IDENTIFICATION AND CHARACTERIZATION OF A LEPIDOPTERAN AMINOACYLASE, A HYDROLASE OF FATTY ACID AMINO ACID CONJUGATES
Open Access
- Author:
- Kuhns, Emily Hohlfeld
- Graduate Program:
- Ecology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 08, 2010
- Committee Members:
- James Homer Tumlinson Iii, Dissertation Advisor/Co-Advisor
James Homer Tumlinson Iii, Committee Chair/Co-Chair
Andrew George Stephenson, Committee Member
Ming Tien, Committee Member
Consuelo M De Moraes, Committee Member
Gary Felton, Committee Member - Keywords:
- Heliothis virescens
Heliothine
Protein Purification
Lepidoptera
Aminoacylase
Liquid Chromatography - Abstract:
- Fatty-acid amino acid conjugates (FACs) in caterpillar oral secretions elicit plant defense responses during herbivory. The levels of these FAC elicitors within the caterpillar appear to be controlled by two enzymes, one in the gut tissue membranes that synthesizes FACs and the second in the gut lumen that hydrolyzes them. Since FACs are persistent despite obvious fitness costs, it is believed that they are important to the performance and/or survival of the caterpillar. The FAC hydrolase was purified by liquid chromatography, and was identified as an aminoacylase by Edman degradation and tandem mass spectroscopy. In mammals, orthologous enzymes are cytosolic and posses several strikingly different characteristics that suggest caterpillars may have evolved a slightly different use for this aminoacylase. Furthermore, closely related species differ in their activity levels of this enzyme, apparently due to different levels of transcription. Two or three isozymes appear to be present in species with high levels of aminoacylase activity, which suggests that the protein may possess post-translational-modifications. In order to further understand the role of caterpillar FAC hydrolase, feeding studies were used to examine the effect of increased hydrolase activity in vivo on the performance and nitrogen uptake of caterpillars reared on sub-optimal diets. Although caterpillar performance was greatly diminished on poor diets, transcript levels of aminoacylase did not appear to be affected. It is still unclear what the role of FACs and aminoacylase in caterpillars is, however the aforementioned experiments have paved the way for future examinations of this metabolic pathway. From this work, we know the nucleotide and amino acid sequences that code for aminoacylase from three species. We have developed primers for amplification of these genes and quantitative real time PCR. Lastly, we have determined that the enzyme abundance varies between closely related species that differ in their life strategies.