Isolation and characterization of a novel molecular weight 11,000 Ca2(+)-binding protein from smooth muscle.
A new low molecular weight calcium-binding protein, designated as SMCaBP-11, has been isolated from chicken gizzard using a phenyl-Sepharose affinity column followed by ion-exchange and gel filtration chromatographies. The isolated protein was homogeneous by the criteria of gel electrophoresis in the absence and presence of sodium dodecyl sulfate (NaDodSO4). Molecular weight studies by both sedimentation equilibrium in 6 M guanidine hydrochloride and 15% polyacrylamide-SDS gels indicated the subunit molecular weight to be 11,000, and since a molecular weight of 21,000 was obtained in native solvents, the protein exists as a dimer in benign medium. The amino acid composition of this protein is similar but distinct from other known low molecular weight Ca2(+)-binding proteins. Ca2(+)-binding assays using Arsenazo III (Sigma) indicated the protein to bind 2 mol of Ca2+/subunit. In non-SDS gels, the protein moved faster in the presence of EDTA, suggesting that Ca2+ binding affects its mobility in a manner similar to other smooth muscle calcium-binding proteins such as calmodulin and 67-kDa calcimedin. Upon binding calcium, the protein underwent a conformational change as revealed by UV difference spectroscopy and circular dichroism studies in the aromatic and far-ultraviolet range. When the protein was excited at 280 nm, the tyrosine fluorescence emission maximum was centered at 306 nm. Ca2+ addition resulted in a nearly 15% decrease in intrinsic fluorescence intensity. Fluorescence titration with Ca2+ exhibited two classes of calcium-binding sites with Kd values of 0.2 and 80 microM, in agreement with UV difference spectral data.(ABSTRACT TRUNCATED AT 250 WORDS)
Mani
R S
RS
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Kay
C M
CM
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D002645:Chickens; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000032:analysis*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D002118:Calcium