Characterization of wild type and mutant chicken gizzard alpha calponin expressed in E. coli.
Calponin is a thin filament-associated protein that is implicated in the regulation and maintenance of smooth muscle contraction. Molecular cloning of chicken gizzard calponin indicated the presence of two isoforms, alpha and beta, the expression of the alpha-isoform being uniformly more abundant in various smooth muscle tissues [Takahashi, K. & Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 13284-13288]. For the long-range goal of understanding of the structure and function of calponin, we have started bacterial expression and site-directed mutagenesis of alpha calponin. The amino acid composition and N-terminal sequence of the recombinant alpha calponin were found to be identical to those deduced from its nucleotide sequence. Recombinant alpha calponin is capable of binding to calmodulin, troponin C, tropomyosin, and actin, and of inhibiting skeletal muscle acto-subfragment-1 ATPase activity. A mutant alpha calponin with a replacement in the putative inhibitory region (residues 146-171) has impaired ability to inhibit the acto-subfragment-1 ATPase activity, suggesting that this region of calponin may be involved in the modulation of the actin-myosin interactions.
Gong
B J
BJ
Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts 02114.
Mabuchi
K
K
Takahashi
K
K
Nadal-Ginard
B
B
Tao
T
T
D000199:Actins / Q000037:antagonists & inhibitors / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002645:Chickens; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005895:Gizzard, Avian / Q000737:chemistry*; D015151:Immunoblotting; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000037:antagonists & inhibitors; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D014335:Tropomyosin / Q000378:metabolism; D014336:Troponin / Q000378:metabolism; D019209:Troponin C; D000096985:Calponins
D000199:Actins; D000251:Adenosine Triphosphatases; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D008840:Microfilament Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014335:Tropomyosin; D014336:Troponin; D019209:Troponin C; D000096985:Calponins; C033025:actin subfragments