Function of neutral endopeptidase on the cell membrane of human neutrophils.
Intact human neutrophils hydrolyzed N-formyl-Met-Leu-[3H]Phe (fMLP) and released Leu-[3H]Phe, cleaving 45-50% of the peptide within 20 min at 37 degrees C. The dipeptide after its release was then hydrolyzed to free amino acids by a dipeptidase (EC 3.4.13.11). This activity, present in plasma membrane-enriched fractions of neutrophil lysates, was also inhibited over 90% by phosphoramidon, an inhibitor of neutral endopeptidase (NEP, EC 3.4.24.11). Dithiothreitol and EDTA inhibited the activity to a comparable degree, suggesting the requirement for a heavy metal cofactor. Bestatin and amastatin, inhibitors of aminopeptidases (but not human kidney NEP), did not inhibit the rate of fMLP degradation but prevented the production of free phenylalanine and enhanced the accumulation of Leu-Phe. Of other inhibitors, alpha 1-antitrypsin and alpha 2-macroglobulin slightly enhanced the rate of fMLP hydrolysis by neutrophils, and others tested were ineffective. Rabbit antiserum to homogeneous human kidney NEP reacted specifically with a 100-kDa protein present in sodium dodecyl sulfate-solubilized neutrophils. The Mr of this protein was slightly larger than that of the kidney enzyme in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antiserum incubated with intact cells specifically inhibited the degradation of fMLP over 70%. First, we confirm that NEP present on the plasma membrane cleaves fMLP at the Met-Leu bond; then the dipeptide Leu-Phe is cleaved by a dipeptidase. Finally, inhibition of NEP completely blocks fMLP-mediated chemotaxis. Thus, the enzyme may play an important role in modulating chemotactic responses.
Painter
R G
RG
Department of Biochemistry, University of Texas Health Center, Tyler 75710.
Dukes
R
R
Sullivan
J
J
Carter
R
R
Erdös
E G
EG
Johnson
A R
AR
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D010450:Endopeptidases / Q000097:blood*; D006020:Glycopeptides / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D015260:Neprilysin; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*
D000900:Anti-Bacterial Agents; D006020:Glycopeptides; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010450:Endopeptidases; D015260:Neprilysin; C008890:phosphoramidon