A Rapid Two-Step Purification of Rat Cystatin C,One Major Inhibitor of Cysteine Proteinases

ABSTRACT

Rat cystatin C was purified to apparent homogeneity from rat urine after induction of a tubular dysfunction with sodium chromate. Twentyfold concentrated urine was chromatographed by a rapid purification procedure. A two-step purification including affinity chromatography on carboxymethyl papain- Sepharose and high-resolution anion exchange chromatography was developped. The purified protein has an apparent molecular mass of 15 kDa and pI of 10.2; its aminoacid composition was similar to human cystatin C. As opposed to previous data, purified urinary rat cystatin C did not contain significant amounts of carbohydrate. Antisera against rat cystatin C, raised in rabbits, partially cross-reacted with human and mouse cystatin C, indicating their antigenic similarities. Like human cystatin C, native rat cystatin C, named slow form, is degraded into a more acidic form, called fast form, by a loss of N-terminal amino acids; fast form displayed a pI of 9.4.     

Affinity Chromatography of Thymidine Kinase from a Rat Colon Adenocarcinoma

Thymidine kinase from a transplantable colon adenocarcinoma, induced by 1, 2-dimethylhydrazine and maintained in CDF rats, was purified by affinity chromatography using thymidine-3′(4-amino-phenylphosphate) coupled to carboxyhexyl-Sepharose. Most of the contaminating protein passed through the column; non-specifically adsorbed protein was washed from the column by 0.1 M KC1 in 0.01 M Tris-HCl, pH 7.5. Thymidine kinase was eluted with 0.1 mM thymidine, 0.1 M KC1 in 0.01 M Tris-HCl, pH 7.5. The purified enzyme accounted for about 267. of the applied activity, the specific activity of the purified material (peak fraction) was 3, 500 nmoles TMP formed per mg protein per 10 min., a 1, 800-fold purification of the applied extract. The preparation is free of nucleoside phosphotransferase, but contains other protein impurities. Purification was completed in less than 1 hour, making this a useful procedure for isolation of this unstable enzyme.     

Thymidine Kinase 1(TK1)重组蛋白的表达、纯化及鉴定

为获得具有免疫原性的TK1重组蛋白。通过构建能够表达TK1蛋白的重组菌BL21-pET32a-TK1,采用大肠杆菌pET32a表达系统,优化IPTG浓度、诱导温度、诱导时间使BL21-pET32a-TK1重组菌表达目的蛋白的作用条件最佳。表达产物用镍离子亲和层析纯化获得TK1蛋白,并用SDS-PAGE和Western blot进行检测。用TK1重组蛋白免疫BALB/c小鼠制备单克隆抗体,检测蛋白质免疫原性。实验结果表明,成功构建能够表达TK1蛋白的重组菌BL21-pET32aTK1,在37℃条件下,IPTG浓度为0.2mmol/L、诱导6h时重组蛋白TK1表达量最高。镍离子亲和层析梯度洗脱在80mmol/L咪唑条件下TK1蛋白纯度最大,灰度分析为87.3%,浓缩后蛋白质浓度为5.96mg/ml。用该蛋白质制备杂交瘤共获得10株稳定分泌TK1抗体的阳性单克隆细胞株,表明TK1重组蛋白具有较好的免疫原性。成功获得可溶性、抗原活性高、免疫原性强的TK1重组蛋白,为肿瘤科学及临床应用研究提供物质支撑。     

A Kinetic Study of the Rat Liver Adenosine Kinase Reverse Reaction

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP → AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP → ATP + inosine + NH₃. The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels.     

Electrophoresis of Adenovirus-Specified Thymidine Kinase

Stimulation of thymidine kinase (TK) activity occurs after abortive or productive infection with human adenoviruses. Striking changes in TK electrophoretic profile accompany this stimulation.     

High-Yield Purification of Glucokinase from Rat Liver

A rapid and reliable method for the purification of rat liver glucokinase was developed. The procedure consists of DEAE-cellulose ion-exchange chromatography, Phenyl-Sepharose hydrophobic interaction chromatography, DEAE-Affi Gel Blue dye-ligand chromatography, and duplicate steps of glucosamine-Sepharose affinity chromatography. Glucokinase was purified to a specific activity of 290 units/mg protein in a yield of 55% in 6 days. The final enzyme preparations were completely homogeneous in most experiments as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The estimated molecular weight (51.000) and sigmoidal saturation function for glucose of purified glucokinase were in good agreement with published data.     

鼠肝中金属硫蛋白的纯化

旨在探索鼠肝中金属硫蛋白(MT)提取工艺并加以改进。按照经典方法工艺提取MT,用中空纤维柱超滤方法加以改进,依据MT自身特点进行分离和鉴定。结果显示,粗品符合MT特点:分子量在6.5kD左右;无280nm特征吸收峰,加酸后紫外吸收(200-300nm)肩峰消失;通过离子交换可以实现MT1、MT2的分离;通过原子吸收测定,含有锌、铜、镉3种金属,锌含量最高,具有重要的病理生理意义。因此,与其它方法比较,改进后方法更适宜实验室制备。     

Adenosine Kinase from Rat Liver: New Biochemical Properties

Adenosine kinase is a well-known enzyme which catalyzes the phosphorylation of adenosine to AMP: Its metabolic and kinetic properties are well studied. Here, we report new properties of rat liver enzyme, demonstrating a new reaction: ADP can be a phosphate donor instead ATP, according to the reaction: adenosine + ADP → 2AMP) demonstrating the efficiency of AdK to phosphorylate adenosine, also starting from ADP. Cells could exploited this property in situations in which ATP levels are strongly decreased and ADP decreases slowly.     

胎肝细胞中一个刺激蛋白激酶C的胰岛素介体

经胰岛素处理的人胎肝细胞,经酸性抽提,热处理,活性炭吸附,阴离子AG1×8柱pH梯度洗脱,Sephadex G10脱盐、C18疏水层析。二次薄层层析分离,得到一个对蛋白激酶C有刺激作用的活性物质。组成分析结果显示,该物质含Ser,Ala,Gly,Yal,Gln五种氨基酸和甘露糖、肌醇两种单糖,质谱测出其分子量为841。该活性物质与胰岛素介体有相似的性质、组成和结构,推测它可能为刺激胎肝细胞蛋白激酶C的胰岛素介体。     

Gangliosides Prevent Ischemia-Induced Down-Regulation of Protein Kinase C in Fetal Rat Brain

Complete obstruction of the maternal blood flow to fetal rats at 20 days of gestation for a period of 10 min causes a significant shift of approximately 22% in protein kinase C (PKC) activity from a cytosolic to a membrane-bound form in the fetal brain. This translocation can be entirely reversed without losses in activity by a single intraperitoneal injection into the gravid rat of either a mixture of disialo- and trisialoganglioside [polysialoganglioside (PSG)] or by GM1 (50 mg/kg of body weight) given 3 h before onset of the ischemic episode. Cessation of blood flow for 15 min followed by a reperfusion period of 24 h results in a 47% loss in total PKC activity. This down-regulation can be almost entirely prevented upon intraperitoneal administration of GM1 3 h before, but also during and even 90 min after the onset of ischemia. The PSG mixture is also effective, particularly when given 3 h before the insult. Down-regulation of PKC is accompanied by an increase in a Ca2(+)-phosphatidylserine-independent kinase [protein kinase M (PKM)] activity, which rises from 30 pmol/min/mg of protein in control animals to a maximal value of 83.1 pmol/min/mg of protein after 15 min of ischemia and 6 h of reperfusion. By 24 h, PKM activity is 46.8 pmol/min/mg of protein. Administration of GM1 blocks completely the appearance of PKM, a result suggesting that PKC down-regulation and PKM activity elevation are intimately associated events and that both are regulated by GM1 ganglioside.     

人胎肝超氧化物歧化酶的研究

采用离子交换和凝胶过滤层析法,从正常人胎肝提取、纯化铜锌超氧化物歧化酶(Cu.Zn-SOD),并对其性质作了研究,结果测得人胎肝组织Cu·Zn-SOD平均含量为6.44unit/g湿重,并发现随着胎龄的增加人胎肝Cu.Zn-SOD含量上升;纯化后的Cu·Zn-SOD在聚丙烯酰胺凝胶电泳图谱上呈现单一区带;其活性受氰化钾抑制;以原子吸收光谱测得其铜、锌含量分别为0.39％和0.1％,采用SDS-聚丙烯酰胺凝胶电泳测得其亚基分子量为16kD;氨基酸自动分析仪测得其亚基的氨基酸残基数为151.5;在紫外吸收光谱上于265nm和258nm处分别出现两个吸收高峰。本研究结果表明,人胎肝Cu.Zn-SOD与成人肝及其他组织的Cu.Zn-SOD理化性质上基本一致。     

肝癌组织中GD3合成酶的纯化

ＧＤ３合成酶是膜嵌合蛋白,定位于高尔基体,经过制作微粒体,Ｔｒｉｔｏｎ增溶、ＡＨ－Ｓｅｐｈａｒｏｓｅ及ＧＭ３－Ｇｌａｓｓｂｅａｄｓｅ及ＧＭ３－Ｇｌａｓｓｂｅａｄｓ亲和层析等步骤,二乙基亚硝胺诱发大鼠肝癌组织中的ＳＴ２被纯化了３１５９７倍,得率为０．３５％。ＳＤＳ－聚丙烯酰胺凝胶电泳后银染色呈１条蛋白着色带,分子量为５５ｋｄ。     

大鼠肝脏过氧化氢酶的分离纯化及性质

根据过氧化氢酶的催化特性,采用盐析,透析,离心等技术,从大鼠肝脏中分离纯化过氧化氢酶,提纯倍数达到26倍,酶活性回收率为57%。为一般实验室分离纯化大鼠过氧化氢酶提供了一种有效的方法。酶学性质研究表明,该酶最适温度为37℃,最适pH值为7.5,在此条件下,以过氧化氢为底物的Km值为56 mmol.L-1。     

人胎肝造血干细胞增殖刺激物的分离及鉴定

经过超滤、ＤＥＡＥ－Ｓｅｐｈａｃｅｌ、ＳｅｐｈａｃｒｙｌＳ－２００和Ｓｕｐｅｒｏｓｅ１２ＨＲ多步分离纯化,从人胎肝细胞原代培养上清中分离到一分子量为３５ｋＤ的单一活性组分,具有造血干细胞增殖刺激活性,定义为ＦＬＳ－４。ＦＬＳ－４可能是一种新型造血干细胞增殖刺激因子,与具有这类活性的ＩＬ－３、ＩＬ－６、ＧＭ－ＣＳＦ、ＦＬＴ３配基和ＳＣＦ等在理化特性或生物学性质上均有所差异,在胎肝造血活跃时期,是启动早期造血干细胞从Ｇ＿０期进入Ｓ期的主要候选活性物质。     

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。     

鼠肝DNA甲基化酶的纯化及物理化学性质的研究

以Ｗｉｓｔａｒ大鼠肝为材料,确立了一个简便的纯化鼠肝ＤＮＡ甲基化酶的程序,包括：细胞的超声破碎、去内源核酸、硫酸铵盐析、磷酸纤维素亲和层析、ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０柱层析及ＳｅｐｈａｄｅｘＧ－１５０凝胶过滤。用不同浓度聚丙烯酰胺凝胶电泳和孔梯度凝胶电泳检测,纯化后的酶已达电泳均一,且酶的比活力提高１１２倍。以聚丙烯酰胺孔梯度凝胶电泳测得其天然酶的分子量为３６５ｋＤ,以ＳＤＳ－聚丙烯酰胺凝胶电泳测得该酶有两种亚基,大亚基为９５ｋＤ,小亚基为８５ｋＤ,推测该酶由两个大亚基和两个小亚基组成。     

胸苷激酶同功酶与肿瘤基因治疗

重点介绍了各种胸苷激酶（ＴＫ）同工酶的差异及其与癌症和细胞周期的关系,并介绍了最近利用ｔｋ基因进行肿瘤基因治疗研究的进展。