A-Glycerol-phosphate dehydrogenase

Figure 5.8 Stopped-flow record of NADH oxidation at 20 °C in a reaction mixture containing dihydroxyacetone phosphate (DHAP) (5 pM), a-glycerol-phosphate dehydrogenase (30 pN), NADH (50 pM), in Tris-HCl buffer (0.1 M) and EDTA (2 mM) adjusted to pH 7.5 with NaOH. One syringe contained DHAP and the other enzyme and NADH. The baseline shown is the end of the reaction. The ratio of the fast step to the slow step shows as the ratio of available substrate to precursor which is slowly converted to substrate (for detail see text and Reynolds et ai, 1971).

Figure 5.10 The observed rate constant for the displacanmt of NADH by NAD from the complex with a-glycerol-phosphate dehydrogenase as a function of the concentration of NAD. (Extracted from the data of Chock Gutfreund, 1988.)...

Figure 5.12 The rate of transfer of NADH from its complex with El to E2 when increasing concentrations of E2 are added to solutions of El-NADH. In the upper curve El is lactate dehydrogenase and E2 is a-glycerol-phosphate dehydrogenase. In the lower curve El is a-glycerol-phosphate dehydrogenase and E2 is lactate dehydrogenase. (Data extracted from Wu et al., 1991.)...

Many enzymes in the mitochondria, including those of the citric acid cycle and pyruvate dehydrogenase, produce NADH, aU of which can be oxidized in the electron transport chain and in the process, capture energy for ATP synthesis by oxidative phosphorylation. If NADH is produced in the cytoplasm, either the malate shuttle or the a-glycerol phosphate shuttle can transfer the electrons into the mitochondria for delivery to the ETC. Once NADH has been oxidized, the NAD can again be used by enzymes that require it. 

FADH is produced by succinate dehydrogenase in the citric acid cycle and by the a-glycerol phosphate shuttle. Both enzymes are located in the inner membrane and can reoxidize FADHj directly by transferring electrons into the ETC. Once FADH2 has been oxidized, the FAD can be made available once again for use by the enzyme. 

All these components are in the inner membrane of the mitochondria as shown in Figure I-I3-3. Succinate dehydrogenase and the a-glycerol phosphate shuttle enzymes reoxidize their FADHj and pass electrons directly to CoQ. 

Adler, A.J., Klucznik, K.M. (1982). Glycerol phosphate dehydrogenase in developing chick retina and brain. J. Neurochem. 38 909-15. 

Link, W.A., Kausehnann, G., Mellstrdm, B., Kubl, D., Naranjo J.R. (2000). Induction of glycerol phosphate dehydrogenase gene expression during seizure and analgesia. J. Neurochem. 75 1419-28. 

Describe the entry of electrons into the respiratory chain at the succinate-Q reductase complex (Complex 11) from flavoproteins such as succinate dehydrogenase (a component of Complex II), glycerol phosphate dehydrogenase, and fatty acyl CoA dehydrogenase by way of FADH.2. Appreciate that Complex II is not a proton pump. 

A TG molecule is synthesized in the fat cells of mammals and plants from L-glycerol-3-phosphate and fatty acid-CoA esters (Fig. 3.9). The L-glycerol-3-phosphate supply is provided by the reduction of dihydroxy acetone phosphate by NAD+-dependent glycerol phosphate dehydrogenase. The dihydroxy acetone phosphate originates from glycolysis. 

The secondary alcohol group of glycerol-3-phosphate is then oxidized by NAD to a ketone. The enzyme that catalyzes this reaction is called glycerol phosphate dehydrogenase. Recall that a dehydrogenase is an enzyme that oxidizes its... 

A coupled enzyme system can also be used. The glycer-aldehyde-3-phosphate formed is converted to dihydroxy-acetone phosphate by triosephosphate isomerase and this is followed by reduction by glycerol phosphate dehydrogenase. The oxidation of NADH in this last stage is measured spectrophotometrically. 

Alternatively, 41 can be formed from glycerol by successive phosphorylation and oxidation effected by a combination of glycerol kinase and glycerol phosphate dehydrogenase, tvith an integrated double ATP/NAD+ cofactor recycling system [148]. 

Dihydroxyacetone phosphate (41) was prepared enzymatically from glycerol 1-phosphate (74) by the action of glycerol phosphate oxidase [177]. Analytical thin-layer chromatography was performed on silica gel plates using a 1 1 mixture of sat. ammonia-ethanol for development, and anisaldehyde stain for detection. The aldolases are commercially available or can be purified in accordance with published procedures [150]. Activity of aldolases (1 unit catalyzes cleavage of 1 pmol L-ketose 1-phosphates (43/45) per minute at 25 °C [150]) and amounts of 41 were determined photometrically by an assay coupled with glycerol phosphate dehydrogenase-catalyzed NADH oxidation [149]. 

A specific example will illustrate some important points about the rate of displacement of a ligand with high afiinity by another with relatively lower affinity. The rate of dissociation of NADH from its complex with the enzyme glycerol phosphate dehydrogenase (E) has been investigated by the displacement of NADH by NAD". At 10 C the two reactions can be described by the following equilibrium constants ... 

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