大鼠正常肝、肝癌前病变组织谷胱甘肽S-转移酶的纯化及部分性质的研究

本文用S-己基谷胱甘肽-琼脂糖-6B亲和层析一步纯化法分别获得电泳纯大鼠正常肝GST_S及含增生结节大鼠肝GST_S。经DE_(52)阴离子交換柱将含增生结节大鼠肝GST_S分离为三个同功酶组份,依次命名为C_(DE)A1及A2,C_(DE)占上柱总GST_S活性84.8％。等电聚焦电泳测定等电点分别为7.8、6.7及6.3。经CM_(52)阳离子交換柱获得五个同功酶组份,依次命名为A_(CM),C1,C2,C3及C4,等电点分别为7.8,7.4,7.9,8.3及8.6。A_(CM)的活性占CM_(52)柱上柱总活性的10％。SDS-PAGE电泳结果和正常大鼠肝GST_S比较,含增生结节大鼠肝GST_S同样出现Ya,Yb及Yc三条区带,而后者的氨基酸组成也与正常大鼠肝GST_S相近,但是和大鼠正常肝组织比较后者GST_S活性明显升高,以阳离子同工酶的活性为主。     

人胎盘谷胱甘肽S-转移酶的纯化和性质

将人胎盘组织粗匀浆经105000×g超速离心后,用S-已基谷胱甘肽-琼脂糖-6B亲和层析一步纯化法获得电泳纯的人胎盘GST(简称GST-π)。其比活性较粗匀浆高194.7倍,回收率为50％。再经DE_(52)交换柱进一步纯化,用KCl浓度梯度洗脱后为单一锐峰,等电聚集电泳呈一条带,等电点(pI)为4.60。GST-π经TSKgel-G3000SW柱高效液相层析,也为单一对称锐峰,测得其分子量为45.2kD;在SDS-PAGE电泳也为单一区带,测得其亚基单位的分子量为22.5kD。GST-π氨基酸组成分析可检出十六种氨基酸,其中以谷氨酸、亮氨酸、丙氨酸、天冬氨酸及甘氨酸含量较高。 GST-π酶动力学研究证明GST-π以GST和CDNB为底物时km值分别为0.16mmol/L和0.55mmol/L,经测定表明,GST-π的最适作用pH值为7.5,在pH6.5—9的范围内较为稳定,体外GST-π在温度超过25℃对容易失活。以GST-π为抗原,得到兔抗人GST-π抗血清,其效价为1:32,与人肝GSTs不发生免疫交叉反应。     

大鼠肝谷胱甘肽转硫酶的制备及其部分性质的研究

本文通过CM-52纤维素柱层析分离得到六种大鼠肝谷胱甘肽转硫酶同工酶;经GSH—亲和层析柱进一步纯化,得到纯酶。讨论了该酶的部分理化性质。抑制实验结果表明,胆酸类化合物对谷胱甘肽转硫酶的抑制作用类型为非竞争性。     

人肝脏特异的F抗原分离纯化及其抗血清制备

取新鲜的人肝脏,充分洗涤,制备匀浆,上清液经Sephadex-G200、DE52层析,聚丙烯酰胺凝胶电泳,得到电泳纯Ｆ抗原,并免疫制备抗血清.     

人心肌胞质天门冬氨酸转氨酶的纯化及性质

从人心肌提纯胞质天门冬氨酸转氨酶同工酶(c-AST)。经匀浆、热处理、硫酸铵分离,进一步用离子交换层析和亲和层析。其比活为220 u/mg。经免疫电泳和PAGE鉴定,达到免疫纯和电泳纯。免疫动物获得较高效价抗体。并对该酶的分子量,等电点和氨基酸组成进行了研究。     

河蚌C-反应蛋白的分离纯化及其性质研究

用合成的CDPC-Sepharose-6B亲和层析吸附剂一步提纯河蚌C-反应蛋白(CRP)。纯化的CRP在SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和等电聚焦电泳鉴定时均显示为一条区带。其等电点(pI)为5.2,分子量约为310,000道尔顿,由六个亚基组成,亚基分子量约为51,000道尔顿。在280nm处的消光系数E_(1cm)~(1mg)/ml=1.2。河蚌CRP与C_多糖(CPS)发生的沉淀反应是钙依赖性的。本文对河蚌CRP的氨基酸组成及其它性质也作了研究。     

肠出血性大肠杆菌(EHEC)O157∶H7紧密粘附素免疫保护性片段(Intimin-C)的克隆表达纯化及部分生物学活性研究

克隆表达并纯化肠出血性大肠杆菌 (EHEC)O15 7∶H7紧密粘附素免疫保护性片段 (Intimin C) ,并对其部分生物学活性进行研究。设计引物采用PCR自O15 7菌基因组扩增紧密粘附素免疫保护性片段 (Intimin C)的编码基因eae C ,T A克隆测序后构建原核表达质粒 pET 2 8a(+) eaeC并转化E .coliBL2 1(DE3) ,诱导表达破菌及包涵体洗涤后采用离子交换柱和亲和层析柱对目的蛋白进行纯化 ,PAGE电泳初测目的蛋白的分子量、滴定法初测目的蛋白的等电点 ,并以纯化蛋白Intimin C免疫家兔制备多抗血清。采用PCR法自O15 7菌基因组扩增出了约 90 0b…     

菠菜叶片蔗糖磷酸合成酶的纯化

经硫酸铵分部沉淀,DEAE-纤维素(DE 52),Sepharose 6B和 AH—4B连续三次柱层析,得到纯化88倍电泳均一的菠菜叶片蔗糖磷酸合成酶。电泳分析该酶分子量为490 kD,是由八个分子量为60 kD的相同亚基组成的寡聚体,等电点为PI=4.l,其最适pH值为6.9。     

钙蛋白酶抑制蛋白功能结构域Ⅳ在大肠杆菌中的表达、纯化及其抗血清的制备

为了研究钙蛋白酶系统在细胞发育及其它生理过程的功能 .应用 PCR从鼠钙蛋白酶抑制蛋白 ( calpastatin) c DNA中扩增了保守的具有功能的结构域 ( 40 4 bp) ,克隆于 p GEX- KG载体 .重组质粒 p GEX- Calp4在大肠杆菌中经 IPTG诱导可表达分子量约 4 5 k D融合蛋白 GST- Galp4 .诱导表达后的菌体超声裂解液经谷胱甘肽 - Sepharose4 B亲和层析柱得到纯化的 GST- Calp4融合蛋白 ,纯度达电泳纯 .纯化的 GST- Calp4免疫兔 8周后 ,抗血清的效价达 1∶ 64 .Western- blot分析表明制备的抗血清确实可以与肌细胞中分子量为 1 4 0 k D左右蛋白 (亦即完整 calpastatin)发生特异的免疫交叉反应 ,此表明实验获得了高特异性多克隆抗体     

人谷胱甘肽硫转移酶A1在乳酸乳球菌中的表达及活性研究

用RTPCR技术从人肝总RNA中分离扩增了人谷胱甘肽硫转移酶A1基因的cDNA序列,克隆至大肠杆菌表达质粒pET23b,采用蛋白表达筛查法及DNA测序证明该cDNA序列完全正确。重组质粒pET23bhgst转化大肠杆菌BL21(DE3),经IPTG诱导获得高效表达的可溶性hGSTA1产物,其表达量约为大肠杆菌可溶性总蛋白的40%。将hGSTA1cDNA亚克隆至乳酸乳球菌表达载体pMG36e,电穿孔法转化乳酸乳球菌MG1363获得hGSTA1乳酸乳球菌表达株。SDSPAGE及Western杂交分析表明该菌株表达预期大小的hGSTA1融合蛋白,经谷胱甘肽琼脂糖亲和层析纯化获得的hGSTA1蛋白具有较高的谷胱甘肽硫转移酶活性。具hGSTA1酶活性的乳酸乳球菌工程菌可望应用于研制防癌保健乳制品。     

Purification and characterization of the individual glutathione S-transferases from sheep liver

The glutathione S-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000g supernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography on S-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathione S-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathione S-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identical Mr of 24,000 whereas a predominant subunit with Mr of 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathione S-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathione S-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathione S-transferases.     

Immunological and sequence interrelationships between multiple human liver and rat glutathione S-transferases

The 13 forms of human liver glutathione S-transferases (GST) (Vander Jagt, D. L., Hunsaker, L. A., Garcia, K. B., and Royer, R. E. (1985) J. Biol. Chem. 260, 11603-11610) are composed of subunits in two electrophoretic mobility groups: Mr = 26,000 (Ha) and Mr = 27,500 (Hb). Preparations purified from the S-hexyl GSH-linked Sepharose 4B affinity column revealed three additional peptides at Mr = 30,800, Mr = 31,200, and Mr = 32,200. Immunoprecipitation of human liver poly(A) RNAs in vitro translation products revealed three classes of GST subunits and related peptides at Mr = 26,000, Mr = 27,500, and Mr = 31,000. The Mr = 26,000 species (Ha) can be precipitated with antisera against a variety of rat liver GSTs containing Ya, Yb, and Yc subunits, whereas the Mr = 27,500 species (Hb) can be immunoprecipitated most efficiently by antiserum against the anionic isozymes as well as a second Yb-containing isozyme (peak V) from the rat liver. The Mr = 31,000 band can be immunoprecipitated by antisera preparations against sheep liver, rat liver, and rat testis isozymes. Human liver GSTs do not have any subunits of the rat liver Yc mobility. Antiserum against the human liver GSTs did not cross-react with the Yc subunits of rat livers or brains in immunoblotting experiments. The human liver GST cDNA clone, pGTH1, selected human liver poly(A) RNAs for the Ha subunit(s) in the hybrid-selected in vitro translation experiments. Southern blot hybridization results revealed cross-hybridization of pGTH1 with the Ya, Yb, and Yc subunit cDNA clones of rat liver GSTs. This sequence homology was substantiated further in that immobilized pGTH1 DNA selected rat liver poly(A) RNAs for the Ya, Yb, and Yc subunits with different efficiency as assayed by in vitro translation and immunoprecipitation. Therefore, we have demonstrated convincingly that sequence homology as well as immunological cross-reactivity exist between GST subunits from several rat tissues and the human liver. Also, the multiple forms of human liver GSTs are most likely encoded by a minimum of three different classes of mRNAs. These results suggest a genetic basis for the subunit heterogeneity of human liver GSTs.     

Comparative biochemistry of mammalian arylsulfatases A and B

1. Arylsulfatases A and B occurred as a major anionic and cationic isozyme, respectively, among eleven eutherian mammalian species. 2. Minor anionic arylsulfatase B isozymes were observed in rodents, dog, whale and pig, and were either monomeric (vole, Mr = 67 +/- 2 kDa), an apparent aggregate (dog, whale, pig; Mr = 192 +/- 10 kDa), or both (rat, mouse; monomeric Mr = 57 +/- 2 kDa; apparent dimeric Mr = 114 +/- 3 kDa). 3. Minor cationic arylsulfatase A isozymes were isolated from the deer, whale and pig. 4. Opossum arylsulfatases A and B were both anionic, had similar relative molecular weights, were not inhibited by silver, and were not precipitated by anti-murine arylsulfatase B nor anti-bovine arylsulfatase A IgG preparations.     

Differential induction of class alpha glutathione S-transferases in mouse liver by the anticarcinogenic antioxidant butylated hydroxyanisole. Purification and characterization of glutathione S-transferase Ya1Ya1.

A novel cytosolic Alpha class glutathione S-transferase (GST) that is not normally expressed in mouse liver was found to be markedly induced (at least 20-fold) by the anti-carcinogenic compound butylated hydroxyanisole. This enzyme (designated GST Ya1 Ya1) did not bind to either the S-hexylglutathione-Sepharose or the glutathione-Sepharose affinity matrices, and purification was achieved by using bromosulphophthalein-glutathione-Sepharose. The purified isoenzyme, which comprises subunits of Mr 25,600, was characterized, and its catalytic, electrophoretic, immunochemical and structural properties are documented. GST Ya1 Ya1 was shown to be distinct from the Alpha class GST that is expressed in normal mouse liver and is composed of 25,800-Mr subunits; the Alpha class isoenzyme that is constitutively expressed in the liver is now designated GST Ya3 Ya3. Hepatic concentrations of GST Ya3 Ya3 were not significantly affected when mice were treated with butylated hydroxyanisole. Both Pi class GST (subunit Mr 24,800) and Mu class GST (subunit Mr 26,400) from female mouse liver were induced by dietary butylated hydroxyanisole. By contrast, hepatic concentrations of microsomal GST (subunit Mr 17,300) were unaffected.     

Specificity of the glutathione S-transferases in the conversion of leukotriene A4 to leukotriene C4

We have synthesized the 5,6-LTA4, 8,9-LTA4, and 14,15-LTA4 as methyl esters by an improved biomimetic method with yields as high as 70-80%. We have investigated the catalytic efficiency of the purified cytosolic glutathione S-transferase (GST) isozymes from rat liver in the conversion of these leukotriene epoxides to their corresponding LTC4 methyl esters. Among various rat liver GST isozymes, the anionic isozyme, a homodimer of Yb subunit, exhibited the highest specific activity. In general, the isozymes containing the Yb subunit showed better activity than the isozymes containing the Ya and/or Yc subunits. Interestingly, all three different LTA4 methyl esters gave comparable specific activities with a given GST isozyme indicating that regiospecificity of GSTs was not the factor in determining their ability to catalyze this reaction. Surprisingly, purified GSTs from sheep lung and seminal vesicles showed little activity toward these leukotriene epoxides, indicating a lack of the counterpart of rat liver anionic GST isozyme in these tissues.     

Expression of glutathione S-transferases in rat brains

The tissue-specific expression of glutathione S-transferases (GSTs) in rat brains has been studied by protein purification, in vitro translation of brain poly(A) RNAs, and RNA blot hybridization with cDNA clones of the Ya, Yb, and Yc subunit of rat liver GSTs. Four classes of GST subunits are expressed in rat brains at Mr 28,000 (Yc), Mr 27,000 (Yb), Mr 26,300, and Mr 25,000. The Mr 26,3000 species, or Y beta, has an electrophoretic mobility between that of Ya and Yb, similar to the liver Yn subunit(s) reported by Hayes (Hayes, J. D. (1984) Biochem. J. 224, 839-852). RNA blot hybridization of brain poly(A) RNAs with a liver Yb cDNA probe revealed two RNA species of approximately 1300 and approximately 1100 nucleotides. The band at approximately 1300 nucleotides was absent in liver poly(A) RNAs. The Mr 25,000 species, or Y delta, can be immunoprecipitated by antisera against rat heart and rat testis GSTs, but not by antiserum against rat liver GSTs. Therefore, the Y delta subunit may be related to the "Mr 22,000" subunit reported by Tu et al. (Tu, C.-P.D., Weiss, M.J., Li, N., and Reddy, C. C. (1983) J. Biol. Chem. 258, 4659-4662). The abundant liver GST subunits, Ya, are not expressed in rat brains as demonstrated by electrophoresis of purified brain GSTs and a lack of isomerase activity toward the Ya-specific substrate, delta 5-androstene-3,17-dione. This is apparently because of the absence of Ya mRNA expression prior to RNA processing. The data on the preferential expression of Yc subunits in rat brains, together with the differential phenobarbital inducibility of the Ya subunit(s) in rat liver reported by Pickett et al. (Pickett, C. B., Donohue, A. M., Lu, A. Y. H., and Hales, B. F. (1982) Arch. Biochem. Biophys. 215, 539-543), suggest that the Ya and Yc genes for rat GSTs are two functionally distinct gene families even though they share 68% DNA sequence homology. The expression of multiple GSTs in rat brains suggests that GSTs may be involved in physiological processes other than xenobiotics metabolism.     

Properties of peroxidases from tobacco cell suspension culture

An enzyme preparation from suspension cultured tobacco cells oxidized IAA only in the presence of added cofactors, Mn²⁺ and 2,4-dichlorophenol, and showed two pH optima for the oxidation at pH 4·5 and 5·5. Effects of various phenolic compounds and metal ions on IAA oxidase activity were examined. The properties of seven peroxidase fractions separated by column chromatography on DEAE-cellulose and CM-Sephadex, were compared. The peroxidases were different in relative activity toward o-dianisidine and guaiacol. All the peroxidases catalysed IAA oxidation in the presence of added cofactors. The pH optima for guaiacol peroxidation were very similar among the seven isozymes, but the optima for IAA oxidation were different. The anionic and neutral fractions showed pH optima near pH 5·5, but the cationic isozymes showed optima near pH 4·5. With guaiacol as hydrogen donor, an anionic peroxidase (A-1) and a cationic peroxidase (C-4) were very different in H₂O₂ concentration requirements for their activity. Peroxidase A-1 was active at a wide range of H₂O₂ concentrations, while peroxidase C-4 showed a more restricted H₂O₂ requirement. Gel filtration and polyacrylamide gel studies indicated that the three cationic peroxidases have the same molecular weight.     

Differential expression of alpha, mu and pi classes of isozymes of glutathione S-transferase in bovine lens, cornea, and retina

Isozyme characterization of glutathione S-transferase (GST) isolated from bovine ocular tissue was undertaken. Two isozymes of lens, GST 7.4 and GST 5.6, were isolated and found to be homodimers of a Mr 23,500 subunit. Amino acid sequence analysis of a 20-residue region of the amino terminus was identical for both isozymes and was identical to GST psi and GST mu of human liver. Antibodies raised against GST psi cross-reacted with both lens isozymes. Although lens GST 5.6 and GST 7.4 demonstrated chemical and immunological relatedness, they were distinctly different as evidenced by their pI and comparative peptide fingerprint. A corneal isozyme, GST 7.2, was also isolated and established to be a homodimer of Mr 24,500 subunits. Sequence analysis of the amino-terminal region indicated it to be about 67% identical with the GST pi isozyme of human placenta. Antibodies raised against GST pi cross-reacted with cornea GST 7.2. Another corneal isozyme, GST 8.7, was found to be homodimer of Mr 27,000 subunits. Sequence analysis revealed it to have a blocked amino-terminus. GST 8.7 immunologically cross-reacted with the antibodies raised against cationic isozymes of human liver indicating it to be of the alpha class. Two isozymes of retina, GST 6.8 and GST 6.3, were isolated and identified to be heterodimers of subunits of Mr 23,500 and 24,500. Amino-terminal sequence analysis gave identical results for both retina GST 6.8 and GST 6.3. The sequence analysis of the Mr 23,500 subunit was identical to that obtained for lens GSTs. Similarly, sequence analysis of the Mr 24,500 subunit was identical to that obtained for the cornea GST 7.2 isozyme. Both the retina isozymes cross-reacted with antibodies raised against human GST psi as well as GST pi. The results of these studies indicated that all three major classes of GST isozymes were expressed in bovine eye but the GST genes were differentially expressed in lens, cornea, and retina. In lens only the mu class of GST was expressed, whereas cornea expressed alpha and pi classes and retina expressed mu and pi classes of GST isozymes.     

Novel immunochemical characteristics of glutathione S-transferase isozymes of rat liver cytosol

Reevaluation of the immunochemical relationships among the individual glutathione S-transferase (GST) isozymes (GSTs 1-1, 1-2, 2-2, 3-3, 3-4, 4-4, and GSTs with isoelectric points of 7.5 and 6.8) of rat liver cytosol was performed utilizing the immunoblot technique. As a result, we found that the respective isozymes of two isozyme classes of rat liver cytosol might possess a common epitope(s) which has been undetected by the Ouchterlony double-diffusion method. The assumption was further supported by the results of the effects of Fab' prepared from some anti-GST antibodies on the enzymatic activity of GSTs.     

Epigenetic formation of lactate dehydrogenase isozymes in the house mouse, Mus musculus.

The origin of mouse lactate dehydrogenase (LDH) sub-bands was investigated by using our miniaturized polyacrylamide gel electrophoretic apparatus. Mouse LDH isozymes are generated by combinations of three types of A subunit, the primary type and two epigenetically modified forms. These are designated A1, A2, and A3 in the order of their electrophoretic mobilities towards the anode. The A1 subunit arises from the covalent binding of molecules of glutathione through disulfide bonds to the original subunit, A3. The A2 subunit arises from the covalent binding of molecules of cysteine through disulfide bonds to the A3 subunit. All isozymes can be explained as tetramers composed of the three kinds of A subunit (A1, A2, or A3) in combination with B subunits to yield a total of 35 isozymes. The kinetic properties of these sub-bands were also examined. There was no difference between A24 and A34 in the Km for pyruvate and for lactate. Thermostability at 56 degrees C was greater for A34 than for A24. The activities of tetramers at the electrophoretic position of A3B1 and A4 in extracts containing all five isozymes were increased by treatment of the extracts with high concentrations of reduced glutathione or cysteine with the concomitant disappearance or decrease in activity of tetramers at the position of B4 and A3B1. These results suggest that, in the presence of reduced glutathione or cysteine, LDH isozymes containing the B subunit are first dissociated and then the A subunits are preferentaially recombined.