Characterization of Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Human Plasma

Demetrius Moutsiakis Departments of Research and Medicine, State University of New York, School of Medicine, Stony Brook, New York, USA; Department of Veterans Affairs Medical Center, State University of New York, School of Medicine, Stony Brook, New York, USA; Department of Medicine, State University of New York, School of Medicine, Stony Brook, New York, USA

Paul Mancuso Departments of Research and Medicine, State University of New York, School of Medicine, Stony Brook, New York, USA

Henry Krutzsch Departments of Research and Medicine, State University of New York, School of Medicine, Stony Brook, New York, USA; Department of Veterans Affairs Medical Center, State University of New York, School of Medicine, Stony Brook, New York, USA; Department of Medicine, State University of New York, School of Medicine, Stony Brook, New York, USA; Department of Pathology, National Cancer Institute, Bethesda, Maryland, USA

William Stetler-Stevenson Departments of Research and Medicine, State University of New York, School of Medicine, Stony Brook, New York, USA; Department of Veterans Affairs Medical Center, State University of New York, School of Medicine, Stony Brook, New York, USA; Department of Medicine, State University of New York, School of Medicine, Stony Brook, New York, USA; Department of Pathology, National Cancer Institute, Bethesda, Maryland, USA

Stanley Zucker Departments of Research and Medicine, State University of New York, School of Medicine, Stony Brook, New York, USA; Department of Veterans Affairs Medical Center, State University of New York, School of Medicine, Stony Brook, New York, USA; Department of Medicine, State University of New York, School of Medicine, Stony Brook, New York, USA

Abstract

In this study, we have identified and characterized metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) in human plasma. Treatment of plasma with trypsin or aminophenylmercuric acetate resulted in activation of latent gelatinolytic activity. Fractionation of plasma by gelatin Sepharose chromatography resulted in the isolation of 72 kDa and 92 kDa gelatinases/type IV collagenases. The 72 kDa gelatinase was purified by gel filtration chromatography. Stromelysin-1 was isolated from plasma by Matrex green A affinity chromatography. Immunoblotting of plasma fractions with antibodies to unique peptide regions of human gelatinases differentiated the 72 kDa gelatinase from the 92 kDa gelatinase. Antibodies to the amino terminal peptides of each enzyme were used to determine that plasma gelatinases circulate as latent proenzymes. Immunoblotting with antibodies directed against human stromelysin identified a 57 kDa stromelysin. T1MP-1 (28 kDa) and TIMP-2 (21 kDa) were also identified by immunoblotting of gelatin Sepharose bound plasma proteins using non-crossreacting antibodies to each protein.

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Characterization of Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Human Plasma

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Characterization of Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Human Plasma

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