Effect of caltropin on caldesmon-actin interaction.
The binding of chicken gizzard caldesmon to actin was studied both in the presence and the absence of caltropin using Airfuge centrifugation experiments, disulfide cross-linking studies, and the fluorescent probe acrylodan (6-acryloyl-2-(dimethylamino)naphthalene). In co-sedimentation studies most of the caldesmon pelleted along with actin. However, when caldesmon in the presence of caltropin was mixed with actin, caldesmon did not pellet along with actin following high speed centrifugation, suggesting that caltropin has significantly weakened its binding to actin. The caltropin effect was noticed even when tropomyosin was included in the reaction mixture. Acrylodan-labeled caldesmon, when excited at 375 nm, had an emission maximum at 515 +/- 2 nm. The addition of actin produced a nearly 70% increase in fluorescent intensity, accompanied by a blue shift in the emission maximum (i.e. lambda em (max) = 505 +/- 2 nm), suggesting that the probe now occupies a more nonpolar environment. Titration of labeled caldesmon with actin indicated a strong affinity (K alpha = approximately 6 x 10(7) M-1). When actin was titrated with labeled caldesmon in the presence of caltropin in a 0.2 mM Ca2+ medium, its affinity for caldesmon was lowered (K alpha = approximately 2 x 10(7) M-1). Caltropin, which is very effective in reversing caldesmon's inhibition of the actin-activated myosin ATPase (Mani, R. S., McCubbin, W. D., and Kay, C. M. (1992) Biochemistry 31, 11896-11901), is shown in the present study to have a pronounced effect on its binding to actin, suggesting a major role for caltropin in regulating caldesmon in smooth muscle.
Mani
R S
RS
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Kay
C M
CM
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002645:Chickens; D011817:Rabbits; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin / Q000378:metabolism
D000199:Actins; D002135:Calcium-Binding Proteins; D002148:Calmodulin-Binding Proteins; D018797:Cell Cycle Proteins; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins; C489133:S100A6 protein, Gallus gallus; D014335:Tropomyosin