Renaturation of phosphorylase kinase activity from sodium dodecyl sulfate-polyacrylamide gels.
Phosphorylase kinase activity is renatured and detected in situ following electrophoresis of the denatured holoenzyme in a sodium dodecyl sulfate-polyacrylamide gel containing phosphorylase b that has been included in the gel polymerization according to the method of R. L. Geahlen et al. [(1986) Anal. Biochem. 153, 151-158]. Among the enzyme's four subunits, only gamma is catalytically active. When extract of rabbit muscle is electrophoresed and renatured in a similar manner, the phosphorylase-conversion activity is also associated only with a protein band that comigrates with the gamma subunit of phosphorylase kinase. This suggests that the gamma subunit of phosphorylase kinase may be the sole activity in rabbit muscle responsible for the phosphorylation of phosphorylase b. In an alternative method for the renaturation of activity from conventional sodium dodecyl sulfate-polyacrylamide gels, the subunits of the enzyme are visualized using 2.5 M KCl, excised from the gel, and eluted by diffusion into buffer containing sodium dodecyl sulfate, which is subsequently removed by acetone precipitation of the eluted subunits. Catalytic activity is recovered when the acetone precipitate of the extracted gamma subunit is dissolved in 6 M guanidine hydrochloride and diluted 50-fold into an activity assay. Inclusion of eluted alpha and beta subunits in the assay inhibits the activity of the gamma subunit, which supports our previous finding that the alpha and/or beta subunits suppress the activity of the catalytic gamma subunit [H. K. Paudel and G. M. Carlson (1987) J. Biol. Chem. 262, 11912-11915].
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology; D010764:Phosphorylase Kinase / Q000302:isolation & purification / Q000378:metabolism*; D010763:Phosphorylase b / Q000378:metabolism; D010766:Phosphorylation; D011489:Protein Denaturation; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate
D012967:Sodium Dodecyl Sulfate; D000255:Adenosine Triphosphate; D010763:Phosphorylase b; D010764:Phosphorylase Kinase; D008274:Magnesium