Subcellular distribution in rat liver of a novel broad-specificity (alpha 1----2, alpha 1----3 and alpha 1----6) mannosidase active on oligomannose glycans.
Recently, the purification to homogeneity was reported of a novel broad-specificity alpha-mannosidase from rat liver microsomal membranes [P. Bonay and R. C. Hughes (1991) Eur. J. Biochem. 197, 229-238]. The enzyme catalyzed the ordered removal of alpha 1----2-, alpha 1----3- and alpha 1----6-linked mannose residues from MannGlcNAc oligosaccharide substrates where n = 4-9. We now show by subcellular fractionation and immunocytochemistry that the novel mannosidase is present in the endoplasmic reticulum, Golgi apparatus and endosomes. Enzyme activity is enriched in a heavy Golgi membrane fraction and to lesser extent in an intermediate density Golgi membrane fraction containing GlcNAc transferase I activity and in a 'late' endosomal fraction. Low levels of enzyme activity were detectable in endoplasmic reticulum membranes and in 'early' endosomes but not in receptor-enriched and ligand-free endosomes. Assays of enzymic activity using Golgi membrane fractions in the absence and presence of Triton X-100 showed that the active site of the enzyme faces the lumen of the membrane vesicles. Antibodies directed against the purified mannosidase showed no immunological cross-reaction to known endoplasmic reticulum and Golgi mannosidases. Conversely, the purified mannosidase was not recognized by antibodies directed against endoplasmic reticulum mannosidase nor Golgi mannosidase IA.
Bonay
P
P
National Institute for Medical Research, London, UK.
Roth
J
J
Hughes
R C
RC
D000818:Animals; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004797:Enzyme-Linked Immunosorbent Assay; D006056:Golgi Apparatus / Q000201:enzymology; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008358:Mannose / Q000378:metabolism*; D008361:Mannosidases / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism*; D011233:Precipitin Tests; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D011134:Polysaccharides; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; D008358:Mannose