Two kinds of myosin phosphatases with different enzymatic properties from fresh chicken gizzard smooth muscle. Purification and characterization.
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 4 mM 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 2 mM 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 mumol/mg/min) using phosphorylated myosin head as the substrate under optimal conditions, CGP-b had a smaller Km (3.7 to 6.7 microM) than CGP-a by about 4-fold. CGP-b showed a lower Vm (1.9 to 8.4 mumol/mg/min) for the isolated myosin light chain than myosin itself, while CGP-a showed rather higher Vm (17 to 32 mumol/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.(ABSTRACT TRUNCATED AT 250 WORDS)
Yoshida
M
M
Department of Chemistry, Faculty of Science, Hokkaido University.
Yagi
K
K
D000818:Animals; D002384:Catalysis; D002645:Chickens; D005895:Gizzard, Avian / Q000201:enzymology*; D007527:Isoenzymes / Q000032:analysis / Q000302:isolation & purification*; D009130:Muscle, Smooth / Q000201:enzymology*; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000302:isolation & purification*
D007527:Isoenzymes; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase