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Cytosolic glycerol-3-phosphate dehydrogenase (G3PDH, EC 1.1.1.8) is an important branch point enzyme connecting lipid metabolism and carbohydrate metabolism. We investigated the dynamic nature of G3PDH by purifying the enzyme from the liver of Richardson's ground squirrel (Urocitellus richardsonii), a hibernating species, and analyzing its structural and functional changes during hibernation. Kinetic parameters of purified G3PDH from ground squirrel liver were characterized at 37, 22 and 5°C and compared between euthermic and hibernating states. Relative to euthermic liver G3PDH, hibernator liver G3PDH had a decreased affinity for its substrate, glycerol-3-phosphate (G3P), at 37°C and 22°C. However, at 5°C, there was a significant increase in the affinity for G3P in the hibernating form of the enzyme, relative to the euthermic form. Furthermore, the structure of G3PDH in the species' hibernating state showed greater thermal stability compared to its structure in the euthermic state. Western blot analysis revealed greater tyrosine phosphorylation in hibernator G3PDH as compared to euthermic G3PDH. In addition, using the protein sequence of the hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and bioinformatics tools, a three-dimensional model of G3PDH was built to identify the potential phosphorylation site ( 83 Tyr) responsible for the differential phosphorylation between euthermic and hibernator G3PDH. The structural and functional changes in G3PDH support the enzyme's function at a low core body temperature experienced during the species hibernating season., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A full-length 2.4-kb cDNA for the FAD-linked glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) was cloned from rat liver using PCR techniques. The cloned gene encodes a protein of 727 amino acids. The calculated molecular mass of 80,898 Da is higher than the apparent molecular mass observed by SDS/PAGE (74,000 Da) of the purified enzyme. This result indicates that the enzyme is synthesized as a precursor with a putative mitochondrial signal sequence. mRNA for this gene was detected in liver, heart, muscle, brain, testes, and pancreas. With the exception of testes, basal expression levels were very low in all tissues examined. However, application of thyroid hormones led to a 10- to 15-fold increase in liver glycerol-3-phosphate dehydrogenase mRNA, whereas hypothyroidism further decreased the mRNA level.

The major isozymic form of sn-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) has been purified from rabbit liver using a simplified three-step chromatographic procedure involving an ion exchange and two affinity chromatography steps. The 1200-fold purified enzyme is electrophoretically homogeneous, nucleotide-free, and possesses a specific activity of 295 units/mg and an isoelectric point of 6.5. A steady-state kinetic analysis was applied to both the forward and reverse reactions. The NADH oxidation reaction was found to adhere to Michaelis-Menten behavior with Km values of 22 micrometer and 75 micrometer for NADH and dihydroxyacetone phosphate, respectively. In the NAD reduction reaction, sigmoidal kinetic patterns were observed when NAD was the variable substrate whereas with sn-glycerol-3-phosphate as the variable substrate, strictly hyperbolic kinetics were observed. The apparent Km values for NAD and glycerol-3-phosphate were 83 and 909 micrometer, respectively. By comparison with published reports, these results demonstrate that the rabbit muscle and liver isozymes of sn-glycerol-3-phosphate dehydrogenase have different kinetic properties and suggest that the liver isozyme is better adapted to participation in glyconeogenesis in vivo.