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Use of a multiple-enzyme/multiple-reagent assay system to quantify activity levels in samples containing mixtures of matrix metalloproteinases.

Publication ,  Journal Article
Rasmussen, FH; Yeung, N; Kiefer, L; Murphy, G; Lopez-Otin, C; Vitek, MP; Moss, ML
Published in: Biochemistry
March 23, 2004

Matrix metalloproteinases (MMPs) are a family of enzymes that are up-regulated in many diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA). Here we report on a novel technique that can be used to simultaneously measure activity levels for a panel of enzymes, such as the MMPs. The technique, termed the multiple-enzyme/multiple-reagent assay system (MEMRAS), relies on the use of reagents such as substrates with varying selectivity profiles against a group of enzymes. When reaction rates are measured by following a change in fluorescence with time, for mixtures of enzymes, an equation with unknown concentrations for each activity is generated for each reagent used. Simultaneously solving the set of equations leads to a solution for the unknown concentrations. We have applied this mathematical technique to measure activity levels for mixtures of MMPs such as collagenase 3 and gelatinase A. In addition, because we were most interested in determining collagenase 3 levels as a potential biological marker for OA, we developed highly selective substrates for this enzyme by using results found in previous bacteriophage substrate-mapping experiments. Some of the best substrates tested have specific activities for collagenase 3 that are 37,000-, 17,000-, 90-, and 200-fold selective over stromelysin 1, collagenase 1, and gelatinases A and B, respectively.

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Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

March 23, 2004

Volume

43

Issue

11

Start / End Page

2987 / 2995

Location

United States

Related Subject Headings

  • p-Dimethylaminoazobenzene
  • Synovial Fluid
  • Substrate Specificity
  • Structure-Activity Relationship
  • Oligopeptides
  • Matrix Metalloproteinases
  • Matrix Metalloproteinase 9
  • Matrix Metalloproteinase 3
  • Matrix Metalloproteinase 2
  • Matrix Metalloproteinase 13
 

Citation

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Rasmussen, F. H., Yeung, N., Kiefer, L., Murphy, G., Lopez-Otin, C., Vitek, M. P., & Moss, M. L. (2004). Use of a multiple-enzyme/multiple-reagent assay system to quantify activity levels in samples containing mixtures of matrix metalloproteinases. Biochemistry, 43(11), 2987–2995. https://doi.org/10.1021/bi036063m
Rasmussen, Fred H., Nolan Yeung, Laura Kiefer, Gillian Murphy, Carlos Lopez-Otin, Michael P. Vitek, and Marcia L. Moss. “Use of a multiple-enzyme/multiple-reagent assay system to quantify activity levels in samples containing mixtures of matrix metalloproteinases.Biochemistry 43, no. 11 (March 23, 2004): 2987–95. https://doi.org/10.1021/bi036063m.
Rasmussen FH, Yeung N, Kiefer L, Murphy G, Lopez-Otin C, Vitek MP, et al. Use of a multiple-enzyme/multiple-reagent assay system to quantify activity levels in samples containing mixtures of matrix metalloproteinases. Biochemistry. 2004 Mar 23;43(11):2987–95.
Rasmussen, Fred H., et al. “Use of a multiple-enzyme/multiple-reagent assay system to quantify activity levels in samples containing mixtures of matrix metalloproteinases.Biochemistry, vol. 43, no. 11, Mar. 2004, pp. 2987–95. Pubmed, doi:10.1021/bi036063m.
Rasmussen FH, Yeung N, Kiefer L, Murphy G, Lopez-Otin C, Vitek MP, Moss ML. Use of a multiple-enzyme/multiple-reagent assay system to quantify activity levels in samples containing mixtures of matrix metalloproteinases. Biochemistry. 2004 Mar 23;43(11):2987–2995.
Journal cover image

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

March 23, 2004

Volume

43

Issue

11

Start / End Page

2987 / 2995

Location

United States

Related Subject Headings

  • p-Dimethylaminoazobenzene
  • Synovial Fluid
  • Substrate Specificity
  • Structure-Activity Relationship
  • Oligopeptides
  • Matrix Metalloproteinases
  • Matrix Metalloproteinase 9
  • Matrix Metalloproteinase 3
  • Matrix Metalloproteinase 2
  • Matrix Metalloproteinase 13