Characterization of neutral endopeptidase 24.11 in dog glomeruli.
Neutral endopeptidase (NEP; also known as neprilysin and enkephalinase; EC 3.4.24.11) is a cell-surface metallopeptidase that is present in many mammalian tissues. It is particularly abundant on the brush-border membranes of the kidney proximal tubule. In this paper, the presence of NEP in purified glomeruli from dog kidney was assessed by measuring phosphoramidon- and thiorphan-sensitive [D-Ala2,Leu5]enkephalin-degrading activity. Using this assay, the Km and kcat. of the glomerular enzyme were found to be identical to those of the tubular enzyme. By Western blotting the apparent M(r) of the glomerular enzyme was found to be 104,000, compared with 94,000 for the tubular enzyme. This might be due to a different glycosylation pattern, since endoglycosidase F treatment of NEP obtained from both tissues yielded deglycosylated enzymes with similar electrophoretic mobilities. The glomerular enzyme also appears to be membrane-bound, since it was retained in the detergent-rich phase after phase separation with Triton X-114. Autoradiography experiments performed with RB104, a new highly selective and potent NEP inhibitor, showed that NEP was expressed in both glomeruli and proximal tubules. The presence in glomeruli of NEP and some other brush-border peptidases (dipeptidyl-dipeptidase IV, aminopeptidase N and angiotensin I-converting enzyme) suggests that cell-surface peptidases might play an important role as regulators of plasma-derived peptides in this part of the nephron.
Landry
C
C
Département de Biochimie, Université de Montréal, Québec, Canada.
Santagata
P
P
Bawab
W
W
Fournié-Zaluski
M C
MC
Roques
B P
BP
Vinay
P
P
Crine
P
P
D000818:Animals; D000911:Antibodies, Monoclonal; D001345:Autoradiography; D015153:Blotting, Western; D004285:Dogs; D016308:Enkephalin, Leucine-2-Alanine / Q000378:metabolism; D006020:Glycopeptides / Q000494:pharmacology; D006031:Glycosylation; D015151:Immunoblotting; D007462:Iodobenzenes / Q000494:pharmacology; D007678:Kidney Glomerulus / Q000201:enzymology*; D007687:Kidney Tubules, Proximal / Q000201:enzymology; D008871:Microvilli / Q000201:enzymology; D015260:Neprilysin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017830:Octoxynol; D011092:Polyethylene Glycols; D015244:Thiorphan / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D007462:Iodobenzenes; C075662:RB 104; D011092:Polyethylene Glycols; D005680:gamma-Aminobutyric Acid; D016308:Enkephalin, Leucine-2-Alanine; D017830:Octoxynol; C010615:Nonidet P-40; D015244:Thiorphan; D015260:Neprilysin; C008890:phosphoramidon