应用SDS-PAGE显示小分子多肽技术的探讨

为探讨显示小分子多肽技术,应用SDSPAGE寻找更简单、快捷的分析小分子多肽的方法。采用8%T,3%C的丙烯酰胺浓缩胶和含6mol/L脲(或含24%的甘油)的16%T,6%C的丙烯酰胺分离胶的双层不连续SDSPAGE和三羟基甘氨酸电泳缓冲液显示蛋白分子量标准(2512～16949kD)和待测样品,并对蛋白分子量标准在这两种分离胶中进行了回归分析。结果表明在这两种条件下均能显示分子量小至2512kD的多肽;多肽样品在含脲和在含甘油的2LTSDSPAGE中均有较好的显带;蛋白分子量标准的直线回归系数分别为r=-0966和r=-0964。它们是显示小分子多肽和估测其相对分子质量及对多肽分子进行进一步分析的更为简便易行的方法。     

用于测定小分子多肽的两种电泳方法的比较

对于小分子多肽的分析目前多采用甘油－ＳＤＳ－ＰＡＧＥ系统,但经实践后发现,此系统电泳时间太长,导致小分子扩散丢失较多,小分子成像不清,后经用尿素代替甘不同,同时降低三层胶的浓度,制成１５.5％Ｔ（Ｃ＝６％）的分离胶、１０％Ｔ（Ｃ＝３％）的间隙胶与４％Ｔ（Ｃ＝３％）的浓缩胶档但节省了电泳时间,且小分子多肽成像清楚。     

应用SDS—PAGE显示小分子多肽技术的探讨

为探讨显示小分子多肽技术,应用SDS－PAGE寻找更简单,快捷的分析小分子多肽的方法。采用8％,T,3％C的丙烯酰胺浓缩胶和含6mol/L脲（或含24％的甘油）的16％T,6％C的丙烯酰胺分离胶 的双层不连续SDS－PAGE和三羟基甘氨酸电泳缓冲液显示蛋白分子量标准（2．512－16．949KD）和待测样品,并对蛋白分子量标准在这两种分离胶中进行了回归分析。结果表明在这两种条件下均能显示分子量小至2．512KD的多肽;多肽样品在含脲和在含甘油的2L－T－SDS－PAGE中均有较好的显带,蛋白分子量标准的直线回归系数分别为r=-0.966和r=-0.964,它们是显示小分子多肽和估测其相对分 子质量及对多肽分子进行进一步分析和更为简便易行的方法。     

Glycine—SDS—PAGE测定小分子多肽分子量

小分子多肽分子量的测定 ,目前多采用Ｔｒｉｃｉｎｅ -ＳＤＳ -ＰＡＧＥ系统[1～ 3 ] ,但系统中的Ｔｒｉｃｉｎｅ价格昂贵 ,加之电泳时间较长 ( 3～ 4ｈ) ,为此 ,作者经过实验摸索 ,建立了Ｇｌｙｃｉｎｅ -ＳＤＳ -ＰＡＧＥ测定小分子多肽的方法。1　材料和方法表 1　制胶配方成分分离胶 (ｍｌ)浓缩胶 (ｍｌ)分离胶单体母液 1 3 3 -浓缩胶单体母液 -0 23 2ｍｏｌ/ＬＴｒｉｓ-ＨＣｌ 1 3 3 -(ｐＨ8 8)0 5ｍｏｌ/ＬＴｒｉｓ-ＨＣｌ -0 3 8(ｐＨ6 8)尿素 1 44ｇ -Ｈ2 Ｏ 0 40 910 %ＳＤＳ 40 μｌ 15 μｌ10 %ＡＰＳ 2 5 μｌ 12 μｌＴ…     

银染SDS-PAGE微量肽图法的改进

近年来,很多学者对微量蛋白质的肽图分析进行了颇为深入的研究,建立了分析灵敏度较高的方法。但对小分子多肽的检测尚存在不少困难。因为小分子多肽在电泳过程中易于扩散,常规SDS-PAGE难于检测。本文采用改进的10～25％SDS-PAGE梯度胶进行检测,并在胶上直接用CNBr裂解,可以分析微克量蛋白质,这样改进之后提高了灵敏度和分辨率,使小分子多肽亦能呈现清晰的图谱。     

SDS-PAGE电泳对小分子多肽的分析

对一些小分子多肽进行电泳分析时,在固定、染色、脱色过程中极易扩散丢失;即使用常规尿素－ＳＤＳ－ＰＡＧＥ系统测定寡肽分子量,银染色后小于８ｋＤａ的标准品也无着色带显示。我们实践摸索出提高分离胶的浓度和交联度,制成２０％Ｔ,６％Ｃ和１５％Ｔ,３％Ｃ的梯度胶,胶中不加尿素,且不需银染均可见小分子多肽着色带的方法。     

有效分离1kDa分子量环脂肽的电泳方法

目的：建立一种有效分离1kDa分子量环脂肽的电泳方法。方法：针对1kDa分子量环脂肽的特点,对凝胶的组分和比例、凝胶的聚合速度等电泳条件进行了优化。结果：改进凝胶组分和比例,使得浓缩胶为7％T、3％C,夹层胶为12％T、3％C,致密胶为16．5％T、6％C;并在胶中加入尿素和甘油;同时加大APS的量使聚合速度加快。由此得到的环脂肽电泳条带清晰、平稳、紧凑,显示分子量为1．05kDa,与质谱结果吻合。结论：新建立的Tricine—SDS—PAGE是一种有效的分离1kDa分子量环脂肽的电泳方法。     

自体软骨细胞修复兔膝关节全层软骨缺损的实验研究

目的：研究自体软骨细胞复合于人脐带Wharton胶支架对兔膝关节全层软骨缺损的修复效果。方法：经自体关节软骨细胞 经体外培养后复合到制备人脐带Wharton 胶取向支架内构建细胞- 支架复合体,选取健康清洁新西兰兔23 只,雌雄不拘,体重 2.5-3.0 kg,取滑车沟中下部制作全层软骨缺损模型后随机分成A、B和C 组。A组(n= 10)：植入自体软骨细胞+人脐带Wharton 胶取向支架复合体;B组(n= 10)：植入单纯人脐带Wharton 胶取向支架;C组(n= 3)：不做任何处理正常兔。分别于术后3 个月和6 个月各处死后取材进行生物力学特性评估检测。结果：压痕实验显示在3 个月时A 和B 组修复区组织刚度分别达到正常软骨的 45.72%和25.25%,且A组刚度明显优于B组,均低于C组( P<0.05);到6 个月时各自达到正常软骨刚度的69.76%和35.14%,同 样A 组刚度明显优于B 组,均低于C 组( P<0.05)且在同期个各组之间均有显著性差异（F=80.309,P<0.05）。结论：体外培养的自 体软骨细胞与人脐带Wharton 胶复合在体内的微环境作用下修复软骨缺损效果良好,为软骨组织工程提供了一种新支架材料。     

一步法电泳分析肌联蛋白亚型和肌球蛋白重链

目的为研究超大分子量肌小节蛋白肌联蛋白(titin)的生理病理功能,在一次电泳过程中同时分离titin各亚型和中分子量肌小节蛋白肌球蛋白重链(myosin heavy chain,MHC)。方法使用16cm×18cm垂直电泳系统,在电泳板下1/3灌注10g/L SDS-PAGE胶,上2/3灌注60g/L SDS-琼脂糖(SDS-VAGE)胶。低温8℃下持续电泳5h,在电泳板上层以SDS-VAGE胶电泳分离titin亚型,下层以SDS-PAGE胶电泳分离MHC。电泳后VAGE胶使用银染法标记titin各亚型,PAGE胶使用考马斯亮蓝染色法标记MHC。结果 titin各亚型得到有效的分离,目标蛋白条带显示清晰,与其分子量大小一一对应,分离效果明确。结论一步法垂直电泳系统可应用于超大分子量蛋白的电泳,同时可分离多个分子量差距大的蛋白,提高蛋白电泳实验效率。     

尖吻蝮蛇小分子多肽对人神经胶质瘤细胞系U251的凋亡及bcl-2/bax基因表达和氧化应激的影响

目的:探讨尖吻蝮蛇小分子多肽对人神经胶质瘤细胞系U251的增殖抑制作用及其机制。方法:MTT法检测2.5、5.0和10.0mg/L尖吻蝮蛇小分子多肽对人神经胶质瘤细胞系U251的增殖抑制作用;RT-PCR法检测尖吻蝮蛇小分子多肽对人神经胶质瘤细胞bcl-2和bax基因表达的影响;分光光度法检测胶质瘤细胞中丙二醛(MDA)的含量和超氧化物歧化酶(SOD)的活性。结果:2.5、5.0和10.0 mg/L尖吻蝮蛇小分子多肽对人神经胶质瘤细胞生长抑制率(49.77%、67.65%和76.42%)高于对照组(P<0.001)。当小分子多肽的剂量增加时,bcl-2 mRNA的表达呈逐渐下降趋势,而bax mRNA的表达则呈逐渐升高趋势,bcl-2/bax亦逐渐减小。2.5、5.0和10.0 mg/L组MDA含量高于对照组(P<0.01),且SOD活性低于对照组(P<0.01);5.0和10.0 mg/L组MDA含量高于2.5mg/L组和对照组(P<0.001),10.0 mg/L组SOD活性低于2.5mg/L组和对照组(P<0.001);10.0 mg/L组SOD活性低于5.0、2.5mg/L组和对照组(P<0.01)。结论:尖吻蝮蛇小分子多肽通过氧化应激使bcl-2/bax mRNA表达降低可能是抑制人神经胶质瘤细胞系U251增殖的重要原因之一。     

Molecular weight estimation of bovine brain mitochondrial monoamine oxidase

The molecular size of bovine brain mitochondrial monoamine oxidase (MAO) was investigated Mitochondria were solubilized with an anionic detergent. Emarl 20C, and fractionated by ammonium sulfate. Ammonium sulfate-fractionated MAO was subjected to detergent-containing gel chromatography and detergent-containing gel electrophoresis. MAO activity appeared as single symmetrical peak in gel chromatography in the presence of 1% Emarl 20C, and the molecular weight was estimated to be 44,000. Polymerization of MAO was observed when gel chromatography was performed in lower (0.1%, 0%) concentrations of Emarl 20C. Activity staining of MAO after electrophoresis on a gel containing 0.1% Emar 20C was successful. The molecular weight of MAO estimated from the mobility of this stained band was 89,000. It is suggested that the molecular weight of MAO is 44,000 and that it recombines in low concentrations of the detergent to form complex particles with molecular weights of 89,000 or more.     

Characterisation of the α1-protease inhibitor system in Thoroughbred horse plasma by horizontal two-dimensional (ISO-DALT) electrophoresis. 1. Protein staining

The isoelectric points and the molecular weights of the major components of the eight Thoroughbred protease inhibitor (Pi) types have been determined by polyacrylamide gel isoelectric focusing and polyacrylamide gel pore gradient (ISO-DALT) electrophoresis respectively. The major Pi proteins focus in the range pH 3.74–4.43 and have molecular weights ranging from 55 000–72 000 daltons. Using the ISO-DALT method of electrophoresis, protein maps for the eight Thoroughbred Pi types have been presented for the first time. None of the homozygous Pi types are identical except for the types S₁ and S₂ which show partial identity. The results do not necessarily support. Juneja et al.s (1979) contention of two closely linked α1 Pi systems based on molecular weight differences. It is suggested that the traditional nomenclature originally proposed by Braend (1970) be maintained to describe the eight Pi alleles in Thoroughbred horse plasma. The ISO-DALT method provides a sensitive technique which is superior to existing techniques for the analysis of the horse Pi system.     

Detection of chitinase activity after polyacrylamide gel electrophoresis

Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.     

Isozymes of alpha-galactosidase from Bacillus stearothermophilus

Two molecular forms of alpha-galactosidase (EC 3.2.1.22) synthesized constitutively by Bacillus stearothermophilus, strain AT-7, have been purified. alpha-Galactosidase I (with the substrate p-nitrophenyl alpha-D-galactopyranoside (PNPG)) has a pH optimum of 6 and half-life at 65 degrees C of > 2 h at low protein concentration. alpha-Galactosidase II has a pH optimum of 7 with PNPG and a half-life at 65 degrees C of about 3 min. The isozymes also differ with respect to their Km with PNPG and melibiose. Both enzymes are inhibited competitively by D-galactose, melibiose, and Tris. With the beta-glycosides cellobiose and lactose either noncompetitive or mixed-type inhibition is observed, with the pattern dependent on both the pH and the isozyme. The two isozymes have similar Arrhenius activation energies (about 20 kcal/mol, 1 kcal = 4.184 kJ). Their molecular weights, estimated by disc gel electrophoresis, are alpha-galactosidase I, 280 000 +/- 30 000 and alpha-galactosidase II, 325 000 +/- 15 000. Dodecyl sulfate gel electrophoresis gave a single band for each enzyme. The respective molecular weights, 81 000 +/- 500 for alpha-galactosidase I and 84 000 +/- 500 for alpha-galactosidase II, suggest that both enzymes consist of four subunits.     

Elementary chain composition of guinea pig thyroglobulin.

Thyroglobulin obtained from guinea pigs was examined by Na dodecyl-SO4-polyacrylamide gel electrophoresis after reduction and alkylation. In contrast to thyroglobulin from other mammalian sources, only three groups of polypeptide chains accounted for 95% or more of the protein. Determinations of the molecular weights of these purified proteins by equilibrium centrifugation in 6 M guanidine HCl gave values of 295,000 (species A), 210,000 (species B), and 110,000 (species C). Molecular weights determined by gel filtration in 6 M guanidine HCl gave similar results. Due to the large size of the polypeptides, satisfactory molecular weights could not be obtained from Na dodecyl-SO4-polyacrylamide gel electrophoresis. Amino acid analysis of the three species was similar to that of whole thyroglobulin. Only slightly higher level of lysine and histidine and a lower level of glutamic acid were seen in species C. The iodine contents were found to range from 0.07 to 0.12 to 0.20% for species A, B, and C, respectively.     

Bacteriophages of Halobacterium halobium: virion DNAs and proteins

The DNAs from bacteriophages Hh-1 and Hh-3 that infect Halobacterium halobium were characterized. Both phages contain linear double-stranded DNA and show no relatedness based on restriction endonuclease fragment patterns. Hh-1 DNA has 67.05% guanine plus cytosine (G+C) and a molecular weight of 24.6 X 10(6), whereas Hh-3 DNA has 62.15% G+C and a molecular weight of 19.4 X 10(6). Polyacrylamide gel electrophoresis indicated that the major proteins of the two phages are of different molecular weights.     

Lectin Receptors in Central Nervous System Myelin

Abstract: Proteins from central nervous system myelin were separated by high-resolution, sodium dodecyl sulfate-pore gradient slab gel electrophoresis and the glycoproteins were detected by autoradiography after direct application of radioiodinated lectins. A surprising heterogeneity of lectin binding proteins was found associated with this highly purified membrane fraction. Iodinated wheat germ agglutinin, which has a monosaccharide specificity for N-acetyl-D-glucosamine and N-acetylneuraminic acid, revealed six major bands and two minor bands. By correlating the molecular weights (M_r) of radioiodinated protein standards with the gel concentration at the position reached by the protein (%T) using the relationship log(M_r) versus log(%T) for gradient gel systems, molecular weight estimates of 128, 300, 109, 800, 75, 300, 48, 800, 26, 100 and 23, 700 were obtained for the major glycoprotein bands and molecular weights of 98, 300 and 86, 600 for the minor bands. When the isolated myelin was extracted with chloroform-methanol-a procedure that removes the major myelin proteins, including the proteolipid protein and most of the basic proteins and hence concentrates the minor high molecular weight proteins-and analyzed after gradient gel electrophoresis, additional glycoproteins of molecular weights 607, 700, 196, 900, 175, 100, 61, 800, 52, 200 and 42, 600 were resolved with this lectin. Radioiodinated soybean agglutinin, which has a specificity for N-acetyl-D-galactosamine and D-galactose, revealed seven bands, three of which were unique to this lectin (19, 600, 19, 100 and 17,000). Iodinated concanavalin A (d -mannose, d -glucose) revealed bands similar to the wheat germ agglutinin as well as additional bands of 40, 300, 37, 300, 35, 700, 21, 800 and 20, 400. The glycoprotein specificity for these lectin binding components was demonstrated by hapten carbohydrate binding inhibition and by organic solvent extraction for removal of glycolipids. Based on these experiments using three lectins with different carbohydrate specificity, 22 lectin-reactive components were identified; however, six of these bands were removed by chloroform-methanol extraction. The variations observed in the lectin binding capacity for these different bands suggest possible carbohydrate heterogeneity for these individual glycoproteins. Although many of these bands may be dissociated subunits (monomeric polypeptides) of oligomeric complexes, the observed multiplicity of these quantitatively minor glycoproteins associated with the purified myelin membrane implies a more intricate molecular organization for the myelin sheath complex than previously believed.     

Conformational and molecular responses to pH variation of the purified membrane adenosine triphosphatase of Micrococcus lysodeikticus.

A preparation of ATPase from the membranes of Micrococcus lysodeikticus, solubilized and more than 95% pure, showed two main bands in analytical polyacrylamide gel electrophoresis. They did not correspond to isoenzymes because one band could be converted into the other by exposure to a mildly alkaline pH value. The conversion was paralleled by changes in molecular weight, circular dichroism and catalytic properties. Denaturation by pH at 25 degrees C was followed by means of circular dichroism, ultracentrifugation and polyacrylamide gel electrophoresis. A large conformational transition took place in the acid range with midpoints at about pH = 3.6 (I = 10(-4) M), 4.3 (I = 0.03 M) and 5.3 (I = 0.1 M). The transition was irreversible. Strong aggregation of the protein occurred in this range of pH. The final product was largely random coil, but even at pH 1.5 dissociation into individual subunits was not complete. However, partial dissociation took place at pH 5 (I = 0.028 M). At this pH value the enzyme was inactive, but 20-30% of the activity could be recovered when the pH was returned to 7.5. In the alkaline region the midpoint of the transition occurred near pH = 11 (I = 0.028 M). The pK of most of the tyrosine residues of the protein was about 10.9. The unfolding was irreversible and the protein was soon converted into peptide species with molecular weights lower than those determined for the subunits by gel electrophoresis in the presence of sodium dodecyl sulphate. Conventional proteolysis did not account for the transformation.     

玉米酯酶同工酶和过氧化物酶同工酶的分子量研究

本试验利用聚丙烯酰胺凝胶梯度电泳分步染色法直接对玉米苗期酯酶同工酶和过氧化物酶同工酶各酶带的分子量进行了比较测定。酯酶同工酶 E_1、E_2、E_3~F、E_3~S、a、b、c 各酶带的分子量分别为<20000,35200、33000、38500、29900、28500、34000道尔顿过氧化物酶同工酶 PX_4~F和 PX_4~S酶带的分子量分别为131000和149000道尔顿。根据酶带在均匀胶和梯度胶中的位置变化对各酶带的生化性质作了初步分析,发现 E_3~F和 E_3~S、PX_4~F 和 PX_4~S 在迁移率上的差异主要是分子量的差异。本文为同工酶的分子量测定提供了一个简便的方法。     

Human prolyl hydroxylase. Purification, partial characterization and preparation of antiserum to the enzyme.

Prolyl hydroxylase was purified from human foetal skin and from a mixture of human foetal tissues by the affinity chromatography procedure using poly(L-proline). The enzyme from both sources was pure, when examined by polyacrylamide gel electrophoresis, as a native protein or in the presence of sodium dodecylsulphate, and enzyme activity recovery varied from 38% to 70% with seven enzyme preparations. The enzyme synthesized from 61.0 mumol to 82.7 mumol hydroxyproline mg protein-1 h-1 degrees C with a saturating concentration of (Pro-Pro-Gly)5 as substrate. The molecular weight of the enzyme was identical with that of the chick prolyl hydroxylase when studied by gel filtration, and the molecular weights of the subunits of the enzyme were about 61000 and 64000 as determined by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The amino acid composition of the human enzyme was very similar to that of the chick prolyl hydroxylase. Antisera to human and chick prolyl hydroxylases were prepared in rabbits. A single precipitin line was seen between the antiserum to human prolyl hydroxylase and the human enzyme in double immunodiffusion, and no cross-reactivity was detected between the human chick enzymes by this technique. However, a distinct cross-reactivity was observed between the human and chick enzymes in inhibition experiments.