Computational and experimental studies of interactions between general anesthetics and proteins.

Persistent Link:
http://hdl.handle.net/10150/187205
Title:
Computational and experimental studies of interactions between general anesthetics and proteins.
Author:
Louria, Dyan Elis.
Issue Date:
1995
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
The molecular mechanism of general anesthesia remains unknown. Potential anesthetic target sites include membrane lipid, protein or possibly both. Computational and experimental studies were done to determine if the functions of isolated protein systems can be effected by anesthetic. A total of six enzymes: bacterial luciferase, acetylcholinesterase, papain, chymotrypsin, hexokinase and glyceraldehyde-3-phosphate dehydrogenase were evaluated for potential anesthetic sensitivity. For the computational studies on bacterial luciferase (V. Harveyi), an optimized structure of the α subunit provided a basis for performing Hyperchem electrostatic potential calculations. A total of 11 anesthetic and 1 convulsant model were used in constructing electrostatic contour plots of the active site. All anesthetics substantially decreased the field gradient at the active site, while the convulsant increased the gradient. For the computational studies on the remaining five enzymes, protein optimizations and docking analyses were performed with the INSIGHTII program. Models for halothane, methoxyflurane and nitrous oxide were built and docked into enzyme active sites. Intermolecular energy data revealed two possible anesthetic sensitive proteins: papain and, to a lesser degree, glyceraldehyde-3-phosphate dehydrogenase. Experimental enzyme assays for the five enzymes were undertaken to determine activity in the presence and absence of halothane. Activities were measured spectrophotometrically over a wide range of substrate concentrations. Substrate concentrations both spanned and exceeded the Km value of the enzyme under analysis. Substrate-activity curves were constructed to evaluate competitive effects on enzyme kinetics (increased Km). Competitive effects on enzyme kinetics were not found for acetylcholinesterase, α-chymotrypsin and hexokinase. A slight but probably not significant effect of halothane on glyceraldehyde-3-phosphate dehydrogenase activity was observed, as shown by the small increase in Km and the tabulated %control values. Papain was found to be inhibited by nearly 50% at saturating substrate concentrations and by approximately one-third at lower substrate concentrations. From these theoretical and experimental studies on anesthetic-protein interactions, two isolated protein systems (bacterial luciferase and papain) were observed to display an anesthetic sensitivity. These studies have served to support the theory that proteins are plausible sites of anesthetic action and thus should be studied as potential anesthetic target sites.
Type:
text; Dissertation-Reproduction (electronic)
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Physiological Sciences; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Hameroff, Stuart R.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleComputational and experimental studies of interactions between general anesthetics and proteins.en_US
dc.creatorLouria, Dyan Elis.en_US
dc.contributor.authorLouria, Dyan Elis.en_US
dc.date.issued1995en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThe molecular mechanism of general anesthesia remains unknown. Potential anesthetic target sites include membrane lipid, protein or possibly both. Computational and experimental studies were done to determine if the functions of isolated protein systems can be effected by anesthetic. A total of six enzymes: bacterial luciferase, acetylcholinesterase, papain, chymotrypsin, hexokinase and glyceraldehyde-3-phosphate dehydrogenase were evaluated for potential anesthetic sensitivity. For the computational studies on bacterial luciferase (V. Harveyi), an optimized structure of the α subunit provided a basis for performing Hyperchem electrostatic potential calculations. A total of 11 anesthetic and 1 convulsant model were used in constructing electrostatic contour plots of the active site. All anesthetics substantially decreased the field gradient at the active site, while the convulsant increased the gradient. For the computational studies on the remaining five enzymes, protein optimizations and docking analyses were performed with the INSIGHTII program. Models for halothane, methoxyflurane and nitrous oxide were built and docked into enzyme active sites. Intermolecular energy data revealed two possible anesthetic sensitive proteins: papain and, to a lesser degree, glyceraldehyde-3-phosphate dehydrogenase. Experimental enzyme assays for the five enzymes were undertaken to determine activity in the presence and absence of halothane. Activities were measured spectrophotometrically over a wide range of substrate concentrations. Substrate concentrations both spanned and exceeded the Km value of the enzyme under analysis. Substrate-activity curves were constructed to evaluate competitive effects on enzyme kinetics (increased Km). Competitive effects on enzyme kinetics were not found for acetylcholinesterase, α-chymotrypsin and hexokinase. A slight but probably not significant effect of halothane on glyceraldehyde-3-phosphate dehydrogenase activity was observed, as shown by the small increase in Km and the tabulated %control values. Papain was found to be inhibited by nearly 50% at saturating substrate concentrations and by approximately one-third at lower substrate concentrations. From these theoretical and experimental studies on anesthetic-protein interactions, two isolated protein systems (bacterial luciferase and papain) were observed to display an anesthetic sensitivity. These studies have served to support the theory that proteins are plausible sites of anesthetic action and thus should be studied as potential anesthetic target sites.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePhysiological Sciencesen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairHameroff, Stuart R.en_US
dc.contributor.committeememberGillies, Robert J.en_US
dc.contributor.committeememberLynch, Ronald M.en_US
dc.contributor.committeememberSecomb, Timothy W.en_US
dc.identifier.proquest9603352en_US
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