Characterization of the myosin-binding subunit of smooth muscle myosin phosphatase.
A myosin phosphatase was purified from chicken gizzard smooth muscle. The holoenzyme is a trimer and consists of 130,000-, 38,000-, and 20,000-Da subunits (in agreement with the results of Alessi et al.: Alessi, D., MacDougall, L. K., Sola, M. M., Ikebe, M., and Cohen, P. (1992) Eur. J. Biochem. 210, 1023-1035). The catalytic subunit, 38,000 Da, is the type 1 delta isoform, and its derived amino acid sequence is identical to the rat isoform. The larger subunit bound to myosin and also interacted with the catalytic subunit. cDNA clones encoding the large subunit were isolated from chicken gizzard cDNA libraries. Overlapping clones indicated the presence of two isoforms, and open reading frames of 2889 and 3012 bases were obtained. These encoded proteins of 963 and 1004 amino acids, with masses of 106,700 and 111,600 Da, respectively. The insert in the larger isoform is in the center of the molecule, at residues 512-552. The N-terminal third of the molecule is composed of eight repeat sequences, similar to the cdc10/SWI6 or ankyrin repeat. Myosin binding and binding to the catalytic subunit are properties of a 58,000-Da fragment that represents the N-terminal part of the molecule.
Shimizu
H
H
First Department of Internal Medicine, Mie University School of Medicine, Japan.
Ito
M
M
Miyahara
M
M
Ichikawa
K
K
Okubo
S
S
Konishi
T
T
Naka
M
M
Tanaka
T
T
Hirano
K
K
Hartshorne
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002645:Chickens; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D005895:Gizzard, Avian / Q000201:enzymology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins