Characterization of the smooth muscle calponin and calmodulin complex.
Calponin interacts with several Ca2+ binding proteins in a Ca(2+)-dependent manner. In order to determine the possible biological relevance of these interactions in smooth muscle function, it is necessary to characterize the strength and stoichiometry of the complexes formed. The interaction between calponin and calmodulin can be monitored through an acrylodan label on a cysteine of calponin. The fluorescently labeled calponin possesses the same biological function and physical behavior in binding to calmodulin as the native calponin. This probe is very environment-sensitive and responds to the calponin-calmodulin interaction by the emission peak blue-shifting 20 nm and by the fluorescent quantum yield increasing 3.5 times at 460 nm. The stoichiometric nature of this complex has been determined using analytical ultracentrifugation and is two calmodulins to one calponin, and the interaction is Ca(2+)-sensitive with a Kd1 of < or = 0.22 microM and a Kd2 of 2.5-3.4 microM. Calmodulin is not the only protein which interacts with calponin in this manner, but rather this interaction seems to be a general feature attributable to all hydrophobic patch exposing proteins, suggesting that it may be nonspecific, occurring because of a generalized mode of interaction. Two other proteins, S-100b from bovine brain and SMCaBP-11 from smooth muscle, had stronger affinities for calponin, and in particular interaction of SMCaBP-11 with calponin may be biologically relevant. In determining the nature of calponin's interaction with these Ca2+ binding proteins, it was apparent there was no effect of Ca2+ upon calponin itself and physical studies could find no evidence that calponin interacts with calcium.
Wills
F L
FL
Department of Biochemistry, University of Alberta, Edmonton, Canada.
McCubbin
W D
WD
Kay
C M
CM
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D008840:Microfilament Proteins; D009124:Muscle Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014422:Turkeys; D000096985:Calponins
D015081:2-Naphthylamine; D002118:Calcium; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D005456:Fluorescent Dyes; D008840:Microfilament Proteins; D009124:Muscle Proteins; D000096985:Calponins; C038518:acrylodan