Characterization of erythrocyte membrane-associated enzymes (glyceraldehyde-3-phosphate dehydrogenase and phosphoglyceric kinase).

The purpose of our study was to determine why several of the glycolytic enzymes of the erythrocyte have the propensity for adhering to erythrocyte membranes while others do not. Two of these membrane-associated enzymes, glyceraldehyde phosphate dehydrogenase (GAPD) and phosphoglyceric kinase (PGK), have been shown to have controlling functions on intra-erythrocytic glycolysis and sodium-potassium transport. In order to test the hypothesis that the membrane-associated fraction of these enzymes consisted of isozymes with increased capacity to become membrane associated, the enzymes from cytosol or membrane sources were partially purified and characterized. Determination of molecular weight by gel filtration chromatography, measurement of certain kinetic parameters, and the curves relating to pH optimal activity indicated that there were no measurable differences between membrane and cytosol PGK and membrane and cytosol GAPD. It was possible to raise inhibitory antibodies to GAPD in certain species of mice, and these antibodies did not distinguish between GAPD isolated from either membrane or cytosol sources. GAPD was firmly bound to membranes and the membrane-associated fraction counted for approximately 60 per cent of total erythrocytic enzyme. Membrane-associated PGK was only loosely adherent, and accounted for only 1 per cent of the total erythrocytic enzyme. The reasons for membrane association have yet to be determined, but these inward facing membrane-associated enzymes appear to function by carrying the organization of the membrane into the cell interior.     

3-磷酸甘油醛脱氢酶活性检测方法的改良及应用

目的 改良3-磷酸甘油醛脱氢酶(GAPDH)活性检测方法,并探讨该方法的应用.方法在经典血清GAPDH检测方法基础上,根据该酶的组织分布特点,将血清样本改为全血样本,并对样本及检测条件作了进一步的改良.在方法学研究的基础上,运用改良后的方法分别检测正常鹌鹑、高嘌呤饮食诱导的模型鹌鹑、正常大鼠及高果糖饮食诱导的模型大鼠全血GAPDH的活性.结果 全血与血清GAPDH酶促反应时间曲线类似,变异系数均小于10%.方法 学应用结果显示,与正常鹌鹑比,高嘌呤饮食诱导的模型鹌鹑在高尿酸血症及其合并脂、糖代谢紊乱状态下(60～140 d)全血GAPDH活性显著降低;与正常大鼠比,高果糖饮食诱导的模型大鼠在高三酰甘油血症及其并发尿酸、糖代谢紊乱下(7～28 d),全血GAPDH活性显著降低.结论 改良方法血样容易采集,样品用量少,操作简便、重复性较好,经济实用,易于推广应用.     

Thermal stability and structure of glyceraldehyde-3-phosphate dehydrogenase from the coral Acropora millepora

Corals are vulnerable to increasing ocean temperatures. It is known that elevated temperatures lead to the breakdown of an essential mutualistic relationship with photosynthetic algae. The molecular mechanisms of this temperature-dependent loss of symbiosis are less well understood. Here, the thermal stability of a critical metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, from the stony coral Acropora millepora was found to increase significantly in the presence of its cofactor NAD⁺. Determination of the structure of the cofactor–enzyme complex (PDB ID 6PX2) revealed variable NAD⁺ occupancy across the four monomers of the tetrameric enzyme. The structure of the fully occupied monomers was compared to those with partial cofactor occupancy, identifying regions of difference that may account for the increased thermal stability.

The thermal stability of a critical coral metabolic enzyme increases significantly in the presence of its cofactor. X-ray crystallography identifies the protein backbone changes associated with cofactor occupancy.     

The inhibition of d-glyceraldehyde 3-phosphate dehydrogenase by specific antiserum

Antibodies to yeast d-glyceraldehyde 3-phosphate dehydrogenase have been produced in rabbits and chickens. The antiserum from either animal inhibits the activity of the yeast enzyme with the formation of a precipitate that has a low residual activity. One combining site of antibody on the enzyme appears to be the point of DPN attachment. The rate of antigen-antibody formation has been studied with varying concentrations of antibody. The activity of yeast hexokinase is not inhibited by the antiserum to the dehydrogenase. Five of six antisera to the yeast enzyme showed no inhibition of the analogous enzyme from rabbit muscle, but one serum strongly inhibited the muscle dehydrogenase in the absence of DPN. No evidence was obtained that the inhibition by this one antiserum was due to an immune reaction, because the inhibition persisted after absorption with the homologous antigen.     

福建畲族葡萄糖-6-磷酸脱氢酶基因突变型

目的研究福建畲族葡萄糖-6-磷酸脱氢酶（G6PD）基因突变型特点，调查其G6PD缺乏症发生率及基因频率。方法用硝基四氮唑蓝（NBT）纸片法进行G6PD缺乏症定性筛查，用突变特异性扩增系统、错配碱基PCR介导酶切位点／限制性内切酶图谱分析、聚合酶链反应．单链构象多态性分析（PCR-SSCP）和DNA测序进行基因突变型鉴定结果在1820名青水畲族和764名穆云畲族人中，分别发现101例和104例酶活性异常，两地的致病基因频率分别为0．0607和0．1706。在穆云乡54例纯畲族血缘的样本中，共检出nt1376G→T突变38例、nt1388G→A突变12例、nt95A→G突变6例，三种基因突变型分别占70．3％，18．5％和11．1％；并发现1例nt1376G→T复合nt95A→G突变、1例nt1376G→T复合nt1388G→A突变。在青水畲族中发现6例nt1024C→T突变，占8．6％；发现1例392G→T突变，结论①nt1376G→T、nt1388G→A是幅建畲族中主要的基因突变型，中华民族具有共同的G6PD基因突变型，因而可能源于共同的祖先。②在畲族人群中发现nt1376G→T、nt1388G→A、nt95A→G、nt1024C→T和nt392G→T 5种基因突变型。③发现nt1376G→T复合nt95A→G突变、nt1376G→T复合nt1388G→A突变④nt95A→G是穆云畲族中另一种常见的基因突变型；1024C→T是青水畲族中常见的一种G6PD基因突变型。⑤明确青水畲族中G6PD基因频率为0．0607，穆云畲族中为0．1706，为上述地区G6PD缺乏症的防治提供决策参考。     

Genetically determined deficiency of glucose 6-phosphate dehydrogenase (type-A-) is expressed in the liver.

G6PD activity was determined in liver biopsy specimens from 31 patients (25 men and six women). The G6PD genotype of the patients was determined by carrying out on lysates of their red blood cells quantitative assays of the enzyme and starch-gel electrophoresis. In 21 subjects with normal G6PD activity in red cells, a relatively wide variation of G6PD activity was found in liver extracts. By contrast, in 10 subjects with G6PD deficiency, the activity of the enzyme in liver extracts was always low. The difference between the distribution of liver G6PD activity values of G6PD-normal and G6PD-deficient subjects was statistically significant (p less than 0.01). We conclude that G6PD deficiency of the African type is also expressed in the liver. These findings may have a bearing on hyperibilirubinemia, which cannot be entirely attributed to hemolysis, often encountered in G6PD-deficient patients.     

我国葡萄糖-6-磷酸脱氢酶缺乏症的分布特征和基因突变

葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenas,G6PD)缺乏症是最常见的遗传性酶缺陷疾病,可导致急性溶血性贫血、新生儿高胆红素血症和慢性非球形红细胞溶血性贫血等疾病,其发生率和基因突变类型有明显的地域和种族特异性,本文主要综述了中国不同民族和地区G6PD缺乏症的分布特点和基因突变.     

样品放置时间对葡萄糖-6-磷酸脱氢酶活性的影响

目的了解样本不同的保存时间和温度对G6PD活性的影响.方法用速率法测定20份全血标本及溶血液制备后放置各时间段的G6PD活性.结果ACD抗凝的全血标本放置室温及4℃冰箱48小时内G6PD活性与即刻测定的结果比较无显著性差异(P＞0.05),而放置72小时G6PD活性均明显下降(P＜0.01,P＜0.05).溶血液4℃冰箱放置2小时结果与放置15分钟时的结果比较无显著性差异(P＞0.05),4小时结果明显偏低(P＜0.05).结论G6PD活性测定时,溶血液制备后4℃冰箱放置不能超过2小时,全血标本应在48小时内完成检测.     

Rapid detection of glucose-6-phosphate dehydrogenase gene mutations by denaturing high-performance liquid chromatography

OBJECTIVES: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide. Different kinds of G6PD mutations may result in variable severity of clinical onset in G6PD-deficient individuals. In this study, a reliable molecular diagnostic method was developed for rapid detection of G6PD gene mutation. DESIGN AND METHODS: Primers were designed to amplify G6PD gene fragments that were subjected to mutation screening using denaturing high-performance liquid chromatography (DHPLC) analysis. Mutations were identified by their distinct elution peak patterns and were confirmed by DNA sequencing. The assay was further validated against 29 samples from individuals with G6PD deficiency. RESULTS: A DHPLC-based assay for G6PD mutation detection was established. The 9 common G6PD mutations in the Taiwanese and Chinese population could be distinguished through the analysis of DNA elution patterns. During the validation test with the 29 G6PD deficiency specimens, two additional rare mutations, T517C and C519G, were unveiled. Overall, the DHPLC-based mutation detection was 100% concordant with the DNA sequencing results. CONCLUSION: Compared to other genotyping techniques, this method requires significantly less technical time to perform and has a greatly increased throughput capacity. Hence, the DHPLC method represents a major technical advance for G6PD genotyping and should benefit G6PD-deficient individuals for proper clinical care.     

Screening of jaundiced neonates for glucose-6-phosphate dehydrogenase deficiency

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is transmitted as an X-linked recessive disorder, and thus female infants are expected to be only rarely affected. Review of the records of 1,478 jaundiced newborn infants (728 boys and 750 girls) screened for G6PD deficiency at the Foothills Provincial Hospital in Calgary showed 41 (5.6%) boys and 17 (2.2%) girls with this disorder. In view of the unexpected and unexplained high frequency of G6PD deficiency in female infants, I recommend that screening for this disorder be done in selected jaundiced infants regardless of sex.     

Microheterogeneity of glucose-6-phosphate dehydrogenase phenotypes

Glucose-6-phosphate dehydrogenase (G-6-PD) is the first enzyme of the pentose phosphate pathway and has been extensively investigated. Several variants of G-6-PD were encountered in different regions of Saudi Arabia during an extensive screening programme. A modified rapid isoelectric focussing procedure using LKB Ampholine PAG plates (pH 5.5-8.5) was used to separate the isoenzymes of G-6-PD. G-6-PD-B+ (the normal enzyme) was separated into five major and three minor fractions. The pattern obtained for G-6-PD-A+, G-6-PD-A-, G-6-PD-Mediterranean and G-6-PD-Mediterranean-like showed that the variants differ significantly in their isoenzyme pattern. In this paper it is shown that isoelectric focussing is a sensitive, powerful and reproducible technique to study microheterogeneity of red cell enzymes.