Abstract
A Dictyostelium discoideum myosin heavy chain kinase has been purified 14,000-fold to near homogeneity. The enzyme has a Mr = 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and greater than 700,000 as determined by gel filtration on Bio-Gel A-1.5m. The enzyme has a specific activity of 1 mumol/min X mg when assayed at a Dictyostelium myosin concentration of 0.3 mg/ml. A maximum of 2 mol of phosphate/mol of myosin is incorporated by the kinase, and the phosphorylated amino acid is threonine. Phosphate is incorporated only into the myosin heavy chains, not into the light chains. The actin-activated Mg2+-ATPase of Dictyostelium myosin is inhibited 70-80% following maximal phosphorylation with the kinase. The myosin heavy chain kinase requires 1-2 mM Mg2+ for activity and is most active at pH 7.0-7.5. The activity of the enzyme is not significantly altered by the presence of Ca2+, Ca2+ and calmodulin, EGTA, cAMP, or cGMP. When incubated with Mg2+ and ATP, phosphate is incorporated into the myosin heavy chain kinase, perhaps by autophosphorylation.
Highlights
The clear cases, phosphorylation of the heavy chain inhibits the actin- supernatant was collected, added to 100 g of packed DE52-cellulose activated ATPase activity of the myosin and its ability to polymerize into bipolar filaments
We have found that the catalytic subunit of smooth muscle phosphatase I rapidly removes 32Pwhich has been incorporated into the Dictyostelium myosin heavy chain by the myosin heavy chain kinase
Dictyostelium myosin is composed of a pair of 210,000dalton heavy chains and two pairs of light chains of 18,000 and 16,000 daltons
Summary
Says for myosin heavy chain kinase activity were performed at 25 ‘C Purification of Dictyostelium Myosin-Previous procedures in a reaction mixture containing 2 mM MgCl2, 10 mM imidazole, 0.5 mM [-y-32P]ATP(50 pCi/pmol) 1 mM dithiothreitol, pH 7.0 (Kbuffer). Chain Kinase specific activity of the kinase present in the initial extract When myosin heavy chain kinase pooled from the hydroxyland the actomyosin supernatant for several different prepa- apatite column was rerun over a 1 x 50-cm column of Biorations consistently gave values of 60-80 and 40-50 pmol/ GelA-1.5m, the kinase activity again eluted earlier than min - mg, respectively (Table I). These values were not altered thyroglobulin and again was correlated with the appearance if the phosphataseinhibitors sodium fluoride and sodium of the 130,000-daltonprotein band (data notshown). Extract" DE52 supernatant" Actomyosin supernatant" Phosphocellulose peak Ammonium sulfate ppt 20Bio-Gel A-1.5m peak Hydroxylapatite peak Aminohexyl-Sepharose 4B peak ml 640 625 1,350 130 mg
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