Subfractionation of rat liver Golgi apparatus: separation of enzyme activities involved in the biosynthesis of the phosphomannosyl recognition marker in lysosomal enzymes.
A highly purified Golgi apparatus preparation from rat liver was subfractionated on a Percoll gradient into two 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 II 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 (Gal-Tase) activities distributed almost equally among the two protein peaks. Controls rule out that the two 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.
D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D006056:Golgi Apparatus / Q000201:enzymology* / Q000648:ultrastructure; D006600:Hexosephosphates / Q000378:metabolism*; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure*; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D008360:Mannosephosphates / Q000378:metabolism*; D008361:Mannosidases / Q000302:isolation & purification*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D017855:Transferases (Other Substituted Phosphate Groups)
D006600:Hexosephosphates; D008360:Mannosephosphates; C027693:mannose-6-phosphate; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase; D010727:Phosphoric Diester Hydrolases; C026645:N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase; D008361:Mannosidases