Trehalase of Dictyostelium discoideum: inhibition by amino-containing analogs of trehalose and affinity purification.
The inhibitory effects of three nitrogen containing analogs of trehalose, validamycin A, MDL 25,637 and castanospermine, on Dictyostelium discoideum trehalase were examined. Prior to this study, the effects of glycohydrolase inhibitors on D discoideum trehalase have not been reported. Validamycin A, MDL 25,637 and castanospermine were found to be potent, reversible, competitive inhibitors of D discoideum vegetative trehalase in vitro with IC50 values of 1 x 10(-9) M, 2 x 10(-8) M and 1.25 x 10(-4) M, respectively. Validamycin A and MDL 25,637 also exhibited time-dependent inhibition of D discoideum trehalase, whereby the potencies of these two inhibitors were observed to increase when pre-incubated with the enzyme for up to 60 min. The competitive natures of validamycin A and MDL 25,637 were also altered during pre-incubation with enzyme such that the compounds behaved as mixed inhibitors under these conditions. Taken together, these results suggest that the inhibitory action of validamycin A and MDL 25,637 on trehalase is of a slow-binding nature. A trehalase-specific affinity resin was synthesized by covalently coupling validamycin A to Sepharose 6B. This resin was used to purify D discoideum trehalase to near homogeneity in a two-step procedure. SDS-PAGE of affinity-purified trehalase, and silver staining or in situ staining for trehalase activity, revealed a major protein species of 42 kDa, exhibiting trehalase activity, and two minor protein species of approximately 45 and 49 kDa. Since validamycin A demonstrates strict binding specificity for trehalase, validamycin A-Sepharose has potential and novel applications in rapid, large scale, purification of trehalases from a variety of species origins.
Temesvari
L A
LA
Department of Biological Sciences, University of Windsor, Ontario, Canada.
Cotter
D A
DA
D000818:Animals; D004023:Dictyostelium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007212:Indolizines / Q000494:pharmacology*; D007294:Inositol / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D012685:Sepharose / Q000737:chemistry; D013402:Sugar Alcohols / Q000494:pharmacology*; D014198:Trehalase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D054836:Indolizidines
D004791:Enzyme Inhibitors; D007212:Indolizines; D007294:Inositol; D012685:Sepharose; D013402:Sugar Alcohols; D014198:Trehalase; C052772:MDL 25637; C030790:validamycin A; C037806:castanospermine; D054836:Indolizidines