated calmodulin. In the presence of Ca2+ and under conditions where beta-endorphin and calmodulin were present at a molar ratio of 2.5:1, the amino groups of lysines 75 and 148 were significantly reduced in reactivity compared to calmodulin alone. At equimolar concentrations of peptide and protein, essentially the same result was obtained except that the magnitudes of the perturbation of these two lysines were less pronounced. With trifluoperazine, at a molar ratio to calmodulin of 2.5:1, significant perturbations of lysines 75 and 148, as well as Lys 77, were also found. These results further substantiate previous observations of a commonality between phenothiazine and peptide binding sites on calmodulin. Lastly, an intriguing difference in Ca2+-mediated reactivities between lysines 75 and 77 of calmodulin is demonstrated. In the Ca2+-saturated form of the protein, both lysines are part of the long connecting helix between the two homologous halves of the protein (Babu, Y. S., Sack, J. S., Greenhough, T. G., Bugg, C. E., Means, A. R., and Cook, W. J. (1985) Nature 315, 37-40). Yet, Lys 75 increases in reactivity some 25-fold, compared to only a 2-fold change for Lys 77, in going from EGTA-treated to Ca2+-saturated calmodulin. Thus, the microenvironment of Lys 75 is markedly altered upon Ca2+ binding, and this linker region between the two globular lobes of the protein appears to be quite important in the interaction of calmodulin with inhibitory molecules and perhaps activatable enzymes.
Highlights
From the Department of Biochemistry and the Reproductive Sciencesand Endocrinology Laboratories, University of Miami School of Medicine, Miami, Florida 33101
Upon binding Ca2+,calmodulin can amino groups of lysines 75 and 148were significantly modulate the activities of a wide spectrum of enzymes in vitro reduced in reactivity compared to calmodulin aloneA. t (4, 5 )
Protective Labeling Procedure-For purposes of illustration, the protective labeling experiment and preparation of radiolabeled lysine-containing peptides is described for experimental reaction mixtures containing,&endorphin and Ca’+-saturated calmodulin relative to a control incubation with calmodulin alone
Summary
Regions of calmodulin at the level of the primary structure which are involved in the interaction with /3-endorphin This procedure is based on the “competitive labeling” technique of Hartley and co-workers (31). Previous studies have shown that the reactivities of amino groups in proteins are a sensitive index of ligand-induced conformational changes (32) and can be used to map interaction sites in associating protein systems (33, 34). Using this approach, we found that P-endorphin binding to calmodulin significantly perturbs just two of the seven lysyi residues in calmodulin. We show that the binding of these molecules occurs in an area on calmodulin which undergoes significant changes in structure when comparing the functionally inactive with the Ca’+-saturated, functionally active form of the protein
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