Inhibition of Smooth Muscle Actomyosin ATPase by Caldesmon Is Associated with Caldesmon-Induced Conformational Changes in Tropomyosin Bound to Actin

The smooth muscle tropomyosin isoforms beta and gamma were isolated in pure form and labeled with N-(1-pyrenyl)iodoacetamide (PIA) on the cysteine residues at either the N- or the C-terminal region (Cys-36 and Cys-190 of beta- and gamma-isoforms, respectively). The effect of caldesmon (CaD) on local...

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Bibliographic Details
Published in:Biochemistry (Easton) 1995-12, Vol.34 (51), p.16815-16820
Main Authors: Horiuchi, Kurumi Y, Wang, Ze, Chacko, Samuel
Format: Article
Language:English
Subjects:
Actins - chemistry
Actins - metabolism
Animals
Base Sequence
Binding Sites - genetics
Calmodulin-Binding Proteins - chemistry
Calmodulin-Binding Proteins - genetics
Calmodulin-Binding Proteins - pharmacology
DNA Primers - genetics
Enzyme Inhibitors - pharmacology
In Vitro Techniques
Molecular Sequence Data
Muscle, Smooth - drug effects
Muscle, Smooth - enzymology
Muscle, Smooth - metabolism
Myosins - antagonists & inhibitors
Myosins - chemistry
Myosins - metabolism
Protein Binding
Protein Conformation - drug effects
Rabbits
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - pharmacology
Sequence Deletion
Spectrometry, Fluorescence
Tropomyosin - chemistry
Tropomyosin - metabolism
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Summary:The smooth muscle tropomyosin isoforms beta and gamma were isolated in pure form and labeled with N-(1-pyrenyl)iodoacetamide (PIA) on the cysteine residues at either the N- or the C-terminal region (Cys-36 and Cys-190 of beta- and gamma-isoforms, respectively). The effect of caldesmon (CaD) on local conformational changes in different regions of the tropomyosin molecule was determined on the basis of changes in the excimer fluorescence (excited dimer of pyrene) formed in homodimers of tropomyosin isoforms. In the absence of actin, excimer fluorescence from the pyrene at Cys-190 of gamma-tropomyosin homodimer decreased in a simple manner on the addition of CaD, whereas the excimer from the Cys-36 of beta-tropomyosin homodimer exhibited a biphasic change, suggesting that additional weak binding sites exist near Cys-36. In the presence of actin, CaD-induced changes in the excimer fluorescence of pyrene-tropomyosin were observed only with Cys-36, and this change was associated with an inhibition of actin-activated myosin ATPase. A competition study with unlabeled tropomyosin isoforms indicated that the different excimer changes exhibited by beta- and gamma-tropomyosin in the presence of CaD were due to conformational changes in different regions of the tropomyosin molecule and not to differences in their affinities for CaD. Experiments with recombinant CaD mutants derived using the baculovirus expression system showed that the inhibition of tropomyosin potentiation of actomyosin ATPase by CaD requires the regions between residues 728-756 and 718-727 on the CaD molecule, although the latter region was sufficient for direct interaction with tropomyosin.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00051a032