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Kinetic Characterization of Embryonic Glyceraldehyde-3-Phosphate Dehydrogenase from C. elegans

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Material Information

Title:
Kinetic Characterization of Embryonic Glyceraldehyde-3-Phosphate Dehydrogenase from C. elegans
Physical Description:
Book
Language:
English
Creator:
Schwabe-Warf, Derek
Publisher:
New College of Florida
Place of Publication:
Sarasota, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Bachelor's ( B.A.)
Degree Grantor:
New College of Florida
Degree Divisions:
Natural Sciences
Area of Concentration:
Chemistry, Biology
Faculty Sponsor:
Walstrom, Katherine

Subjects

Genre:
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, territorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Glyceraldehyde-3-phosphate dehydrogenase (GPD) is a glycolytic enzyme that catalyzes the conversion of glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG). In this reaction, a phosphate is added onto G3P in order to create a high energy phosphoryl energy compound. Later in glycolysis, 1,3-BPG is converted to pyruvate, which can undergo fermentation to regenerate nicotinamide adenine dinucleotide (NAD+) or continue into the Krebs cycle when oxygen is present. NAD+ and phosphate are the two additional substrates used in this reaction. Caenorhabditis elegans (C. elegans) is a nematode that has served as a model organism in the elucidation of several fundamental facets of molecular biology, genetics, and biochemistry. C. elegans has four GPD isozymes denoted as GPD one through four. In this study, the isolation and kinetic characterization of one of the four isozymes of glyceraldehyde-3-phosphate dehydrogenase denoted as GPD-1 was conducted. GPD-1, along with GPD-4, are the primary GPD isozymes expressed during the embryonic stage of the nematode. The significance of having four GPD isozymes is poorly understood at the present. However, kinetics evaluation should provide an avenue for comparison with the previously characterized GPD-3 isozyme. Rosetta cells (a strain of competent E. coli) were used in conjunction with the pTXB3 plasmid to express the gpd-1 gene. Subsequently, the IMPACT system from New England Biolabs was utilized to purify GPD-1. Kinetics were evaluated at pH 8.5 using a Cary-14 spectrometer. The Km values were found to be 0.9 ± 0.1 mM, 0.022 ± 0.002 mM, and 1.9 ± 0.1 mM for NAD+, G3P, and phosphate, respectively. Specific activity values ranged between 24.7 ± 0.15 and 39.1 ± 0.15 μmol/min*mg. In comparison with the kinetics values reported for GPD-3, the KmNAD+ and KmPi were lower than GPD-1. The KmG3P value for GPD-3 was higher than GPD-1. This suggests differential regulation since GPD-1 is found in the cytoplasm while GPD-3 is predominantly bound to muscle sarcomeres. The differences in Km values are hypothesized to result from the cytotoxicity of G3P in the cytoplasm, which requires rapid removal by GPD-1. In the muscle, NAD+ and phosphate are tightly bound to GPD-3 to facilitate rapid muscle movement.
Statement of Responsibility:
by Derek Schwabe-Warf
Thesis:
Thesis (B.A.) -- New College of Florida, 2014
General Note:
RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
General Note:
Online version not currently available.
Bibliography:
Includes bibliographical references.
General Note:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
General Note:
Faculty Sponsor: Walstrom, Katherine

Record Information

Source Institution:
New College of Florida
Holding Location:
New College of Florida
Rights Management:
Applicable rights reserved.
Classification:
S.T. 2014 S39
System ID:
AA00024805:00001

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