Glycosylation variants of endopeptidase-24.11 ('enkephalinase').
Anion exchange chromatography resolves two charge variants of rat kidney endopeptidase-24.11 (designated NEP 1 and NEP 2); each was purified to homogeneity using immunoaffinity chromatography. In addition to charge differences the subunit molecular weights of NEP 1 and NEP 2 differ and are 89 and 96 kDa, respectively. Isoelectric focusing resolved 8-10 pl species in the pH range of 5.95-6.20 for NEP 1 and 5.46-6.06 for NEP 2. Removal of sialic acid residues converted the multiple pl species to one form with a pl of 6.32 for NEP 2, and two forms with pls of 6.27 and 6.32 for NEP 1. Endoglycosidase H or F, capable of removing high-mannose and biantennary branched N-linked oligosaccharides, produced a 2-3 kDa decrease in the molecular weight of both NEP 1 and NEP 2. Peptide-N-glycosidase F, capable of removing all classes of N-linked oligosaccharides, produced 8 and 11 kDa decreases in NEP 1 and NEP 2, respectively. Removal of all N-linked and O-linked oligosaccharides with trifluoromethanesulfonic acid resulted in 10 and 15 kDa decreases in NEP 1 and NEP 2, respectively. Tryptic epitope maps demonstrated that NEP 2 was cleaved at a slower rate than NEP 1. These analyses demonstrate that rat kidney NEP exhibits sialic acid microheterogeneity resulting in two distinct change variants. The data also indicate that NEP 2 contains more N- and O-linked carbohydrate mass than NEP 1 and may contain a larger polypeptide backbone giving rise to molecular weight differences between these enzyme forms.
Vida
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Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038.
Hersh
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LB
D000818:Animals; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D006031:Glycosylation; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification; D007668:Kidney / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D015260:Neprilysin / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats
D007527:Isoenzymes; D046911:Macromolecular Substances; D015260:Neprilysin