Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase.
Human neutrophils contain a neutral metalloproteinase which degrades denatured collagens and potentiates the action of interstitial collagenase. This gelatinase is rapidly secreted from neutrophils stimulated with phorbol myristate acetate. The secreted enzyme has been purified by a combination of chromatography on DEAE-cellulose and gelatin-Sepharose. The purified enzyme was latent and had a specific activity of 24,000 units. Estimated molecular weight obtained by gel filtration was 150,000-180,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed three bands with relative molecular weights of 225,000, 130,000, and 92,000. Electrophoresis in the presence of a reducing agent revealed a single band of Mr = 92,000. All the proteins seen on the unreduced gel were found to contain proteolytic activity against gelatin and native type V collagen. Polyclonal antibodies were prepared against the Mr = 130,000 and 92,000 proteins. When analyzed by immunoblotting, both antibodies recognized all three proteins. Furthermore, the identical three proteins were identified by the antibodies when crude culture medium was immunoblotted. The purified enzyme was inhibited by EDTA and 1,10-phenanthroline but not by serine or thiol proteinase inhibitors, suggesting that the enzyme is a metalloendoproteinase. The enzyme had little or no activity against common protein substrates such as bovine serum albumin or casein. Native type I collagen was not cleaved under conditions where native type V collagen was extensively degraded.
Hibbs
M S
MS
Hasty
K A
KA
Seyer
J M
JM
Kang
A H
AH
Mainardi
C L
CL
D002478:Cells, Cultured; D002845:Chromatography; D003094:Collagen / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D018093:Gelatinases; D006801:Humans; D007124:Immunoenzyme Techniques; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D010434:Pepsin A / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D003094:Collagen; D004492:Edetic Acid; D010434:Pepsin A; D018093:Gelatinases