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Identification and partial characterization of three calcium‐ and zinc‐independent gelatinases constitutively present in human circulation
Author(s) -
Makowski Gregory S.,
Ramsby Melinda L.
Publication year - 1998
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/15216549800204592
Subject(s) - gelatinases , leupeptin , chemistry , gelatinase , pmsf , calcium , matrix metalloproteinase , zymography , serine protease , biochemistry , microbiology and biotechnology , protease , biology , enzyme , organic chemistry
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 μM and 500 μM 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 μM). 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 pl 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.