Date of Award
1-1-1986
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biochemistry and Molecular Biology
Abstract
The cytoplasmic and mitochondrial isozymes of phosphoenolpyruvate carboxykinase (PEPCK) catalyze the reversible decarboxylation and phosphorylation of OAA to PEP with GTP or ITP serving as the phosphoryl donor. The forward reaction is an essential component of the gluconeogenic pathway. The purpose of this research was to compare the effects of metal activation on the forward (decarboxylating) and reverse (carboxylating) reactions of the PEPCK isozmes purified from rabbit liver.A purification procedure for rabbit cytoplasmic PEPCK was developed. The final enzyme preparation was homogeneous as established by SDS-PAGE. Pure cytoplasmic PEPCK and pure mitochondrial PEPCK from rabbit liver exhibit identical electrophoretic behavior on SDS-PAGE. The molecular weight of both isozymes is approximately 64,500. Results with anion-exchange chromatography on QAE-Sephadex and SynChropak AX-300 showed that cytoplasmic PEPCK is more negatively charged than mitochondrial PEPCK. The isoelectric point, as established by chromatofocusing, is 5.0 for cytoplasmic PEPCK and 5.8 for mitochondrial PEPCK.A new method of assaying PEPCK which relies on the separation of IDP and ITP by reverse phase HPLC was developed. This method is compatible with the inclusion of iron in the assay. The assays were linear up to a 15% conversion of IDP to ITP and a 30% conversion of ITP to IDP.In the forward direction both PEPCK isozmes exhibited substantial activity with Mg('2+) as the sole cation. The addition of micromolar concentrations of Mn('2+) to cytoplasmic PEPCK activated the enzyme up to 5-fold. Activation with Fe('2+) was somewhat lower than with Mn('2+). Mitochondrial PEPCK was not activated by micromolar concentrations of Mn('2+) or Fe('2+) in the forward direction.In the reverse direction both isozymes exhibited very low activity in the presence of Mg('2+) alone. Micromolar concentrations of Fe('2+) or Mn('2+) added to the assay resulted in maximal 70 to 80-fold activation. Plots of activity versus metal concentration yielded sigmoidal curves with a lag in activation at the lower metal concentrations for cytoplasmic PEPCK and hyperbolic curves with no lag for mitochondrial PEPCK. The results indicate that Fe('2+) or Mn('2+) could play a role in the regulation of either isozyme.
Recommended Citation
Jacoby, Gertrude Hanke, "Comparative Studies Of The Metal Activation Of The Cytoplasmic And Mitochondrial Forms Of Phosphoenolpyruvate Carboxykinase In Rabbit Liver (Gluconeogenesis)." (1986). Theses and Dissertations. 8636.
https://commons.und.edu/theses/8636