Purification and characterization of the catalytic subunit of cAMP-dependent protein kinase from the bivalve mollusc Mytilus galloprovincialis

Abstract

The catalytic subunit of cyclic AMP-dependent protein kinase was isolated from mantle tissue of the sea mussel Mytilus galloprovincialis and purified to apparent homogeneity using a simple two-step procedure. The purified enzyme had a molecular weight of 40 ± 1.5 kDa on electrophoresis under denaturing conditions, Stokes' radius 25.8 Å and isoelectric point (pI) 8.5. Kinetic experiments showed that the mussel enzyme used Mg 2+ as preferred divalent cation (it was inactive in the presence of Mn 2+ and the apparent K m values for MgATP and histone II-A were 43 ± 7 μM and 1.72 ± 0.65 mg/ml, respectively. Mussel C-subunit was inhibited by the regulatory subunit (type RII) of cAMP-dependent protein kinase from mammals and also by the inhibitor peptide PKI(5–24) with a I 50 of 6.5 ± 1.6 nM. When RII-subunit from porcine heart was incubated, in the presence of [ γ- 32P]ATP, with C-subunit from mussel or porcine, a comparable rate of regulatory subunit phosphorylation was achieved. These physical and biochemical properties lead to the conclusion that the catalytic subunit of cAMP-dependent protein kinase from a bivalve mollusc is closely related to its counterparts from mammalian sources.