Neutral proteinase inhibitors in PMN leukocytes. I. Purification and characterization of a neutral proteinase inhibitor from bovine neutrophils.
An inhibitor of neutral proteinases was isolated from the cytosol of bovine leukocytes by anion exchange chromatography on Mono Q and gel filtration on a HPLC TSK column. The gel filtration resulted in two fractions with inhibitory activity which could be identified by sodium dodecyl sulphate-poly-acrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions as dimer and monomer of the inhibitor. The latter was shown to be homogeneous in SDS-PAGE with an apparent molecular mass of 40 kDa, with calibrated HPLC a molecular mass of 36.5 kDa has been determined. Isoelectric focusing followed by Western blot analysis revealed four bands in the pH range of 5.0 to 5.9. The inhibitor was found in bovine polymorphonuclear neutrophils (PMN), whereas lymphocytes and monocytes lacked this protein. No immunological cross-reactivity between the described cell-derived PMN-inhibitor (PMN-I) and alpha 1-proteinase inhibitor was detectable. The mechanism of inhibition for the serine proteinases chymotrypsin, trypsin, pancreatic elastase and leukocyte elastase was studied. PMN-I could not bind to PMS-chymotrypsin. The reaction of the serine proteinases with the PMN-I was characterized by the determination of the association rate constant kon.
Max-Planck-Institut für Immunbiologie, Freiburg.
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P
Hintz
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M
Jochum
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H
Mossmann
D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007457:Iodine Radioisotopes; D007962:Leukocytes / Q000032:analysis; D008623:Mercaptoethanol; D009504:Neutrophils / Q000201:enzymology*; D011480:Protease Inhibitors / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D013552:Swine
D007457:Iodine Radioisotopes; D011480:Protease Inhibitors; D008623:Mercaptoethanol