Purification and characterization of a Ca(2+)-binding 450-kDa protein (MCBP-450) in the plasma membrane-enriched fraction from a molluscan smooth muscle.
In accordance with physiological and electronmicroscopic evidence that, in the anterior byssal retractor muscle (ABRM) of a common mussel Mytilus edulis, Ca2+ activating the contractile system is accumulated at the inner surface of the plasma membrane and at the membrane of sarcoplasmic reticulum (Ebashi, S. and Endo, M. (1968) Prog. Biophys. Mol. Biol. 18., 123-183; Suzuki, S. and Sugi, H. (1982) in The role of calcium in biological systems, Vol. I (Anghileri, L.J. and Tuffet-Anghileri, A.M., eds.), pp. 201-207, CRC Press, Boca Raton), we have found a high-molecular-mass (450 kDa) Ca(2+)-binding protein (MCBP-450) in the membrane fractions of the ABRM by 45Ca autoradiography of proteins transferred to nitrocellulose membrane (Rüegg, J. C. (1971) Physiol. Rev. 51, 201-248). MCBP-450, purified to electrophoretic homogeneity, exhibited Ca(2+)-dependent changes in mobility, tryptophan fluorescence, UV absorption and CD spectrum, indicating its Ca(2+)-dependent conformational changes. MCBP-450 has a high content of aspartic and glutamic acid (23.8%) and a high content of basic residues (27%). It has a high capacity Ca(2+)-binding site, which binds about 38 mol of Ca2+ per mol with an adissociation constant of 10(4) M-1, and a low-capacity Ca(2+)-binding site, which binds about 7 mol of Ca2+ per mol with an association constant of 10(5) M-1. These characteristics of MCBP-450 are consistent with the view that it is actually involved in regulating the contraction-relaxation cycle in the ABRM.
Yamanobe
T
T
Central Laboratory of Analytical Biochemistry, School of Medicine, Teikyo University, Tokyo, Japan.
Sugi
H
H
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002462:Cell Membrane / Q000378:metabolism*; D008974:Mollusca; D009130:Muscle, Smooth / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
D000596:Amino Acids; D002135:Calcium-Binding Proteins