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Characterization of Rat Pulmonary Carboxylesterase

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Characterization of Rat Pulmonary Carboxylesterase Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 1999 Characterization of rat pulmonary carboxylesterase Timothy J. Wallace Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Biochemistry, Biophysics, and Structural Biology Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/5616 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. Virginia Commonwealth University School of Medicine This is to certify that the dissertation prepared by Timothy J Wallace entitled "Characterization of Rat Pulmonary Carboxylesterase" has been approved by his committee as satisfactory completion of the dissertation requirement fo r· the degree of Doctor of Philosophy. Jack L. Haar, Ph.D., Dean, School of Graduate Studies cZS: L'79:2 Date ,�( © Timothy 1. Wallace 1999 All Rights Reserved Characterization of Rat Pulmonary Carboxylesterase A dissertation submitted in partial fulfillment of the requirements fo r the degree of Doctor of Philosophy at Virginia Commonwealth University By Timothy 1. Wallace BA University of Virginia, May 1991 Director: W. McLean Grogan Professor Department of Biochemistry and Molecular Biophysics Virginia Commonwealth University Richmond, Virginia May, 1999 11 Acknowledgement In loving memory of Margaret O'Neill Wallace. 1II Table of Contents Page List of Tables .......................... ..................................................................................... ..v VI List of Figures ..... .......... ......................... ... .............................. ........ ........................... .. List of Abbreviations .................................. ................ IX Abstract. ..................................................................................................................... ....x Introduction ............................. ............ .... ........... .... ..... .......... ....... ... .......... ..... ............. ... I Physiological function. ................. ................ ... ........ ........ ........ ....... ........ .... .... 3 Xenobiotic metabolism .............. ......... .................. ............ .... .............. .............. .4 Tissue distribution......................................................... ......... ............. ..5 Hormonal regulation ..................... ..... ..... .... .................................................. .. .....6 Biochemical characterization ......... .......................... ....... .... ............... ............ ..-.. ... 8 Biochemical properties ................ ...... .............. ......... ...................... ... ................10 Molecular characterization .................. ........ ......... ............ .............................: ..... 15 Pulmonary carboxyl esterase .......................... ......... .... .........................................22 Rationale fo r study ...................... ........ .... ............... ................................... ... ... 25 Materials and Methods ................ .............. ... ...... .... .... ...... ........ .................. ............ ... 27 Preparation of total RNA from lung tissue..... ....................... ... 27 Reverse transcriptase polymerase chain reaction............................. ....... 27 Cloning and sequencing oflung carboxylesterase ........ ............. ..................... .....28 Confirmation of cDNA by alternative cloning... ............... .... 29 Eukaryoticexpression system.. ....... .... ................. ....30 In situ hybridization...... .............. ........................................................ .31 Northern blot analysis.. .............. ........................... .... 32 Inhibition oflung carboxylesterase by organophosphorus compounds... .... 33 Homologous recombination.................. .............. ............... ............ ...33 Purification of recombinant products.............................................. ...34 PCR analysis of recombinant virus....................................................... .. 34 IV Preparation of high-titer viral stocks .............................. ... ............................ ....35 Time course expression of recombinant protein ..................... ............... .. ..36 Processing time point samples ................... ...................................... .. .... 36 Large-scale expression of recombinant protein . ..... ................ .....37 Column purificationof recombinant protein ................................................... ....37 Site-directed mutagenesis ........................... ....... .................... .... .... ................. ..39 Confirmationof the mutated lung carboxyl esterase sequence .......................... ...41 Transfection of the mutated lung carboxylesterase cDNAs ................................ 43 General Methods..... ....................... ...................... ................................ .44 Results . ........................................................................................ .......... ................. ... .47 Cloning the rat lung carboxylesterase cDNA. ................................................. ....47 Verification of the lung carboxyl esterase .......................................................... .47 Cellular localization of the lung carboxyl esterase .................................................52 Organophosphorus inhibition of the lung carboxylesterase ..................................59 Homologous recombination and purification of recombinant products .................59 Preparation of high titer stocks ...........................................................................62 Time course expression of recombinant protein ....................................... ............69 Large-scale expression and purification of recombinant protein ... .................. ....70 Determination of specific activity of the purified protein ......... ....................... ....77 Determination of enzymatic kinetic parameters of the purified protein ..... ............77 Determination of the pH optima fo r the purified protein .................................� .... 77 Site-directed mutagenesis of the lung carboxylesterase cDNA. ....................... .... 87 Discussion .................................................................................................................. ... 99 Literature Cited ............................................................................................................ 121 Vita...... ........................................................................................................................ 131 v List of Tables Table Page Specific activities of various carboxylesterases................................... ... 11 2 Kinetics of hydrolysis of p-nitrophenyl acetate................................................ 13 3 Comparison of conserved motifs in carboxylesterases ............................... ..... ....17 4 Kinetic determinations fo r lung carboxylesterase ..... ....... ............................ ... ...82 5 Sequence differences between hepatic CEH and lung carboxylesterase ......... .. ..88 VI List of Figures Figure Page A proposed mechanism fo r the action of carboxylesterases....... .......................... 19 2 The eDNA nucleotide sequence and the deduced amino acid sequence of the rat lung carboxylesterase ........................................ ............... .. .49 3 The cloning strategy used to determine the eDNA sequence of the rat lung carboxylesterase .............................................. ......................... ..51 4 Lung carboxyl esterase activity in transfected CoS-7 cells........ ..................... ...54 5 Northernblot analysis of rat lung RNA probed with lung carboxylesterase cDNA. .......................... ...................... .................... ...56 6 In Situ hybridization in rat lung tissue sections ..................................... ....58 7 Paraoxon inhibition of rat lung carboxyl esterase. ....................................... .61 8 Confirmation of cloning lung carboxyl esterase insert into pBluBacHis2B ....64 9 PCR analysis of recombinant virus................................................................. .66 10 The time-dependent infection ofthe SF9 cells by the viral stock ..... ....... ... 68 II Measurement of the 00280 of the nickel column elution of the recombinant lung carboxyl esterase ....................................................... ....72 VII 12 SDS-PAGE electrophoresis of recombinant lung carboxylesterase stained with Coomasie R-250 .............................. ............. ......74 13 SDS- PAGE electrophoresis of recombinant lung carboxylesterase stained with silver stain ........................................................ ....76 14 Lineweaver-Burk double reciprocal plot, IIv vs. I/[S] ................. ................. ....79 15 Lineweaver-Burk double reciprocal plot, IIv vs. II[S] .................. ............... .....81 16 pH Optima curve of the hydrolysis of p-nitrophenyl acetate by the purifiedrecombinant lung carboxylesterase. .................. ...84 17 pH Optima curve of the hydrolysis of p-nitrophenyl caprylate by the purifiedrecombinant lung carboxylesterase ............ .. ...... 86 18 Specificacitvities
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