Purification of a lipid-activated and Ca2+-independent protein kinase from the mantle tissue of Mytilus galloprovincialis Lmk.
A phospholipid-sensitive Ca2+-independent protein kinase (p105) was purified to homogeneity from mantle tissue of the mussel Mytilus galloprovincialis Lmk., employing consecutively DE-52 cellulose, Sephacryl S-200 and Biogel HTP chromatographies. The purified enzyme appeared as a single band on 10% SDS-PAGE, and had a molecular weight of 105 kDa. The positive Western blotting of the purified eluate for anti-human-PKCdelta and PKCepsilon suggests that the enzyme from mussel mantle may be an ancestral nPKC isoform, with the kinetic properties of the enzyme very close to those of PKCepsilon isoform of vertebrates. Western blotting of samples from different steps of purification using specific mouse anti-p105, showed two protein bands in samples from the initial steps. However, only one band was detected in the Biogel-HTP eluate, the most purified fraction. The purification steps did not affect the presence of P-serine in p105. No P-tyrosine peptides were detected in any of the purification steps. These results open a new field of work on the study of several molecular processes related to energetic metabolism and reproduction in molluscs, whose regulation is associated with the activation of protein kinases.
Mercado
Luis
L
Departamento de Bioquimica y Biología Molecular, Universidad de Santiago de Compostela, Facultad de Veterinaria, Lugo, Spain.
Cao
Asunción
A
Barcia
Ramiro
R
Ramos-Martínez
Juan Ignacio
JI
D000818:Animals; D049872:Bivalvia / Q000187:drug effects / Q000201:enzymology*; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004075:Diglycerides / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D051379:Mice; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism / Q000494:pharmacology; D011494:Protein Kinases / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification*
D004075:Diglycerides; D010446:Peptide Fragments; D010718:Phosphatidylserines; D011494:Protein Kinases; D002118:Calcium