糖基化3－磷酸甘油醛脱氢酶的分离纯化及糖基代位点的探讨

应用糖基化蛋白亲和层析技术对兔肌及人红细胞的３－磷酸甘油醛脱氢酶的分离分析表明,兔肌非糖基化ＧＡＰＤＨ的比活为１８０—２００单位,而糖基化ｇＧＡＰＤＨ的为４０—５０单位,并占该酶蛋白总量的４０％。人类红细胞糖基化ｇＧＡＰＤＨ的活力占其总活力的５５％左右。以上结果表明：哺乳动物体内存在糖基化３－磷酸甘油醛脱氢酶。由于（１）糖基化明显影响ＧＡＰＤＨ的活力;（２）糖基化酶活性部位的巯基（Ｃｙｓ－１４９）空间位置发生了改变;（３）糖基化影响活性部位的空间构象及（４）ＯＰＴ对糖基化及非糖基化酶的修饰无论在动力学上还是在ＫＩ淬灭时都有明显差异,因此,糖基化的位点可能与赖氨酸残基有关,并且接近或位于酶的活性部位。     

糖基化3－磷酸甘油醛脱氢酶的含糖量及其构象变化

通过对ＧＡＰＤＨ及ｇＧＡＰＤＨ含糖量、ＣＤ、荧光及ＤＴＮＢ的修饰表明：用间氨基苯硼酸琼脂糖（ｍ－ＡＰＢＡ－ＳｅｐｈａｒｏｓｅＣＬ６Ｂ）亲和层析法分离的兔肌ｇＧＡＰＤＨ每分子含有１．８９个糖基。ｇＧＡＰＤＨ及ＧＡＰＤＨ的远紫外ＣＤ谱差别较小,但近紫外差别较明显。两者内源荧光在不同浓度的ＧｕＨＣｌ溶液中的变化亦有一定差异。ＤＴＮＢ对酶活性部位巯基的修饰表明,ｇＧＡＰＤＨ的ＤＴＮＢ修饰的快相一级动力学常数大于ＧＡＰＤＨ动力学常数一个数量级。以上结果提示：糖基化导致酶分子及活性部位的空间结构改变,糖基化位点可能发生在酶活性部位附近。     

糖基化3－磷酸甘油醛脱氢酶的含糖量及其构象变化

通过对ＧＡＰＤＨ及ｇＧＡＰＤＨ含糖量、ＣＤ、荧光及ＤＴＮＢ的修饰表明：用间氨基苯硼酸琼脂糖（ｍ－ＡＰＢＡ－ＳｅｐｈａｒｏｓｅＣＬ６Ｂ）亲和层析法分离的兔肌ｇＧＡＰＤＨ每分子含有１．８９个糖基。ｇＧＡＰＤＨ及ＧＡＰＤＨ的远紫外ＣＤ谱差别较小,但近紫外差别较明显。两者内源荧光在不同浓度的ＧｕＨＣｌ溶液中的变化亦有一定差异。ＤＴＮＢ对酶活性部位巯基的修饰表明,ｇＧＡＰＤＨ的ＤＴＮＢ修饰的快相一级动力学常数大于ＧＡＰＤＨ动力学常数一个数量级。以上结果提示：糖基化导致酶分子及活性部位的空间结构改变,糖基化位点可能发生在酶活性部位附近。     

邻苯二甲醛对糖基化3－磷酸甘油醛脱氢酶的修饰及荧光性质的研究

ＯＰＴ修饰ＧＡＰＤＨ及ｇＧＡＰＤＨ的荧光衍生物与Ｔｒｐ残基之间存在非辐射的能量传递。在不同浓度的ＧｕＨＣｌ溶液中,糖基化和非糖基化酶ＯＰＴ衍生物的荧光的变化具有一定的差异。特别是两者的荧光在碘化钾溶液中的淬来有明显的不同。ＯＰＴ修饰动力学研究表明,ｇＧＡＰＤＨ的修饰速度快于ＧＡＰＤＨ的修饰速度。以上结果提示：糖基化的位点可能在赖氨酸残基上,并且被糖基化的残基可能位于或靠近活性部位。     

邻苯二甲醛对糖基化3－磷酸甘油醛脱氢酶的修饰及荧光性质的研究

ＯＰＴ修饰ＧＡＰＤＨ及ｇＧＡＰＤＨ的荧光衍生物与Ｔｒｐ残基之间存在非辐射的能量传递。在不同浓度的ＧｕＨＣｌ溶液中,糖基化和非糖基化酶ＯＰＴ衍生物的荧光的变化具有一定的差异。特别是两者的荧先在碘化钾溶液中的淬灭有明显的不同。ＯＰＴ修饰动力学研究表明,ｇＧＡＰＤＨ的修饰速度快于ＧＡＰＤＨ的修饰速度。以上结果提示：糖基化的位点可能在赖氨酸残基上,并且被糖基化的残基可能位于或靠近活性部位。     

用双水相萃取分离纯化磷酸甘油酸激酶和磷酸甘油醛脱氢酶

用聚乙二醇（ＰＥＧ）／羟丙基淀粉（ＲｅｐｐａｌＰＥＳ）双水相体系两步法从黄豆中分离磷酸甘油酸激酶（ＰＧＫ）和磷酸甘油醛脱氢酶（ＧＡＰＤＨ）。ＰＧＫ在上相收率及ＧＡＰＤＨ在下相收率均在８０％以上。放大采用离心倾析机（ｄｅｃａｎｔｅｒ）连续处理匀浆液,用离心萃取器（ｓｅｐａｒａｔｏｒ）完成双水相体系的萃取两相分离。整个工艺具有处理量大、接触时间短、酶收率高的优点     

糖基化对乙型肝炎表面抗原疫苗的影响

哺乳动物（ＣＨＯ）细胞表达的三种糖基化程度不同的乙肝表面抗原（ＨＢｓＡｇ）Ａ（含ＧＰ３０,ＧＰ２７,Ｐ２３）,Ｂ（含ＧＰ２７及Ｐ２３）,Ｃ（只有Ｐ２３）,在免疫原性、放置后抗原的稳定性以及对不同单克隆抗体亲和力等方面都有明显的差异;（１）免疫ＢＡＬＢ／Ｃ小鼠后血清中抗体工（ＥＤ５０）。平均结果为Ａ＝１：８０,Ｂ＝１：１１７,Ｃ＝１：１４,表明有糖基的ＨＢｓＡｇ疫苗对小鼠的免疫力明显高于无糖基的ＨＢ     

南方鲇碱性磷酸酶的分离纯化及部分性质的研究

用正丁醇抽提,硫酸铵分级沉淀,ＤＥＡＥ－纤维素和ＳｅｐｈａｃｒｙｌＳ－２００柱层析,从南方鲇（Ｓｉｌｕｒｕｓ　ｍｅｒｉｄｉｏｎａｌｉｓ　Ｃｈｅｎ）肠粘膜中提取出碱性磷酸酶（ＡＫＰ）。提纯倍数为３９．５０倍,比活为６８．３５μ／ｍｇ蛋白,提取酶液经ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ只呈现一条区带。该酶的分子量为１３２１４０,Ｎ末端氨基酸为门冬氨酸,最适ｐＨ为１０．１０,７．５＞ｐＨ＞１１．５时不稳定,最适温度为４０℃左右,对热不很稳定,以磷酸苯二钠为底物其Ｋ_ｍ值为１．７２×１０~（－３）ｍｏｌ／Ｌ。Ｍｇ~（２＋）、Ｍｎ~（２＋）为该酶的激活剂,ＫＨ_２ＰＯ_４、Ｌ－ＣｙＳ、ＭＥ、ＤＦＰ、ＥＤＴＡ－Ｎａ_２为抑制剂。选用ＫＨ_２ＰＯ_４和ＤＦＰ作抑制类型的判断,结果表明,ＫＨ_２ＰＯ_４属竞争性掏剂,其抑制常数为２．３ｍｍｏｌ／Ｌ;ＤＦＰ为非竞争性抑制剂,抑制常数为１．０５ｍｍｏｌ／Ｌ。     

高原红细胞增多症患者红细胞二磷酸甘油酸和氧亲和力变化的探讨

在海拔４３００ｍ地区,对１８名移居汉族、２４名世居藏族和２１名高原红细胞增多症（ＨＡＰＣ）患者测定了２,３—二磷酸甘油酸（２,３—ＤＰＧ）和肺通气功能,并进行了血气分析。结果显示：ＨＡＰＣ患者的全血和红细胞内２,３—ＤＰ６浓度均显著高于健康组,但世居、移居健康组之间无明显差异。ＨＡＰＣ组的红细胞２,３—ＤＰＧ和Ｐｄｏ＿２呈显著负相关（ｒ＝—０．７７１,Ｐ＜０．０１）,而在健康组无显著相关（ｒ＝—０．２６,Ｐ＞０．０５）。ＨＡＰＣ组与健康组相比,ｐＨ、Ｐａｏ＿２和Ｓａｏ＿２降低,Ｐａｃｏ＿２和肺泡动脉氧分压差增高。ＨＡＰＣ病人Ｐ＿（５０）为３．７５±０．６６ｋＰａ,健康组为３．４０±０．１２ｋＰａ（Ｐ＜０．０５）,Ｐ＿（５０）与２,３－ＤＰＧ呈正相关（ｒ＝０．５９２,Ｐ＜０．０５）。ＨＡＰＣ组最大呼气中段流量和５０％肺活量最大呼气量明显低于健康组（Ｐ＜０．０１）。本研究提示：①ＨＡＰＣ患者的低氧血症可能与血中２,３－ＤＰＧ浓度过高有关;②轻度肺功能异常亦可促使红细胞进一步增多。     

甘油醛-3-磷酸脱氢酶与辅酶类似物ATP及底物磷酸结合特性的晶体学研究

气相扩散共晶生长法培养出Ｐ．ｖｅｒｓｉｃｏｌｏｒ龙虾肌ＡＴＰ－Ｄ－甘油醛－３－磷酸脱氢酶（ＡＴＰ－ＧＡＰＤＨ）的晶体。用同步辐射Ｘ光源－磷光储屏－Ｗｅｉｓｓｅｎｂｅｒｇ照相机系统收集了一套２．０分辨率的衍射数据。用同晶差值傅立叶法解析了其结构。精化后的结构模型最终Ｒ因子为０．１９７,与标准键长、键角的均方根偏差为０．０１６°和３．２０°。ＰｖＡＴＰ－ＧＡＰＤＨ结构总体上和Ｐｖａｐｏ－ＧＡＰＤＨ相似。ＡＴＰ分子的占有率较低,并表现出一定程度的无序性,提示ＡＴＰ与酶蛋白结合的稳定性较低,表明ＮＡＤ＋的尼克酰胺核苷部分与蛋白质分子的作用在辅酶与蛋白质的稳定结合中起关键作用。ＡＴＰ－ＧＡＰＤＨ中每个亚基只有一个磷酸结合位点（Ｐｉ）。认为无机磷酸结合位点Ｐｉ的形成不依赖于ＮＡＤ＋,而底物磷酸结合位点ＰＳ的形成则依赖于ＮＡＤ＋的存在。     

Glycation-induced matrix stability in the rabbit achilles tendon

Connective tissue susceptibility to nonenzymatic glycation was examined following 0, 2, 4, 6, 8, and 10 weeks of incubating the rabbit Achilles tendon in phosphate-buffered saline containing ribose (glycated). The biomechanical integrity of the glycated tendons was then compared to control tendons incubated in phosphate-buffered saline (non-glycated) at each time interval, while the biochemical stability of both groups of tendons was determined by examining collagen extractability and the formation of pentosidine at 8 weeks. Whereas there were no significant biomechanical differences between control and glycated tendons at 0- and 2-week intervals (P > 0.05), moderately significant increases in maximum load, energy to yield, and toughness of glycated tendons were observed at 4 weeks. Beyond 4 weeks of incubation, the differences between glycated and non-glycated tendons became highly significant, as glycated tendons withstood more load and tensile stress (P < 0.01 for each variable), attained significantly higher modulus of elasticity (P < 0.01), absorbed more energy (P < 0.01), and became tougher (P < 0.01) than controls. These differences in the biomechanical indices of the effects of glycation were stable between the 6th and 10th week of glycation. The maximum increases in the biomechanical measurements as a result of glycation were 29% for maximum load, 125% for stress, 19% for strain, 106% for Young's modulus of elasticity, 14% for energy to yield, and 57% for toughness. Biochemical analysis showed a 61% reduction in the extractability of neutral salt-soluble collagen, a 48% decrease in acid-soluble collagen, and a 29% decline in pepsin-soluble collagen in glycated tendons (P < 0.01). In contrast, there was a 28% increase in the amount of insoluble collagen and significantly higher amounts of pentosidine (P < 0.01) in glycated tendons. Collectively, these biomechanical and biochemical results suggest that nonenzymatic glycation may explain the altered stability of connective tissue matrix induced by the processes of diabetes and aging.     

Relative quantification of glycated Cu-Zn superoxide dismutase in erythrocytes by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESIMS) was used for relative quantification of glycated Cu-Zn superoxide dismutase (SOD-1) in human erythrocytes. SOD-1 samples were prepared from erythrocytes by removing hemoglobin using hemoglobind gel followed by ethanol and chloroform extraction. The reproducibility in measurement of the relative percentage of glycated protein was good, and the standard deviation of each measurement was 4.0%. From the mass spectral analysis of a mixture of commercial SOD-1 and in vitro partially glycated SOD-1 in several ratios, it was found that free and glycated SOD-1 have the same ionization efficiencies. The percentage of glycation on SOD-1 was measured in 30 individuals, including patients with diabetes mellitus. The glycation levels ranged from 4.5% to below the detection limit. The SOD-1 sample extracted from erythrocytes was fractionated by Glyco-Gel B chromatography, and the separated fractions were analyzed by MS. The mass spectra of absorbed fraction showed significant amounts of non-specific binding of non-glycated proteins to Glyco-Gel B.     

Inhibitory effect of glycation on catalytic activity of alanine aminotransferase

Non-enzymatic glycation is a common post-translational modification of tissue and plasma proteins which can impair their functions in living organisms. In this study, the authors have demonstrated for the first time an inhibitory effect of in vitro glycation on the catalytic activity of alanine aminotransferase (ALT, EC 2.6.1.2), a pyridoxal phosphate enzyme with several lysine residues in the molecule. The porcine heart enzyme was incubated with 50 mmol/l D-fructose, D-glucose, D,L-glyceraldehyde, or D-ribose in 0.1 mol/l phosphate buffer (pH 7.4) at 25°C for up to 20 days. The strongest glycation effect was shown by D,L-glyceraldehyde, which caused complete enzyme inhibition within 6 days. After 20 days of incubation, the ALT activity in samples with D-fructose and D-ribose was less than 7% of the initial enzyme activity. A statistically significant effect of D-glucose on the enzymatic activity of ALT was not found. Incubation of ALT with D-fructose, D,L-glyceraldehyde and D-ribose minimized its catalytic activity both in the glycated and non-glycated fractions of the samples. Markedly higher activity was found in the glycated fraction with glucose. The inhibitory effect of glycation of ALT with D-fructose and D-ribose was found to be more intensive in the presence of L-alanine and weaker in the presence of 2-oxoglutarate. The findings suggest that glycation of the e-amino group of Lys313 as a crucial part of the catalytic site of ALT may contribute to ALT inactivation in the presence of glycating sugars. Nevertheless, glycation of lysine residues outside the active center of ALT seems to be primary.     

Non-enzymatic glycation of antithrombin III in vitro

Non-enzymatic glycation of antithrombin III (AT-III) has been proposed as a significant contributor to the increased incidence of thrombo-occlusive events in diabetics. AT-III, isolated from normal human plasma by means of heparin affinity and ion-exchange chromatography, was incubated with 0-0.5 M glucose in neutral phosphate buffer at 37 degrees C. The extent of non-enzymatic glycation could be monitored by uptake of radioactivity as well as by binding to a phenylboronate affinity resin, which effectively retards AT-III containing ketoamine-linked glucose. Non-enzymatically glycated AT-III (approx. 1 mol glucose/mol protein) bound heparin nearly as efficiently as non-glycated AT-III. The two AT-III preparations were equally active in inhibiting thrombin cleavage of chromogenic substrate. Following incubation with [14C]glucose, structural analyses of cyanogen-bromide-cleaved peptides of enzymatically glycated AT-III showed that the [14C]glucose adducts were distributed over many sites on the molecule. This lack of specificity contrasts with the restricted sites of modification on hemoglobin, albumin and ribonuclease A, and explains why non-enzymatic glycation of AT-III has little if any effect on its function.     

Docking and molecular dynamics simulation studies on glycation-induced conformational changes of human paraoxonase 1

Human paraoxonase 1 (huPON1) is a calcium-dependent esterase responsible for hydrolysis of a wide variety of substrates including organophosphates, esters, lactones, and paraoxon. Although its natural substrate is unknown, the action of PON as an antioxidant is well documented. Because recent reports have suggested glycation may induce reduced PON activity in diabetes, we investigated the structural features of huPON1 and its glycated mutant by template-based modeling, docking, and molecular dynamics (MD) simulations. Our results corroborated the importance of the His115–His134 dyad in both the lactonase and paraoxonase activity of huPON1. Structural alterations in the glycated model reflected weak interactions between the docked substrate and the active site cleft. We also used MD simulation to gain insight into glycation-induced conformational changes of huPON1 and the implication of this on depleted enzymatic activity. The catalytic calcium found on the surface interacts with the side chain oxygen of residues, including Asn224, Asn270, Asn168, Asp269, and Glu53, and this interaction with the respective residues undergoes minor displacement on glycation. The root-mean-square fluctuation had high motional flexibility in the non-glycated model whereas the conformation of the glycated structure was comparatively stable. Our findings emphasize the consequence of glycation-induced alterations and their effect on overall enzymatic activity.     

Effect of limited solid-state glycation on the conformation of lysozyme by ESI-MSMS peptide mapping and molecular modeling

Although protein glycation has been implicated in the alteration of protein functionality, both in vivo (in biological systems) and in vitro (in food systems), the effect of the protein-bound glycan moiety on the structure/conformation of proteins that result in the modification of functionality is not clear. In this article, we report a study of the conformational changes of glycated lysozyme using LC-ESI-MSMS peptide mapping, and molecular modeling. A comparison of the RP-HPLC of the tryptic digests of unglycated and glycated lysozyme showed markedly different chromatographic profiles. Analysis of the peptide composition of the chromatographic fractions of the tryptic digests revealed that glycation of lysozyme resulted in the modification of its conformation. Glycation-induced changes in the conformation of lysozyme resulted in the exposure of its active site region to increased proteolytic activity of trypsin. Molecular simulation of triglycated lysozyme also showed that limited glycation of lysozyme caused reorientation of the active site residues (Arg 45, Arg 68, Asn 44, and Trp 62) and increased solvent accessibility into the active site region of the protein. The results of the modeling experiment corroborated the results of the RP-HPLC and ESI-MSMS peptide mapping.     

Quantitative analysis of glycation and its impact on antigen binding

Glycation has been observed in antibody therapeutics manufactured by the fed-batch fermentation process. It not only increases the heterogeneity of antibodies, but also potentially affects product safety and efficacy. In this study, non-glycated and glycated fractions enriched from a monoclonal antibody (mAb1) as well as glucose-stressed mAb1 were characterized using a variety of biochemical, biophysical and biological assays to determine the effects of glycation on the structure and function of mAb1. Glycation was detected at multiple lysine residues and reduced the antigen binding activity of mAb1. Heavy chain Lys100, which is located in the complementary-determining region of mAb1, had the highest levels of glycation in both stressed and unstressed samples, and glycation of this residue was likely responsible for the loss of antigen binding based on hydrogen/deuterium exchange mass spectrometry analysis. Peptide mapping and intact liquid chromatography-mass spectrometry (LC-MS) can both be used to monitor the glycation levels. Peptide mapping provides site specific glycation results, while intact LC-MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing. Capillary isoelectric focusing (cIEF) can also be used to monitor glycation because glycation induces an acidic shift in the cIEF profile. As expected, total glycation measured by intact LC-MS correlated very well with the percentage of total acidic peaks or main peak measured by cIEF. In summary, we demonstrated that glycation can affect the function of a representative IgG1 mAb. The analytical characterization, as described here, should be generally applicable for other therapeutic mAbs.