Abstract
Glycaemia in humans, rats and other mammals is regulated by a glucose sensor mechanism that uses the unique glucose kinetics of glucokinase (HK4) along with the glucokinase regulator protein (GKRP). This glucose sensor mechanism is absent in various species including bovine and other ruminants but does not appear to hinder glycaemic regulation, whereas in humans this absence can cause disease. Experiments were performed on the bovine liver to investigate how the HK4 sensor mechanism is bypassed in the liver and if there is a secondary hyperglycaemic regulator mechanism to control hyperglycaemia in animals that have lost the HK4-dependent glucose regulatory mechanism. The glucokinases/hexokinases catalytic mechanism and substrate specificity were investigated using recombinantly expressed proteins and assaying them.The results of spectrophotometric enzyme assays and novel NMR enzyme assays suggest that there is a glucose-dependent activity in the liver mimicking glucokinase kinetics in a coupled assay, but is ATP-independent and not the result of glucokinase (HK4). Evidence suggests this activity arises from a highly active cytosolic glucose dehydrogenase (G1DH) that is ATP-independent but responsive to polyphosphate. This activity may be a novel hyperglycaemic regulatory mechanism and/or a novel glucose sensor, capable of substituting for the absence of the glucokinase mechanism in the bovine liver. Novel NMR assay on recombinant PPGK and bovine liver cytosol also suggests this activity arises from a glucose dehydrogenase.
To investigate glucokinases/hexokinases multiple sequence alignment and molecular modelling were used which indicated that there are specific changes in a region on the glucokinase resulting in the loss of sigmoidal kinetics as evident by bovine glucokinase results in this work. Preliminary attempts to purify the bovine GDH using a series of chromatographic steps resulted in a highly purified preparation, analysis suggests the GDH from bovine liver cytosol is a 55 kDa protein. Results of multiple sequence alignment and molecular modelling of bacterial hexokinases, to investigate the phosphorylation donor specificity, suggest this specificity is due to increased affinity towards the adenosine site of the ATP to the exclusion of polyphosphate.
| Date of Award | 24 Mar 2025 |
|---|---|
| Original language | English |
| Supervisor | Dougie Clarke (Main Supervisor) |