Characterization of meprin, a membrane-bound metalloendopeptidase from mouse kidney.
Meprin is an intrinsic protein of the brush border, a specialized plasma membrane, of the mouse kidney. It is a metalloendopeptidase that contains 1 mol of zinc and 3 mol of calcium per mol of the 85,000-Mr subunit. The enzyme is isolated, and active, as a tetramer. The behaviour of the enzyme on SDS/polyacrylamide gels in the presence and absence of beta-mercaptoethanol indicates that the subunits are of the same Mr (approx. 85,000) and held together by intersubunit S--S bridges. Eight S-carboxymethyl-L-cysteine residues were detected after reduction of the enzyme with beta-mercaptoethanol and carboxymethylation with iodoacetate. The enzyme is a glycoprotein and contains approx. 18% carbohydrate. Most of the carbohydrate is removed by endoglycosidase F, indicating that the sugar residues are N-linked. The isoelectric point of the enzyme is between pH 4 and 5, and the purified protein yields a pattern of evenly spaced bands in this range on isoelectric focusing. The peptide-bond specificity of the enzyme has been determined by using the oxidized B-chain of insulin as substrate. In all, 15 peptide degradation products were separated by h.p.l.c. and analysed for their amino acid content and N-terminal amino acid residue. The prevalent peptide-bond cleavages were between Gly20 and Glu21, Phe24 and Phe25 and between Phe25 and Tyr26. Other sites of cleavage were Leu6-Cysteic acid7, Ala14-Leu15, His10-Leu11, Leu17-Val18, Gly8-Ser9, Leu15-Tyr16, His5-Leu6. These results indicate that meprin has a preference for peptide bonds that are flanked by hydrophobic or neutral amino acid residues, but hydrolysis is not limited to these bonds. The ability of meprin to hydrolyse peptide bonds between small neutral and negatively charged amino acid residues distinguishes it from several other metalloendopeptidases.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D006026:Glycoside Hydrolases; D007328:Insulin / Q000378:metabolism; D007525:Isoelectric Focusing; D007668:Kidney / Q000201:enzymology*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008565:Membrane Proteins / Q000302:isolation & purification*; D008623:Mercaptoethanol; D051379:Mice; D015260:Neprilysin; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000378:metabolism
D000596:Amino Acids; D007328:Insulin; D008565:Membrane Proteins; D010446:Peptide Fragments; D010455:Peptides; D008623:Mercaptoethanol; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D010450:Endopeptidases; D015260:Neprilysin