A Human Neutrophil-Dependent Pathway for Generation of Angiotensin II: Purification and Physicochemical Characterization of the Plasma Protein Substrate

Human neutrophils contain a neutral protease, previously designated ``neutral peptide-generating protease,'' which generates a smooth muscle contractile activity from a plasma protein substrate. The plasma protein substrate has been purified to homogeneity from fresh citrated human plasma by 45% (wt/vol) ammonium sulfate precipitation of contaminating proteins, Affi-Gel Blue affinity chromatography, hydroxylapatite chromatography, phenyl-Sepharose hydrophobic chromatography, and Sephacryl S-200 gel filtration. The purified product produced a single stained protein on alkaline disc gel electrophoresis and elicited a monospecific goat antiserum. Purification was approximately 330- to 350-fold, and overall recovery was 6-11% of substrate protein in starting plasma as determined by quantitative radial immunodiffusion assay. The substrate has an isoelectric point of pH 4.6-5.1 and is a single polypeptide chain glycoprotein of Mr62,000-67,000 by sodium dodecyl sulfate electrophoresis. The mean(± SD) concentration of this plasma protein substrate in normal serum is 120± 22μ g/ml. The plasma protein substrate of the neutrophil neutral protease may be identical to human angiotensinogen (renin substrate) because the physicochemical characteristics are similar and the peptide product is recognized by antibody to angiotensin II.