Subcellular distribution of "intersecting' beta-N-acetylglucosaminyltransferase in Dictyostelium discoideum. A likely marker for the Golgi apparatus.
Glycoprotein processing in Dictyostelium discoideum is characterized by enzyme catalyzed steps not reported in other organisms. One of these is the formation of a beta 1 --> 4 linkage between GlcNAc and the mannose linked to the core mannose in the alpha 1 --> 6 position of N-glycosides. A simple and sensitive assay for this GlcNAc transferase activity, using a tri-mannose acceptor and a low concentration of UDP-GlcNAc, was developed. Homogenates of the organism were subjected to sub-cellular fractionation by centrifugation in discontinuous sucrose gradients. The specific activity was enriched 4-5-fold in a crude membrane fraction. The transferase was purified 10-12-fold in a membrane fraction that bands on top of 1.1 M sucrose. This fraction was also enriched in nucleotidyldiphosphatase. The enriched fraction was deficient in glucose-6-phosphatase, an endoplasmic reticulum marker. Approx. 80% of the transferase activity was latent, and unavailable to protease. Purified membranes were either subjected to phase separation in Triton X-114, or sodium carbonate extraction or sonication. In each case, the transferase behaved as an intrinsic membrane protein. Several secreted and lysosomal proteins are modified by the enzyme. These data support the idea that the GlcNAc transferase is present as an integral Golgi membrane protein and that at least the catalytic center of the transferase is on the lumenal side of the vesicles.
Capasso
J M
JM
Department of Biological Sciences, University of Arkansas, Fayetteville 72701, USA. jcapasso@uafsysb.uark.edu
Capasso
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Kaplan
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D017766:Acid Anhydride Hydrolases / Q000032:analysis; D000818:Animals; D015415:Biomarkers; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004023:Dictyostelium / Q000201:enzymology* / Q000648:ultrastructure*; D006023:Glycoproteins / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology*; D008345:Manganese / Q000494:pharmacology; D008565:Membrane Proteins / Q000032:analysis; D017351:N-Acetylglucosaminyltransferases / Q000032:analysis* / Q000378:metabolism
D015415:Biomarkers; D006023:Glycoproteins; D008565:Membrane Proteins; D008345:Manganese; D017351:N-Acetylglucosaminyltransferases; D017766:Acid Anhydride Hydrolases