Agonist and antagonist properties of calmodulin fragments

• Published in: The Journal of biological chemistry Vol. 259; no. 7; pp. 4419 - 4426
• Main Authors: Newton, D L, Oldewurtel, M D, Krinks, M H, Shiloach, J, Klee, C B
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 10-04-1984; American Society for Biochemistry and Molecular Biology
• Subjects: 3',5'-Cyclic-AMP Phosphodiesterases - metabolism; Amino Acids - analysis; Analytical, structural and metabolic biochemistry; Animals; Binding and carrier proteins; Biological and medical sciences; Calcium - pharmacology; Calmodulin - pharmacology; Calmodulin-Binding Proteins; Cattle; Chromatography, High Pressure Liquid; Egtazic Acid - pharmacology; Fundamental and applied biological sciences. Psychology; Kinetics; Male; Peptide Fragments - isolation & purification; Peptide Fragments - pharmacology; Phosphoprotein Phosphatases - metabolism; Proteins; Testis; Trypsin - metabolism; High performance; Binding protein; Reversed phase chromatography; Trypsin; Purification; Calcium; Peptide fragment; Enzymatic digestion; Molecular interaction; Liquid chromatography; Calmodulin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)43063-8

Limited proteolysis of calmodulin with trypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetracetic acid (EGTA) or Ca2+ was performed according to a modification of the method of Drabikowski et al. (Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Biochim. Biophys. Acta 485, 124-133). The resulting peptides were purified by reverse-phase high performance liquid chromatography. Tryptic digests in EGTA yielded peptides 1-106, 1-90, and 107-148 with yields of 9, 47, and 61%, respectively. The digests performed with Ca2+ yielded peptides 1-77 and 78-148 in 35 and 45% yield. Analysis by high performance liquid chromatography indicated that the purified fragments contained less than 0.1% contamination by calmodulin, thus allowing a definitive study of the ability of these fragments to activate, or interact with, calmodulin-regulated enzymes and anti-calmodulin drugs. Each of the fragments, except 107-148, bound to a phenothiazine affinity column in a Ca2+-dependent manner. Thus, calmodulin contains two interaction sites for phenothiazines: one on the NH2-terminal half (fragment 1-77) and one on the COOH-terminal half (fragment 78-148). None of the fragments activates the protein phosphatase, calcineurin, or prevents its stimulation by calmodulin, nor does any of the fragments stimulate Ca2+-dependent cAMP phosphodiesterase. A single cleavage in the middle of the calmodulin molecule results in the rapid dissociation of the two resultant fragments and a loss of ability to activate cAMP phosphodiesterase. One fragment, 78-148, interacts with phosphodiesterase and prevents its activation by calmodulin (Ki: 1.5 +/- 0.4 X 10(-6) M). The same fragment, 78-148, can fully activate phosphorylase kinase but with a lower affinity than calmodulin (Kuznicki, J., Grabarek, Z., Brzeska, H., Drabikowski, W., and Cohen, P. (1981) FEBS Lett. 130, 141-145). Thus, peptide 78-148 behaves as a calmodulin agonist or antagonist or as neither, depending on the enzyme under study.