UBC Theses and Dissertations
Developmental regulation of alkaline phosphatase in Dictyostelium discoideum Mohandas, Devaki Velayudhan
The membrane bound alkaline phosphatase activity' in vegetative cells of Dictydstelium discdideum was found to exhibit a 8 to 10 fold increase in specific activity when incubated at 50°C. This activation was reversed on transfer of the preparation to 0°C. Similar activation of the vegetative enzyme was achieved by dialysing the crude membrane preparation, suggesting the removal of a low molecular weight inhibitor. Alkaline phosphatase solubilized from the membranes using Triton X-100 was similarly activated by 50°C treatment or dialysis and the 50°C activation was reversed by incubation at 0°C. Both the dialysed vegetative membrane and the dialysed Triton X-100 extract were inhibited by the addition of concentrated dialysate. This inhibition could be relieved by subsequent dialysis. A 620 fold purified alkaline phosphatase preparation was obtained from crude vegetative membranes by affinity and ion exchange chromatography after solubilization of the enzyme using Triton X-100-5'-nucleotidase activity copurified along with the alkaline phosphatase activity in all the fractionation steps employed. The membrane bound 5'-nucleotidase activity was not activated either by incubation at 50°C or by dialysis and the activity was far less stable than the alkaline phosphatase. However, the Triton X-100 extracted 5'-nucleotidase was activated by dialysis to the same extent as the alkaline phosphatase and both activities in the partially purified preparation were equally inhibited by the dialysates from vegetative membranes. These results suggest that both alkaline phosphatase and 5'-nucleotidase are due to a single protein but the interaction of the two substrates, AMP and pNPP, with the enzyme are different and are markedly influenced by conformational changes induced by the inhibitor. Unlike the vegetative enzyme, the alkaline phosphatase activity in the culminating membrane was not markedly activated by incubation at 50°C or by dialysis. Since the vegetative enzyme was activated by both treatments to levels similar to those found in culminating cells, it was proposed that the developmental increase in alkaline phosphatase in D.discoideum was due to the unmasking of already existing enzyme by the removal of inhibitor. However, the alkaline phosphatase activity of culminating cells differed from that of vegetative cells in chromatography on DEAE-Sephacel and conA-Sepharose and it was less stable in low concentrations of Tris-Cl and in SDS. In contrast, the culminating enzyme was more stable in high concentrations of Tris-Cl. These results suggest that the vegetative enzyme is modified during development. This modification appears to be slight, since the vegetative and culminating enzymes migrate identically in SDS polyacrylamide electrophoretic gels and the enzymes from the two developmental stages were similar in pH optima, in inhibition by phosphate and in inhibition by concentrated dialysate.
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