Identification and partial characterization of three calcium- and zinc-independent gelatinases constitutively present in human circulation.
Three constitutive gelatinases in human plasma were identified and characterized relative to known matrix metalloproteinase (MMP) gelatinases: MMP-2 (fibroblast 72-kDa) and MMP-9 (neutrophil 92-, 130-, and 225-kDa). Substrate gel electrophoresis (gelatin zymography) revealed an apparent Mw of 78-, 82-, and 89-kDa for these gelatinases. Densitometry revealed that MMP-9 and MMP-2 were highly calcium sensitive requiring 50-150 microM and 500 microM calcium for half-maximal activity, respectively. Of the new gelatinases, only the 89-kDa form demonstrated slight calcium activation. The three gelatinases were unaffected by known MMP inhibitors: EDTA (5 mM), 1,10-phenanthroline (2 mM), and pepstatin (18 microM). Serine and thiol protease inhibitors (leupeptin, aprotinin, PMSF, TLCK, TPCK, antichymostatin, antipain) were also ineffective. Solution-phase IEF revealed that the 78- and 82-kDa forms focused at neutral pI 6.72-7.95 whereas the 89-kDa focused at an acidic pI 4.89-5.18 (similar to neutrophil and fibroblast forms). The data indicate that these gelatinases are not MMPs or partially activated MMPs. Their role in normal and pathological conditions is not known.
Makowski
G S
GS
Department of Laboratory Medicine, University of Connecticut Health Center, Farmington 06030, USA.
Ramsby
M L
ML
D002118:Calcium / Q000494:pharmacology*; D002614:Chelating Agents / Q000494:pharmacology; D017364:Collagenases / Q000097:blood / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000201:enzymology; D005780:Gelatin / Q000378:metabolism; D018093:Gelatinases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D061965:Matrix Metalloproteinase Inhibitors; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000097:blood / Q000737:chemistry; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology; D011480:Protease Inhibitors / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
D002614:Chelating Agents; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D005780:Gelatin; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D015032:Zinc; D002118:Calcium