Purification and characterization of a multisubunit phosphatase from turkey gizzard smooth muscle. The effect of calmodulin binding to myosin light chain kinase on dephosphorylation.
A phosphatase that is active in dephosphorylating the isolated 20,000-Da light chain of myosin, as well as the enzyme myosin light chain kinase, has been purified to apparent homogeneity from turkey gizzards. The enzyme has a molecular weight of 165,000 by sedimentation-equilibrium centrifugation under nondenaturing conditions and is composed of three subunits (Mr = 60,000, 55,000, and 38,000) in a 1:1:1 molar ratio. The properties of the holoenzyme, as well as the purified catalytic subunit (Mr = 38,000) were compared using myosin light chains, intact myosin, and myosin light chain kinase as substrates. Although the holoenzyme is active in dephosphorylating the isolated myosin light chains and the enzyme myosin light chain kinase, the holoenzyme does not dephosphorylate myosin. On the other hand, the catalytic subunit of the holoenzyme dephosphorylates all three substrates. When myosin light chain kinase, which has been phosphorylated at two sites is used as substrate, both sites are rapidly dephosphorylated by the phosphatase in the absence of bound calmodulin. If calmodulin is bound to the diphosphorylated kinase, only one site is dephosphorylated. Interestingly, the single site dephosphorylated when calmodulin is bound to myosin light chain kinase is the site that is not phosphorylated when the calmodulin-myosin kinase complex is phosphorylated by cAMP-dependent protein kinase.
D000818:Animals; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D005895:Gizzard, Avian / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification* / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D014422:Turkeys
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D046911:Macromolecular Substances; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase