Design and Use of Fluorescent Assays to Study Plasminogen Activator and Other Serine Proteases Involved in Biological Control
Leytus, Steven Phillip
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Permalink
https://hdl.handle.net/2142/70507
Description
Title
Design and Use of Fluorescent Assays to Study Plasminogen Activator and Other Serine Proteases Involved in Biological Control
Author(s)
Leytus, Steven Phillip
Issue Date
1982
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
In order to study the role of serine proteases in the wide variety of physiological functions which they serve, sensitive and quantitative assays need to be devised. Because the xanthene dyes rhodamine and fluorescein are among the most fluorescent compounds known and, as such, can be detected at very low concentrations, derivatives of these fluorescent dyes were designed as fluorogenic substrates for the amidase and esterase activity of serine proteases. As a consequence of their unique spectral and biochemical properties, these derivatives represent the most sensitive synthetic substrates for serine proteases yet described.
Using these fluorogenic substrates, sensitive and quantitative assays were developed and used to study plasminogen activation by transformed cells. In addition, a fortunate observation led to the discovery of a specific and efficient plasminogen activator activity in E. coli.
Taking advantage of the excellent kinetic properties and sensitivity of these fluorogenic substrates, an experimentally simple and accurate method for obtaining the second-order rate constant for inactivation of serine proteases by fast-acting, irreversible inhibitors was also devised. The method was based on formal analysis of an irreversible active site-directed substrate competing with an irreversible active site-directed inhibitor for the active site of a serine protease.
As an example of the wide applicability of these fluorogenic substrates, rhodamine-based dipeptide substrates were adapted for use in a fluorescent single cell assay for transformed cells. Using fluorescence microscopy, it was shown that plasminogen activator-producing transformed cells could be identified at the single cell level. This technique has a number of important applications and should also prove useful in studying the process of cellular transformation.
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