人心肌酸性铁蛋白的分离纯化及临床应用

人心肌匀浆经热变性、酸化、硫酸铵盐析、超离心、ＳｅｐｈａｒｏｓｅＣＬ－４Ｂ柱层析和制备等电聚焦分离,得到酸性铁蛋白。经鉴定,所得酸性铁蛋白ｐＩ为５．０,Ｈ亚基分子量为２１ｋＤ,Ｌ亚基为１９ｋＤ,ＰＡＧＥ分析呈单一区带。制备了兔抗人酸性铁蛋白抗血清,用该抗血清建立的人酸性铁蛋白放射免疫分析可检测出８０％甲胎蛋白阴性肝癌病人。     

甲基单胞菌Methylomonas sp.GYJ3中甲烷单加氧酶羟基化酶组分…

从Ｍｅｔｈ ｙｌｏｍｏｎａｓ ｓｐ．ＧＹＪ３菌株中经ＤＮＥＡＥ－ＳｅｐｈａｒｏｓｅＣｌ－６Ｂ阴离子交换层析和ＳｅｐｈａｃｒｙｌＳ３００凝胶层析分离出纯化出甲烷加氧酶羟基酶组分,经ＨＰＬＣ分析,纯度大于９０％,分子量为２４０ｋＤ,纯化们数为３．９,比活为２２５ｎｍｏｌ环氧丙烷每分钟毫克蛋白,ＳＤＳ－ＰＡＧＥ表明,羟基化酶由三个亚基组成,亚基分子量为５６、４３、２７ｋＤ．ＩＣＰＡＥＳ测定羟基化酶的Ｆｅ     

人心肌酸性铁蛋白的分离纯化及临床应用

人心肌匀浆经热变性,酸化,硫酸铵盐析,超离心、Ｓｅｐｈａｒｏｓｅ ＣＬ－４Ｂ柱层析和制备等电聚焦分离,得到酸性铁蛋白,经鉴定,所得酸性铁蛋白ｐＩ为５．０,Ｈ亚基分子量为２１ｋＤ,Ｌ亚基为１９ＫＤ,ＰＡＧＥ分析呈单一区带,制备了兔抗人酸性铁蛋白抗血清,用该抗血清建立的人酸性铁蛋白放射免疫分析可检测出８０％甲胎蛋白阴性肝癌病人。     

玉米花粉32kD蛋白的纯化及性质

利用丙酮粉、硫酸铵分段盐析及离子交换层析技术,从玉米花粉细胞质中分离纯化了一种３２ｋＤ的可溶性蛋白,其ＧＴＰａｓｅ活性大于ＡＴＰａｓｅ。ＳＤＳ－ＰＡＧＥ表明达银染电泳纯。等电聚焦电泳测得等电点为５．２５。免疫印迹鉴定表明该蛋白与抗牛脑动蛋白或动力蛋白的抗体无免疫交叉反应。最大紫外吸收波长为２８２ｎｍ,ＣＤ谱分析说明具有球蛋白特征。     

玉米花粉32kD蛋白的纯化及性质

利用丙酮粉,硫酸铵分段盐析及离子交换层析技术,从玉米花粉细胞质中分离纯化了一种３２ｋＤ的可溶性蛋白,其ＧＴＰａｓｅ活性大于ＡＴＰａｓｅ。ＳＤＳ－ＰＡＧＥ表明达银染电泳纯。等电聚焦电泳测得等电点为５．２５。免疫印迹鉴定表明该蛋白与抗牛脑动蛋白或动力蛋白的抗体无免疫交叉反应。最大紫外吸收波长为２８２ｎｍ,ＣＤ谱分析说明具有球蛋白特征。     

林生山黧豆谷氨酸脱羧酶的分离纯化及部分性质的研究

以林生山黧豆为材料,利用硫酸铵分段盐析,丙酮沉淀,ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＦＦ离子交换柱层析,ＳｅｐｈａｃｒｙｌＳ３００凝胶过滤柱层析及ＦＰＬ－ＭｏｎｏＱ柱层析技术,以聚酰胺薄膜层析荧光定量法为酶活力检测手段,分离纯化了谷氨酰羧酶,达到电泳银染纯,纯化后的林生山黧豆谷氨酸脱羧酶活力达３７５．０９Ｕ．ｍｇ＾－１,纯化保数３８．２倍,经ＳＤＳ－ＰＡＧＥ测定,其亚基分子量为７０ｋＤ,经工ＰＡＧＥ确定     

系统感染TMV的番茄叶胞外β-1,3-葡聚糖酶

系统感染ＴＭＶ （ｔｏｂａｃｃｏ ｍ ｏｓａｉｃ ｖｉｒｕｓ）的番茄（Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ Ｍｉｌｌ．）叶胞外蛋白提取液经冰冻干燥浓缩、－ ２０℃丙酮沉淀、ＣＭ－Ｓｅｐｈａｄｅｘ Ｃ－２５离子交换层析、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－２５离子交换层析和Ｓｅｐｈａｄｅｘ Ｇ－７５凝胶层析纯化,获得ＰＡＧＥ均一的β－１,３－葡聚糖酶．ＳＤＳ－ＰＡＧＥ证明,它包含分子量为３６ ｋＤ 和２７ ｋＤ的两个同工酶．以昆布多糖为底物,酶的最适ｐＨ 在４．８—５．２之间,在ｐＨ ４—８稳定;酶的最适温度在３０—４０℃之间,在４０℃保温１ｈ 后酶活性不变;Ｋｍ 值为９．２ ｍ ｇ／ｍ Ｌ．在系统感染ＴＭＶ 的番茄叶胞外蛋白提取液中,有分子量为２２ ｋＤ、２７ ｋＤ和３６ ｋＤ的３个β－１,３－葡聚糖酶同工酶     

蚯蚓体内一种纤溶酶原激活剂(e-PA)的分离纯化

为获得一种高效,低廉的溶栓药物,从赤子爱胜蚓（Ｅｉｓｅｎｉａｆａｅｔｉｄａ）体内分离纯化出一种可体外激活纤溶酶原从而间接降解纤维蛋白的酶（ｅ－ＰＡ）．纯化过程包括：粗品的盐析,离子交换层析,凝胶过滤层析及疏水相互作用层析．该组份是由二个亚基通过疏水相互作用维系在一起的．通过凝胶过滤层析,可测得全酶的分子量为４５０００;ＳＤＳ电泳显示大、小亚基的分子量分别是２６０００与１８０００;而质谱法测得的大、小亚基的分子量分别为２４５５６．７与１５５４６．６．对大小亚基进行了氨基酸组成分析,结果显示大亚基不含Ｌｙｓ而小亚基不含Ｃｙｓ．测定了大亚基Ｎ端２５个氨基酸序列：ＶＩＧＧＴＮＡＳＰＧＥＩＰＷＱＬＳＱＱＲＱＳＧＳＷ．并与部分已知蛋白质序列进行了比较．ｅ－ＰＡ在纤维蛋白平板上表现有三种不同的纤溶活性     

牛肾上腺皮质LDL受体的纯化和抗体的制备

牛肾上腺皮质ＬＤＬ受体经Ｔｒｉｔｏｎ Ｘ－１００增溶,ＤＥＡＥ３２离子交换柱和ＬｐＢ Ｓｅｐｈａｒｏｓｅ亲和柱层析,在ＳＤＳ－ＰＡＧＥ中有三条区带,分别在原点;Ｍｒ １６０ｋＤ;Ｍｒ１２５ｋＤ处。进一步用８％ＳＤＳ－ＰＡＧＥ纯化三个区带的蛋白质分别免疫新西兰大白兔所得的抗体,应用免疫印迹和ＥＣＬ非同位素标记法可对牛肾上腺皮质和人皮肤纤维细胞膜上的ＬＤＬ受体进行测定。     

柑叶片蔗糖酶的分离纯化及其部分性质的研究

柑（Ｃｉｔｒｕｓｒｅｔｉｃｕｌａｔａ Ｂｌａｎｃｏ）幼叶中存在高活性的酸性蔗糖酶。经硫酸铵盐析、ＤＥＡＥ－琼脂糖离子交换层析、ＳｅｐｈａｃｒｙｌＳ－２００ 凝胶层析纯化,活性回收率６．４％ ,纯化倍数１７９．２ 倍。纯化的酶经聚丙烯酰胺凝胶电泳显示单一蛋白带,ＳＤＳ－ＰＡＧＥ显示１ 条蛋白带,其亚基分子量４０ ｋＤ。用ＳｅｐｈａｃｒｙｌＳ－２００ 凝胶层析法测得分子量为８０ ｋＤ。推测该酶由两个相同亚基构成。以蔗糖为底物测定该酶的表观Ｋｍ 为１．６×１０－ ２ ｍ ｏｌ·Ｌ－ １,Ｖｍ ａｘ为１００ ｍ ｇ 还原糖·ｍ ｇ－ １蛋白质·ｈ－ １。最适ｐＨ ５．０,酸碱稳定区在ｐＨ ４．５—５．５ 之间。最适温度５５℃     

豌豆铁蛋白的纯化及其抗血清的制备

干豌豆种子粗提物经MgCl2 盐析、AcA2 2 凝胶过滤和DEAE 纤维素阴离子交换柱层析等方法进行纯化 ,以邻菲咯啉显色法检测铁蛋白 ,最后获得纯的铁蛋白 .纯化的铁蛋白在PAGE上显示一条带 ,SDS PAGE显示该蛋白仅含 2 8kD一条亚基 .纯化的豌豆铁蛋白免疫兔 7周后 ,琼脂糖双扩散法检测抗血清效价达 1∶32 .用分级盐析法纯化抗血清 ,纯化后的抗体用琼脂糖双扩散法对大豆铁蛋白粗提物有免疫交叉反应     

Purification, quaternary structure and immunological properties of phosphorylase kinase from chicken skeletal muscle

Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme as determined by gel filtration on Sepharose 4B is close to that of rabbit skeletal muscle phosphorylase kinase (i. e., approximately 1300 000). The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 140 000 beta, 129 000; gamma', 44 000; delta, 17 000 (cf. the Mr values of the alpha- and gamma-subunits of the rabbit muscle isoenzyme are 146 000 and 42 000). The four subunits, alpha', beta, gamma' and delta, were found to exist in equimolar amounts as shown by a densitometric analysis of acrylamide gels; hence, the subunit formula of the chicken skeletal muscle isoenzyme is (alpha' beta gamma' delta)4. Rabbit antisera against a mixture of alpha'- and beta-subunits of chicken phosphorylase kinase yield a single precipitin line with this enzyme, do not show cross reactions of identity with the rabbit muscle enzyme but strongly inhibit the activity of the chicken enzyme and partially inhibit the activity of the rabbit muscle isoenzyme.     

Purification and characterization of a multicatalytic high-molecular-mass proteinase from rat skeletal muscle.

A proteolytic enzyme was purified from the post-myofibrillar fraction of rat skeletal muscle. The purification procedure consisted of fractionation of the muscle extract by (NH4)2SO4, chromatography on DEAE-Sephacel, fast protein liquid chromatography on Mono Q and gel filtration on Sepharose 6B. The enzyme preparation appeared to be homogeneous as judged by disc electrophoresis in polyacrylamide gels and by immunoelectrophoresis. The isoelectric point of the proteinase is at 5.1-5.2. The enzyme has an Mr of about 650 000 and dissociates into eight subunits of Mr 25 000-32 000 when subjected to electrophoresis in sodium dodecyl sulphate/polyacrylamide gels. The proteinase contains hydrolytic activity against N-blocked tripeptide 4-methyl-7-coumarylamide substrates with an arginine or phenylalanine residue adjacent to the leaving group. Maximum activity with the first group of substrates was at pH 10.5, and this activity was inhibited by leupeptin, chymostatin and Ca2+. Maximum activity with the latter group of substrates was at pH 7.5, and was also inhibited by the two microbial inhibitors, but was activated by Ca2+ ions. By using [14C]methylcasein as a substrate, maximum activity was observed at pH9.0, and this proteolytic activity was not affected by leupeptin, was enhanced by chymostatin and inhibited by Ca2+. Similar effects were observed when benzyloxycarbonyl-Leu-Leu-Glu 2-naphthylamide was used as a substrate. These enzymic activities were abolished by p-hydroxymercuribenzenesulphonic acid or mersalyl acid, whereas a small activation was observed with cysteine or dithiothreitol.     

小鼠腹水型肝癌H22a细胞浆胞内磷蛋白磷酸酶的纯化和性质

 磷蛋白磷酸酶是磷酸化/脱磷酸化作用中重要的调节酶。本文建立了小鼠腹水型肝癌细胞胞浆内磷蛋白磷酸酶(PrP)的纯化方法。用~(32)P-酪蛋白为底物测定活力。经纯化的酶纯度提高1380倍,聚丙烯酰胺梯度凝胶电泳检查,只有一条泳带。用凝胶过滤法和聚丙烯酰胺梯度凝胶电泳法测得该酶分子量为200,000。该酶催化~(32)P-酪蛋白脱磷酸化反应的最适pH7.2,对热不稳定。     

Phosphorylase kinase from chicken skeletal muscle. Quaternary structure, regulatory properties and partial proteolysis

Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross-react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 mumol min-1 mg-1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2-4-fold by calmodulin and troponin C, with concentrations for half-maximal activation of 2 nM and 0.1 microM respectively. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol 32P/mol alpha beta gamma delta monomer) and autophosphorylation (up to 8 mol 32P/mol alpha beta gamma delta monomer) increased the activity 1.5-fold and 2-fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2-fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken gamma subunits and some similarities in the structure of the rabbit red muscle and chicken alpha'.     

Purification and detection of multiple forms of histidine decarboxylase in rat gastric mucosa (author's transl)]

A specific histidine decarboxylase from rat gastric mucosa has been obtained at high purity and good yield (purification about 600-fold). The purification procedure included double (NH4)2SO4 fractionation, ion-exchange chromatography, preparative isoelectric focusing in a granulated gel and gel filtration. Only the specific histidine enzyme was obtained by that procedure; DOPA decarboxylase, a non-specific enzyme, was absent in our final preparation. Each step of the purification was visualized by polyacrylamide gel electrophoresis and analytical isoelectric focusing. The purified enzyme was apparently homogenous by criteria of electrophoresis and gel filtration and has a molecular weight of 94 000. Several protein bands appeared after isoelectric focusing and the enzyme activity was localized in 3 distinct peaks. The gastric enzyme consists of 3 active forms which could be distinguished by their isoelectric points: 5.4, 5.75 and 6. Moleculare weights estimated by SDS polyacrylamide gel electrophoresis were 97 000, 93 000 and 90 000, and no subunits were observed. Pyridoxal phosphate was required as a coenzyme and resolution of the holoenzyme agreed with a portion of the coenzyme tightly bound to the apoenzyme. The purified enzyme was stable at low ionic strength, near neutral pH; concentrated reducing agents inhibit the enzyme.     

Carbonic anhydrase from spinach leaves. Isolation and some chemical properties.

Spinach carbonic anhydrase has been purified by modification and extension of a published method (Pocker, Y., and Ng. J. S. U. (1973) Biochemistry 12, 5127-5134), using (NH4)2SO4 precipitation and chromatography on DEAE-cellulose, agarose, and DEAE-Sephadex. The enzyme so obtained was homogeneous by criteria of both standard and sodium dodecyl sulfate polyacrylamide gel electrophoresis and of constant specific activity throughout the elution profile on DEAE-Sephadex chromatography. The enzyme has an apparent Mr of 212,000 by gel filtration on Sephadex G-200, a Mr of 26,000 by sodium dodecyl sulfate electrophoresis, and each of the subunits contains approximately 1 g atom of zinc. These data and the excellent correlation between the number of lysine and arginine residues per subunit, and the number of tryptic peptides obtained by peptide mapping, suggest that spinach carbonic anhydrase is an octamer consisting of identical or very similar subunits. Its amino acid composition is similar to parsley carbonic anhydrase; both contain large numbers of half-cystine residues relative to erythrocyte carbonic anhydrases. The spinach enzyme is devoid of disulfide bonds. The enzyme is stable around neutrality at -14 degrees, as a suspension in saturated (NH4)2SO4 solution.     

Enzymes regulating glycogen metabolism in swine subcutaneous adipose tissue. I. Phosphorylase and phosphorylase phosphatase.

Glycogen phosphorylase from swine adipose tissue was purified nearly 700-fold using ethanol precipitation, DEAE-cellulose adsorption, AMP-agarose affinity chromatography, and agarose gel filtration. The purified enzyme migrated as one major and several minor components during polyacrylamide gel electrophoresis. Activity was associated with the major component and at least one of the minor components. The molecular weight of the disaggregated, reduced, and alkylated enzyme, estimated by polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate, was 90,000. Stability of the purified enzyme was considerably increased in the presence of AMP. The isoelectric pH of the enzyme in crude homogenates was 6.3. The sedimentation coefficient of the purified enzyme (7.9 S) and that in crude homogenates (7.3 S) was determined by sucrose density gradient sedimentation. Optimal pH for activity was between pH 6.5 and 7.1. Apparent Km values for glycogen and inorganic phosphate were 0.9 mg/ml and 6.6 mM, respectively. The Ka for AMP was 0.21 mM. Enzyme activity was increased by K2SO4, KF, KCl, and MgCl2 and decreased by NaCl, Na2SO4, D-glucose, and ATP. Inhibition by glucose was noncompetitive with the activator AMP; inhibition by ATP was partially competitive with AMP. The purified enzyme was activated by incubation with skeletal muscle phosphorylase kinase. Enzyme in crude homogenates was activated by the addition of MgCl2 and ATP; activation was not blocked by addition of protein kinase inhibitor, suggesting that phosphorylase kinase in homogenates of swine adipose tissue is present largely in an activated form. Deactivation of phosphorylase a by phosphorylase phosphatase was studied using enzyme purified approximately 200-fold from swine adipose tissue by ethanol precipitation, DEAE-cellulose chromatography, and gel filtration. The Km of the adipose tissue phosphatase for skeletal muscle phosphorylase a was 6 muM. The purified swine adipose tissue phosphorylase, labeled with 32-P, was inactivated and dephosphorylated by the adipose tissue phosphatase. Dephosphorylation of both skeletal muscle and adipose tissue substrates was inhibited by AMP and glucose reversed this inhibition. Several lines of evidence suggest that AMP inhibition was due to an action on the substrate rather than on the enzyme. We have previously reported that the system for phosphorylase activation in rat fat cells differs in some important characteristics from that in skeletal muscle. However, both swine fat phosphorylase and phosphorylase phosphatase have major properties very similar to those described for the enzymes from skeletal muscle.     

alpha-L-arabinofuranosidase from Ruminococcus albus 8: purification and possible role in hydrolysis of alfalfa cell wall.

An alpha-L- arabinofuranosidase has been purified from the extracellular broth of cultures of Ruminococcus albus 8. The purification procedure utilized gel filtration, (NH4)2SO4 precipitation, and isoelectric focusing. The purified enzyme appeared to be homogeneous when chromatographed on disc and analytical isoelectric focusing gels. The estimated molecular weight of the native protein was 305,000 to 310,000. Sodium dodecyl sulfate-gel electrophoresis analysis suggested that the native protein is a tetramer composed of 75,000-molecular-weight subunits. The enzyme appeared to have no metal cofactor requirement but was sensitive to several sulfhydryl reagents. The pH optimum with p-nitrophenyl-alpha-L-arabinofuranoside as the substrate was 6.9 and the Km was 1.3 mM. Several lines of evidence indicated that the enzyme is a glycoprotein. When assayed against alfalfa cell wall material, the enzyme hydrolyzed only small amounts of arabinose from the substrate. When assayed together with hemicellulolytic or pectinolytic enzymes against the same substrate, the arabinosidase significantly enhanced the hydrolytic action of the glycanases .     

Purification and some properties of alkaline pullulanase from a strain of bacillus no. 202-1, an alkalophilic microorganism.

Pullulanase (pullulan 6-glucanohydrolase EC 3.2.1.41) was purified about 290-fold from the culture fluid of Bacillus No. 202-1 by DEAE-cellulose adsorption, acetone fractionation, (NH4) 2SO4 precipitation and DEAE--cellulose column chromatography followed by Sephadex G-200 molecular sieve chromatography. The enzyme gave a single band of protein by disc polyacrylamide gel electrophoresis. The molecular weight was estimated as 92 000 by sodium dodecyl sulfate gel electrophoresis. The isolectric point was lower than pH 2.5. The optimum pH for enzyme action was about 8.5-9.0. The action of the enzyme on amylopectin and glycogen resulted in increase in the iodine coloration of 85% and 70%, respectively. The enzyme completely hydrolyzed 1,6-alpha-glucosidic linkages in amylopectin, glycogen and pullulan.