Two Kinds of Myosin Phosphatases with Different Enzymatic Properties from Fresh Chicken Gizzard Smooth Muscle. Purification and Characterization1

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Abstract
Two kinds of myosin phosphatases were purified from fresh chicken gizzard smooth muscle. Alkaline phosphatase (CGP-a), which requires Mg2+, was most active at pH 8.6 with 2 to 4mM Mg2+, and was essentially the same as the phosphatase we reported previously (J. Biochem. 99, 1027–1036 (1986)). On the other hand, neutral phosphatase (CGP-b), was most active at pH 7.5 with 0 to 2mM Mg2+, and was similar to SMP-IV reported by Pato and Kerc (J. Biol. Chem. 260, 12359–12366 (1985)). Although both phosphatases showed similar Vm (4.8 to 13 μmol/mg/min) using phosphorylated myosin head as the substrate under optimal conditions, CGP-b had a smaller Km (3.7 to 6.7 μM) than CGP-a by about 4-fold. CGP-b showed a lower Vm (1.9 to 8.4 μmol/mg/min) for the isolated myosin light chain than myosin itself, while CGP-a showed rather higher Vm (17 to 32 μmol/mg/min). Although the activity of CGP-a decreased monotonically with increase of ionic strength, that of CGP-b increased slightly with increase in NaCl until 0.1 M and then decreased. Those results suggest that CGP-b may be the effective myosin phosphatase in vivo. The analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that both phosphatases were composed of a single polypeptide having a molecular weight of 37,000. The tetrameric structure was assumed for both phosphatases, because the molecular weight in the native state was estimated as 140,000 or 145,000 for CGP-a or CGP-b, respectively. A dimeric form of each enzyme was also found and purified, and showed the same enzymatic properties as the corresponding tetramer form. We assume tentatively that CGP-b and CGP-a may be active and inactive forms of the myosin phosphatase, respectively, which are interconvertible by some mechanism, e.g., phosphorylation or methylation.

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