Turkey gizzard caldesmon: molecular weight determination and calmodulin binding studies.
Sedimentation equilibrium and sedimentation velocity measurements demonstrate that turkey gizzard caldesmon is an elongated molecule of molecular mass 75 +/- 2 kDa. The frictional ratio (2.14) is consistent with a prolate ellipsoid of axial ratio 24, corresponding to an apparent length and width of 516 and 21.5 A, respectively. As was previously determined for chicken gizzard caldesmon [Graceffa, P., Wang, C.-L.A., & Stafford, W.F. (1988) J. Biol. Chem. 263, 14196-14202], this molecular weight is appreciably smaller than the value (approximately 135,000) estimated from the results of NaDodSO4 gel electrophoresis experiments. However, a significant difference between the true molecular weights of turkey and chicken gizzard caldesmons--75,000 versus 93,000--also points to probable molecular weight variations within the subclass. Binding measurements, based on perturbation of the intrinsic tryptophan fluorescence of caldesmon in the presence of calmodulin, show that the interaction between the two proteins is strongly ionic strength and temperature dependent. Dissociation constants of 0.075 and 0.38 microM were determined in solutions containing 0.1 and 0.2 M KCl, respectively, at 24.3 degrees C. Fluorescence emission spectra and fluorescence anisotropy excitation spectra indicate that the tryptophanyl residues of caldesmon are located in solvent-accessible regions of the molecule, where they exhibit a high degree of mobility even when calmodulin is bound.
Malencik
D A
DA
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-6503.
Ausio
J
J
Byles
C E
CE
Modrell
B
B
Anderson
S R
SR
D000818:Animals; D001923:Brain Chemistry; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005454:Fluorescence Polarization; D005895:Gizzard, Avian / Q000378:metabolism*; D007202:Indicators and Reagents; D008970:Molecular Weight; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000032:analysis; D014422:Turkeys / Q000378:metabolism*; D014461:Ultracentrifugation
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D007202:Indicators and Reagents; D014364:Tryptophan