| Summary: | Evidence is presented that a single enzyme is responsible for the hydrolysis of pNPP and AMP in Dictyostelium discoideum membranes. The two activities have identical mobilities when subjected to polyacrylamide gel electrophoresis under non-denaturing conditions.- The pH optima of hydrolysis of AMP or pNPP are highly dependent upon the concentration of the substrate and are identical when similar concentrations are used. pKm vs pH plots gave identical inflection points for AMP and pNPP and suggested that a functional group of pKa=8.5 is involved in the binding of either substrate to the enzyme. The plots also suggested that while only one such functional group is required for pNPP binding, there are two involved in the binding of AMP. Further evidence for the existence of only one enzyme was provided by the use of mutants with an altered Km for both pNPP and AMP. Furthermore, both AMPase and pNPPase
activities in the mutant were unstable whereas both activities were stable in the wild-type strain. A previous demonstration that AMPase activity was selectively released from the membrane by co-incubation with a phospholipase C preparation was invalidated by the finding that the phospholipase C preparation contained an AMP specific hydolytic activity. The enzyme that hydrolyzed both AMP and pNPP was shown to be mechanistically an alkaline phosphatase. Kinetic evidence was obtained implicating Tris as a co-substrate in the hydrolysis of pNPP, and a transphosphorylation to Tris was demonstrated. Futhermore, biphasic kinetics of a double-displacement reaction was detected by stopped flow spectrophotometry. The hydrolysis of substrates via a double displacement mechanism and the consequent transphosphoryla-tion to a variety of low molecular weight alcohols is a distinguishing feature of alkaline phosphatases. The inhibition of the alkaline phosphatase with orthophosphate was competitive at low ionic strength but irreversible at high ionic strength.
The alkaline phosphatase activity in D.discoideum is developmentally regulated and increases markedly during culmination. Attempts were made to quantitate the levels of alkaline phosphatase by immunological methods and by active site labelling with ³²Pi. Problems were encountered with both these methods and it could not be established with confidence whether the increase in alkaline phosphatase activity during development is due to the previously proposed hypothesis that unmasking of pre-existing enzyme may account for the developmental increase in activity. The biological process that activates alkaline phosphatase in vivo is unknown. However, two plausible methods of activating the enzyme were found. The vegetative cell enzyme is activated at pH 5.5 and by exposure to carbohydrate binding proteins. === Science, Faculty of === Microbiology and Immunology, Department of === Graduate
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