Studies on the effect of lysosomotropic agents on the release of Gal beta 1-4GlcNAc alpha-2,6-sialytransferase from rat liver slices during the acute-phase response.

The mechanism of release of Gal beta 1-4GlcNAc alpha-2,6-sialyltransferase (CMP-N-acetylneuraminate: beta-galactoside alpha-2,6-sialytransferase, EC 2.4.99.1) from rat liver during the acute-phase response is due to the action of a cathepsin D-like proteinase that cleaves the trans-Golgi membrane-bound enzyme from a membrane anchor; this allows a major portion of the enzyme containing the catalytic site to escape into the extracellular space [Lammers & Jamieson (1988) Biochem. J. 256, 623-631]. The release of sialytransferase was most effective at pH 5.6, suggesting that release of sialyltransferase from the Golgi in whole cells is dependent on maintaining an acidic environment in the trans-Golgi compartment of the hepatocyte. Golgi membranes contain a proton pump that maintains the acidic pH in these compartments [Glickman, Croen, Kelly & Al-Awquati (1983) J. Cell Biol. 97, 1303-1308; Yamashiro, Tycko & Maxfield (1984) Cell (Cambridge, Mass.) 37, 789-800; Zhang & Schneider (1983) Biochem. Biophys. Res. Commun. 114, 620-625; Anderson & Pathak (1985) Cell (Cambridge, Mass.) 40, 635-643]. Lysosomotropic agents, such as NH4Cl, chloroquine and methylamine can penetrate acidic compartments of the cell, such as the Golgi complex, raise the pH, and thus affect proteolytic cleavage events. The present paper describes the effect of lysosomotropic agents on the release of sialyltransferase from the hepatocyte using liver slices as a whole-cell system. Slices were prepared from control rats and rats suffering from the acute-phase response, where release of sialyltransferase is increased substantially [Lammers & Jamieson (1988) Biochem. J. 256, 623-631; Kaplan, Woloski, Hellman & Jamieson (1983) J. Biol. Chem. 258, 11505-11509]. Release of sialyltransferase was almost abolished in presence of 50 mM-NH4Cl, 50 mM-methylamine or 1 mM-chloroquine. Inhibition of release of sialyltransferase was reversed when the lysosomotropic agents were removed from the medium, showing that these agents are not cytotoxic to the cells under the conditions used. The secretion of rat alpha 1-acid glycoprotein, which is not subject to proteolytic processing in the Golgi complex, was not found to be substantially affected by the presence of lysosomotropic agents. The results suggest that proteolytic cleavage of the catalytic site of sialyltransferase is a process that is significantly affected by the intra-Golgi pH.