Studies on the Golgi apparatus. Cumulative inhibition of protein and glycoprotein secretion by d-galactosamine

Abstract

1. The administration of d-galactosamine leads to inhibition of protein and glycoprotein secretion by rat liver. To test the secretory function, the secretion times for galactose-and fucose-containing glycoproteins were determined; they were lengthened from 6 to 9min and from 8 to 13min respectively. 2. The Golgi apparatus was enriched 100–120-fold relative to the homogenate. A new linked-assay system for the marker enzyme, UDP-galactose–N-acetyl-d-glucosamine galactosyltransferase, is presented. The activity of the enzyme was measured spectrophotometrically by following the formation of UDP coupled to nicotinamide nucleotide reduction. The Michaelis constants were calculated to be 0.11mm for UDP-galactose with N-acetyl-d-glucosamine as exogenous acceptor and 19mm for N-acetyl-d-glucosamine itself. 3. The physiological substrate of the galactosyltransferase, UDP-galactose, can be replaced by UDP-galactosamine, which accumulates after d-galactosamine administration. Under conditions in vitro the rate of d-galactosamine transfer to an endogenous acceptor protein of the Golgi fraction reaches 9% of that with d-galactose; this finding is noteworthy, because normally a non-acetylated amino sugar does not occur in glycoproteins. 4. The albumin content of the Golgi-rich fraction was diminished to 55% of the reference value 6h after the injection of 375mg of d-galactosamine hydrochloride/kg body wt. The transfer of d-[1-14C]galactose to an endogenous acceptor protein fell to 60% compared with Golgi-rich fractions from untreated animals. Analysis of the Golgi-rich fraction by polyacrylamide-gel electrophoresis showed a decrease or loss of several protein bands. 5. Protein synthesis can be restored by up to 80% if the UTP pool, decreased after d-galactosamine administration, is filled up by several injections of uridine. 6. From the results presented it can be concluded that the disturbed secretion of proteins and glycoproteins was due to a cumulative effect of galactosamine by: (a) inhibition of protein synthesis leading to a diminution of the endogenous acceptor pool of the galactosyltransferase; (b) inhibition of the galactosyltransferase activity by galactosamine metabolites and (c) replacement of UDP-galactose by UDP-galactosamine.