Purification of Dictyostelium myosin II heavy chain kinase A based on the increase in negative charge accompanying hyperphosphorylation.
The initial step in the purification of Dictyostelium myosin II heavy chain kinase A (MHCK A) is chromatography over phosphocellulose. Fractions containing MHCK A are pooled and chromatographed over a Mono Q column (Pharmacia LKB Biotechnology) equilibrated in 0.15 M KCl. Under these conditions MHCK A and most of the contaminating proteins elute in the flowthrough. The addition of Mg2+ and ATP to the Mono Q flowthrough results in the phosphorylation, within 15 min, of MHCK A to a level of 10 mol of phosphate per mole of 130-kDa kinase subunit. The hyperphosphorylated MHCK A binds to Mono Q columns in the presence of 0.15 M KCl and can be eluted, as a single homogeneous band, by a salt gradient to 0.35 M KCl. A similar purification procedure may prove useful for other proteins which can be highly phosphorylated. Hyperphosphorylation is shown to have no effect on the position at which MHCK A elutes from gel filtration columns (apparent M(r) greater than 700,000).
Medley
Q G
QG
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Lee
S F
SF
Côté
G P
GP
D000818:Animals; D000837:Anion Exchange Resins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004023:Dictyostelium / Q000201:enzymology*; D004563:Electrochemistry; D008970:Molecular Weight; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry / Q000302:isolation & purification*; D015800:Protozoan Proteins; D012117:Resins, Synthetic
D000837:Anion Exchange Resins; D015800:Protozoan Proteins; D012117:Resins, Synthetic; C042855:Mono Q; D010770:Phosphotransferases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase