Purification and characterization of a Mn2+/phospholipid-dependent protein phosphatase from pig brain membranes.
A Mn2+/phospholipid-dependent protein phosphatase has been identified and characterized from brain membranes. The phosphatase contains three subunits with molecular weights of 64,000, 54,000, and 35,000 in a 1:1:1 molar ratio. On gel filtration, the enzyme has an apparent molecular weight of approximately 180,000. The phosphatase was active on many substrates, including p-nitrophenyl phosphate, phosphotyrosine, phosphothreonine, phosphorylase a, myelin basic protein, histones, type 1 phosphatase inhibitor-2, microtubule tau protein, and synapsin I. To dephosphorylate phosphoproteins, the phosphatase was dependent on such acidic phospholipids as phosphatidylinositol and phosphatidylserine but not on neutral phospholipids such as phosphatidylcholine and phosphatidylethanolamine. The phospholipid-mediated activation of the phosphatase was time and dose dependent and could be reversed by Triton X-100 or gel filtration. Kinetic study further indicates that phospholipid was able to increase the Vmax of the phosphatase but had no effect on the Km value for substrates, suggesting a direct interaction of phospholipids with the phosphatase. Conversely, in order to dephosphorylate phosphoamino acids such as phosphotyrosine and phosphothreonine, this phosphatase was entirely dependent on Mn2+. Phospholipids had no effect on the dephosphorylation of phosphoamino acids, whereas Mn2+ had no effect on the dephosphorylation of phosphoproteins. It is concluded that this Mn2+/phospholipid-dependent membrane phosphatase has two distinct activation mechanisms. The enzyme requires Mn2+ to dephosphorylate micromolecules, whereas acidic phospholipids are needed to dephosphorylate macromolecules. This suggests that Mn2+ and phospholipids may play a role in regulating the substrate specificity of this multisubstrate membrane phosphatase.
Yu
J S
JS
Institute of Life Science, National Tsing Hua University, Taiwan, Republic of China.
Yang
S D
SD
D000818:Animals; D001921:Brain / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008345:Manganese / Q000502:physiology*; D008566:Membranes / Q000201:enzymology; D010743:Phospholipids / Q000502:physiology*; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis / Q000302:isolation & purification*; D013379:Substrate Specificity; D013552:Swine
D010743:Phospholipids; D008345:Manganese; D010744:Phosphoric Monoester Hydrolases