Calcium-dependent regulation of the caldesmon-heavy meromyosin interaction by caltropin.
The binding of chicken gizzard caldesmon to smooth muscle heavy meromyosin (HMM) was studied using caldesmon-Sepharose 4B affinity chromatography, far-ultraviolet circular dichroism (CD), and the fluorescent probe acrylodan. When HMM was applied to a caldesmon-Sepharose column in the presence of 40 mM NaCl, most of the protein was retained on the column, and HMM could be eluted by increasing the NaCl level to 0.5 M; this interaction was not Ca(2+)-dependent. Far-UV CD studies indicated an interaction between caldesmon and HMM since the experimentally observed ellipticity values at 222 and 207 nm deviated from the theoretical values for the complex, and this interaction was also not Ca(2+)-sensitive. Addition of HMM to a caldesmon-caltropin complex induced a conformational change suggesting the formation of a ternary complex for which Ca2+ was essential. Acrylodan-labeled caldesmon, when excited at 375 nm, had an emission maximum at 515 +/- 2 nm. Addition of HMM resulted in a nearly 20% decrease in fluorescence intensity with little or no shift in the emission maximum. Titration of HMM with labeled caldesmon indicated a strong affinity for HMM [K(a) was on the order of (4.5 +/- 0.5) x 10(7) M-1], and this interaction was observed both in the presence and in the absence of calcium. When HMM was titrated with labeled caldesmon in the presence of caltropin in a 0.2 mM Ca2+ medium, its affinity for caldesmon was lowered nearly 3-fold [K(a) approximately (1.50 +/- 0.5) x 10(7) M-1].(ABSTRACT TRUNCATED AT 250 WORDS)
Mani
R S
RS
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Kay
C M
CM
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000494:pharmacology*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002645:Chickens; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D005895:Gizzard, Avian / Q000737:chemistry; D009130:Muscle, Smooth / Q000737:chemistry; D015879:Myosin Subfragments / Q000378:metabolism*; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins; D013050:Spectrometry, Fluorescence
D002135:Calcium-Binding Proteins; D002148:Calmodulin-Binding Proteins; D018797:Cell Cycle Proteins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins; C489133:S100A6 protein, Gallus gallus; C038518:acrylodan; D015081:2-Naphthylamine; D002118:Calcium