Stability of glucose-6-phosphate dehydrogenase in human cells cultivated in vitro]

It was shown that the thermal stability of glucose-6-phosphate dehydrogenase in human diploid cells is much higher than in human heteroploid cell lines HeLa and T-9. The purified enzymes from human diploid cells and from HeLa and T-9 cells possess similar thermal stabilities. Mixing of T-9 extracts with the purified enzyme preparations revealed that the non-stability factors of the dehydrogenase are present in the T-9 extracts. An addition of NADP- and NADPH-containing buffers and crystalline NADP to the heteroploid cell extracts stabilizes the enzyme. The thermal stability of the enzyme from "in vitro" cultivated human cells depends on the concentration of the coenzyme. It was also demonstrated that glucose-6-phosphate dehydrogenase stability in HeLa and T-9 extracts is the same at low concentrations of the coenzyme and after addition of crystalline NADP. However, at NADP concentration of 10(-3) M the enzyme stability in HeLa and T-9 extracts is different. It is assumed that the destabilizing factors are the enzymes possessing the nucleotidases activity, which is different in various cell lines.     

The effect of hydrophobization of glucose-6-phosphate dehydrogenase with progesterone on its thermal inactivation

Thermal inactivation of glucose-6-phosphate dehydrogenase (G6PDH) and its conjugates with progesterone containing 3, 7 and 35 molecules of the modifier was studied in bidistilled water over a temperature range 35-47 degrees. At different temperatures and initial concentrations of the enzyme and its modified forms, thermal inactivation is described by the equation of the first order up to a significant degree of enzyme deactivation. The effective Kin values are decreased with the increase of the native G6PDH concentration and changed in a complicated manner with the increase of the conjugate concentration depending on the enzyme modification degree, which reflects a great role of the enzyme hydrophobicity in its inactivation. The role of hydrophobicity of the modified G6PDH in changes of its specific activity is discussed.     

Human erythrocyte glucose-6-phosphate dehydrogenase. Identification of a reactive lysyl residue labelled with pyridoxal 5'-phosphate

Human erythrocyte glucose-6-phosphate dehydrogenase contains a reactive lysyl residue, which can be labelled with pyridoxal 5'-phosphate. The binding of one mole of pyridoxal 5'-phosphate per mole of enzyme subunit produces substantial inactivation. The substrate glucose-6-phosphate prevents the loss of activity, suggesting that the reaction site is close to the substrate-binding site. A tryptic peptide containing the pyridoxal-5'-phosphate-binding lysyl residue has been isolated and characterised. The reactive lysyl residue has been identified in the glucose-6-phosphate dehydrogenase amino acid sequence. Comparison with glucose-6-phosphate dehydrogenase from other sources shows a high homology with a peptide containing a reactive lysyl residue, isolated from the enzyme from Saccharomyces cerevisiae; glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides also contains a region highly homologous with the sequence around the reactive lysyl residue in the human enzyme. The results of this communication provide the first direct evidence for the association of an essential catalytic function with a specific region of the molecule of human erythrocyte glucose-6-phosphate dehydrogenase.     

Oxidative inactivation of the calcium-stimulated neutral proteinase from human red blood cells by divicine and intracellular protection by reduced glutathione

Calpain, the micromolar Ca2+-requiring form of Ca2+-stimulated neutral proteinase purified from human red cells, is remarkably inactivated during autoxidation of divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism. Inactivation of purified calpain is produced, in decreasing order of efficiency, by transient, probably semiquinonic species arising from autoxidation of divicine, by the H2O2 that is formed upon autoxidation itself, and by quinonic divicine, respectively. Purified procalpain, the millimolar Ca2+-requiring form that can be converted to the fully active calpain form by a variety of mechanisms, is less susceptible than calpain itself to inactivation by the same by-products of divicine autoxidation. When intact red cells are exposed to autoxidizing divicine, procalpain undergoes a significant loss of activity. At 1 mM divicine, intracellular inactivation is observed with procalpain only, while the activity of a number of red cell enzymes is unaffected. Inactivation of procalpain is consistently greater in red cells from glucose-6-phosphate dehydrogenase-deficient subjects than in normal cells. Restoration of normal levels of glucose-6-phosphate dehydrogenase activity by means of entrapment of homogeneous human glucose-6-phosphate dehydrogenase in the deficient red cells results in normal stability of intracellular reduced glutathione; decreased susceptibility of procalpain to inactivation by autoxidizing divicine. These findings suggest that in the glucose-6-phosphate dehydrogenase-deficient red cells the procalpain-calpain system is a major target of divicine cytotoxicity.     

Evidence contrary to the protein error hypothesis for in vitro senescence.

A strain of diploid fibroblasts, obtained from the skin of a male infant, was cultured in vitro and cells were tested throughout their lifespan for the appearance of altered glucose-6-phosphate dehydrogenase (G-6-PD) detected either by thermostability studies or by immunotitration. No significant difference was found in the proportion of thermolabile enzyme in 31 young cultures (4.8 +/- 1%, S.E.), in comparison with that in 19 old cultures (4.9 +/- 1%, S.E.). Old cultures had ceased active cell division (49-60 doublings); DNA replication, measured by [3H]thymidine uptake over a period of 24 hours, was limited to less than 5% of these cells. Young cells (5-22 doublings) had a [3H]thymidine labeling index of 75-85%. Titration of G-6-PD activity in extracts of young and old cells with neutralizing antibody directes specifically against G-6-PD failed to detect an increment of enzymatically defective G-6-PD in old cells. The thermostability studies were capable of detecting altered G-6-PD in skin fibroblasts from a female heterozygous for a thermolabile mutant of G-6-PD, and in fibroblasts treated with a proline analogue, azetidine carboxylic acid. The immunotitration technique was also capable of detecting catalytically altered G-6-PD from the thermolabile mutant and G-6-PD inactivated with N-ethylameimide. These findings argue against a protein error catastrophe as the cause of in vitro clonal senescence.     

Gd(+)Cuiabá, a new rare glucose-6-phosphate dehydrogenase variant presenting normal activity

Summary A 33-year-old Brazilian male of Portuguese extraction was found to have a new glucose-6-phosphate dehydrogenase variant, herein named Gd(+)Cuiabá. The enzyme variant is characterized by normal activity, normal electrophoretic mobility, high K_m, for glucose-6-phosphate, high K_i for NADPH, decreased thermal stability, normal utilization of substrate analogues and normal pH curve.     

Correcting a potential defect in an enzymatic cycle for NADP

An enzymatic cycle for NADP which uses as one of its enzymes glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides has occasionally caused trouble due to failure to completely heat-kill this enzyme before the indicator step. It was found that a very small increase in pH was the cause of this. It was also found that the other two proteins present in the reagent greatly increase heat inactivation of the enzyme. The inactivation problem is completely overcome by keeping the pH below 7.2.     

Isolation and properties of glucose-6-phosphate dehydrogenase from human cultivated cells]

A new procedure for purification of glucose-6-phosphate dehydrogenase resulting in an electrophoretically homogenous preparation made up of 5.10(8) cells (390 mg of protein) is proposed. The enzyme yield is more than 20%. The molecular weights of a subunit and a native enzyme are 55000 and 220000, respectively. The isoelectric point for the protein lies at 4,8. The kinetics of the enzyme thermal inactivation obey the first order equation with the inactivation rate constant of 6.10(-3) min-1.     

Detection of a new anomalous variant of glucose-6-phosphate dehydrogenase in human erythrocytes]

Kinetic and electrophoretic properties of 230--300 fold purified preparations of glucose-6-phosphate dehydrogenase (G6PD) from red cells of donors and patients with acute drug hemolytic anemia due to G6PD deficiency were studied. A new abnormal variant of G6PD isolated from red cell of a patient with acute drug hemolytic anemia, which was not described in literature, has been discovered. The abnormal enzyme differs from the normal by decreased Michaelis constant for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP), by increased utilization of analogues of substrates--2-deoxy-glucose-6-phosphate and particularly deamino-NADP, by low thermal stability, by the character of pH-dependence, by the appearance of a single band of G6PD activity in polyacrylamide gel electrophoresis.     

Glucose metabolism and template synthesis in the mitotic cycle of human diploid fibroblasts

The correlation between the rates of protein and nucleic acid synthesis and the activity of the key enzymes of glycolysis (hexokinase, phosphofructokinase) and pentose phosphate cycle (glucose-6-phosphate dehydrogenase) in the mitotic cycle of human diploid fibroblasts synchronized by double thymidine block was studied. It was found that the removal of the thymidine block is followed by short-term (presumably, non-specific) simultaneous stimulation of matrix syntheses, as well as by glycolytic and pentose phosphate cycle enzyme syntheses. By the beginning of the S-phase, all the processes appear to be inhibited, followed by gradual activation of glycolysis and pentose phosphate cycle reactions. The implementation of the cell cycle is concomitant with stepwise transitions of protein and hexokinase synthesis rates and ATP content to one of the following levels--basal, intermediate or maximal. Changes in the activity of glucose-6-phosphate dehydrogenase in the course of the cell cycle appear as oscillations, those in phosphofructokinase as alternative states. At stage M, the oscillatory processes are temporarily quenched, whereas the ATP content occupies an intermediate level. In contrast with diploid fibroblasts, in transformed T9 cells the enzyme activity is much higher, and the fluctuations in activity throughout the cell cycle are less noticeable. Presumably, in transformed cells the enzyme activity is at the maximum level and is not prone to effector regulation.     

Effect of estrogen on synthesis of glucose-6-phosphate dehydrogenase in R3230AC mammary tumors and uteri.

The effect of estrogen on synthesis of glucose-6-phosphate dehydrogenase (D-Glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) in the R3230AC mammary adenocarcinoma of ovariectomized Fischer rats was investigated. Enzyme synthesis was estimated by techniques using immunochemica precipitation and isolation of enzyme protein from tissues of rats that had been given radioactive leucine prior to sacrifice. The antibody-enzyme complex was dissociated and glucose-6-phosphate dehydrogenase was isolated after electrophoresis on sodium dodecyl sulfate-acrylamide gels. Administration of estradiol-17beta produced a two-fold increase in glucose-6-phosphate dehydrogenase activity, which was preceded by a five-fold increase in specific synthesis of glucose-6-phosphate dehydrogenase in R3230AC tumors. At least a 15-fold increase in enzyme synthesis was observed in the uterus. The rate of enzyme degradation (t 1/2) in the tumor was estimated at 17 h. These data indicate that the estrogen-induced increase in glucose-6-phosphate dehydrogenase activity was due to a de novo increase in enzyme synthesis.     

Localization of Glucose-6-phosphate Dehydrogenase Activity on Ribosomes of Granular Endoplasmic Reticulum, in Peroxisomes and Peripheral Cytoplasm of Rat Liver Parenchymal Cells

Glucose-6-phosphate dehydrogenase activity has been localized ultrastructurally in fixed tissues. Activity was found in particular in association with ribosomes of granular endoplasmatic reticulum. Biochemical studies indicated that glucose-6-phosphate dehydrogenase activity is also present in the cytoplasm and in peroxisomes. Fixation may be held responsible for selective inactivation of part of glucose-6-phosphate dehydrogenase activity. In the present study, we applied the ferricyanide method for the demonstration of glucose-6-phosphate dehydrogenase activity in unfixed cryostat sections of rat liver in combination with the semipermeable membrane technique and in isolated rat liver parenchymal cells. Isolated liver parenchymal cells were permeabilized with 0.025% glutaraldehyde after NADP⁺ protection of the active site of glucose-6-phosphate dehydrogenase. This treatment resulted in only slight inactivation of glucose-6-phosphate dehydrogenase activity. The composition of the incubation medium was optimized on the basis of rapid light microscopical analysis of the formation of reddish-brown final reaction product in sections. With the optimized method, electron dense reaction product was observed in cryostat sections on granular endoplasmic reticulum, in mitochondria and at the cell border. However, the ultrastructural morphology was rather poor. In contrast, the morphology of incubated isolated cells was preserved much better. Electron dense precipitate was found on ribosomes of the granular endoplasmic reticulum, in peroxisomes and the cytoplasm, particularly at the periphery of cells. In conclusion, our ultrastructural study clearly demonstrates that it is essential to use mildly-fixed cells to allow detection of glucose-6-phosphate dehydrogenase activity in all cellular compartments where activity is present.     

Protein hydrophobicity and stability support the thermodynamic theory of protein degradation

Rat liver enzymes were used to study the relationship between their in vivo half-lives and their apparent hydrophobicity or their resistance to inactivation by mechanical shaking. The apparent hydrophobicity of these enzymes, measured as the percent of the protein recovered from an octyl-Sepharose column, is correlated with their known half-lives (r = 0.75, P less than 0.01). The presence of specific ligands which are known to increase compactness by impeding unfolding of proteins decreased the apparent hydrophobicity of fructose-1,6-bisphosphatase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase. Resistance of enzymes to inactivation by mechanical shaking correlated well with their in vivo half-lives (r = 0.90, P less than 0.01). When the shaking experiments were done in the presence of substrates, fructose-1,6-bisphosphatase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase were protected from inactivation.     

Catalases protect cellular proteins from oxidative modification in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae cells had higher antioxidant enzyme activities under growth in ethanol than that in glucose as a carbon and energy source. The correlations between catalase activity and protein carbonyl level (r(2)=0.857), between catalase and glucose-6-phosphate dehydrogenase activities (r(2)=0.924) and between protein carbonyl levels and glucose-6-phosphate dehydrogenase activity (r(2)=0.988) under growth in ethanol were found. Growing in ethanol the strain deficient in cytosolic and peroxisomal catalases had 7.1-fold higher level of carbonyl proteins than that of wild-type strain. Our data suggest that in vivo catalases may protect glucose-6-phosphate dehydrogenase against oxidative inactivation.     

Purification of a new high activity form of glucose-6-phosphate dehydrogenase from rat liver and the effect of enzyme inactivation on its immunochemical reactivity.

A new form of cytoplasmic glucose-6-phosphate dehydrogenase (E.C.1.1.1.49) was purified from rat liver by protamine sulfate precipitation, ammonium sulfate fractionation, ion exchange chromatography with diethylaminoethyl cellulose, and affinity chromatography with Cibacron blue agarose and NADP agarose. This form of the enzyme has a specific activity of over 600 units/mg of protein and gives essentially a single band by polyacrylamide gel electrophoresis. The form of the enzyme isolated by this purification method is 3 times more active than the form purified from liver by previously reported procedures. The relative mass of this pure glucose-6-phosphate dehydrogenase enzyme was determined by disc gel electrophoresis to be 269,000. This high activity glucose-6-phosphate dehydrogenase enzyme, after inactivation by reaction with palmityl-CoA, was no longer precipitated by specific rabbit and goat antisera to this purified enzyme. Thus, the possibility still exists that starved fat-refed animals contain glucose-6-phosphate dehydrogenase (G6PD) enzyme protein in an inactivated form no longer detectable by either enzyme activity or immunoprecipitation.     

Hormonal regulation of translatable mRNA of glucose-6-phosphate dehydrogenase in primary cultures of adult rat hepatocytes

The quantity of translatable mRNA of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) in primary cultures of adult rat hepatocytes subjected to different hormonal conditions was determined with a reticulocyte-lysate, cell-free system. The level of glucose-6-phosphate dehydrogenase mRNA was about 5-fold higher in the presence of insulin than in its absence. This increase of glucose-6-phosphate dehydrogenase mRNA reached a maximum 12 h after the addition of insulin. The maximum level of induction of glucose-6-phosphate dehydrogenase mRNA required 10(-8) M insulin. Glucagon and triiodothyronine had no effect on the glucose-6-phosphate dehydrogenase mRNA level. The increase of glucose-6-phosphate dehydrogenase activity correlated with the increase in level of mRNA of this enzyme. This suggests that the changes in glucose-6-phosphate dehydrogenase activity in response to the above hormonal changes are primarily due to changes in the amount of mRNA coding for this enzyme.     

Modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with the 2',3'-dialdehyde derivative of NADP+ (oNADP+)

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is irreversibly inactivated by the 2,3'-dialdehyde of NADP+ (oNADP+) in the absence of substrate. The inactivation is first order with respect to NADP+ concentration and follows saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inhibitor followed by covalent modification (KI = 1.8 mM). NADP+ and NAD+ protect the enzyme from inactivation by oNADP+. The pK of inactivation is 8.1. oNADP+ is an effective coenzyme in assays of glucose-6-phosphate dehydrogenase (Km = 200 microM). Kinetic evidence and binding studies with [14C] oNADP+ indicate that one molecule of oNADP+ binds per subunit of glucose-6-phosphate dehydrogenase when the enzyme is completely inactivated. The interaction between oNADP+ and the enzyme does not generate a Schiff's base, or a conjugated Schiff's base, but the data are consistent with the formation of a dihydroxymorpholino derivative.     

Properties of cathepsin D from unfertilized eggs, embryos and skeletal muscles of the loach

The action and some properties of cathepsin D, partly purified from unfertilized loach eggs, embryos and skeletal muscles were determined. The enzyme from embryo cells displays the activity maximum at pH 3.0 and pH 4.8 while enzyme from skeletal muscles--only at pH 3.0. Cathepsin D purified from all three sources splits actively hemoglobin, albumin, alpha-glycerophosphate dehydrogenase, pyruvate kinase and practically does not influence casein, hexokinase, glucose-6-phosphate dehydrogenase. The enzyme is comparatively thermolabile and its activity decreases in the presence of thiol compounds. The main part of cathepsin D in skeletal muscle cells and in embryo cells is precipitated after differential centrifugation of homogenates (25000 g; 60 min).     

Structural defects underlying protein dysfunction in human glucose-6-phosphate dehydrogenase A(-) deficiency

The enzyme variant glucose-6-phosphate dehydrogenase (G6PD) A(-), which gives rise to human glucose-6-phosphate dehydrogenase deficiency, is a protein of markedly reduced structural stability. This variant differs from the normal enzyme, G6PD B, in two amino acid substitutions. A further nondeficient variant, G6PD A, bears only one of these two mutations and is structurally stable. In this study, the synergistic structural defect in recombinant G6PD A(-) was reflected by reduced unfolding enthalpy due to loss of beta-sheet and alpha-helix interactions where both mutations are found. This was accompanied by changes in inner spatial distances between residues in the coenzyme domain and the partial disruption of tertiary structure with no significant loss of secondary structure. However, the secondary structure of G6PD A(-) was qualitatively affected by an increase in beta-sheets substituting beta-turns related to the lower unfolding enthalpy. The structural changes observed did not affect the active site of the mutant proteins, since its spatial position was unmodified. The final result is a loss of folding determinants leading to a protein with decreased intracellular stability. This is suggested as the cause of the enzyme deficiency in the red blood cell, which is unable to perform de novo protein synthesis.     

Molecular cloning of DNA sequences complementary to rat liver glucose-6-phosphate dehydrogenase mRNA. Nutritional regulation of mRNA levels

The nutritional regulation of rat liver glucose-6-phosphate dehydrogenase was studied using a cloned DNA complementary to glucose-6-phosphate dehydrogenase mRNA. The recombinant cDNA clones were isolated from a double-stranded cDNA library constructed from poly(A+) RNA immunoenriched for glucose-6-phosphate dehydrogenase mRNA. Immunoenrichment was accomplished by adsorption of polysomes with antibodies directed against glucose-6-phosphate dehydrogenase in conjunction with protein A-Sepharose and oligo(dT)-cellulose chromatography. Poly(A+) RNA encoding glucose-6-phosphate dehydrogenase was enriched approximately 20,000-fold using these procedures. Double-stranded cDNA was synthesized from the immunoenriched poly(A+) RNA and inserted into pBR322 using poly(dC)-poly(dG) tailing. Escherichia coli MC1061 was transformed, and colonies were screened for glucose-6-phosphate dehydrogenase cDNA sequences by differential colony hybridization. Plasmid DNA was purified from clones which gave positive signals, and the identity of the glucose-6-phosphate dehydrogenase clones was verified by hybrid-selected translation. A collection of glucose-6-phosphate dehydrogenase cDNA plasmids with overlapping restriction maps was obtained. Northern blot analysis of rat liver poly(A+) RNA using nick-translated, 32P-labeled cDNA inserts revealed that the glucose-6-phosphate dehydrogenase mRNA is 2.3 kilobases in length. RNA blot analysis showed that refeeding fasted rats a high carbohydrate diet results in a 13-fold increase in the amount of hybridizable hepatic glucose-6-phosphate dehydrogenase mRNA which parallels the increase in enzyme activity. These results suggest that the nutritional regulation of hepatic glucose-6-phosphate dehydrogenase occurs at a pretranslational level.