Subcellular localization and release of human neutrophil gelatinase, confirming the existence of separate gelatinase-containing granules.
An e.l.i.s.a. was developed using specific polyclonal rabbit antibodies against human neutrophil gelatinase. This assay, in contrast to the functional assay, is independent of activation of gelatinase, and is specific for the detection of gelatinase in both its reduced and unreduced forms. Using this assay, we were able to demonstrate a difference between the subcellular localization of gelatinase on the one hand, and the subcellular localization of vitamin B-12-binding protein, lactoferrin and cytochrome b558 on the other hand. The latter three co-localized in fractions of slightly higher density than gelatinase on a two-layer Percoll density gradient. Furthermore, the release of gelatinase exceeded the release of vitamin B-12-binding protein as well as lactoferrin by a factor of 3-6 following stimulation with formylmethionyl-leucyl-phenylalanine, leukotriene B4 and other soluble stimuli. Thus, although gelatinase has previously been found to co-localize with lactoferrin on immuno-electron microscopy, we confirm the existence of gelatinase-rich and lactoferrin- and vitamin B-12-binding-protein-poor granules, that are lighter and mobilized more easily than specific granules. These gelatinase-containing granules are not the store of cytochrome b558.
Kjeldsen
L
L
Department of Haematology L, University Hospital, Copenhagen, Denmark.
Bjerrum
O W
OW
Askaa
J
J
Borregaard
N
N
D003594:Cytoplasmic Granules / Q000201:enzymology*; D004797:Enzyme-Linked Immunosorbent Assay; D018093:Gelatinases; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007781:Lactoferrin / Q000032:analysis; D007975:Leukotriene B4 / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D010434:Pepsin A / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D010972:Platelet Activating Factor / Q000494:pharmacology; D011485:Protein Binding; D013347:Subcellular Fractions / Q000201:enzymology; D014805:Vitamin B 12 / Q000378:metabolism; D015054:Zymosan / Q000494:pharmacology
D010972:Platelet Activating Factor; D007975:Leukotriene B4; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D015054:Zymosan; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D007781:Lactoferrin; D010434:Pepsin A; D018093:Gelatinases; D014805:Vitamin B 12