Mannose 6-phosphate-independent endocytosis of beta-glucuronidase. II. Purification of a cation-dependent receptor from bovine liver.
A new binding protein, which recognizes a specific peptide sequence from pronase digested bovine beta-glucuronidase, has been isolated from bovine liver membranes. Prior work has shown that this peptide (IIIb2) contains a Ser-X-Ser sequence, where X might be a posttranslational modified Trp. This receptor was detergent-extracted from total bovine liver membranes and purified by affinity chromatography on a bovine beta-glucuronidase-Sepharose and a IIIb2 peptide-Sepharose column. Binding of bovine beta-glucuronidase to the isolated receptor requires divalent cations, and their presence was necessary to maintain the receptor-ligand complex. Only the peptide sequence containing the fraction IIIb2 was able to impair the binding of the bovine enzyme to the receptor, no other peptide from bovine beta-glucuronidase had an effect on binding. When analyzed by SDS-PAGE under reducing conditions, two bands were observed, a major band of 78 kDa and a faint band of 72 kDa. Rabbit antibodies against this binding protein revealed the presence of the 78 kDa protein in membranes from bovine liver, human and bovine fibroblasts. These antibodies impaired human fibroblasts endocytosis of the bovine but not of the human beta-glucuronidase, which is taken up by a 300 kDa receptor that recognizes phosphomannosyl moieties in the enzyme.
González-Noriega
A
A
Departamento de Biología Celular, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México. P.O. Box 70228, Ciudad Universitaria, 04510 D.F., México, Mexico. gonor@servidor.unam.mx
Michalak
C
C
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001692:Biological Transport; D002413:Cations, Divalent; D002417:Cattle; D002460:Cell Line; D004705:Endocytosis / Q000187:drug effects; D005966:Glucuronidase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D008360:Mannosephosphates / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D011402:Pronase; D011956:Receptors, Cell Surface / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D000906:Antibodies; D002413:Cations, Divalent; D008360:Mannosephosphates; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; C027693:mannose-6-phosphate; D005966:Glucuronidase; D011402:Pronase