Phosphorylation of C6- and C3-positions of glucosyl residues in starch is catalysed by distinct dikinases

Glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) are required for normal starch metabolism. We analysed starch phosphorylation in Arabidopsis wild-type plants and mutants lacking either GWD or PWD using (31)P NMR. Phosphorylation at both C6- and C3-positions of glucose moieties in starch was drastically decreased in GWD-deficient mutants. In starch from PWD-deficient plants C3-bound phosphate was reduced to levels close to the detection limit. The latter result contrasts with previous reports according to which GWD phosphorylates both C6- and C3-positions. In these studies, phosphorylation had been analysed by HPLC of acid-hydrolysed glucans. We now show that maltose-6-phosphate, a product of incomplete starch hydrolysis, co-eluted with glucose-3-phosphate under the chromatographic conditions applied. Re-examination of the specificity of the dikinases using an improved method demonstrates that C6- and C3-phosphorylation is selectively catalysed by GWD and PWD, respectively.     

Apparent Absence of a Translocase in the Cerebral Glucose-6-Phosphatase System

In the hepatocyte endoplasmic reticulum, a substrate transporter could provide a means of regulating hydrolysis of glucose-6-phosphate by specifically modulating access of the substrate to the hydrolase. Several characteristics of the cerebral microsomal enzyme suggest that such an hypothesis is untenable in the brain. These are: (a) the inability of the enzyme in either untreated or detergent-disrupted brain microsomes to distinguish between glucose-6-phosphate and mannose-6-phosphate; (b) the close agreement of the apparent Km values for either substrate in intact or disrupted microsomal preparations; (c) the constancy of the latency toward both substrates over a wide concentration range; (d) the inability of nonpenetrating, covalently-linking reagents [e.g., 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)] to affect the accessibility of the hydrolase to its substrate; (e) the absence of a putative transporter polypeptide, such as that of the liver, in experiments where tritiated H2DIDS, polyacrylamide gel electrophoresis, and radioautography are applied to brain microsomes.     

Fluorescent labeling of proteins with amine-specific 1,3,2-(2H)-dioxaborine polymethine dye

A novel water-soluble amine-reactive dioxaborine trimethine dye was synthesized in a good yield and characterized. The potential of the dye as a specific reagent for protein labeling was demonstrated with bovine serum albumin and lysozyme. Its interaction with proteins was studied by fluorescence spectroscopy and gel electrophoresis. The covalent binding of this almost nonfluorescent dye to proteins results in a 75- to 78-fold increase of its emission intensity accompanied by a red shift of the fluorescence emission maximum by 27 to 45 nm, with fluorescence wavelengths of labeled biomolecules being more than 600 nm. The dye does not require activation for the labeling reaction and can be used in a variety of bioassay applications.     

Regulation of specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in Penicillium chrysogenum

Abstract The specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase changed when Penicillium chrysogenum was grown on different carbon sources. In the presence of 2% lactose, the activities of these enzymes were approximately 25–35% lower than those in media containing 2% glucose or 2% fructose. We assume that an increase in cAMP concentration was responsible for the observed decreases in the enzyme activities, because a higher cAMP concentration could be detected when the mycelium was grown in a medium containing solely lactose as carbon source. The likely role played by cAMP in the regulation was also demonstrated by the addition of either cAMP or caffeine to the medium.     

New insights into the organisation and intracellular localisation of the two subunits of glucose-6-phosphatase

Glucose-6 phosphatase (G6Pase), a key enzyme of glucose homeostasis, catalyses the hydrolysis of glucose-6 phosphate (G6P) to glucose and inorganic phosphate. A deficiency in G6Pase activity causes type 1 glycogen storage disease (GSD-1), mainly characterised by hypoglycaemia. Genetic analyses of the two forms of this rare disease have shown that the G6Pase system consists of two proteins, a catalytic subunit (G6PC) responsible for GSD-1a, and a G6P translocase (G6PT), responsible for GSD-1b. However, since their identification, few investigations concerning their structural relationship have been made. In this study, we investigated the localisation and membrane organisation of the G6Pase complex. To this aim, we developed chimera proteins by adding a fluorescent protein to the C-terminal ends of both subunits. The G6PC and G6PT fluorescent chimeras were both addressed to perinuclear membranes as previously suggested, but also to vesicles throughout the cytoplasm. We demonstrated that both proteins strongly colocalised in perinuclear membranes. Then, we studied G6PT organisation in the membrane. We highlighted FRET between the labelled C and N termini of G6PT. The intramolecular FRET of this G6PT chimera was 27%. The coexpression of unlabelled G6PC did not modify this FRET intensity. Finally, the chimera constructs generated in this work enabled us for the first time to analyze the relationship between GSD-1 mutations and the intracellular localisation of both G6Pase subunits. We showed that GSD1 mutations did neither alter the G6PC or G6PT chimera localisation, nor the interaction between G6PT termini. In conclusion, our results provide novel information on the intracellular distribution and organisation of the G6Pase complex.     

葡萄糖-6-磷酸异构酶早期诊断类风湿关节炎的临床意义

目的：探讨血清葡萄糖6-磷酸异构酶（GPI）早期诊断类风湿关节炎（RA）的临床意义。方法：用ELISA法检测105例RA组、51例风湿病组、42例非风湿病组及40例健康对照组的血清GPI浓度,其中RA组分为早期组和中晚期组。同时收集RA患者类风湿因子（RF）、血沉（EsR）、免疫球蛋白、C-反应蛋白（CRP）、补体（C3、C4）、关节炎部位数等相关临床指标。结果：GPI对早期RA和中晚期RA诊断的敏感性分别为70．03％,和79．41％;特异性分别为89．89％和90．91％;二者敏感性、特异性比较均无显著差异;在RA患者中,GPI结果与RF、CRP、ESR、IgA、IgG、关节炎部位数均有相关性（P〈0．05）,与c3、C4、IgM无相关性;RF诊断RA的敏感性80．95％,特异性为78．19％,与GPI比较,二者敏感性无显著差异,特异性有差异（P〈0．05）,二者同时检测诊断RA的敏感性为69．52％,特异性达93．99％。结论：GPI诊断早期RA具有较好的敏感性和特异性,与RF联合检测对RA诊断具有很高的特异性,且可能成为判断RA病情活动的指标之一。     

Pentamidine activates T1 the hepatic microsomal glucose 6-phosphate transport protein of the glucose-6-phosphatase system

The mechanism of activation of hepatic microsomal glucose-6-phosphatase (EC 3.1.3.9) in vitro by pentamidine has been investigated in both intact and fully disrupted microsomes. The major effect of pentamidine is a 4.7-fold reduction in the K_m of glucose-6-phosphatase activity in intact diabetic rat liver microsomes. The site of action of pentamidine is T₁ the hepatic microsomal glucose 6-phosphate transport protein. The activation of T₁ by pentamidine may contribute to the disturbed blood glucose homeostasis see in many patients after administration of the drug pentamidine.     

G6P-capturing molecules in the periplasm of Escherichia coli accelerate the shikimate pathway

Escherichia coli, the most studied prokaryote, is an excellent host for producing valuable chemicals from renewable resources as it is easy to manipulate genetically. Since the periplasmic environment can be easily controlled externally, elucidating how the localization of specific proteins or small molecules in the periplasm affects metabolism may lead to bioproduction development using E. coli. We investigated metabolic changes and its mechanisms occurring when specific proteins are localized to the E. coli periplasm. We found that the periplasmic localization of β-glucosidase promoted the shikimate pathway involved in the synthesis of aromatic chemicals. The periplasmic localization of other proteins with an affinity for glucose-6-phosphate (G6P), such as inactivated mutants of Pgi, Zwf, and PhoA, similarly accelerated the shikimate pathway. Our results indicate that G6P is transported from the cytoplasm to the periplasm by the glucose transporter protein EIICB^Glc, and then captured by β-glucosidase.     

高等植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个酶.在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位.结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生.讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料.     

Investigation of the Mutation Points and Effects of Some Drugs on Glucose-6-phosphate Dehydrogenase-deficient People in the Erzurum Region

We have carried out a systematic study of the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency on three samples of 1,183 children aged 0.5–6 years from Erzurum, in eastern Anatolia. Total genomic DNAs were isolated from the blood samples of a healthy person and the three persons determined with G6PD deficiency by examining the enzyme activity and hemoglobin ratio. Then PCR amplification of the entire coding region in eight fragments was carried out followed by Agarose gel electrophoresis. The 540-bp PCR fragment containing exons VI-VII and the 550 bp PCR fragment containing exons XI-XIII were digested with EcoRI and with NIaIII, respectively. SSCP techniques for eight fragments (exons II, III-IV, V, VI-VII, VIII, IX, X, and XI-XIII) were employed to determine the mutations on the exons of the G6PD gene. A mutation occurred on the region of the exons 6 and 7 of one person (person-1) and exon 5 of two G6PD-deficient persons (person 2 and 3) examined. The sequential approach described is fast and efficient and could be applied to other populations.

Effects of analgesic drugs on G6PD were studied on the purified enzyme (ammonium fractionation, dialysis and 2',5' ADP-Sepharose 4B affinity chromatography) for the healthy person and G6PD-deficient persons 1, 2 and 3. The effects of remifentanil hydrochloride, fentanyl citrate, alfentanil hydrochloride and pethidine hydrochloride, as analgesic drugs, on G6PD activity were tested. Although remifentanil hydrochloride, fentanyl citrate (I₅₀ values; 1.45 mM and 6.1 mM, respectively) inhibited the activity of the enzyme belonging to the healthy person, they did not alter enzyme activity on two of the three persons with G6PD deficiency. Other drugs (alfentanil hydrochloride and pethidine hydrochloride) did not effect the enzyme activity of the healthy or G6PD-deficient children.     

Effects of cadmium and zinc ions on purified lamb kidney cortex glucose-6-phosphate dehydrogenase activity

Glucose-6-phosphate dehydrogenase (G-6-PD) is the first enzyme in the pentose phosphate pathway. Cadmium is a toxic heavy metal that inhibits several enzymes. Zinc is an essential metal but overdoses of zinc have toxic effects on enzyme activities. In this study G-6-PD from lamb kidney cortex was competitively inhibited by zinc both with respect to glucose-6-phosphate (G-6-P) and NADP⁺ with Ki values of 1.066 ± 0.106 and 0.111 ± 0.007 mM respectively whereas cadmium was a non-competitive inhibitor with respect to both G-6-P and NADP⁺ Ki values of 2.028 ± 0.175 and 2.044 ± 0.289 mM respectively.     

Molecular mechanism of glucose-6-phosphate utilization in the dinoflagellate Karenia mikimotoi

Phosphorus (P) is an essential nutrient for marine phytoplankton as for other living organisms, and the preferred form, dissolved inorganic phosphate (DIP), is often quickly depleted in the sunlit layer of the ocean. Phytoplankton have developed mechanisms to utilize organic forms of P (DOP). Hydrolysis of DOP to release DIP by alkaline phosphatase is believed to be the most common mechanism of DOP utilization. Little effort has been made, however, to understand other potential molecular mechanisms of utilizing different types of DOP. This study investigated the bioavailability of glucose-6-phosphate (G6P) and its underlying molecular mechanism in the dinoflagellate Karenia mikimotoi. Suppression Subtraction Hybridization (SSH) was used to identify genes up- and down-regulated during G6P utilization compared to DIP condition. The results showed that G6P supported the growth and yield of K. mikimotoi as efficiently as DIP. Neither DIP release nor AP activity was detected in the cultures grown in G6P medium, however, suggesting direct uptake of G6P. SSH analysis and RT-qPCR results showed evidence of metabolic modifications, particularly that mitochondrial ATP synthase f1 gamma subunit and thioredoxin reductase were up-regulated while diphosphatase and pyrophosphatase were down-regulated in the G6P cultures. All the results indicate that K. mikimotoi has developed a mechanism other than alkaline phosphatase to utilize G6P.     

Invalidity of Criticisms of the Deoxyglucose Method Based on Alleged Glucose-6-Phosphatase Activity in Brain

The observations made by Sacks et al. [Neurochem. Res. 8, 661-685 (1983)] on which they based their criticisms of the deoxyglucose method have been examined and found to have no relationship to the conclusions drawn by them. (1) The observations of Sacks et al. (1983) of constant concentrations of [14C]deoxyglucose and [14C]deoxyglucose-6-phosphate, predominantly in the form of product, reflects only the postmortem phosphorylation of the precursor during the dissection of the brain in their experiments. When the brains are removed by freeze-blowing, the time courses of the [14C]deoxyglucose and [14C]deoxyglucose-6-phosphate concentrations in brain during the 45 min after the intravenous pulse are close to those predicted by the model of the deoxyglucose method. (2) Their observation of a reversal of the cerebral arteriovenous difference from positive to negative for [14C]deoxyglucose and not for [14C]glucose after an intravenous infusion of either tracer is, contrary to their conclusions, not a reflection of glucose-6-phosphatase activity in brain but the consequence of the different proportions of the rate constants for efflux and phosphorylation for these two hexoses in brain and is fully predicted by the model of the deoxyglucose method. (3) It is experimentally demonstrated that there is no significant arteriovenous difference for glucose-6-phosphate in brain, that infusion of [32P]glucose-6-phosphate results in no labeling of brain, and that the blood-brain barrier is impermeable to glucose-6-phosphate. Glucose-6-phosphate cannot, therefore, cross the blood-brain barrier, and the observation by Sacks and co-workers [J. Appl. Physiol. 24, 817-827 (1968); Neurochem. Res. 8, 661-685 (1983)] of a positive cerebral arteriovenous difference for [14C]glucose-6-phosphate and a negative arteriovenous difference for [14C]glucose cannot possibly reflect glucose-6-phosphatase activity in brain as concluded by them. Each of the criticisms raised by Sacks et al. has been demonstrated to be devoid of validity.     

Selective synthesis of 2',3'-cyclic nucleotide 3'-phosphodiesterase isoform 2 and identification of specifically phosphorylated serine residues

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is a protein found abundantly in the cytoplasmic compartments of CNS myelin. Two isoforms of this protein, CNP1 and CNP2, are detectable. They differ by a 20-amino acid extension exclusive to CNP2. Additionally, CNP2 is essentially the only isoform to be phosphorylated in vivo. In this study, we examine the phosphorylation of CNP2 in transfected cells. CNP2 was selectively expressed ectopically in 293T cells and labeled with 32P. Immunoprecipitation of labeled CNP2 and tryptic phosphopeptide mapping analyses identified serines 9 and 22 as the major sites of phosphorylation. Only serine 22 was phosphorylated initially in oligodendrocyte-enriched cultures of neonatal rat brain glial cells. However, 4beta-phorbol 12,13-dibutyrate (PDB) induced the phosphorylation of serine 9, thereby producing the same pattern seen in 293T cells. These results suggest that serine 9 is phosphorylated by a PDB-sensitive kinase, likely protein kinase C, and that serine 22 appears to be constitutively phosphorylated.     

Investigation of the effects of some sulfonamide derivatives on the activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase from human erythrocytes

In this study, the in vitro effects of some sulfonamide derivatives, which are carbonic anhydrase inhibitors, on the enzymes activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase were investigated. For this purpose, these three enzymes were purified from human erythrocytes. Purification procedure composed of four steps; preparation of the hemolysate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography, and gel filtration chromatography on Sephadex G-200. 5-(3α-Hydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (1), 5-(3α,12α-Dihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (2), 5-(3α,7α,12α-Trihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (3), 5-(3α,Acetoxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (4), 5-(3α,7α,12α-Triacetoxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (5), 5-(3,7,12-Trioxo-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (6), acetazolamide, and dorzolamide were tested in this experiment. Compounds 3, 5, and dorzolamide showed inhibitory effects on the activity of 6-phosphogluconate dehydrogenase, and I₅₀ values and K_i constants were calculated as 0.0601 mM, 0.00253 mM, and 1.41 mM and 0.0878 ± 0.0274 mM, 0.0042 ± 0.0009 mM, and 3.1446 ± 0.2081 mM, respectively. Glutathione reductase was also inhibited by 1 and 2. I₅₀ values and K_i constants were 0.0471 mM and 0.0723 ± 0.0388 mM for 1 and 0.0045 mM and 0.0061 ± 0.0014 mM, for 2. If these sulfonamide derivatives are proposed as drugs, some of which are being used in glaucoma treatment such as acetazolamide and dorzolamide, these results should be taken into consideration concerning via these enzymes.     

还原型谷胱甘肽治疗儿童葡萄糖-6-磷酸脱氢酶缺乏症中的临床效果研究

目的:研究还原型谷胱甘肽治疗儿童葡萄糖-6-磷酸脱氢酶(G-6-P-D)缺乏症并发急性溶血的临床疗效,为临床治疗提供参考。方法:选取我院2015年6月-2017年6月因葡萄糖-6-磷酸脱氢酶(G-6-P-D)缺乏症并发急性溶血的患儿78例并将其随机分为两组,每组39例。对照组予以停用氧化类药物,卧床休息,水化、碱化尿液,贫血严重者输注去白红细胞治疗;观察组在对照组基础上加用还原型谷胱甘肽治疗。观察和比较两组患儿第1天、第2天、第3天小便恢复率以及平均恢复时间,血清总胆红素第3天、第5天恢复率、平均恢复时间及平均住院时间。结果:治疗后,观察组第1天、第2天、第3天小便恢复率分别为51.3%、92.3%、100%,对照组分别为25.6%、64.1%、89.7%,观察组第1天、第2天、第3天小便恢复率均显著高于对照组(P0.05);观察组及对照组小便恢复正常平均时间分别为1.8±0.7天、2.6±0.9天,观察组明显短于对照组(P0.05);观察组第3天、第5天血清总胆红素恢复率分别为71.8%、100%,对照组为46.2%、97.4%;观察组和对照组血清总胆红素恢复正常平均时间分别为3.6±0.9天、4.1±1.0天;平均住院时间分别为2.3±0.6天、2.8±0.6天;观察组小便及血清总胆红素平均恢复时间(P0.05)、平均住院时间均显著短于对照组(P0.05)。结论:在儿童葡萄糖-6-磷酸脱氢酶缺乏并发急性溶血中应用还原型谷胱甘肽可增强其治疗疗效,缩短治疗疗程。     

Mutual stimulation of temperature and light effects on C(4) phosphoenolpyruvate carboxylase in leaf discs and leaves of Amaranthus hypochondriacus

This article reports marked modulation of the activity and regulatory properties of phosphoenolpyruvate carboxylase (PEPC) by temperature and light in leaf discs as well as leaves of Amaranthus hypochondriacus. The activity of PEPC increased by 1.7-fold at 45 degrees C over 25 degrees C. Warm temperature also stimulated the photoactivation of PEPC. The activation by light of PEPC was 1.9-fold at 25 degrees C and increased to 2.2-fold at 45 degrees C. The sensitivity of PEPC to its inhibitor malate was less and the activation by glucose-6-phosphate (G-6-P) or inorganic phosphate (Pi) was more at 45 degrees C than that at 25 degrees C. These effects of temperature were quite pronounced in light. Similar responses were observed when detached leaves were exposed to varying ambient temperature (dry heat). The activity of PEPC increased by 1.6-fold at 45 degrees C over 25 degrees C in the dark. The activation of PEPC by light was 2.1-fold at 25 degrees C and increased to 2.6-fold at 45 degrees C. Inhibition by malate was less and activation by G-6-P or Pi was more at 45 degrees C than that at 25 degrees C. Thus, there was a marked modulation of not only the activity but also the regulatory properties of the enzyme by temperature and light, independently as well as cooperatively with each other. Further experiments suggested that PEPC was able to memorize to a significant extent the changes induced by warm temperature and that these changes were complemented by subsequent illumination. These effects were not due to changes in PEPC protein levels. We conclude that temperature and light can modulate PEPC activity and regulatory properties not only individually but also in a significantly cooperative manner with each other. As significant increases in temperature are common during daytime in tropical or subtropical conditions, we suggest that the synergistic effects of temperature and light are quite relevant in optimizing the activity of PEPC in leaves of C(4) plants.     

Evidence for the expression of both the hydrolase and translocase components of hepatic glucose-6-phosphatase in murine pancreatic islets

Glucose-6-phosphatase (G6Pase) is a multicomponent enzyme system which regulates the catalysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate. G6Pase can antagonize glucose phosphorylation, a step prerequisite in the regulation of insulin secretion from pancreatic beta cells, and G6Pase activity is increased in islets isolated from animal models of type II diabetes. Using RT-PCR with hepatic G6Pase catalytic subunit primers, we demonstrate that the sizes of amplified products from ob/ob mouse islets are identical to those from liver cDNA. This was confirmed by PCR-based cloning and sequencing of the hepatic G6Pase catalytic subunit open reading frame from islet cDNA. The expression in islets of the G6P transporter, G6PT1, was also demonstrated, suggesting that all of the identified hepatic G6Pase system genes are expressed in pancreatic islets. Finally, the expression of islet-specific G6Pase-related protein (IGRP) in pancreatic islets was confirmed and its expression in liver was also observed.     

Measurement of glucose-6-phosphate dehydrogenase and glutathione reductase activity in patients with paracoccidioidomycosis treated with ketoconazole

Hemoglobin rates, hematocrit and glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase activities were measured in 38 patients with paracoccidioidomycosis treated with ketoconazole or sulfadoxin, and in 13 normal individuals.Ketoconazole-treated patients showed reduced G6PD and glutathione reductase activities. One of these patients was found to be G6PD-deficient and suffered a hemolytic episode during treatment, which, however, did not require interruption of therapy.The authors suggest that patients showing an erythrocyte enzyme defect should be monitored hematologically during treatment with ketoconazole. They also suggest that ketoconazole is an oxidant drug in addition to being a possible inhibitor of antioxidant erythrocyte enzymes.