Purification and Characterization of a Human Neutrophil Neutral Protease: THE NEUTRAL PEPTIDE-GENERATING PROTEASE

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Abstract

A human neutrophil neutral protease which generates a low molecular weight peptide from a plasma protein substrate and cleaves the basic amino acid ester substrates α-N-p-tosyl-l-arginine methyl ester HCl, α-N-benzoyl-l-arginine-methyl ester HCl, and α-N-carbobenzoxy-l-lysine-p-nitrophenyl ester has been purified to homogeneity and distinguished from the known lysosomal neutrophil proteases. The starting activity was obtained from purified human neutrophils by homogenization, sedimentation by low-speed centrifugation, and high salt elution of the insoluble material. Purification was achieved by aprotinin-affinity chromatography, precipitation at low ionic strength, and gel filtration. The overall recovery, relative to the activity in the starting eluate of the neutrophil fraction, was ≅50% with a 200- to 400-fold increase in specific activity. After treatment with diisopropylfluorophosphate to eliminate autodegradation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced and unreduced protein gave a single protein band of 29,000-30,000 mol wt. The isoelectric point determined in sucrose gradients ranged from pH 7.8 to 8.3 with a peak at pH 8.0. This neutrophil protease, like cathepsin G and elastase, is composed of a single polypeptide chain of ≅30,000 mol wt, but differs from cathepsin G and elastase in its less cationic isoelectric point and its failure to cleave synthetic substrates presenting an aromatic amino acid ester linkage and alanyl peptide bonds, respectively.

Authors

Jonathan S. Coblyn, K. Frank Austen, Bruce U. Wintroub