Zonal rotor purification and characterization of 'mannosomes': A tubular membrane system in gastropod mollusc digestive gland

Mannitol oxidase (manox) is an H2O2-generating oxidase apparently unique to molluscs and especially abundant in alimentary tissues. In the digestive gland it is localized to an organelle ('mannosome') that forms an unusual tubular membrane system. We have developed a novel centrifugation procedure for >100-fold purification of these membranes in 20% yield from ~30 g of digestive gland of the slug Arion ater. Mannosomes from several other gastropod species are also substantially purified by the procedure. Four successive density gradient separations are employed which minimize structural damage by exploiting near isosmotic conditions early on and by completely avoiding traumatic pelleting and resuspension. Plasma membrane contamination is reduced by digitonin-induced density perturbation. The purified preparation is characterized by a predominant 68-kDa integral membrane protein and retains the in situ appearance of hexagonally arranged tubules with an enveloping outer membrane.