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The operation of GAPDH as a metabolic enzyme differs between the cytoplasm and nucleus
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- Author / Creator
- Tang, Helen SH
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Metabolic enzymes are active in both the nucleus and cytoplasm. While metabolism in cytoplasm is widely acknowledged for energy production, nuclear-localized metabolic enzymes are thought to modulate availability of metabolites used in epigenetics. A thorough investigation of this regulatory axis requires a deeper understanding of the native environment and enzymology of these nuclear-localized proteins. Xenopus laevis oocytes provide several unique advantages with allowed us to develop a top-down method for biochemical analysis of near-native enzyme activity.
The cytoplasm and nucleus of X. laevis oocytes can be separated while maintaining compartmental integrity in mineral oil. By homogenizing the harvested nuclei and cytoplasms in a near-native buffer, we were able to compare the native enzyme kinetics between the cytoplasmic and nuclear-localized enzymes. We focused on enzymes that produce or consume NAD(P)H which can be readily detected by non-invasive spectrophotometry, and our chosen enzyme is glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
GAPDH is a classic glycolytic enzyme that is highly abundant in both the cytoplasm and nucleus. Using native-western blotting, we identified that GAPDH is present in the catalytically active homotetramers in both the cytoplasm and nucleus. Robust GAPDH activity can also be detected in whole cytoplasmic and nuclear homogenates of X. laevis oocytes. Taking advantage of published quantitative analysis of X. laevis oocytes, we were able to compare the kinetic behaviour of GAPDH by analyzing the same amounts of nuclear and cytoplasmic enzyme in the near-native reaction buffer. Our results demonstrate that the operation of GAPDH as a metabolic enzyme differs between the cytoplasm and nucleus, providing the first definitive evidence of catalytic regulation of a metabolic enzyme by nuclear as compared to cytoplasmic localization. -
- Graduation date
- Fall 2022
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- Type of Item
- Thesis
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- Degree
- Master of Science
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- License
- This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.