| Title: |
Thiol-Based Modulation and Partial Purification from Rabbit Brain, of Glyceraldehdye-3-Phosphate Dehydrogenase |
| Authors: |
Spak, Nathan J. |
| Contributors: |
University of Scranton. Department of Chemistry |
| Publisher Information: |
University of Scranton |
| Publication Year: |
2024 |
| Collection: |
The University of Scranton Digital Collections |
| Subject Terms: |
University of Scranton -- Dissertations; Academic theses; Thiols; Modulation spectroscopy; Purification; Rabbits; Brain |
| Time: |
2020-2029 |
| Description: |
Earlier work by the Foley research group, using redox phenylarsine oxide (PAO)-affinity chromatography, identified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as one of several metabolic enzymes containing closely-spaced pairs of thiols that can be oxidized to presumed intrachain disulfide bonds and located these thiols to the catalytic cysteine residue at position 150 and a nearby cysteine at 154. The impact of this reversible oxidation on the structure and activity of the enzyme, and the possibility that prior conformational changes may be necessary for intrachain disulfide bond formation to take place remain to be investigated. Using the PAO-affinity method, the present study confirmed that GAPDH from rabbit brain tissue contains closely-spaced thiols that can undergo a reversible oxidation to form a presumed intrachain disulfide bond involving the conserved C150 and C154. Enzyme assays revealed that the activity of purified GAPDH from rabbit muscle was slightly stimulated by the disulfide bond-reducing agent DTT and by low, but not high, concentrations of a second disulfide bond-reducing agent, tris(2-carboxyethyl) phosphine (TCEP). The increases in GAPDH activity by the disulfide bond-reducing agents were associated with the formation of apparent interchain disulfide bonds involving the monomers of GADPH. Free PAO, in the presence of TCEP to maintain thiols in the reduced state, produced mixed and unexpected results on GAPDH activity. Specifically, PAO slightly increased the activity of native GAPDH but, notably, this effect was shifted to a slight inhibition of activity when the enzyme was subject to a freeze-thaw cycle prior to the assay. Finally, experiments found that phosphocellulose cation exchange chromatography coupled to ammonium sulfate precipitation may be a useful initial step in purifying GAPDH from rabbit brain. These results suggest that the significance of thiol modifications for GAPDH structure and activity may extend beyond intrachain crosslinking of C150 and ... |
| Document Type: |
text |
| File Description: |
application/pdf |
| Language: |
English |
| Relation: |
Master of Science in Biochemistry; University of Scranton Archives; University of Scranton Masters and Honors Theses; University of Scranton Masters Theses; MT_Spak_N_2024; http://digitalservices.scranton.edu/u?/p15111coll1,1467 |
| Availability: |
http://digitalservices.scranton.edu/u?/p15111coll1,1467 |
| Rights: |
http://rightsstatements.org/vocab/InC/1.0/ ; The author of this work retains the copyright. The University of Scranton does not have permission from the author to provide access to this thesis in the Library's Digital Collections. The print thesis is available for review in the University Archives reading room. |
| Accession Number: |
edsbas.7D473D19 |
| Database: |
BASE |