Identification and characterization of the major stilbene-disulphonate- and concanavalin A-binding protein of the porcine renal brush-border membrane as aminopeptidase N.
A 130 kDa glycoprotein (GP 130) was purified from porcine renal brush-border membranes by affinity chromatography using immobilized 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonate (SITS)- and concanavalin A-Sepharose. GP 130 was the major concanavalin A-binding protein in porcine renal brush-border membranes and also bound Ricinus communis (castor-bean) and wheat-germ agglutinins. Endo-beta-N-acetylglucosaminidase F reduced the molecular mass of GP 130 by 20 kDa as determined by SDS/PAGE, whereas endo-beta-N-acetylglucosaminidase H reduced the molecular mass by 5 kDa, showing that GP 130 contained both complex and high-mannose carbohydrate structures. Western-blot analyses using an antibody raised against GP 130 showed that it was localized to the brush-border membrane fraction and was present in a membrane fraction of the pig kidney cell line LLC-PK1. The N-terminal sequence and amino acid composition of GP 130 showed that GP 130 is similar to rat kidney zinc peptidase and human intestinal aminopeptidase N. GP 130 had aminopeptidase N enzymic activity and was inhibited by bestatin (Ki = 36 microM), 1,10-phenanthroline (Ki 30 microM), Zn2+ (Ki 26 microM), Cu2+ (Ki 260 microM), pre-incubation with EDTA and by a polyclonal antibody against GP 130. Bicarbonate and iodide blocked the binding of GP 130 to the SITS-affinity resin, showing that GP 130 has an anion-binding site. Neither these anions nor stilbene disulphonates affected the aminopeptidase N activity of GP 130.
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Department of Medicine, University of Toronto, Canada.
Reithmeier
R A
RA
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000118:Acetylglucosaminidase / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D000838:Anions; D001665:Binding Sites; D015153:Blotting, Western; D018826:CD13 Antigens; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D003208:Concanavalin A / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D037102:Lectins / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008871:Microvilli / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D037121:Plant Lectins; D013552:Swine; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D000596:Amino Acids; D000838:Anions; D037102:Lectins; D037121:Plant Lectins; C017707:Ricinus communis agglutinin-1; D014909:Wheat Germ Agglutinins; D003208:Concanavalin A; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D006026:Glycoside Hydrolases; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000626:Aminopeptidases; D018826:CD13 Antigens; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid