Alkaline phosphatase from Paramecium cilia and cell bodies: purification and characterization.
A soluble alkaline phosphatase was purified 10 000-fold in an overall yield of 8% from both of the cilia and cell bodies of the protozoan Paramecium tetraurelia. The concentration in cilia (1.7 microM) was 6-fold higher than in cell bodies, although the latter contained most of the activity due to their much greater volume. The purified protein showed a single (36 kDa) protein staining band on SDS-PAGE. This value, in conjunction with the apparent molecular mass of 66 kDa for the native enzyme (gel filtration) suggests a dimeric structure. The specific activity of the purified phosphatase ranged from 10 to 70 mumols.min-1.mg-1 at the pH-optimum of 8.0 and the Km for p-nitrophenyl phosphate was 81 microM. Basal enzyme activity was inhibited by metal chelators and stimulated up to 12-fold by addition of divalent cations. Mg2+ acted as a non-essential mixed-type activator with a half-maximal effect at 7 microM. Ca2+ was inhibitory, the extent of inhibition was dependent on the concentration of Mg2+ in the assay. Furthermore, the kinetics of inhibition by Ca2+ varied with the Mg2+ concentration. Phosphate, pyrophosphate, and SH-group blocking agents also strongly inhibited. The enzyme did not dephosphorylate Tyr- or Ser-/Thr-phosphoproteins. The Paramecium enzyme is not of lysosomal origin and its properties are quite different from all known phosphatases. It is a novel type of phosphatase since it (i) shows F(-)-inhibition like Ser/Thr-phosphatases but (ii) is inhibited by vanadate and molybdate like Tyr-phosphatases, and (iii) inhibition by Ca2+ has not been reported for any other phosphatase.
Klumpp
S
S
Pharmazeutisches Institut der Universität, Tübingen, F.R.G.
Schultz
J E
JE
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002384:Catalysis; D002413:Cations, Divalent; D002614:Chelating Agents; D002923:Cilia / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010247:Paramecium / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D013379:Substrate Specificity
D002413:Cations, Divalent; D002614:Chelating Agents; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D000469:Alkaline Phosphatase; D010749:Phosphoprotein Phosphatases; D002118:Calcium