Characterization of rat pulmonary carboxylesterase

Author

Timothy J. WallaceFollow

DOI

https://doi.org/10.25772/9NPQ-ET82

Defense Date

1999

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biochemistry and Molecular Biology

First Advisor

William M. Grogan

Abstract

The 1839 bp cDNA for rat pulmonary carboxylesterase was cloned by reverse transcription polymerase chain reaction (RT PCR) from total rat lung RNA using specific primers derived from the 5' and 3' untranslated regions of rat hepatic cholesteryl ester hydrolase (CEH). The unique cDNA was sequenced and found to have 99% homology with hepatic CEH. This homology extends to the predicted amino acid sequences which show only six amino acid residue differences in the coding region: three conserved and three nonconserved changes. However, the catalytic activitites of the two proteins are dramatically different. While CEH hydrolyzes cholesterol oleate, the pulmonary carboxylesterase has no activity towards this substrate. The active recombinant lung carboxylesterase was purified using a baculovirus expression system. The substrate specifities were determined using p-nitrophenyl acetate, p-nitrophenyl caprylate and cholesterol oleate. Also, the K_M, V_max and pH optima were determined for each substrate. Comparison of the substrate specificities of the recombinant pulmonary carboxylesterase with the recombinant CEH further establish the critical role of the six amino acid residues in determining the differences in the catalytic activities of these two proteins. Cumulative mutations were made in the lung carboxylesterase sequence to those of the hepatic CEH sequence, in order to determine the role(s) of these six amino acid residues in conferring cholesterol oleate hydrolytic activity. These studies showed that GIn₁₈₆ is vital for activity towards hydrophilic substrates, while the region around amino acid residue 500 consisting of Ser₄₉₁, Lys₄₉₂, Asn₅₀₆ and Asn₅₀₄ may be responsible for the absence of catalytic activity towards hydrophobic substrates.

Comments

Scanned, with permission from the author, from the original print version, which resides in University Archives.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

10-17-2018