Cycloheximide-resistance in Chinese hamster ovary cells and human fibroblast cells

Summary A total of 3000 men living in Yamaguchi were screened for glucose-6-phosphate dehydrogenase (G6PD) deficiency using Beutler's spot test and three types of starch gel electrophoresis. These electrophoresis used a phosphate buffer system at pH 7.0, a TRIS-EDTA-borate buffer system at pH 8.6, and a TRIS-hydrochloride buffer system at pH 8.8. Fifteen G6PD-deficient variants were found at the rate of 0.5% and classified into four groups. As new variants, G6PD Konan, Kamiube, and Kiwa were identified. These three variants had a mild to moderate G6PD deficiency and were not associated with any clinical signs. G6PD Konan had fast electrophoretic mobility as compared with normal levels, G6PD Kiwa had slightly elevated electrophoretic mobility, and G6PD Kamiube had normal electrophoretic mobility. These three variants had normal levels of Km G6P, Km NADP, and Ki NADPH, normal utilizations of both 2-deoxy-G6P and deamino-NAPD, normal heat stability, and a normal pH curve. The other variant was G6PD Ube, which we had previously found in Yamaguchi (Nakashima et al., 1977). One boy with G6PD Ube was Korean.     

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.     

Red cell glucose 6 phosphate dehydrogenase genotypes of the population of two West African villages

Summary Blood samples from 1109 individuals, residents of two villages in The Gambia, West Africa, have been examined for red cell G6PD. Using both starch gel electrophoresis and a spectrophotometric assay, preliminary phenotypes were assigned to the 519 males and 590 females. The G6PD genotypes were established by reference to the family trees of the two village populations. In addition to the G6PD alleles B⁺, A⁺ and A^-, a fourth allele, representing a new variant of human G6PD was discovered. A significant difference in the frequency of G6PD deficiency was discovered between the two villages, despite their being of the same tribal origin and only five miles apart.     

Purification and characterization of glucose 6-phosphate dehydrogenase from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver, using a simple and rapid method, and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: homogenate preparation and 2', 5'-ADP Sepharose 4B affinity gel chromatography, which took 7-8 hours. Thanks to the two consecutive procedures, the enzyme, having specific activity of 38 EU/mg protein, was purified with a yield of 44.39% and 1310 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. Optimal pH, stable pH, optimal temperature, Km and, Vmax values for NADP+ and glucose 6-phosphate (G6P) were also determined for the enzyme. In addition, molecular weight and subunit molecular weights were found by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography respectively.     

Glucose 6-phosphate dehydrogenase: isoenzymatic pattern in Oesophagostomum venulosum, Trichuris ovis and T. suis.

In the present communication we have studied the isoenzymatic pattern activity of the glucose 6-phosphate dehydrogenase (G6PD) in Oesophagostomum venulosum, Trichuris ovis and T. suis, parasites of Capra hircus (goat), Ovis aries (sheep) and Sus scrofa domestica (pig) respectively, by polyacrylamide gel electrophoresis. Different phenotypes have been observed in the G6PD isoenzymatic pattern activity in males and females of Oesophagostomum venulosum. Furthermore, G6PD activity has been assayed in Trichuris ovis collected from Ovis aries and Capra hircus. No differences have been observed in the isoenzymatic patterns attending to the different hosts. All the individuals exhibited one single band or two bands; this suggests a monomeric condition for G6PD in T. ovis. In T. suis the enzyme G6PD appeared as a single electrophoretic band in about 85.7% of the individuals.     

Genetic heterogeneity at the glucose-6-phosphate dehydrogenase locus in southern Italy: a study on a population from the Matera district

Summary Glucose-6-phosphate dehydrogenase (G6PD) has been analyzed by gel electrophoresis and by quantitative assay in an unselected sample of 1524 schoolboys from the province of Matera (Lucania) in southern Italy. We have identified 43 subjects with a G6PD variant. Of these, 31 had severe G6PD deficiency, nine had mild to moderate deficiency, and three had a non-deficient electrophoretic variant. The overall rate of G6PD deficiency was 2.6%. The frequency of G6PD deficiency, ranging from 7.2% on the Ionian Coast to zero on the eastern side of the Lucanian Apennines, appears to be inversely related to the distance of each town examined from the Ionian Coast, suggesting that this geographic distribution may reflect, at least in part, gene flow from Greek settlers. Biochemical characterization has shown that most of the G6PD deficiency in this population is accounted for by G6PD Mediterranean. In addition, we have found several examples of two other known polymorphic variants (G6PD Cagliari and G6PD A^–): three new polymorphic variants. G6PD Metaponto (class III), G6PD Montalbano (class III), and G6PD Pisticci (class IV); and two sporadic variants, G6PD Tursi (class III) and G6PD Ferrandina (class II). These data provide further evidence for the marked genetic heterogeneity of G6PD deficiency within a relatively narrow geographic-area and they prove the presence in the Italian peninsula of a sene (Gd ^A–) regarded as characteristically African.     

Altered aggregational properties in a genetic variant of human glucose 6-phosphate dehydrogenase

The Tel Hashomer variant of human G6PD migrates as two prominent components during electrophoresis in several gel systems in which red cell G6PD from other males migrates predominantly as a single band. Since human males normally have but one X-chromosome, the extra band of this variant seemed an exception to earlier biochemical and genetic observations suggesting that human red cell G6PD is determined by a locus on the X chromosome. Results of the present studies indicate that the Tel Hashomer variant is unusually susceptible to the formation of a complex which has a higher molecular weight than normal G6PD and which represents the slow electrophoretic component. The conditions of formation and disruption of this complex in crude and purified Tel Hashomer preparations suggest that it results from the formation of disulfide bridges between molecules of Tel Hashomer G6PD.Supported by U.S. Public Health Service Research Grants AM-11065 and FR-5406 and Research Career Development Award 5 K3 AM 7992.     

High Expression of G6PD Increases Doxorubicin Resistance in Triple Negative Breast Cancer Cells by Maintaining GSH Level

Resistance to doxorubicin (DOX) remains a big challenge to breast cancer treatment especially for triple negative breast cancer (TNBC). Our previous study revealed that the antioxidant system plays an important role in conferring metastasis derived DOX resistance. In this study, we used two-dimensional difference gel electrophoresis (2D-DIGE) proteomics to compare the expression profiles of two generations of TNBC cell lines which have increased metastatic ability in nude mice and exhibited resistance to DOX. Through careful analyses, one antioxidant protein: glucose-6-phosphate dehydrogenase (G6PD) was identified with 3.2-fold higher level in metastatic/DOX-resistant 231-M1 than its parental 231-C3 cells. Analyses of clinical data showed that TNBC patients with higher G6PD levels exhibited lower overall survival than patients with lower G6PD level. Reducing G6PD expression by siRNA or inhibiting its activity with dehydroepiandrosterone (DHEA) significantly increased DOX''s cytotoxicity in both cell lines. Importantly, inhibiting G6PD''s activity with DHEA dramatically increased the apoptotic rate of 1.25 µM DOX from 2% to 54%. Our results suggest that high level of G6PD can help TNBC to resist DOX-induced oxidative stress. Thus, inhibiting G6PD shall be a good strategy to treat DOX-resistant TNBC.     

PCR—DGGE法研究葡萄糖-6-磷酸脱氢酶缺乏症基因突变

目的：应用PCR-DGGE法和DNA测序分析云南籍G6PD缺乏症患者基因突变类型和特点、方法应用硝基四氮唑蓝（NBT）纸片法进行G6PD缺乏症定性筛查,G6PD／6PGD比值法验证,应用PCR—DGGE法和DNA测序分析46例云南籍G6PD缺乏症患者基因突变类型和特点。结果：46例云南籍G6PD缺乏症样本中有30例经PCR—DGGE法分析G6PDexon12发现有异常电泳条带,DNA测序证实26例（56、52％）为nt-1388G→A,4例（8.7％）nt-1376G→T．而PCR—DGGE法分析G6PDexon2未发现有异常电泳条带的样本出现。结论：（1）nt-1388G→A（56．52％）、nt-1376G→T（8．7％）是云南省主要的基因突变型也是中国人中最常见的两种突变型,揭示中华民族有着共同的起源;（2）所检样本中未发现nt95A→G。（3）应用PCR—DGGE法结合DNA测序检测G6PD缺乏症患者的基因型,阳性检出率高,方法简便、快捷、灵敏、结果准确可靠。     

Autosomal determination of erythrocyte glucose 6-phosphate dehydrogenase in domestic chickens and ring-necked pheasants

Erythrocyte glucose 6-phosphate dehydrogenase isozymes of domestic chickens, ring-necked pheasants, and their hybrids were studied, using the starch gel zone electrophoresis technique. In domestic chickens G6PD isozymes were represented by two fast-moving bands and an indistinct third band, whereas in ring-necked pheasants a slow-moving broad band which seemed to consist of two closely apposed G6PD isozymes was observed. The F₁ hybrids showed three distinct bands combining the characteristic mobility pattern of the two parents, which seemed to indicate that both parental alleles are expressed in F₁ hybrids. Since both male and female hybrids exhibited strikingly similar isozyme patterns representing both sire and dam, it was assumed that the genes controlling the production of G6PD in chicken and pheasant red blood cells are located on the autosomes.This study was supported in part by a research grant from the National Research Council of Canada.     

The structural subunit of glucose-6-phosphate dehydrogenase (baker's yeast).

The subunit molecular weight of glucose-6-phosphate dehydrogenase (G6PD) from baker's yeast has been evaluated. The subunit molecular weight value is shown to be 25,500 daltons by analytical ultracentrifugation, SDS-polyacrylamide gel electrophoresis, and the number of peptides produced by CNBr cleavage. The number of NADP binding sites was determined to be one per 25,500 dalton unit.     

Evaluation of DNA damage in leukocytes of G6PD-deficient Iranian newborns (Mediterranean variant) using comet assay

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited disease, which causes neonatal hemolytic anemia and jaundice. Recent studies of our group showed that the Mediterranean variant of this enzyme (Gd-Md) is the predominant G6PD in Iranian male infants suffering from jaundice; this variant is classified as severe G6PD deficiency. Considering the importance of G6PD reaction and its products NADPH and glutathione (GSH) against oxidative stress, we hypothesized the failure of detoxification of H(2)O(2) in G6PD-deficient white blood cells that could probably induce primary DNA damage. For the evaluation of DNA damage, we analyzed mononuclear leukocytes of 36 males suffering from the Gd-Md deficiency using alkaline single cell gel electrophoresis (SCGE) or comet assay. The level of DNA damage was compared with the level of basal DNA damage in control group represented by healthy male infant donors (of the same age group). Visual scoring was used for the evaluation of DNA damages. The results showed that the mean level of the DNA strand breakage in mononuclear leukocytes of 36 male G6PD-deficient (Gd-Md) infants was significantly higher (P < 0.001) than those observed in the normal lymphocytes. In conclusion, this investigation indicates that the mononuclear leukocytes of the Gd-Md samples may be exposed to DNA damage due to oxidative stress. This is the first report using comet assay for evaluation of DNA damage in severe G6PD deficiency samples.     

Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.     

Purification and characterization of glucose 6-phosphate dehydrogenase from sheep erythrocytes and inhibitory effects of some antibiotics on enzyme activity

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep erythrocytes, using a simple and rapid method. The purification consisted of three steps; preparation of haemolysate, ammonium sulphate fractionation and 2', 5'-ADP Sepharose 4B affinity chromatography. The enzyme was obtained with a yield of 37.1% and had a specific activity of 4.64 U/mg proteins. Optimal pH, stable pH, molecular weight, and KM and Vmax values for NADP+ and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. The overall purification was about 1,189-fold. A temperature of +4 degrees C was maintained during the purification process. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done in 4% and 10% acrylamide concentration for stacking and running gel, respectively. SDS-PAGE showed a single band for enzyme. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm. In addition, in vitro effects of gentamicin sulphate, penicillin G potassium, amicasin on sheep red blood cell G6PD enzyme activity were investigated. These antibiotics showed inhibitory effects on enzyme activity. I50 values were determined from Activity%-[Drug] graphs and Ki values and the type of inhibition (noncompetitive) were determined by means of Lineweaver-Burk graphs.     

Association of red cell glucose-6-phosphate dehydrogenase with haemoglobinopathies

A total of 1,112 randomly selected Saudi Arabs, of both sexes, living in Jeddah and the surrounding areas were screened for the phenotypic distribution of red cell glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). They were also investigated for haemoglobin and for thalassaemia. Phenotyping of the haemoglobins and the red cell enzymes was carried out by starch gel electrophoresis and the dye-decolouration screening test, while the investigation for thalassaemia was carried out by globin-chain biosynthesis, followed by column chromatography. The red cell Gd- alleles were significantly associated with the sickle-cell gene in both the males (chi 2(1): AS-28.80; SS-4.89) and females (chi 2(1): AS-10.99; SS-13.16). A similar association was also observed between G6PD deficiency and thalassaemias in males (chi 2(1): alpha-thalassaemia - 3.13; beta-thalassaemia - 11.06) and females (chi 2(1): alpha-thalassaemia - 6.63). However, no such association was detected between red cell 6PGD types and haemoglobin genes. The results suggest that the red cell G6PD deficiency, sickle-cell and thalassaemia genes might have evolved as a result of the same ecological factor, probably malaria.     

Glucose-6-phosphate dehydrogenase from Plasmodium berghei: kinetic and electrophoretic characterization

Evidence is given for the existence of a parasite-specific glucose-6-phosphate dehydrogenase (G6PD) in Plasmodium berghei by characterization of its kinetic and electrophoretic properties. From infected rat erythrocytes the parasites were isolated, washed, and lysed. G6PD was purified by affinity chromatography with 2'5'-ADP-Sepharose 4B, although the separation of the malaria-specific enzyme from that of the host cell was not complete. Malarial G6PD significantly differed from the red cell enzyme with respect to its electrophoretic properties. In cellulose acetate electrophoresis, a band with catodic mobility was observed in addition to the anodically mobile host cell enzyme at pH 7.0. The subunits of the parasite-specific G6PD have a molecular weight of 55 kDa in contrast to 59 kDa of red cell G6PD subunits. The enzyme from P. berghei shows no cross-reactivity with polyclonal antibodies against G6PD from rat erythrocytes. Thus, a close evolutionary relationship between both proteins and the presence of proteolytic modifications could be excluded. The Km value for G6P of malarial G6PD is increased by one order of magnitude compared with the host cell enzyme.     

Glucose-6-phosphate dehydrogenase isozymes in fish--a comparative study

The electrophoretic distribution and substrate specificities of isozymes of glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) were studied in seven species of teleost fish. The fish examined included two species of bonefish, Albula neoguinaica and A. glossodonta (Albulidae, Anquilliformes) (Shaklee and Tamaru, '81), and five representatives of the order Perciformes: two species of butterflyfish, Chaetodon miliaris and C. aurega (Chaetodontidae); a goatfish, Upeneus arge (Mullidae); a goby, Bathygobius fuscus (Gobiidae); and a snapper, Pristipomoides filamentosus (Lutjanidae). After horizontal starch gel electrophoresis, gel slices were stained using a variety of substrates and cofactors. In all species except the goby, two groups of isozymes were distinguished, corresponding to the mammalian G6PD (specific for glucose-6-phosphate (G6P) and NADP+) and H6PD (capable of utilizing galactose-6-phosphate and in certain cases other monosaccharide phosphates in addition to G6P). None of the five visible isozymes in the goby was specific for G6P. In each of the other species a single G6P- and NADP+-specific isozyme was noted, having the most rapid mobility toward the anode. In addition, it was found that all of the isozymes in all of the fish examined could catalyze the oxidation of fructose-6-phosphate at a rate comparable to that for G6P, suggesting that glucose-6-phosphate dehydrogenase can obviate the role of glucosephosphate isomerase in monosaccharide metabolism.     

Purification and Characterization of Glucose 6-phosphate Dehydrogenase from Sheep Erythrocytes and Inhibitory Effects of some Antibiotics on Enzyme Activity

Glucose 6-phosphate dehydrogenase (d -glucose 6-phosphate: NADP + oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep erythrocytes, using a simple and rapid method. The purification consisted of three steps; preparation of haemolysate, ammonium sulphate fractionation and 2′, 5′-ADP Sepharose 4B affinity chromatography. The enzyme was obtained with a yield of 37.1% and had a specific activity of 4.64 U/mg proteins. Optimal pH, stable pH, molecular weight, and K M and V max values for NADP + and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. The overall purification was about 1,189-fold. A temperature of +4°C was maintained during the purification process. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done in 4% and 10% acrylamide concentration for stacking and running gel, respectively. SDS-PAGE showed a single band for enzyme. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm. In addition, in vitro effects of gentamicin sulphate, penicillin G potassium, amicasin on sheep red blood cell G6PD enzyme activity were investigated. These antibiotics showed inhibitory effects on enzyme activity. I 50 values were determined from Activity %-[Drug] graphs and K i values and the type of inhibition (noncompetitive) were determined by means of Lineweaver-Burk graphs.     

Functional expression of human glucose-6-phosphate dehydrogenase in Escherichia coli

The complete coding sequence for human glucose-6-phosphate-dehydrogenase (G6PD) was inserted downstream from the tac promoter of a plasmid, pJF118EH, which also carries the lacIq repressor gene. When Escherichia coli strains (that are unable to grow on glucose due to the absence of functional zwf (G6PD-) and pgi genes) were transformed with this plasmid (pAC1), they were able to grow on glucose as sole carbon source. The rate of growth on glucose was faster in the presence of the inducer of the tac promoter, isopropyl-beta-D-thiogalactopyranoside (IPTG). Extracts of the transformed cells contained a G6PD activity that was not detectable in the parental strains and that was inducible by IPTG. The G6PD activities from normal E. coli and from pAC1-transformed cells comigrated with human G6PD when subjected to electrophoresis on agarose gels. However, when denatured, the G6PD produced by pAC1 was, like the human enzyme, distinguishable from the E. coli-encoded enzyme on the basis of its immunoreactivity with antibody specific for human G6PD. Therefore, human G6PD can be expressed in E. coli and can function to complement the bacterial enzyme deficiency.