Dictyostelium myosin light chain kinase. Purification and characterization.
A Dictyostelium myosin light chain kinase has been purified approximately 15,000-fold to near homogeneity. The purified kinase is a single polypeptide of approximately 34 kDa that phosphorylates only the 18-kDa Dictyostelium myosin regulatory light chain and itself among substrates tested. The enzyme was purified largely by ammonium sulfate fractionation and hydrophobic (butyl) interaction chromatography. Analysis using polyclonal antibodies raised against the purified 34-kDa protein confirms that this protein is responsible for myosin light chain kinase activity. Protein microsequence of the 34-kDa protein reveals conserved protein kinase sequences. The purified Dictyostelium myosin light chain kinase exhibits a Km for Dictyostelium myosin of 4 microM and a Vmax of 8 nmol/min/mg. Unlike other characterized myosin light chain kinases, this enzyme is not regulated by calcium/calmodulin. Western blot analysis demonstrates that the purified kinase is not a proteolytic fragment that has lost calcium/calmodulin regulation. The Dictyostelium myosin light chain kinase activity is not directly regulated by cyclic nucleotides. However, this kinase undergoes an intramolecular autophosphorylation that activates the enzyme.
Tan
J L
JL
Department of Cell Biology, Stanford University School of Medicine, California 94305.
Spudich
J A
JA
D000595:Amino Acid Sequence; D000906:Antibodies; D015153:Blotting, Western; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004023:Dictyostelium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D000906:Antibodies; D009219:Myosin-Light-Chain Kinase