TREHALASE OF DICTYOSTELIUM-DISCOIDEUM - INHIBITION BY AMINO-CONTAINING ANALOGS OF TREHALOSE AND AFFINITY PURIFICATION

The inhibitory effects of three nitrogen containing analogs of trehalo se, validamycin A, MDL 25 637 and castanospermine, on Dictyostelium di scoideum trehalase were examined. Prior to this study, the effects of glycohydrolase inhibitors on D discoideum trehalase have not been repo rted. Validamycin A, MDL 25 637 and castanospermine were found to be p otent, reversible, competitive inhibitors of D discoideum vegetative t rehalase 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 exhibit ed rime-dependent inhibition of D discoideum trehalase, whereby the po tencies of these two inhibitors were observed to increase when pre-inc ubated with the enzyme for up to 60 min. The competitive natures of va lidamycin A and MDL 25 637 were also altered during pre-incubation wit h enzyme such that the compounds behaved as mixed inhibitors under the se conditions. Taken together, these results suggest that the inhibito ry action of validamycin A and MDL 25 637 on trehalase is of a slow-bi nding nature. A trehalase-specific affinity resin was synthesized by c ovalently coupling validamycin A to Sepharose 6B. This resin was used to purify D discoideum trehalase to near homogeneity in a two-step pro cedure. SDS-PAGE of affinity-purified trehalase, and silver staining o r in situ staining for trehalase activity, revealed a major protein sp ecies of 42 kDa, exhibiting trehalase activity, and two minor protein species of approximately 35 and 49 kDa. Since validamycin A demonstrat es strict binding specificity for trehalase, validamycin A-Sepharose h as potential and novel applications in rapid, large scale, purificatio n of trehalases from a variety of species origins.