Purification and characterization of a Ca2+-binding protein in Lumbricus terrestris.

A Ca2+-binding protein which is capable of activating mammalian Ca2+-activatable cyclic nucleotide phosphodiesterase has been purified from Lumbricus terrestris and characterized. This protein and the Ca2+-dependent protein modulator from bovine tissues have many similar properties. Both proteins have molecular weights of approximately 18,000, isoelectric points of about pH 4, similar and characteristic ultraviolet spectra, and similar amino acid compositions. Both proteins bind calcium ions with high affinity. However, the protein from Lumbricus terrestris binds 2 mol of calcium ions with equal affinity, Kdiss = 6 X 10(-6) M, whereas the Ca2+-dependent protein modulator from bovine tissues binds 4 mol of calcium ions with differing affinities. Although the Ca2+-binding protein of Lumbricus terrestris activates the Ca2+-activatable cyclic nucleotide phosphodiesterase from mammalian tissues, we have failed to detect the existence of a Ca2+-activatable phosphodiesterase activity in Lumbricus terrestris. The activation of phosphodiesterase by the Ca2+-binding protein from Lumbricus terrestris is inhibited by the recently discovered bovine brain modulator binding protein (Wang, J. H., and Desai, R. (1977) J. Biol. Chem. 252, 4175-4184). Since the modulator binding protein has been shown to associate with the mammalian protein modulator to result in phosphodiesterase inhibition, it can be concluded that the Lumbricus terrestris Ca2+-binding protein also associates with the bovine brain modulator binding protein. Attempts to demonstrate the existence of a similar modulator binding protein in Lumbricus terrestris have been unsuccessful.

A Ca'+-binding protein which is capable of activating mammalian Ca'+-activatable cyclic nucleotide phosphodiesterase has been purified from Lumbricus terrestris and characterized.
This protein and the Ca"*-dependent protein modulator from bovine tissues have many similar properties. Both proteins have molecular weights of approximately 18,000, isoelectric points of about pH 4, similar and characteristic ultraviolet spectra, and similar amino acid compositions. Both proteins bind calcium ions with high affinity. However, the protein from Lwnbricus terrestris binds 2 mol of calcium ions with equal affinity, Kdlss = 6 x lo-" M, whereas the Ca'+-dependent protein modulator from bovine tissues binds 4 mol of calcium ions with differing affinities. Although the Ca'+-binding protein of Lumbricus terrestris activates the Ca'+-activatable cyclic nucleotide phosphodiesterase from mammalian tissues, we have failed to detect the existence of a Ca'+-activatable phosphodiesterase activity in Lumbricus terrestris. The activation of phosphodiesterase by the Ca'+-binding protein from Lumbricus terrestris is inhibited by the recently discovered bovine brain modulator binding protein (Wang, J. H., and Desai, R. (1977) J. Biol. Chem. 252, 4175-4184). Since the modulator binding protein has been shown to associate with the mammalian protein modulator to result in phosphodiesterase inhibition, it can be concluded that the Lumbricus terrestris Caz+-binding protein also associates with the bovine brain modulator binding protein. Attempts to demonstrate the existence of a similar modulator binding protein in Lumbricus terrestris have been unsuccessful.
A Ca"-activatable cyclic nucleotide phosphodiesterase has been reported in many mammalian tissues (1, 2). Ca'+-linked regulation of this phosphodiesterase is mediated by a specific C&+-dependent modulator protein (3)(4)(5) which in the presence of Ca"+ binds to the phosphodiesterase and stimulates enzyme activity (6-9). The protein modulator has also been shown to mediate the Ca')+ activation of brain adenylate cyclase (10) and a similar mechanism appears to be involved in this activation (11).

* This investigation is supported by Medical Research Council of Canada Grants
MT-2381 (to J. H. W.) and MT-2907 (to F. C. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The protein modulator has been purified from several mammalian tissues (12-15) as well as from electroplax of the electric eel (16). Based primarily on the similarity in physical, chemical, and Ca'+-binding properties of the protein modulator and troponin C, the Ca'+-binding subunit of the regulatory protein of muscle, we have suggested that these two proteins are homologous proteins (17,18 Acrylamide Gel Electrophoresis -Discontinuous gel electrophoresis was carried out according to Davis (25). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis was carried out by the method of Weber and Osborn (26). Sodium dodecyl sulfate-urea gels were carried out according to Swank and Munkres (27). In all gel systems 0.05% Coomassie blue in 25% isopropyl alcohol and 10% acetic acid was used as stain (28). Gels were destained in 50% methanol, 7r/z% acetic acid.
Isoelectric Focusing -The isoelectric point was measured by analytical isoelectric focusing in polyacrylamide gels according to the method of Vesterberg (29). Ampholytes from pH 3.5 to 6.0 were used.
AnaZytical Ultracentrifugation -Analytical ultracentrifugation was carried out with a Beckman-Spinco model E analytical ultracentrifuge.
Sedimentation velocity experiments were run at 53,000 rpm and 19.8" using Schlieren optics. Sedimentation equilibrium runs were carried out at 19.8" and at a rotor speed of 12,933 rpm. Both Rayleigh and Schlieren optics were used. The buffer density was measured with a pycnometer. The partial specific volume, U, of the protein sample was calculated from the amino acid composition of the protein according to Cohn and Edsall (30    ATPase. This observation has raised the possibility that the Ca'+-dependent protein modulator found in these lower forms of animal is in fact troponin C. Several observations, however, appear to argue against such a possibility: (a) troponin C from skeletal muscle of rabbit is at best a poor substitute for protein modulator in phosphodiesterase activation, (b) modulator activity is mostly found in the supernatant of the low ionic strength extract of the animal; (cl the modulator in crude extracts is eluted from DEAE-cellulose column at the same position as the purified modulator, suggesting that the protein exists as a single protein in the extract whereas troponin C is a subunit of a protein complex, troponin; (d) animal species whose muscle is controlled by a myosin-linked rather than a troponin-linked Ca'+ regulation also have high amounts of the modulator activity in their muscle extracts. However, none of the above observations argue against a possibility that the protein modulator plays the role of troponin C as one of its functions in these animals. Such a possibility should be further studied. Several investigators have shown the great abundance of the protein modulator activity over that of Ca'+-activatable

Ill2
Ca"+-binding Protein in Lumbricus terrestris phosphodiesterase in various mammalian tissues, animal species, and cultured cells (15,22,(43)(44)(45). This observation has led to the suggestion that the protein modulator has function(s) in addition to the regulation of cyclic nucleotide metabolism. The failure to detect Ca"'-activatable phosphodiesterase in earthworm extract under a variety of assay conditions further supports this suggestion. Although the function or functions of the protein modulator in the lower animals is not yet known, it appears to require the conservation of the modulator structure since this more primitive modulator has retained the structural feature for both the Ca"'-dependent activation of bovine phosphodiesterase and specific interaction with bovine brain modulator binding protein. It seems plausible to suggest from the present results that the prot,ein modulator has a more fundamental function which is expected to be operative in all animal and plant species and that the regulation of cyclic nucleotide met,abolism by the protein modulator is a more recent development in the animal kingdom.