GP-3, A NEWLY CHARACTERIZED GLYCOPROTEIN ON THE INNER SURFACE OF THE ZYMOGEN GRANULE MEMBRANE, UNDERGOES REGULATED SECRETION

We have recently reported the cloning of the rat zymogen granule membr ane glycoprotein GP-3 and the related pancreatic secretory lipase (Wis hart, M. J., Andrews, P. C., Nichols, R., Blevins, G. T., Logsdon, C. D., and Williams, J. A (1993) J. Biol. Chem. 268, 10303-10311). Specif ic antipeptide antibodies were generated against both GP-3 and secreto ry lipase and used for the biochemical and physiological characterizat ion of GP-3. Western blotting confirmed that GP-3 was found exclusivel y in zymogen granule membranes and was absent from zymogen granule con tent which contains the majority of secretory lipase. Extraction of zy mogen granule membranes with Triton X-114 showed GP-3 to be significan tly more hydrophobic than lipase. The GP-3 amino acid sequence contain s one potential N-linked glycosylation site at Asn-336. The loss of co ncanavalin A labeling after both chemical deglycosylation with trifluo romethanesulfonic acid and enzymatic deglycosylation with N-glycanase showed GP-3 to possess a small N-linked oligosaccharide side chain. Di gestion of intact and permeabilized zymogen granules with the nonspeci fic protease Pronase localized GP-3 to the inner surface of zymogen gr anule membranes. Since GP-3 is resident on the inner surface of the zy mogen granule membrane, it should appear on the outer cellular surface after exocytosis. Although membrane attachment of GP-3 was resistant to treatment with phosphatidylinositol-specific phospholipase C, we ob served that GP-3 is released into the pancreatic juice and that secret ion of GP-3 was greatly enhanced by cholecystokinin.