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Purification and characterization of a myosin heavy chain kinase from Dictyostelium discoideum
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.
Insights
A Dictyostelium discoideum myosin heavy chain kinase was purified and characterized. This kinase phosphorylates myosin heavy chains, inhibiting actin-activated Mg2+-ATPase activity.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Myosin heavy chain kinase plays a crucial role in regulating myosin function.
- Understanding the specific mechanisms of this kinase is essential for comprehending muscle contraction and cell motility.
Purpose of the Study:
- To purify and characterize the Dictyostelium discoideum myosin heavy chain kinase.
- To investigate the effects of this kinase on myosin heavy chains and its enzymatic activity.
Main Methods:
- Purification of the enzyme using affinity chromatography.
- Biochemical assays to determine enzyme kinetics and substrate specificity.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration for molecular weight determination.
Main Results:
- The myosin heavy chain kinase was purified 14,000-fold with a molecular weight of 130,000 Da (SDS-PAGE) and >700,000 Da (gel filtration).
- The enzyme specifically phosphorylates threonine residues on myosin heavy chains, inhibiting actin-activated Mg2+-ATPase activity by 70-80%.
- Optimal activity was observed at pH 7.0-7.5 with 1-2 mM Mg2+; activity was unaffected by Ca2+, calmodulin, EGTA, cAMP, or cGMP.
Conclusions:
- The purified kinase is a specific regulator of Dictyostelium myosin function.
- Phosphorylation by this kinase significantly impacts myosin's ATPase activity, suggesting a role in modulating cell motility.