ZONAL ROTOR PURIFICATION AND CHARACTERIZATION OF MANNOSOMES - A TUBULAR MEMBRANE SYSTEM IN GASTROPOD MOLLUSK DIGESTIVE GLAND

Mannitol oxidase (manox) is an H2O2-generating oxidase apparently uniq ue to molluscs and especially abundant in alimentary tissues. In the d igestive gland it is localized to an organelle (''mannosome'') that fo rms an unusual tubular membrane system. We have developed a novel cent rifugation procedure for >100-fold purification of these membranes in 20% yield from similar to 30 g of digestive gland of the slug Arion at er. Mannosomes from several other gastropod species are also substanti ally purified by the procedure. Four successive density gradient separ ations are employed which minimize structural damage by exploiting nea r isosmotic conditions early on and by completely avoiding traumatic p elleting and resuspension. Plasma membrane contamination is reduced by digitonin-induced density perturbation. The purified preparation is c haracterized by a predominant 68-kDa integral membrane protein and ret ains the in situ appearance of hexagonally arranged tubules with an en veloping outer membrane. (C) 1998 Academic Press.