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 KM, Vmax 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.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-6707 |
| Date | 01 January 1999 |
| Creators | Wallace, Timothy J. |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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