A novel affinity column for purification of glycerol phosphate dehydrogenase

An affinity column was synthesized and utilized to partially purify glycerolphosphate dehydrogenase (L-glycerol-3-phosphate: NAD⁺ oxidoreductase, E.C.1.1.1.8) from rat skeletal muscle. The novelty of the column resides in the fact that the ligand used, 6-phosphogluconic acid, is neither an inhibitor nor a substrate of the enzyme when free in solution but when immobilized on an agarose matrix, glycerol phosphate dehydrogenase binds to it with a high degree of specificity. The bound enzyme could be eluted by either increasing the ionic strength or by addition of its natural substrate, α-glycerol phosphate. Using a combination of these methods and ammonium sulfate precipitation GPDH was purified about 250 fold with a 75% yield within 24 hours.     

Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection.

Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids.     

Reversible inhibition of the hydrocortisone induction of glycerol phosphate dehydrogenase by cytochalasin B in rat glial C6 cells

The hydrocortisone (HC) induction of glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) in rat glial C6 cells was inhibited reversibly and in a dose-dependent manner by cytochalasin B (CB). CB had no effect on basal level GPDH, total cellular RNA, DNA or protein content nor did it act as a general inhibitor of the rate of protein synthesis. CB did not appear to be acting via dissociation of microtubules since colcemid had no effect on the induction process. The addition of an alternate energy source (sodium pyruvate) did not relieve the CB inhibition of GPDH induction suggesting that CB is not exerting its effect by blocking glucose utilization. The inhibition by CB is not dependent on the temporal sequence of the induction process since it specifically inhibited GPDH induction at any time it was added. CB did not alter the rate of degradation of GPDH in these cells and direct measurements of the specific rate of synthesis of GPDH demonstrated that CB decreased the induced rate of GPDH synthesis by about 60%. The site of inhibition was more precisely defined by experiments which demonstrated a 60% decrease in specific nuclear binding of ³H-HC even though total cellular uptake of ³H-HC was unaffected. This effect on nuclear binding of HC is sufficient to account for the decreased accumulation of GPDH activity in CB-treated cells.     

Induction of glycerol phosphate dehydrogenase gene expression during seizure and analgesia

Using mRNA differential display, we found that the gene for NAD(+)-dependent glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) is induced in rat brain following seizure activity. Northern blot and in situ hybridization analysis confirmed the differential display results; they also showed, in a separate model of neuronal activation, that after thermal noxious stimulation of the hind-paws, a similar increase in GPDH mRNA occurs in the areas of somatotopic projection in the lumbar spinal cord. Surprisingly, administration of analgesic doses of morphine or the nonsteroidal antiinflammatory drugs aspirin, metamizol (dipyrone), and indomethacin also increased GPDH mRNA levels in rat spinal cord. The opioid receptor antagonist naloxone completely blocked morphine induction of GPDH but had no effect on GPDH induction by noxious heat stimulation or metamizol treatment, implicating different mechanisms of GPDH induction. Nevertheless, in all cases, induction of the GPDH gene requires adrenal steroids and new protein synthesis, as the induction was blocked in adrenalectomized rats and by cycloheximide treatment, respectively. Our results suggest that the induction of the GPDH gene upon peripheral noxious stimulation is related to the endogenous response to pain as it is mimicked by exogenously applied analgesic drugs.     

Reduction by lead of hydrocortisone-induced glycerol phosphate dehydrogenase activity in cultured rat oligodendroglia

Summary Time- and dose-dependent toxic effects of lead (Pb) acetate on astroglia, oligodendroglia, and meningeal fibroblasts cultured from immature rat brain were measured. Cultures were exposed for 3 d to Pb (1,10, and 100 μM) and then examined immediately (Day 0) or 3 or 10 d after Pb treatment was discontinued. The percentages of astroglia and fibroblasts excluding dye were unaffected by Pb, whereas the percentage of oligodendroglia excluding dye decrease significantly (P<0.01) at all time points after exposure to 100 μM Pb. Lead (100 μM) also reduced the total cell numbers of astroglia, oligodendroglia, and meningeal fibroblasts. Amino acid incorporation into protein by oligodendroglia was stimulated after exposure to 100 μM Pb at all time points and also by 1 and 10 μM on Day 3. Incorporation was stimulated in astroglia only on Day 0 by 10 and 100 μM. Hydrocortisone-stimulated glycerolphosphate dehydrogenase (GPDH) activity was assayed in oligodendroglia cultures. A significant decrease in specific activity was seen after a 4-d exposure to lead. Because oligodendroglia are responsible for myelin synthesis in the central nervous system, and GPDH may synthesize a precursor for myelin lipid synthesis, it was proposed that the hypomyelination observed in lead-intoxicated neonatal rats may result partially from a primary toxic effect on oligodendroglia. GPDH activity was not inhibited by Pb in mixed glial cultures containing both astroglia and oligodendroglia. This result suggests that astroglia in culture have the ability to delay the lead-induced inhibition of oligodendroglial GPDH activity and supports the hypothesis that astroglia in culture serve a protective function. This work was supported by Environmental Protection Agency Grant R811500 and by U. S. Department of Agriculture Project M-6839 Animal Health Formula Funding Project 6652. This work was carried out by J.-N. Wu in partial fulfillment of the requirements for a Master of Science degree in Veterinary Public Health at Texas A&M University.     

Enzyme regulation by reversible zinc inhibition: glycerol phosphate dehydrogenase as an example

Since cellular zinc is not freely available as the inorganic ion, zinc proteins must acquire their metal from some other source. But how, when, and where they acquire it is unknown. Metallothionein can participate in the controlled delivery of zinc by binding it with high stability and by mobilizing it through a novel biochemical mechanism that critically depends on the redox activity of the zinc-sulfur bond. Thus, metallothionein activates zinc-depleted alcohol (sorbitol) dehydrogenases by glutathione-modulated zinc transfer. In addition to its catalytic, co-catalytic, and/or structural roles in a myriad of enzymes, zinc also inhibits some enzymes that are not necessarily zinc enzymes, e.g. glyceraldehyde and glycerol phosphate dehydrogenases, and aldehyde dehydrogenase. Zinc inhibits glycerol phosphate dehydrogenase with an IC(50) value of 100 nM. Zinc binding is slow at low pH, but instantaneous at high pH. Thionein, the apoprotein of metallothionein, re-activates the zinc-inhibited enzyme. Tight inhibition by zinc and activation of glycerol phosphate dehydrogenase by thionein, a biological chelating agent, provide further support that modulation of zinc binding by metallothionein and thionein is a physiological mechanism of enzyme regulation. Since glycerol phosphate dehydrogenase is a key enzyme in energy metabolism, the effect of zinc is expected to elicit significant physiological responses.     

Potentiometric quantitation of glycerol using immobilized glycerol dehydrogenase

Glycerol dehydrogenase was immobilized in polyacrylamide gel layered over a small platinum screen and used to catalyze the oxidation of glycerol. In the presence of NAD(+) and potassium ferricyanide, the coupling reaction generated a measurable electrical potential which was found to be Nernstian with respect to the glycerol concentration range of 10(-4)M to 10(-1)M. The reproducibility of the measurement and the optimal conditions for glycerol determination were described.     

Interferon inhibits the accumulation of glycerol phosphate dehydrogenase mRNA in oligodendrocytes and C6 cells

We investigated the effect of rat interferon-/ (IFN) on the expression of glycerol phosphate dehydrogenase (E.C.1.1.1.8; GPDH), in both C6 cells and pure cultures of oligodendrocytes. IFNs are naturally produced inhibitors of cell growth that can also affect differentiated cell functions. GPDH is a biochemical marker for oligodendrocytes and is known to be developmentally regulated and steroid inducible. GPDH activity is induced by hydrocortisone (HC) 3.5 fold in C6 cells and 5 fold in oligodendrocytes compared to untreated cultures. A pretreatment of these cells with 75 U/ml of rat IFN-/ resulted in an inhibition of the HC induction of GPDH enzymatic activity by 50% and 40% in C6 cells and oligodendrocytes respectively. We also found that IFN impaired the accumulation of GPDH mRNA in both cell types. These results demonstrate that IFNs are capable of modifying the cellular response to hormones in cells of neuroepithelial origin, and suggest the possibility that IFNs may be able to influence the development and function of the brain.Special issue dedicated to Dr. Paola S. Timiras     

Distribution of glycerol dehydrogenase and glycerol kinase activity in halophilic archaea

Abstract A constitutive NAD⁺-dependent glycerol dehydrogenase activity was detected in Halobacterium salinarium and Halobacterium cutirubrum . Optimal activity was found at 3 M KCl and pH 8–10. No glycerol dehydrogenase activity could be demonstrated in representatives of the genera Haloferax and Haloarcula , even when grown in the presence of glycerol, or in Halobacterium saccharovorum and Halobacterium sodomense . Glycerol kinase activity was shown to be present constitutively in all halophilic archaea examined. The finding that glycerol dehydrogenase is found only in part of the halophilic arachaea makes dihydroxyacetone an improbable candidate as the precursor for the glycerol moiety of halobacterial lipids.     

Normal thyroid thermogenesis but reduced viability and adiposity in mice lacking the mitochondrial glycerol phosphate dehydrogenase

The mitochondrial glycerol phosphate dehydrogenase (mGPD) is important for metabolism of glycerol phosphate for gluconeogenesis or energy production and has been implicated in thermogenesis induced by cold and thyroid hormone treatment. mGPD in combination with the cytosolic glycerol phosphate dehydrogenase (cGPD) is proposed to form the glycerol phosphate shuttle, catalyzing the interconversion of dihydroxyacetone phosphate and glycerol phosphate with net oxidation of cytosolic NADH. We made a targeted deletion in Gdm1 and produced mice lacking mGPD. On a C57BL/6J background these mice showed a 50% reduction in viability compared with wild-type littermates. Uncoupling protein-1 mRNA levels in brown adipose tissue did not differ between mGPD knockout and control pups, suggesting normal thermogenesis. Pups lacking mGPD had decreased liver ATP and slightly increased liver glycerol phosphate. In contrast, liver and muscle metabolites were normal in adult animals. Adult mGPD knockout animals had a normal cold tolerance, normal circadian rhythm in body temperature, and demonstrated a normal temperature increase in response to thyroid hormone. However, they were found to have a lower body mass index, a 40% reduction in the weight of white adipose tissue, and a slightly lower fasting blood glucose than controls. The phenotype may be secondary to consequences of the obligatory production of cytosolic NADH from glycerol metabolism in the mGPD knockout animal. We conclude that, although mGPD is not essential for thyroid thermogenesis, variations in its function affect viability and adiposity in mice.     

Conversion of glycerol to 1,3-dihydroxyacetone by glycerol dehydrogenase co-expressed with an NADH oxidase for cofactor regeneration

Objectives

To investigate the efficiency of a cofactor regeneration enzyme co-expressed with a glycerol dehydrogenase for the production of 1,3-dihydroxyacetone (DHA).

Results

In vitro biotransformation of glycerol was achieved with the cell-free extracts containing recombinant GlyDH (glycerol dehydrogenase from Escherichia coli), LDH (lactate dehydrogenase form Bacillus subtilis) or LpNox1 (NADH oxidase from Lactobacillus pentosus), giving DHA at 1.3 g l^?1 (GlyDH/LDH) and 2.2 g l^?1 (GlyDH/LpNox1) with total turnover number (TTN) of NAD⁺ recycling of 6039 and 11100, respectively. Whole cells of E. coli (GlyDH–LpNox1) co-expressing both GlyDH and LpNox1 were constructed and converted 10 g glycerol l^?1 to DHA at 0.2–0.5 g l^?1 in the presence of zero to 2 mM exogenous NAD⁺. The cell free extract of E. coli (GlyDH–LpNox) converted glycerol (2–50 g l^?1) to DHA from 0.5 to 4.0 g l^?1 (8–25 % conversion) without exogenous NAD⁺.

Conclusions

The disadvantage of the expensive consumption of NAD⁺ for the production of DHA has been overcome.