Baculovirus-directed expression of the gamma-subunit of phosphorylase kinase: purification and calmodulin dependence.
A recombinant baculovirus containing a cDNA encoding the gamma-subunit of phosphorylase kinase from mouse skeletal muscle was constructed. Cultures of Sf-9 insect cells infected with the gamma-baculovirus produce an intact and soluble gamma-protein. A purification procedure is presented that yields a sample of gamma-protein which is devoid of interfering enzyme activity and which is not associated with calmodulin from the insect cells. The isolated gamma sample has a Km for phosphorylase b of 36 (+/- 6, S.E.M) microM at pH 8.2 and 140 (+/- 25) microM at pH 6.8. These values are similar to those reported for the activated phosphorylase kinase holoenzyme isolated from skeletal muscle tissue. However, the Vmax. of the baculovirus-expressed gamma is 65 and 80% of that of the activated holoenzyme at pH 6.8 and 8.2 respectively. These results indicate that one or more of the regulatory subunits alpha, beta, or calmodulin stimulate the activity of the catalytic subunit gamma in the activated holoenzyme. Addition of calmodulin to the baculovirus-expressed gamma stimulates its activity 1.5-2.0 fold at pH 6.8 in both the presence and absence of calcium. At pH 8.2, calmodulin has only minor stimulatory affects. The stimulation by calmodulin at pH 6.8 results from an increase in the Vmax of gamma with little effect on its Km. This result is unlike that for most calmodulin-stimulated kinases which bind calmodulin only in the presence of calcium and exhibit a decrease in their Km upon binding calmodulin. The change in Vmax. of gamma in the presence of calmodulin and in the absence of calcium presents a novel mechanism for the regulation of a calmodulin-stimulated kinase.
Lanciotti
R A
RA
Department of Biochemistry and Anaerobic Microbiology, Virginia Tech., Blacksburg 24061.
Bender
P K
PK
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005822:Genetic Vectors; D051379:Mice; D009036:Moths; D010764:Phosphorylase Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D002147:Calmodulin; D018076:DNA, Complementary; D011994:Recombinant Proteins; D010764:Phosphorylase Kinase