Glycosidase inhibitors from algae.

tion of Eagle’s minimum essential medium supplemented with 10% (v/v) horse serum [3], with or without tunicamycin (0.1 pg/ml) or castanospermine (10 pg/ml). Washed explants (typically 100 pg of protein) were homogenized in hyptonic Tes buffer, pH 7.0 [4], and centrifuged ( lo5 g, for 15 min). The supernatant (containing soluble proteins) was removed. A portion of the washed pellet was extracted with Chaps (20 g/l) to release 50-60‘%, of the membrane-bound proteins. The remaining membrane fraction, and the soluble fraction, were assayed for marker enzymes: Na’ ,K’-ATPase (plasma membrane), hcxosaminidasc (lysosomes), a-mannosidase 11 (Golgi complex), a-glucosidase pH 6.5 (rough endoplasmic reticulum), a-glucosidase pH 4.0 (lysosomes) and lactate dehydrogenase (cytosol). Glycoprotein composition was assessed by lcctin affinity chromatography on insolubilized concanavalin A, followcd by SDS/polyacrylamide-gel electrophoresis (PAGE) 151. Gross morphology and ultrastructure of the explants were assessed by phase-contrast microscopy and transmission electron microscopy, rcspcctively [ 31. After 14 days in the presence o f tunicamycin, the specific activity of lactate dehydrogenase was little affected, whereas that of hexosaminidase, a-mannosidase I I and a-glucosidase (pH 6.5) was severely reduced (to 15%, 42% and 28% of control values, respectively). As expected, tunicamycin depressed the levels of N-linked glycoproteins, but not of non-glycoproteins. Table 1 shows the cffect of castanospermine on the marker enzymes of cultured ( 10 day) cerebellar explants. As expected, the alkaloid markedly reduced the activity of the membrane-bound a-glucosidase involved in processing of nascent Winked oligosaccharide chains in the rough endoplasmic reticulum; the lysosomal isoenzyme was also inhibited. The non-glycoprotein lactate dehydrogenase was not affected, as expected. The activity of the other three (glycoprotein) marker enzymes was moderately reduced. Analysis of the Winked glycoprotein components revealed, as expected, no significant differences between the control and the castanospermine-treated explants. By phase-contrast microscopy, control explants of ccrebellum maintained for 1014 days were pale, with bundles of nerve fibres and a mat of heterogeneous cells extending from them. Electron microscopy revealed well-differentiated healthy neurons, principally granule cells, Purkinje cells, and putative Golgi cells. Large neurons were rich in rough endoplasmic reticulum, Golgi elements, and pale mitochondria. The neuropil contained numerous simplc synapses. Castanospermine-treated explants appeared phase dark and were surrounded by many macrophages, and a rich mat of cells. Electron microscopy revealed a mixture of healthy and degenerating neurons, and abundant simple synapses. Apparently healthy neuronal cytoplasm contained numerous Golgi elements surrounded by smooth and coated vesicles, numerous lysosomes, and dark mitochondria often in prominent clusters.