A neutrophil-dependent pathway for the generation of a neutral peptide mediator. II. Subcellular localization of the neutrophil protease.
The human neutrophil neutral peptide-generating protease was associated with the plasma membrane marker 5'-nucleotidase on sucrose density gradient centrifugation of sonicates of granule-free fractions following homogenization and velocity sedimentation. The two activities were also associated on sucrose density gradient fractionation of plasma membranes obtained by hypotonic lysis in EDTA containing buffers, a technique which minimizes aggregation. Treatment of fractions containing these enzymatic activities with 1-0 M NaCl separated the neutral peptide-generating proteasein to the eluate while leaving the 5'-nucleotidase in the pellet. Gel filtration of the solubilized neutral peptide-generating protease through Sephadex G-100 in 1-0 M NaCl demonstrated that the protease had an approximate mol. wt of 20,000 while filtration in physiological salt concentrations yielded activity only in the excluded volume. In both cases, there was complete recovery of neutral peptide-generating activity suggesting that the filtration characteristics of the protease were determined by the salt concentration. The solubilized purified protease, the whole cell sonicates, and the intact cells interacted with heat-inactivated plasma to yield the same product, a neutral peptide with a 1000 molecular weight and an isoelectric point of 7-2-7-6. The neutral peptide-generating protease in each instance was inhibited in dose-response fashion by alpha-1-antitrypsin, LBTI, and DFP. Only 30-60% of the protease sites were functional on intact cells as revealed by substrate cleavage or were available to inhibitors. The neutrophil protease which generates neutral peptide is an extrinsic plasma membrane protein with an approximate mol. wt of 20,000 which functions as an ectoenzyme.
D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D006801:Humans; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D009708:Nucleotidases / Q000032:analysis; D010452:Peptide Biosynthesis; D010447:Peptide Hydrolases / Q000032:analysis*; D011480:Protease Inhibitors; D013347:Subcellular Fractions / Q000201:enzymology
D011480:Protease Inhibitors; D009708:Nucleotidases; D010447:Peptide Hydrolases