Identification of the 72-kDa (MMP-2) and 92-kDa (MMP-9) gelatinase/type IV collagenase in preparations of laminin and Matrigel.
EDTA inhibitable type IV collagenolytic activity copurified with laminin preparations from the Engelbreth-Holm-Swarm (EHS) tumor. Several gelatinolytic and type IV collagenolytic matrix metalloproteinase (MMP) species were visualized in EHS laminin from three different sources by gelatin and type IV collagen substrate gel electrophoresis. Incubation with 4-aminophenylmercuric acetate and trypsin suggested that laminin contained both active and latent MMPs. EHS-derived reconstituted basement membrane, Matrigel, was found to possess an MMP profile identical to that of laminin. The presence of 72-kDa (MMP-2) and 92-kDa (MMP-9) gelatinases/type IV collagenases was demonstrated in laminin and Matrigel preparations by Western blot analysis. A rough quantitation of MMP-2 and MMP-9 in 30 micrograms of laminin and 100 micrograms of Matrigel was between 0.3 and 0.6 ng. The presence of these contaminants must be considered in experiments addressing the effects of EHS laminin or Matrigel on cell behavior and, in particular, stimulation of cellular proteolytic activity.
Mackay
A R
AR
National Cancer Institute, National Institutes of Health, Bethesda, MD.
Gomez
D E
DE
Cottam
D W
DW
Rees
R C
RC
Nason
A M
AM
Thorgeirsson
U P
UP
D000818:Animals; D015153:Blotting, Western; D003094:Collagen / Q000032:analysis*; D017364:Collagenases / Q000032:analysis*; D004338:Drug Combinations; D004591:Electrophoresis, Polyacrylamide Gel; D018093:Gelatinases / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D007797:Laminin / Q000032:analysis*; D051379:Mice; D008970:Molecular Weight; D009374:Neoplasms, Experimental / Q000737:chemistry / Q000201:enzymology; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D011509:Proteoglycans / Q000032:analysis*; D019714:Tissue Inhibitor of Metalloproteinases; D014357:Trypsin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004338:Drug Combinations; D006023:Glycoproteins; D007797:Laminin; D011509:Proteoglycans; D019714:Tissue Inhibitor of Metalloproteinases; C059485:matrigel; C010024:4-aminophenylmercuriacetate; D003094:Collagen; D014357:Trypsin; D017364:Collagenases; D018093:Gelatinases; D010662:Phenylmercuric Acetate