The membrane-bound alkaline phosphatase and 5'-nucleotidase activities of vegetative cells of Dictyostelium discoideum.
Earlier reports suggested that the adenosine monophosphate (AMP)- and the p-nitrophenyl phosphate (pNPP)-hydrolyzing activities of Dictyostelium discoideum membrane preparations are due to different proteins. These results have been apparently contradicted by the recent purification to homogeneity of the two activities from culmination phase cells as a single protein [D. R. Armant and C. L. Rutherford (1981) J. Biol. Chem. 256, 12710-12718]. Results presented here from studies on the activities of vegetative cells support the concept of a single protein. Nondenaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Triton X-100 extracts of cell membrane preparations of D. discoideum showed identical migration of pNPPase and AMPase activities. Furthermore, the previously reported different pH optima of the two activities was due to the fact that pH optima are dependent upon the substrate concentration, and the selective solubilization of AMPase from membrane preparations by phospholipase C can probably be accounted for by the finding that phospholipase C preparations from the same commercial source contain 5'-nucleotidase activity. Moreover, there are alterations in the Km and the stability of both AMPase and pNPPase in a strain with a mutationally altered alkaline phosphatase, further supporting the concept that the two activities are due to a single protein. Both substrates serve as transphosphorylation donors demonstrating that the enzyme activity is mechanistically an alkaline phosphatase.
Bhanot
P
P
Weeks
G
G
D015720:5'-Nucleotidase; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D055598:Chemical Phenomena; D002621:Chemistry; D004023:Dictyostelium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009708:Nucleotidases / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011489:Protein Denaturation; D013379:Substrate Specificity; D013873:Thionucleotides / Q000378:metabolism; D010738:Type C Phospholipases
D013873:Thionucleotides; C014308:adenosine 5'-phosphorothioate; D000249:Adenosine Monophosphate; C037185:5'-deoxy-5'-thioadenosine 5'-monophosphate; D009708:Nucleotidases; D000469:Alkaline Phosphatase; D015720:5'-Nucleotidase; D010738:Type C Phospholipases