Subfractionation of rat liver Golgi apparatus: separation of enzyme activities involved in the biosynthesis of the phosphomannosyl recognition marker in lysosomal enzymes.

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Abstract
Al highly purified Golgi apparatus preparation from rat liver was subfractionated on a Percoll gradient into 2 major protein peaks of similar size that migrated at densities of 1.028 and 1.051 g/ml. The lighter protein peak contained 70-80% of the total activities of the oligosaccharide-processing enzymes .alpha.-1,2-mannosidase and mannosidase III and of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyl-1-phosphotransferase (.alpha.-N-acetylglucosaminylphosphotransferase), an enzyme involved in the biosynthesis of the mannose 6-phosphate recognition marker of lysosomal enzymes. These enzyme activities were enriched 2-fold in specific activity over that of the heavy protein peak. In contrast, 80% of the .alpha.-N-acetylglucosaminylphosphodiesterase, an enzyme that exposes 6-phosphomonoesters of mannose on the oligosaccharide chains of lysosomal enzymes, migrated in a region of slightly higher density than did the protein peak of density 1.051 g/ml. Sialyltransferase (SiaTase) and galactosyltransferase (GalTase) activities distributed almost equally among the 2 protein peaks. Controls rule out that the 2 protein peaks were the result of aggregation/deaggregation and that enzyme activities were altered by Percoll per se. Lysosomal enzyme activities migrated in a region essentially devoid of Golgi apparatus-associated enzyme activities. These results suggest a physical separation within the Golgi apparatus of some of the enzymes involved in the biosynthesis and processing of the oligosaccharides on glycoproteins, including those responsible for the formation of the mannose 6-phosphate recognition marker on lysosomal enzymes.