Nov 14, 1983 - Head, J. F., Masure, H. R., and Kaminer, B. (1982) FEBS Lett. 137, 71-. 26. Niggli, V., Penniston, J. T., and Carafoli, E. (1979) J. Biol. Chem. 254 ...
THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 259, No. 24, Issue of December 25, pp. 15172-15177 1984 Printed in d.S.A.
0 1984 by The American Society of Biological Chemists, Inc.
Stimulation of the Purified Erythrocyte Ca2+-ATPase by Tryptic Fragments of Calmodulin* (Received for publication, November 14,1983,and in revised form, June 11,1984)
Danilo Guerini, Joachim Krebs, and Ernest0 Carafoli From the Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Uniuersitatstrasse 16, CH-8092 Zurich, Switzerland
Highly purified tryptic peptides of calmodulin have stimulated further by calmodulin (14, 15). been obtained by high-performance liquid chromatog- Structurally, calmodulin is a highly conserved molecule raphy. Trypticcleavage of calmodulin in the presence with a high degree of internal homology (1, 6). Drabikowski of Ca2+results in two main fragments which have been and his co-workers showed that under controlled conditions identified by analysis of the amino acid composition as calmodulin can be cleaved by trypsin into large fragments 1-77 and 78-148. IntheabsenceofCa2+,trypsin (16) with the cleavage points differing depending on whether cleavage yields fragments 1-106,l-90, and 107-148. Caz+ was present or not (17). Attempts have been made to Only fragments 78-148 and 1-106 are still able to use these fragments to characterize the interaction between stimulate the purified Ca2+-ATPase of erythrocytes, calmodulin and some of its targetenzymes (16-20). However, albeit much less efficiently on a molar basis, than intactone severe drawback in some of these studies is the possible calmodulin. On the other hand, the same fragments contamination by intact calmodulin, particularly if convenwereunabletostimulatethecalmodulin-dependent cyclic nucleotide phosphodiesterase, even at 1000-fold tional purification techniques are used. Since the interaction molar excess (shown also by Newton,D. L., Oldewur- between calmodulin fragments and the target enzymes is tel, M. D., Krinks, M. H., Shiloach, J., and Klee, C. B. weaker than with the native protein (and it may even be (1984) J. Biol. Chem. 259,4419-4426). This points to nonexistent), it is clear that the contamination by intact the importance oP the carboxyl-terminal half of cal- calmodulin in the system should be kept to a minimum (i.e. modulin and especially of Ca2+-binding region 111 in
