The Purification and Charachterization of C. elegans Cytoplasmic Malate Dehydrogerase

Wei Gu

Date of Award

2011

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Keywords

C. elegans, Malate Dehydrogenase, Enzymes, Kinetics, Impact

Area of Concentration

Chemistry

Abstract

Malate dehydrogenases (MDH) catalyze the reversible oxidation of malate to oxaloacetate, with concurrent reduction of NAD+ to NADH. C. elegans is a nematode worm which has been widely studied as a model organism for over half a century, due to the ease of growing and manipulating them. Eukaryotic organisms such as C. elegans have mitochondrial and cytoplasmic isozymes of MDH, which are both important to cellular metabolism. C. elegans mitochondrial MDH was recently characterized, while the cytoplasmic MDH was still a hypothetical protein. Elucidating the kinetic profile of cytoplasmic MDH in C. elegans could reveal more about the development of the two isozymes, as well as the enzyme�s activity in more evolutionarily distant organisms, such as mammals and humans. The putative gene for C. elegans cytoplasmic malate dehydrogenase (MDH-1), F46E10.10, was ligated into the plasmid vector pMXB10 and transformed into E. coli Rosetta (DE3) cells. Under the Intein Mediated Purification with an Affinity Chitin-binding Tag system, the MDH-1-intein fusion protein was over-expressed by IPTG induction. This fusion protein contains a self-cleaving peptide and a chitin-binding domain. By applying lysed cells to a chitin column, the fusion protein will remain bound to the chitin. Treating the column with DTT will cause the intein tags to cleave, allowing MDH-1 to be collected by elution. The MDH-1 samples were dialyzed repeatedly to remove residual DTT, which interferes with protein concentration determination. The samples then underwent enzyme assays which tracked the change in NADH associated with conversion of oxaloacetate to malate. When the concentration of NADH was held constant at 60 ?M and oxaloacetate concentrations were varied from 10 to 300 ?M, MDH-1 had a Vmax of 3.33 ?M NADH converted per minute, a Km of 37 ?M oxaloacetate, and a specific activity of 4.7 units/mg. When the concentration of oxaloacetate was held constant at 150 ?M and NADH concentrations were varied from 10 to 300 ?M, MDH-1 had a Vmax of 4.7 ?M NADH converted per minute, a Km of 80 ?M NADH, and a specific activity of 6.7 units/mg.

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