Catalytic subunit of cAMP-dependent protein kinase from a catch muscle of the bivalve mollusk Mytilus galloprovincialis: purification, characterization, and phosphorylation of muscle proteins.
cAMP-dependent protein kinase (PKA) plays a crucial role in the release of the catch state of molluskan muscles, but the nature of the enzyme in such tissues is unknown. In this paper, we report the purification of the catalytic (C) subunit of PKA from the posterior adductor muscle (PAM) of the sea mussel Mytilus galloprovincialis. It is a monomeric protein with an apparent molecular mass of 40.0+/-2.0kDa and Stoke's radius 25.1+/-0.3A. The protein kinase activity of the purified enzyme was inhibited by both isoforms of the PKA regulatory (R) subunit that we had previously characterized in the mollusk, and also by the inhibitor peptide PKI(5-24). On the other hand, the main proteins of the contractile apparatus of PAM were partially purified and their ability to be phosphorylated in vitro by purified PKA C subunit was analyzed. The results showed that twitchin, a high molecular mass protein associated with thick filaments, was the better substrate for endogenous PKA. It was rapidly phosphorylated with a stoichiometry of 3.47+/-0.24mol Pmol(-1) protein. Also, catchin, paramyosin, and actin were phosphorylated, although more slowly and to a lesser extent. On the contrary, myosin heavy chain (MHC) and tropomyosin were not phosphorylated under the conditions used.
Departamento de Bioquímica e Bioloxía Molecular, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain.
Pablo
P
Béjar
J Antonio
JA
Villamarín
D000818:Animals; D020134:Catalytic Domain; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D009124:Muscle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D009132:Muscles / Q000201:enzymology; D049878:Mytilus / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation
D009124:Muscle Proteins; D010446:Peptide Fragments; C518603:protein kinase inhibitor peptide (5-24); D017868:Cyclic AMP-Dependent Protein Kinases