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|Title:||Hydroperoxide stimulation of prostaglandin endoperoxide synthase|
|Author(s):||Pendleton, Robert Brian|
|Doctoral Committee Chair(s):||Lands, William E.M.|
|Department / Program:||Physics|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
Health Sciences, Radiology
|Abstract:||Hydroperoxide is required to activate prostaglandin endoperoxide synthase (PES) cyclooxygenase by formation of an enzyme-radical species (derived from a PES peroxidase intermediate), and is generated by the cyclooxygenase reaction, thereby causing a positive feedback in the rate of oxygenation (hydroperoxide amplification). A kinetic model that integrates the cyclooxygenase and peroxidase activities was proposed. Numerical integration of the kinetic rate equations was found to accurately predict both the time-course of oxygen consumption, and the levels of prostaglandin G$\sb2$ (PGG$\sb2$) hydroperoxide, during oxygenation of arachidonic acid (20:4n-6).
15-Hydroperoxyeicosatetraenoic acid (15-HPETE), was found to be a highly effective activator of the cyclooxygenase activity using a kinetic assay based upon the ability of hydroperoxide to overcome the antagonistic effects of sodium cyanide. Phospholipid triglyceride, and cholesterol ester hydroperoxides were ineffective, but became highly effective after enzymatic hydrolysis, and therefore, PES requires an non-esterified hydroperoxide for activation. The mono-hydroperoxy positional isomers of oxidized arachidonate had decreasing effectiveness as the hydroperoxide group was located nearer to the acid terminus, and the cyclic endoperoxy-hydroperoxides PGG$\sb2$ and PGG$\sb3$ had only one sixth the activity of 15-HPETE. Unexpectedly, the oxygenation of 20:5n-3 was found to be more responsive to 15-HPETE activator than the oxygenation of 20:4n-6.
Using sodium cyanide or glutathione peroxidase as hydroperoxide antagonists the hydroperoxide amplification process was found to be impaired in the reaction with eicosapentaenoate (20:5n-3). The rate constant for the initial reaction between enzyme and hydroperoxide was found to be equal for both PGG$\sb2$ and PGG$\sb3$ and therefore the impaired reactivity of 20:5n-3 probably did not involve the steps leading to the radical-enzyme intermediate. Using OXYSIM, a computer program for numerically integrating the proposed kinetic equations and a systematic process for adjusting the kinetic constants, it was found that the impaired hydroperoxide amplification of the 20:5n-3 reaction could be accounted for by a 4.6-fold decreased rate of oxygen insertion, and a 2.2-fold increased rate of cyclooxygenase inactivation.
These data provide a greater understanding of hydroperoxide and prostaglandin metabolism, and a mechanistic rational for the therapeutic effects of dietary 20:5n-3 in certain inflammatory and thromboembolic diseases where prostaglandin overproduction is believed to be pathologic.
|Rights Information:||Copyright 1990 Pendleton, Robert Brian|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9026293|
This item appears in the following Collection(s)
Dissertations and Theses - Biophysics and Computational Biology
Dissertations and Theses - Physics
Dissertations in Physics
Graduate Dissertations and Theses at Illinois
Graduate Theses and Dissertations at Illinois