Purification and characterization of a novel 12,000-Da calcium binding protein from smooth muscle.
A new low molecular weight calcium binding protein, designated 12-kDa CaBP, 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 and has a molecular weight of 12,000 based on sodium dodecyl sulfate-gel electrophoresis. The amino acid composition of this protein is similar to but distinct from other known low molecular weight Ca2+ binding proteins. Ca2+ binding assays using Arsenazo III (Sigma) indicated that the protein binds 1 mol of Ca2+/mol of protein. The 12-kDa CaBP underwent a conformational change upon binding Ca2+, as revealed by uv difference spectroscopy and circular dichroism studies in the aromatic and far-ultraviolet range. Addition of Ca2+ to the 12-kDa CaBP labeled with 2-p-toluidinylnaphthalene-6-sulfonate (TNS) resulted in a sevenfold increase in fluorescence intensity, accompanied by a blue shift of the emission maximum from 463 to 445 nm. Hence, the probe in the presence of Ca2+ moves to a more nonpolar microenvironment. Like calmodulin and other related Ca2+ binding proteins, this protein also exposes a hydrophobic site upon binding calcium. Fluorescence titration with Ca2+ using TNS-labeled protein revealed the presence of a single high affinity calcium binding site (kd approximately 1 x 10(-6) M).
Mani
R S
RS
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Kay
C M
CM
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000494:pharmacology; D002645:Chickens; D002845:Chromatography; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian / Q000737:chemistry; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D002148:Calmodulin-Binding Proteins; D002118:Calcium