Purification and identification of 91-kDa neutrophil gelatinase. Release by the activating peptide interleukin-8.
Human neutrophils were found to release a 91-kDa gelatinase that is serologically related to tumor-derived gelatinolytic enzymes, as evidenced by immunoprecipitation. In order to identify the neutrophil gelatinase, the activity in conditioned medium from human neutrophil suspensions was purified by affinity chromatography on a gelatin substrate. The 91-kDa active enzyme was further separated from other stainable protein bands by classical SDS PAGE and blotting to a solid support. Amino-terminal sequence analysis of blotted proteins showed that the 91-kDa enzyme is a truncated form of tumor-derived 92-kDa gelatinase (type IV collagenase), lacking eight residues at the NH2-terminus. Sequence analysis of enzymatically inactive cleavage products of this neutrophil gelatinase demonstrated that the gelatin-binding part of the molecule is restricted to the amino-terminal third. Exocytosis of gelatinase-containing granules from neutrophils occurred spontaneously within 6 h after neutrophil plating. When the cells were triggered with the phorbol ester phorbol 12-myristate 13-acetate, a strong secretagogue, rapid gelatinase release was observed. When granulocytes were stimulated with the neutrophil-activating peptide interleukin-8, maximal exocytosis occurred within 1 h. The almost immediate release of neutrophil gelatinase after stimulation of the cells with a chemotactic factor might play a key role in remodeling of the extracellular matrix during granulocyte movement in response to chemotactic stimuli.
Masure
S
S
Rega Institute for Medical Research, University of Leuven, Belgium.
Proost
P
P
Van Damme
J
J
Opdenakker
G
G
D000595:Amino Acid Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D018093:Gelatinases; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D016209:Interleukin-8 / Q000494:pharmacology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010434:Pepsin A / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007375:Interleukin-1; D016209:Interleukin-8; D010446:Peptide Fragments; D010434:Pepsin A; D018093:Gelatinases; D013755:Tetradecanoylphorbol Acetate