Quantitative Reverse Zymography: Analysis of Picogram Amounts of Metalloproteinase Inhibitors Using Gelatinase A and B Reverse Zymograms

Oliver, Gary W.; Leferson, Jill D.; Stetler-Stevenson, William G.; Kleiner, David E.

doi:10.1006/abio.1996.9895

Cited by 153 publications

(103 citation statements)

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“…4. The aliquots were diluted and analyzed for MMP inhibitor activity using the thiopeptolide assay (1:5 (w/w) enzyme:substrate) and reverse zymography (1:100 (w/w) enzyme:substrate), as described previously (16,17).…”

Section: Methodsmentioning

confidence: 99%

“…Reverse zymograms were quantitated by scanning the gels and optical integration of the band densities using the NIH Image program as described (17). Aliquots (40 l) of the aminopeptidase digest samples containing 12 g of digested AlaϩTIMP-2 were also electrophoresed on a 4 -20% acrylamide gradient gel following reduction with 2-mercaptoethanol and heating at 95°C for 2 min.…”

Section: Methodsmentioning

confidence: 99%

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Biophysical and Functional Characterization of Full-length, Recombinant Human Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Produced in Escherichia coli

Wingfield¹,

Sax²,

Stahl³

et al. 1999

Journal of Biological Chemistry

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101

106

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Matrix metalloproteinases (MMPs) function in the remodeling of the extracellular matrix that is integral for many normal and pathological processes. The tissue inhibitor of metalloproteinases family, including tissue inhibitor of metalloproteinases-2 (TIMP-2), regulates the activity of these multifunctional metalloproteinases. TIMP family members are proteinase inhibitors that contain six conserved disulfide bonds, one involving an amino-terminal cysteine residue that is critical for MMP inhibitor activity. TIMP-2 has been expressed in Escherichia coli, folded from insoluble protein, and functionally characterized. The wild type protein inhibited gelatinase A (MMP-2), whereas a variant with an alanine appended to the amino terminus (Ala؉TIMP-2) was inactive. Removal of amino-terminal alanine by exopeptidase digestion restored protease inhibitor activity. This confirms the mechanistic importance of the amino-terminal amino group in the metalloproteinase inhibitory activity, as originally suggested from the x-ray structure of a complex of MMP-3 with TIMP-1 and a complex of TIMP-2 with MT-1-MMP. The Ala؉TIMP-2 variant exhibited conformational, pro-MMP-2 complex formation and fibroblast growth modulating properties of the wild type protein. These findings demonstrate that Ala؉TIMP-2 is an excellent biochemical tool for examining the specific role of MMP inhibition in the multiple functions ascribed to TIMPs.Studies of disease states, such as arthritis and tumor progression, have demonstrated that the matrix metalloproteinases (MMPs) 1 and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), are critical effectors of extracellular matrix turnover. In recent years, the role of MMPs and TIMPs in a variety of pathologic entities has greatly expanded. This suggests that therapeutic targeting of these proteases may be beneficial not only in cancer and arthritis but also in a wide spectrum of pathologic conditions, ranging from rather common pathologic entities, such as nonhealing wounds (e.g. decubitus ulcers), to rare conditions such as pulmonary lymphangiomyomatosis (1, 2). Although current attempts to modulate MMP activities are based principally on substrate analog approaches, an alternative strategy is to utilize analogs of the endogenous inhibitors, the TIMPs.The TIMPs form a family of at least four members, the best characterized of which are TIMP-1 and -2 (2, 3). TIMP-2 (21 kDa) is a nonglycosylated protein that shares a 66% sequence identity with the glycosylated TIMP-1 (28.5 kDa); each protein contains 12 conserved cysteine residues that form six disulfide bonds essential for maintaining functional and structural integrity. The initial stage of TIMP inhibition of MMPs involves the formation of binary complexes. The amino-terminal cysteine appears to play a key role in blocking catalytic activity. The crystal structure of an MMP-TIMP-1 complex between the catalytic domain of stromelysin-1 (MMP-3) and human TIMP-1 showed that Cys-1 bidentally coordinates the active site zinc atom, and the Thr-2 s...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Biophysical and Functional Characterization of Full-length, Recombinant Human Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Produced in Escherichia coli

Wingfield¹,

Sax²,

Stahl³

et al. 1999

Journal of Biological Chemistry

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101

106

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“…The substrate degrading enzymes MMP-9 and MMP-2 were identified as clear bands in the blue background. For reverse zymography, concentrated media was electrophoresed in 15% polyacrylamide reverse zymograms containing 2.5 mg/mL gelatin and 0.15 μg/mL MMP-2 (Oliver et al, 1997). TIMP activity was visualized as dark zones of gelatinase inhibition against a pale, partially digested background.…”

Section: Zymographymentioning

confidence: 99%

Epigenetic inactivation of the tissue inhibitor of metalloproteinase-2 (TIMP-2) gene in human prostate tumors

et al. 2007

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Gene silencing via CpG island methylation in the promoter region is one of the mechanisms by which tumor suppressor genes are inactivated in human cancers. Previous studies have shown that the TIMP-2 gene, which is an endogenous inhibitor of matrix metalloproteinases involved in cell invasion and tumorigenesis, is downregulated or silenced in a variety of human cancer cell lines. Here, we investigated the mechanism underlying TIMP-2 expression in prostate cancer cell lines and primary prostate tumor samples. We observed a strong correlation between promoter hypermethylation and lost expression of TIMP-2 gene, which was supported by other results demonstrating that promoter demethylation by 5-aza-2′-deoxycytidine and trichostatin A reactivated TIMP-2 and restored its expression in TIMP-2-silenced metastatic prostate cell lines. These result were further substantiated by a chromatin immunoprecipitation assay, showing the preferential binding of MeCP2 to methylated CpG island in TIMP-2-silenced metastatic prostate cell lines. In vitro Matrigel invasion assays showed that reexpression of TIMP-2 after a combined treatment with 5-aza and TSA in metastatic prostate cells resulted in a significant reduction of tumor cell invasion. Furthermore, CpG methylation of TIMP-2 promoter was also shown in primary prostate tumors that expressed decreased TIMP-2 protein levels. These results suggest that the downregulation of the TIMP-2 gene is associated with promoter methylation and that this may play an important role in prostate cancer progression during the invasive and metastatic stages of the disease.

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“…Reverse zymographies were performed using a published method (Oliver et a!., 1997) with a few modifications. Ten microliters of nonreducing sample buffer (0.4 mollL Tris pH 6.8, 5% SDS, 20% glycerol, 0.05% bromophenol blue) were added to the samples.…”

Section: Reverse Zymographymentioning

confidence: 99%

Early Appearance of Activated Matrix Metalloproteinase-9 after Focal Cerebral Ischemia in Mice: A Possible Role in Blood—Brain Barrier Dysfunction

Gasche

Fujimura

Morita-Fujimura

et al. 1999

J Cereb Blood Flow Metab

371

226

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Summary: During cerebral ischemia blood-brain barrier (BBB) disruption is a critical event leading to vasogenic edema and secondary brain injury. Gelatinases A and B are matrix metalloproteinases (MMP) able to open the BBB, The current study analyzes by zymography the early gelatinases expression and activation during permanent ischemia in mice (n = 15).ProMMP-9 expression was significantly (P < 0.001) increased in ischemic regions compared with corresponding contralateral regions after '1 hours of ischemia (mean 694.7 arbitrary units[AU], SD ± 238.4 versus mean 107.6 AU, SD ± 15.6) and remained elevated until 24 hours (mean 745,7 AU, SD ± 157.4). Moreover, activated MMP-9 was observed 4 hours after the initiation of ischemia. At the same time as the appearance Blood-brain barrier (BBB) integrity protects the neu ronal microenvironment (Cserr and Bundgaard, 1986). When this integrity is lost, inflammatory cells and fluid penetrate the brain, causing edema and cell death (Fish man, 1975), Impermeability of the BBB is maintained by microvascular endothelial cells through their tight junc tions (Siflinger-Birnboim et aI., 1987) and basal lamina. The latter is composed of type IV collagen, fibronectin, Received October 20, 1998; final revision received January 19, 1999; accepted January 20, 1999. Supported by National Institutes of Health grants NS 25372, NS 14543, NS 36147 and NOI NS 82386. Y. Gasche is a research fellow supported by the Geneva University Hospital Fund. P.H. Chan is a recipient of the Jacob Javits Neuroscience Investigator Award.Address correspondence and reprint requests to Dr. Pak H. Chan, Neurosurgical Laboratories, Stanford University, 701B Welch Road, Room 148, Palo Alto, CA 94304, U.S.A.Abbreviations used: BBB, blood-brain barrier; EDTA, ethylenedi amine tetraacetic acid; MMP, metalloproteinase; MMP-2, gelatinase A; MMP-9, gelatinase B; PBS, phosphate-buffered saline; pMCAO, per manent middle cerebral artery occlusion; ROS, reactive oxygen spe cies; SDS, sodium dodecyl sulfate; TIMP, tissue inhibitor of metallo proteinase; tMCAO, transient middle cerebral artery occlusion; TNCA, Tris HCI-NaCI-CaCI2• 1020 of activated MMP-9, we detected by the Evan's blue extrava sation method a clear increase of BBB permeability, Tissue inhibitor of metalloproteinase-1 was not modified during per manent ischemia at any time. The ProMMP-2 was significantly (P < 0.05) increased only after 24 hours of permanent ischemia (mean 213.2 AU, SD ± 60.6 versus mean 94.6 AU, SD ± 13.3), and no activated form was observed. The appearance of acti vated MMP-9 after 4 hours of ischemia in correlation with BBB permeability alterations suggests that MMP-9 may play an active role in early vasogenic edema development after stroke.

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Quantitative Reverse Zymography: Analysis of Picogram Amounts of Metalloproteinase Inhibitors Using Gelatinase A and B Reverse Zymograms

Cited by 153 publications

References 10 publications

Biophysical and Functional Characterization of Full-length, Recombinant Human Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Produced in Escherichia coli

Biophysical and Functional Characterization of Full-length, Recombinant Human Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Produced in Escherichia coli

Epigenetic inactivation of the tissue inhibitor of metalloproteinase-2 (TIMP-2) gene in human prostate tumors

Early Appearance of Activated Matrix Metalloproteinase-9 after Focal Cerebral Ischemia in Mice: A Possible Role in Blood—Brain Barrier Dysfunction

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