Purification and Characterization of a Human Neutrophil Neutral Protease

Abstract

A B S T R A C T 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 a-N-p-tosyl-L-arginine methyl ester HC1, a-N-benzoyl-L-arginine-methyl ester HC1, and a-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 lowspeed 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 m50% 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 ofa 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.Dr.

A human neutrophil-dependent pathway for generation of angiotensin II. Purification of the product and identification as angiotensin II.

“…Although the contractile peptide was slightly basic, it was designated neutral peptide to distinguish it from the more basic kinin peptides (1). Each component of this pathway has now been purified to homogeneity and characterized (2)(3)(4). The neutrophil enzyme is a neutral protease of 29,000-30,000 mol wt (2) that has been localized to the neutrophil granule by noncytotoxic release from cytocholasin-B treated human neutrophils and by subcellular fractionation of purified neutrophils (3).…”

“…The enzyme and substrate of this system, previously designated the "neutral" peptide-generating pathway, have been purified to homogeneity and characterized. The neutrophil enzyme is a neutral protease whose physicochemical characteristics and synthetic substrate specificity are identical to leukocyte cathepsin G (2,3). The plasma protein substrate is a glycoprotein with physicochemical characteristics similar to angiotensinogen, the substrate of renin (4).…”

Identification of a human neutrophil angiotension II-generating protease as cathepsin G.

“…Isolation of human neutrophil angiotensin II-generating protease and cathepsin G. Human angiotensin II-generating protease was purified to homogeneity from the low speed sediment of homogenized neutrophils by elution in 1.0 M NaCl, aprotinin-Sepharose affinity chromatography, precipitation in low ionic strength buffer at pH 8.0, and Sephadex G-100 gel filtration (2).…”

“…The peptide, Received for publication 3 July 1981 and in revised form 8 September 1981. designated neutral peptide, is -1,000 mol wt and is distinguished from kinins by its nearly neutral isoelectric point and its inactivation by trypsin as well as by chymotrypsin (1). The neutrophil neutral peptide-generating protease was purified to homogeneity and is a 29,000-30,000-mol wt single polypeptide chain, serine, neutral protease, inhibitable by diiso-propylfluorophosphate, by al-antitrypsin, and by soybean and lima bean trypsin inhibitors (1,2). This protease is distinguished from collagenase by its lower molecular weight and from elastase by its inability to digest orcein-elastin, to hydrolyze N-succinyl-(L-ala)3-p-nitroanilide (suc-[ala]3-pNA),' or to be inhibited by the elastase inhibitor acetyl-(L-alanine)2-proline-valine chloromethyl ketone (2).…”

“…The neutrophil neutral peptide-generating protease was purified to homogeneity and is a 29,000-30,000-mol wt single polypeptide chain, serine, neutral protease, inhibitable by diiso-propylfluorophosphate, by al-antitrypsin, and by soybean and lima bean trypsin inhibitors (1,2). This protease is distinguished from collagenase by its lower molecular weight and from elastase by its inability to digest orcein-elastin, to hydrolyze N-succinyl-(L-ala)3-p-nitroanilide (suc-[ala]3-pNA),' or to be inhibited by the elastase inhibitor acetyl-(L-alanine)2-proline-valine chloromethyl ketone (2). The plasma protein substrate for neutral peptide generation was recently purified to homogeneity and shown to be a 62,000-67,000-mol wt single polypeptide chain glycoprotein, with an isoelectric point of pH 4.6-5.0 (3), and an amino-terminal animo acid sequence identical to the covalent structure of angiotensin I (4).…”