Baculovirus-mediated overexpression of the phosphorylase b kinase holoenzyme and alpha gamma delta and gamma delta subcomplexes.
Recombinant baculoviruses were created and used to coexpress rat phosphorylase kinase (Phk) alpha, gamma, and delta subunits and rabbit beta subunit in insect cells. Coexpression allowed creation of the (alphabetagammadelta)4 hexadecamer, the alphagammadelta heterotrimer, and the gammadelta heterodimeric subcomplexes. Neither the individual alpha, beta, or gamma subunit nor any complex containing the beta subunit other than the hexadecameric holoenzyme was obtained in soluble form. The expressed complexes exhibited pH- and [Ca2+]-dependent specific activities that were similar to those of the Phk holoenzyme purified from rabbit skeletal muscle (SkM Phk). SkM Phk, expressed Phk, and the alphagammadelta subcomplex were activated by exogenous calmodulin and underwent Ca(2+)-dependent autophosphorylation. In some of these features there were subtle differences that could likely be attributed to differences in the covalent modification state of the baculovirus-driven expressed protein. Our results provide an important avenue to probe the detailed characterization of the structure of Phk and the function of the individual domains of the subunits using baculovirus-mediated expression of Phk and Phk subcomplexes.
Kumar
Priyadarsini
P
Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, California 95616, USA.
Brushia
Robert J
RJ
Hoye
Elaine
E
Walsh
Donal A
DA
D000818:Animals; D016367:Baculoviridae / Q000201:enzymology* / Q000235:genetics*; D002118:Calcium / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry; D002460:Cell Line; D004789:Enzyme Activation; D020035:Holoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009036:Moths; D018482:Muscle, Skeletal / Q000201:enzymology; D010764:Phosphorylase Kinase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D021122:Protein Subunits / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D002147:Calmodulin; D020035:Holoenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; D010764:Phosphorylase Kinase; D002118:Calcium