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Research Database PMU-SQQUID

Expressing functional domains of mouse calponin: involvement of the region around alanine 145 in the actomyosin ATPase inhibitory activity of calponin.
el-Mezgueldi, M; Strasser, P; Fattoum, A; Gimona, M;
Biochemistry. 1996; 35(12):3654-3661
Originalarbeiten (Zeitschrift)

PMU-Authors

Gimona Mario
Strasser Peter

Abstract

Previously, we attributed the binding of F-actin to the 38-residue stretch of gizzard calponin encompassing the sequence A145-Y182 and postulated the hexapeptide motif VKYAEK, representing residues 142-147, as a putative actin-binding site [Mezgueldi, M., Fattoum, A., Derancourt, J. & Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951]. Herein, the nature of the ATPase inhibitory amino acids of calponin and their relative position within the actin binding domain was investigated by expressing the following fragments of mouse calponin with or without substitution or deletion of the hexapeptide V142-K147: amino acids 1-228 (CaP1-228), 45-228 (CaP45-228), 131-228 (CaP131-228), and CaP1-228 with substitution of A145 with S (CaP1-228A145S) or deletion of V142-K147 (CaP1-228de1142-147). All the recombinant fragments displayed most of the biochemical properties of the smooth muscle purified calponin including (a) expected electrophoretic mobility, (b) heat stability, (c) binding to actin, tropomyosin and calmodulin, and (d) zero-length cross-linking to actin switched by calmodulin in a calcium-dependent fashion. However, while the wild-type recombinant fragments inhibit the acto-S-1 ATPase activity to the same extent as do the parent calponin, modulation of the hexapeptide by either substitution or deletion strongly affect the inhibitory activity with only slightly decreasing actin binding capacity. The data indicate that the stretch VKYAEK is crucial for ATPase inhibition by calponin but represents only part of the actin-binding domain. These results are discussed in terms of multiple contact sites between actin and calponin.


Useful keywords (using NLM MeSH Indexing)

Actins/metabolism*

Amino Acid Sequence

Animals

Binding Sites

Calcium/metabolism

Calcium-Binding Proteins/chemistry*

Calcium-Binding Proteins/genetics

Calcium-Binding Proteins/metabolism

Calcium-Binding Proteins/pharmacology

Calmodulin/metabolism

Calmodulin-Binding Proteins/chemistry

Calmodulin-Binding Proteins/genetics

Calmodulin-Binding Proteins/metabolism

Cloning, Molecular

Cross-Linking Reagents/pharmacology

Electrophoresis, Polyacrylamide Gel

Enzyme Inhibitors/chemistry*

Enzyme Inhibitors/metabolism

Enzyme Inhibitors/pharmacology

Ethyldimethylaminopropyl Carbodiimide/pharmacology

Mice

Microfilament Proteins

Molecular Sequence Data

Myosins/antagonists*

inhibitors*

Myosins/metabolism

Peptide Fragments/genetics

Peptide Fragments/metabolism

Peptide Fragments/pharmacology

Recombinant Proteins/metabolism

Succinimides/pharmacology

Tropomyosin/metabolism