Characterization of Caenorhabditis elegans protein F36A2.3

Damien Clark

Date of Award

2015

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Keywords

Caenorhabditis elegans, Chemistry, Proteins, Biology, C. elegans

Area of Concentration

Chemistry

Abstract

Caenorhabditis elegans protein F36A2.3 was hypothesized to be a malate dehydrogenase (MDH), an enzyme involved in several metabolic pathways in nematodes. Using NCBI BLAST to analyze conserved domains in the F36A2.3 protein sequence, multiple conserved malate/L-lactate dehydrogenase domains were detected. An investigation using SDS-PAGE gel electrophoresis was done to determine protein solubility, and enzyme kinetic assays were performed to further investigate the function of protein F36A2.3. Kinetic assays were performed using 200 μM substrate and 100 μM coenzyme. Four substrates were tested: oxaloacetate, pyruvate, phenylpyruvate, and 2- oxobutyrate. NADH and NADPH were tested with these substrates to determine whether oxidation of these coenzymes occurred. Assays were performed with each of the four substrates with both coenzymes to determine if there was a marked difference in enzymatic activity for either coenzyme in a buffer containing 0.1 M Potassium Phosphate pH 7.5, and 0.1 M KCl. Assays were also performed both with and without 5 mM magnesium. When analyzing these assays, it was observed that there was notable activity with phenylpyruvate and NADPH both with and without magnesium as well as for pyruvate and NADPH both with and without magnesium. Activity was also observed with phenylpyruvate and NADH with magnesium. Activity for phenylpyruvate and pyruvate indicates an enzymatic preference for monocarboxylates, however no significant activity was noted for 2-oxobutyrate.

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