A novel Ca2+ binding protein associated with caldesmon in Ca2+-regulated smooth muscle thin filaments: evidence for a structurally altered form of calmodulin.
Smooth muscle thin filaments are made up of actin, tropomyosin, the inhibitory protein caldesmon and a Ca2+-binding protein. Thin filament activation of myosin MgATPase is Ca2+-regulated but thin filaments assembled from smooth muscle actin, tropomyosin and caldesmon plus brain or aorta calmodulin are not Ca2+-regulated at 25 degrees C/50 mM KCl. We isolated the Ca2+-binding protein (CaBP) from smooth muscle thin filaments by DEAE fast-flow chromatography in 6 M urea and phenyl sepharose chromatography using sheep aorta as our starting material. CaBP combines with smooth muscle actin, tropomyosin and caldesmon to reconstitute a normally regulated thin filament at 25 degrees C/50 mM KCl. It reverses caldesmon inhibition at pCa5 under conditions where CaM is largely inactive, it binds to caldesmon when complexed with actin and tropomyosin rather than displacing it and it binds to caldesmon independently of [Ca2+]. Amino acid sequencing, and electrospray mass spectrometry show the CaBP is identical to CaM. Structural probes indicate it is different: calmodulin increases caldesmon tryptophan fluorescence but CaBP does not. The distribution of charged species in electrospray mass spectrometry and nozzle skimmer fragmentation patterns are different indicating a less stable N-terminal lobe for CaBP. Brief heating abolishes these special properties of the CaBP. Mass spectrometry in aqueous buffer showed no evidence for the presence of any covalent or non-covalently bound adduct. The only remaining conclusion is that CaBP is calmodulin locked in a metastable altered state.
Notarianni
G
G
Imperial College School of Medicine at National Heart and Lung Institute, London, UK.
Gusev
N
N
Lafitte
D
D
Hill
T J
TJ
Cooper
H S
HS
Derrick
P J
PJ
Marston
S B
SB
D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000302:isolation & purification*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000502:physiology*; D009130:Muscle, Smooth / Q000378:metabolism* / Q000648:ultrastructure; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification; D017434:Protein Structure, Tertiary / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D014335:Tropomyosin / Q000378:metabolism
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D020033:Protein Isoforms; D014335:Tropomyosin; D002118:Calcium