Purification and characterization of the plasmodial phosphatase that hydrolyses the phosphorylated light chain of Physarum myosin II from Physarum polycephalum.
A phosphatase was purified through a combination of ion-exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6. The apparent molecular mass of the purified enzyme was estimated at approximately 50 kDa by means of analytical gel filtration. The enzyme was purified 340-fold to a final phosphatase activity of 400 pkat/mg of protein. Among the phosphorylated compounds tested for hydrolytic activity at pH 7.6, the enzyme showed no activity towards nucleotides. At pH 7.6, hydrolytic activity of the enzyme against PMLC was detected; at pH 5.0, however, no hydrolytic activity towards PMLC was observed. The Km of the enzyme for PMLC was 10 microM, and the V(max) was 1.17 nkat/mg of protein. Ca(2+) (10 microM) inhibited the activity of the enzyme, and Mg(2+) (8.5 microM) activated the dephosphorylation of PMLC. Mn(2+) (1.6 microM) highly stimulated the enzyme's activity. Based on these results, we concluded that the enzyme is likely to be a phosphatase with hydrolytic activity towards PMLC.
Okada
Chisa
C
Department of Chemical and Biological Sciences, Graduate School of Science, Japan Women's University, Mejirodai, Bunkyoku, Tokyo 1128681, Japan.
Nakamura
Akio
A
Tomioka
Shigeo
S
Kohama
Kazuhiro
K
Kaneko
Takako S
TS
D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D024462:Myosin Type II / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D016839:Physarum polycephalum / Q000201:enzymology*; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D020033:Protein Isoforms; D015800:Protozoan Proteins; D010744:Phosphoric Monoester Hydrolases; D024462:Myosin Type II