Expressed phosphorylase b kinase and its alphagammadelta subcomplex as regulatory models for the rabbit skeletal muscle holoenzyme.
Understanding the regulatory interactions among the 16 subunits of the (alphabetagammadelta)(4) phosphorylase b kinase (PhK) complex can only be achieved through reconstructing the holoenzyme or its subcomplexes from the individual subunits. In this study, recombinant baculovirus carrying a vector containing a multigene cassette was created to coexpress in insect cells alpha, beta, gamma, and delta subunits corresponding to rabbit skeletal muscle PhK. The hexadecameric recombinant PhK (rPhK) and its corresponding alphagammadelta trimeric subcomplex were purified to homogeneity with proper subunit stoichiometries. The catalytic activity of rPhK at pH 8.2 and its ratio of activities at pH 6.8 versus pH 8.2 were comparable to those of PhK purified from rabbit muscle (RM PhK), as was the hysteresis (autoactivation) in the rate of product formation at pH 6.8. Both the rPhK and alphagammadelta exhibited only a very low Ca(2+)-independent activity and a Ca(2+)-dependent activity similar to that of the native holoenzyme with [Ca(2+)](0.5) of 0.4 microM for the RM PhK, 0.7 microM for the rPhK, and 1.5 microM for the alphagammadelta trimer. The RM PhK, rPhK, and alphagammadelta subcomplex were also all activated through self-phosphorylation. Using cross-linking and limited proteolysis, the alpha-gamma intersubunit contacts previously observed within the intact RM PhK complex were also observed within the recombinant alphagammadelta subcomplex. Our results indicate that both the rPhK and alphagammadelta subcomplex are promising models for future structure-function studies on the regulation of PhK activity through intersubunit contacts, because both retained the regulatory properties of the enzyme purified from skeletal muscle.
Boulatnikov
Igor G
IG
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
Peters
Jennifer L
JL
Nadeau
Owen W
OW
Sage
Jessica M
JM
Daniels
Patrick J
PJ
Kumar
Priyadarsini
P
Walsh
Donal A
DA
Carlson
Gerald M
GM
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D023421:Models, Animal; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000378:metabolism; D010764:Phosphorylase Kinase / Q000378:metabolism*; D010766:Phosphorylation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D020035:Holoenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; D010764:Phosphorylase Kinase