Purification and identification of 91‐kDa neutrophil gelatinase

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 NH 2 ‐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.