Quantitative reverse zymography: analysis of picogram amounts of metalloproteinase inhibitors using gelatinase A and B reverse zymograms.
Matrix metalloproteinases are a growing family of neutral pH optima, zinc atom-dependent endopeptidases that collectively degrade all components of the extracellular matrix. This family of related proteases is further defined by their inhibition of protease activity by a class of low-molecular-weight endogenous inhibitors known as tissue inhibitors of metalloproteinases or TIMPs. Reverse zymography is an electrophoretic technique used to identify TIMP inhibitory activity within acrylamide gels. Previous methods have generally used biochemically complex sources of proteolytic activity (such as cell culture conditioned media) copolymerized with a proteinase substrate in the gel to identify the zones of inhibited proteolysis. We describe a novel system for reverse zymography using purified recombinant human gelatinase A or gelatinase B in place of conditioned media. These reverse zymograms using recombinant gelatinase have sensitivities for TIMPs that are favorable in comparison to immunoblotting techniques but have the benefit of visualizing multiple inhibitors simultaneously. We have developed and characterized these methods for the evaluation of inhibitors and have shown them to be highly sensitive, convenient, and reproducible. Both systems detect TIMPs 1, 2, and 3 simultaneously, but with differential sensitivities for TIMPs 1 and 2. Using gelatinase A the system can detect as little as 1 pg of rTIMP-2, but the limit of detection for rTIMP-1 is 40 pg. Gelatinase B shows less differential activity in that the limits of detection are 60 and 40 pg for TIMP-2 and TIMP-1, respectively. We demonstrate how these varied sensitivities of the gelatinases for the TIMPs can contribute to potential pitfalls in systems using uncharacterized reagents (i.e., conditioned media).
Oliver
G W
GW
Extracellular Matrix Pathology Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-1500, USA.
Leferson
J D
JD
Stetler-Stevenson
W G
WG
Kleiner
D E
DE
D017364:Collagenases; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D018093:Gelatinases; D006023:Glycoproteins / Q000032:analysis*; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D008666:Metalloendopeptidases; D011506:Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D019716:Tissue Inhibitor of Metalloproteinase-2; D019714:Tissue Inhibitor of Metalloproteinases
D006023:Glycoproteins; D011506:Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D019716:Tissue Inhibitor of Metalloproteinase-2; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9