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The Purification and Charachterization of C. elegans Cytoplasmic Malate Dehydrogerase

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004504/00001

Material Information

Title: The Purification and Charachterization of C. elegans Cytoplasmic Malate Dehydrogerase
Physical Description: Book
Language: English
Creator: Gu, Wei
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2011
Publication Date: 2011

Subjects

Subjects / Keywords: C. elegans
Malate Dehydrogenase
Enzyme
Kinetics
Impact
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

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.
Statement of Responsibility: by Wei Gu
Thesis: Thesis (B.A.) -- New College of Florida, 2011
General Note: Online version not currently available.
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, 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.
Local: Faculty Sponsor: Walstrom, Katherine

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2011 G89
System ID: NCFE004504:00001

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004504/00001

Material Information

Title: The Purification and Charachterization of C. elegans Cytoplasmic Malate Dehydrogerase
Physical Description: Book
Language: English
Creator: Gu, Wei
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2011
Publication Date: 2011

Subjects

Subjects / Keywords: C. elegans
Malate Dehydrogenase
Enzyme
Kinetics
Impact
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

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.
Statement of Responsibility: by Wei Gu
Thesis: Thesis (B.A.) -- New College of Florida, 2011
General Note: Online version not currently available.
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, 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.
Local: Faculty Sponsor: Walstrom, Katherine

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2011 G89
System ID: NCFE004504:00001

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