Phosphoenolpyruvate carboxykinase and gluconeogenesis in cotyledons of Cucurbita pepo

1. The aim of this work was to investigate the role of phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) in the conversion of fat to sugar by the cotyledons of seedlings of Cucurbita pepo. 2. The enzyme was partially purified from the cotyledons of 5-day-old seedlings. The Michaelis constants for oxaloacetate and ATP were 56 and 119 micron, respectively. The decarboxylation reaction was optimum at pH 7.4. A range of intermediary metabolites did not affect the activity of the enzyme, but 3-mercaptopicolinic acid at micron concentrations was an effective inhibitor. 3. Centrifugation of extracts of 5-day-old cotyledons sedimented appreciable proportions of the ribuloseibisphosphate carboxylase, isocitrate lyase and fumarate hydratase present but very little of the phosphoenolpyruvate carboxykinase. 4. Measurements of phosphoenolpyruvate carboxykinase of cotyledons during germination showed that the maximum catalytic activity exceeded, and changed coincidently with, the rate of gluconeogenesis. 5. 3-Mercaptopicolinic acid inhibited gluconeogenesis from [1-14C]- and [2-14C]acetate supplied to excised cotyledons. The detailed distribution of 14C indicated inhibition of the conversion of oxaloacetate to phosphoenolpyruvate. 6. It is concluded that in marrow cotyledons phosphoenolpyruvate carboxykinase is in the soluble phase of the cytoplasm and catalyses a component reaction of gluconeogenesis.     

Evidence for the existence of phosphoenolpyruvate carboxykinase ferroactivator activity in adult and fetal guinea pigs.

Newborn (24--72 h) guinea pig liver cytosolic phosphoenolpyruvate carboxykinase (PEPCK) activity is increased by incubation of the cytosol with the metal salts FeCl2, MnCl2, CoCl2 and CdCl2. FeCl2 at 30 micromol/l concentration is the most effective activator causing a 3.5-fold increase in activity. Purified rat liver cytosolic PEPCK is activated by 30 mumol/l FeCl2 in the presence of liver cytosol of fetal and newborn guinea pigs. These results confirm the existence of PEPCK ferroactivator in the guinea pig which has properties similar to the one found in rat liver. The tissue distribution of ferroactivator activity parallels that of cytosolic PEPCK, being highest in the gluconeogenic organs liver and kidney. Hepatic PEPCK ferroactivator activity can be demonstrated by day 45 of gestation, increasing linearly in specific activity to adult levels at term (65 days). The distribution and development of the ferroactivator is consistent with the hypothesis that it may play a role in the physiologic control of PEPCK.     

Phosphoenolpyruvate carboxykinase is a nuclear enzyme in rats and chickens

The presence of phosphoenolpyruvate carboxykinase (PEPCK) in the nuclei of chicken liver cells was confirmed using two experimental designs. PEPCK was found to be enriched in the nuclear fraction of rat liver, a species whose hepatic PEPCK is reported to be predominantly cytosolic. We suggest that PEPCK plays a role in nuclear synthesis of N-acetyl-neuraminic acid.     

Mechanism of plasma glutathione peroxidase production in bovine adipocytes

CD97, an epidermal growth factor (EGF)-TM7 receptor, is not restricted to hematopoetic and carcinoma cells but is also found on smooth muscle cells (SMC). We have examined its location and biochemical structure in various normal and tumorigenic SMC-containing tissues. SMC of the urinary bladder, lung bronchi and bronchioles, myometrium, and gastrointestinal tract were immunohistologically stained by using monoclonal antibodies (mabs) to the CD97 stalk region (CD97^stalk). Mabs directed against an N-glycosylation-dependent epitope within the EGF-domains (CD97^EGF) did not bind to normal SMC. Vascular SMC, which was also CD97^EGF-negative, showed further CD97 heterogeneity. Only a few, if any, SMC from the aorta or elastic arteries of the systemic circulation were positive for CD97 mRNA and therefore also for CD97^stalk. CD97^stalk-positive SMC were slightly more numerous in muscular and peripheral arteries. In contrast, most venous SMC expressed CD97^stalk. A comparison with other SMC molecules revealed a similar but not identical staining pattern for CD97^stalk and desmin. Further CD97 heterogeneity was observed during SMC transformation. All leiomyomas (n=5) and nine out of 21 leiomyosarcomas were positive for both CD97^stalk and CD97^EGF. As expected, CD97^EGF-positive SMC tumors expressed partly N-glycosylated CD97. Seven out of 21 leiomyosarcomas were completely devoid of CD97. Thus, CD97 showed variable expression in vascular and biochemical modification in tumorigenic SMC, suggesting that the function of the molecule is specific for the SMC subtype. This study was supported by a joint grant from the German Research Council (DFG; project AU 132/3-1) and by the Interdisziplinary Center of Clinical Research (IZKF) Leipzig at the Faculty of Medicine, University of Leipzig (project D6). E. Wandel is a fellow of the IZKF.     

Mechanism of action of lignins and lignin model compounds with horseradish peroxidase

Horseradish peroxidase displayed a ping-pong kinetic reaction mechanism with lignin model compounds and lignins. Oxidation of the α carbon on acetosyringone or acetovanillone failed above pH 6.5, while conversion of α-methylsyringyl (or guaiacyl) alcohol to acetosyringone (or vanillone) occurred optimally at pH 7.8. Small MW fragments were not formed from lignins at pH 6.4 and 7.8. These observations provide evidence for the growing concept that freely soluble peroxidase is not a lignolytic enzyme.     

Phylogenetic distribution of glutathione peroxidase.

1. The enzyme glutathione peroxidase (E.C.1.11.1.9), known to be a selenoprotein from mammalian sources, was detected in the following vertebrates: fish, frog, salamander, and turtle. 2. Among invertebrates, the enzyme was detected in crayfish and snail but not in insects or earthworm. 3. No plant tissues or microorganisms showed any evidence of the enzyme activity. 4. The presence of the enzyme activity in so many animal groups implies the widespread occurrence of genetic information for the specific assimilation of the selenium atom.     

Reaction of cyanide with glutathione peroxidase

An oxidized form of ovine erythrocyte GSH peroxidase (Form C) that contains bound glutathione in equimolar ratio to the enzyme selenium is inactivated by cyanide. When Form C was treated with 1 or 10 mM KCN at pH 7.5, there was a rapid increase in ultraviolet absorption at 250 nm, S-cyanoglutathione was released, and the enzyme was reduced, as shown by inactivation with iodoacetate (1 mM, pH 7.5) and uptake of label from [¹⁴C]iodoacetate in equimolar ratio to enzyme selenium. These observations suggest that glutathione is bound to enzyme selenium by a selenenyl-sulfide linkage (E-Se-SG) which is cleaved by cyanide to release a selenol and S-cyanoglutathione; spontaneous oxidation of the selenol to a labile oxidized form of GSH peroxidase leads to irreversible inactivation.     

A mechanistic mathematical model for the catalytic action of glutathione peroxidase

Abstract

Glutathione peroxidase (GPx) is a well-known seleno-enzyme that protects cells from oxidative stress (e.g., lipid peroxidation and oxidation of other cellular proteins and macromolecules), by catalyzing the reduction of harmful peroxides (e.g., hydrogen peroxide: H₂O₂) with reduced glutathione (GSH). However, the catalytic mechanism of GPx kinetics is not well characterized in terms of a mathematical model. We developed here a mechanistic mathematical model of GPx kinetics by considering a unified catalytic scheme and estimated the unknown model parameters based on different experimental data from the literature on the kinetics of the enzyme. The model predictions are consistent with the consensus that GPx operates via a ping-pong mechanism. The unified catalytic scheme proposed here for GPx kinetics clarifies various anomalies, such as what are the individual steps in the catalytic scheme by estimating their associated rate constant values and a plausible rationale for the contradicting experimental results. The developed model presents a unique opportunity to understand the effects of pH and product GSSG on the GPx activity under both physiological and pathophysiological conditions. Although model parameters related to the product GSSG were not identifiable due to lack of product-inhibition data, the preliminary model simulations with the assumed range of parameters show that the inhibition by the product GSSG is negligible, consistent with what is known in the literature. In addition, the model is able to simulate the bi-modal behavior of the GPx activity with respect to pH with the pH-range for maximal GPx activity decreasing significantly as the GSH levels decrease and H₂O₂ levels increase (characteristics of oxidative stress). The model provides a key component for an integrated model of H₂O₂ balance under normal and oxidative stress conditions.     

Vitamin E supplementation and glutathione peroxidase activity.

Both excess dietary vitamin E and vitamin E deficiency in rats can significantly depress the activity of GSH peroxidase in liver and plasma of rats. Of all the six levels of vitamin E tested in this study, the dietary level of vitamin E found to maintain the maximum activity of GSH peroxidase in tissues of rats was somewhere between 25 and 250 IU/kg diet. This study conclusively indicates that the excess dietary vitamin E represses GSH peroxidase activity.     

Testis glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase activities in aminoguanidine-treated diabetic rats

Severe steroidogenic and spermatogenic alterations are reported in association with diabetic manifestations in humans and experimental animals. This study was planned to determine whether oxidative stress is involved in diabetes-induced alterations in the testes. Diabetes was induced in male rats by injection of 50 mg/kg of streptozotocin (STZ). Ten weeks after injection of STZ, levels of selenium and activities of selenium dependent-glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) were measured in rat testis. Lipid and protein oxidations were evaluated as measurements of testis malondialdehyde (MDA) and protein carbonyl levels, respectively. Testis sulfydryl (SH) levels were also determined. The control levels of GPx and PHGPx activities were found to be 46.5 +/- 6.2 and 108.8 +/- 19.8 nmol GSH/mg protein/min, respectively. Diabetes caused an increase in testis GPx (65.0 +/- 21.1) and PHGPx (155.9 +/- 43.1) activities but did not affect the levels of selenium or SH. However, the testis MDA and protein carbonyl levels as markers of lipid and protein oxidation, respectively, did not increase in the diabetic group. Aminoguanidine (AG) treatment of diabetic rats returned the testis PHGPx activity (136.5 +/- 24.9) to the control level but did not change the value of GPx activity (69.2 +/- 17.4) compared with diabetic group. MDA and protein carbonyl levels in testis were not affected by AG treatment of diabetic rats, but interestingly AG caused SH levels to increase. The results indicate that reactive oxygen radicals were not involved in possible testicular complications of diabetes because diabetes-induced activations of GPx and PHGPx provided protection against oxidative stress, which was reported to be related to some diabetic complications.     

A pseudogene for human glutathione peroxidase.

Glutathione peroxidases (GPx) serve a bioprotective function in the reduction of peroxides to less toxic substances. Both cellular and secreted forms of the protein have been reported, as well a number of distinct cDNA sequences. Previous efforts have described three distinct loci on human chromosomes 3, 21 and X which hybridize to a GPX cDNA and these authors have speculated that only the chromosome 3 locus encodes a functional GPX gene. This conclusion was based on mapping studies showing a precise deletion of intron sequences in the GPX loci on chromosomes 21 and X despite strong conservation among these sequences in both the coding and 3'-untranslated regions. To pursue this issue, we have isolated the chromosome 21 GPX locus by molecular cloning and determined its nucleotide sequence. Consistent with the expectations of McBride et al. [Biofactors 4 (1988) 285-292], the sequence does reveal a highly conserved processed pseudogene. It is suggested that a retrotransposed copy of the GPX gene integrated into chromosome 21 and may have maintained activity prior to the accumulation of inactivating mutations.     

Functional studies of an HIV-1 encoded glutathione peroxidase

In an alternate reading frame overlapping the viral envelope gene, HIV-1 has been shown to encoded a truncated glutathione peroxidase (GPx) module. Essential active site residues of the catalytic core regions of mammalian GPx sequences are conserved in the putative viral GPx (vGPx, encoded by the env-fs gene). Cells transfected with an HIV-1 env-fs construct show up to a 100% increase in GPx enzyme activity, and are protected against the loss of mitochondrial transmembrane potential and subsequent cell death induced by exogenous oxidants or mitochondrial reactive oxygen species. An intact vGPx gene was observed to be more common in HIV-1-infected long-term non-progressors, as compared to HIV-1 isolates from patients developing AIDS. An antioxidant/antiapoptotic protective role of the vGPx is also consistent with the observation that -1 frameshifting induced by the HIV-1 env-fs sequence AAAAAGA (which contains a potential "hungry" arginine codon, AGA) increases during arginine deficiency, which has been associated with increased oxidative stress. Under arginine-limited conditions, nitric oxide synthase generates superoxide, which rapidly combines with NO to form peroxynitrite, which can cause activated T-cells to undergo apoptosis. Thus, biosynthesis of the HIV-1 GPx as an adaptive response to low arginine conditions might delay oxidant-induced apoptotic cell death, providing an enhanced opportunity for viral replication.     

Human catalytic antibodies with glutathione peroxidase activity

In order to generate catalytic antibodies with glutathione peroxidase (GPx) activity, we prepared GSH-S-DNP butyl ester and GSH-S-DNP benzyl ester as the haptens. Two ScFvs that bound specifically to the haptens were selected from the human phage-displayed antibody library. The two ScFv genes were highly homologous, consisting of 786 bps and belonging to the same VH family-DP25. In the premise of maintaining the amino acid sequence, mutated plasmids were constructed by use of the mutated primers in PCR, and they were over-expressed in E. coli. After the active site serine was converted into selenocysteine with the chemical modifying method, we obtained two human catalytic antibodies with GPx activity of 72.2U/micromol and 28.8U/micromol, respectively. With the aid of computer mimicking, it can be assumed that the antibodies can form dimers and the mutated selenocysteine residue is located in the binding site. Furthermore, the same Ping-Pong mechanism as the natural GPx was observed when the kinetic behavior of the antibody with the higher activity was studied.     

Micromethods in single muscle fibers. 2. Determination of glutathione reductase and glutathione peroxidase

This paper extends the previous study for systems which control intracellular oxidative events in muscle and describes procedures suitable to assay glutathione peroxidase (GSHPx), glutathione reductase (GR), and total glutathione (GSH + GSSG) after fiber typing of individual muscle fibers. In human skeletal muscle, both GR and GSHPx activities were relatively low when compared to those of other tissue. No difference was found among fiber types (I, IIA, and IIB) with regard to GR activity, but in contrast GSHPx activity was significantly lower in type IIB fibers than in the other types. These results suggest that type IIB fibers may have a reduced ability to cope with hydroperoxides generated during oxidative stress, which, in turn, could lead to increased damage to membrane structures by lipid peroxidation or oxidation of sensitive intracellular thiol (-SH) enzymes by hydrogen peroxide. The Km of skeletal muscle GR for GSSG was 27 microM and for NADPH was 22 microM. If one assumes approximately 95% of total glutathione is present in the reduced state, then GSSG concentration would be of the order of 0.3 mmol/kg and under these conditions skeletal muscle GR would be efficient in all muscle fiber types.