毛花猕猴桃蛋白酶的提纯和性质

用硫酸铵盐析,磷酸盐除果胶和两次ＤＥＡＥ纤维素柱层析等方法,从毛花猕猴桃（ＡｃｔｉｎｉｄｉａｅｒｉａｎｔｈａＢｅｎｔｈ．）无细胞提取液中提纯蛋白酶,在聚丙烯酰胺凝胶电泳上呈现一条带,纯酶的活力为３２５Ｕ／ｍｇ,比活力提高８９倍,总收率约为３７％。用ＳＤＳ聚丙烯酰胺凝胶电泳测定分子量为２３ｋＤ,用等电聚焦电泳测定等电点为４８。酶的最适ｐＨ为３８,最适温度为４３℃左右。纯酶制剂对其底物牛血红蛋白的Ｋｍ值为５５６×１０－３ｍｍｏｌ。酶的紫外吸收光谱最大值为２７８ｎｍ。二巯基苏糖醇、巯基乙醇、Ｌ半胱氨酸盐酸盐等还原剂对该酶有明显的激活作用,而碘乙酸、对氯汞苯甲酸对其有抑制作用,这表明该酶属于巯基酶类。     

蓖麻蚕卵天冬氨酸蛋白酶纯化及性质

采用硫酸铵沉淀,离子交换层析和离子交换凝胶过滤法,从蓖麻蚕卵母细胞中纯化出一种蛋白酸疼。该蛋白酶活性能被天冬氨酸蛋白酶特性异性抑制剂胃肽抑制,初步鉴定该酶为天冬氨酸蛋白酶,经ＳＤＳ－聚丙烯配角安凝胶电泳测得电泳测得分子量为９０ｋＤ,由凝胶过滤估计分子量在３６０ｋＤ,推测该酶由四亚基组成。     

红曲霉葡萄糖淀粉酶肽段P_A的提纯及性质的研究

红曲霉葡萄糖淀粉酶能水解淀粉产生葡萄糖。我们已对它进行了提纯、结晶,及电子显微镜观察,并测定了它的一些基本性质。该酶的两个主要分子型E_3和E_4的肽图谱绝大部分是相同的,只有个别肽段不同。我们用胰凝乳蛋白酶水解红曲霉葡萄糖淀粉酶的两个型,两者的产物中都有一个P_A肽段,其凝胶电泳迁移率和纸层析R_f值相同。本文报道我们用垂直板型凝胶制备电泳及凝胶层析法提纯P_A肽段,并对其性质进行的研究结果。     

黄姑鱼肌肉胆碱酯酶的纯化及其某些性质

用亲和层析方法纯化了黄姑鱼(Nibea albiflora)肌肉胆碱酯酶。结合在膜上的胆碱酯酶用两种非离子型表面活性剂的混合液抽提、硫酸铵分级沉淀、再经一次亲和层析,共提纯11,800倍,最高比活力达2180活力单位/毫克蛋白,总活力回收约20%。纯化后的酶用聚丙烯酰胺凝胶电泳,除部分酶蛋白留在原点外,在凝胶内为单一蛋白染色区带,具有酶活力。SephadexG-200凝胶过滤得两个酶活力峰。亲和吸附剂在八个月内反复使用近20次,性能不变。对酶的一般性质作了初步的研究。根据酶的底物专一性、受真性酶专一性抑制剂抑制作用的敏感性及过量底物抑制作用的存在,可以认为黄姑鱼肌肉胆碱酯酶基本上具有真性酶的特征。     

黄姑鱼肌肉胆碱酯酶的纯化及其某些性质

用亲和层析方法纯化了黄姑鱼(Nibea albifiora)肌肉胆碱酯酶。结合在膜上的胆碱酯酶用两种非离子型表面活性剂的混合液抽提、硫酸铵分级沉淀、再经一次亲和层析,共提纯11,800倍,最高比活力达2180活力单位/毫克蛋白,总活力回收约20%。纯化后的酶用聚丙烯酰胺凝胶电泳,除部分酶蛋白留在原点外,在凝胶内为单一蛋白染色区带,具有酶活力。SephadexG-200凝胶过滤得两个酶活力峰。亲和吸附剂在八个月内反复使用近20次,性能不变。对酶的一般性质作了初步的研究。根据酶的底物专一性、受真性酶专一性抑制剂抑制作用的敏感性及过量底物抑制作用的存在,可以认为黄姑鱼肌肉胆碱酯酶基本上具有真性酶的特征。     

BamHI DNA甲基化酶的提纯及物理化学性质

 通过硫酸铵盐析,DEAE-纤维素柱层析,磷酸纤维素亲和层析及SephadexG-100凝胶过滤法,从噬淀粉芽孢杆菌HI(Bacillus amyloliguefaciens HI)提纯了DNA甲基化酶。用聚丙烯酰胺凝胶电泳检查,已达电泳均一,比活力提高了326倍。并用聚丙烯酰胺梯度凝胶电泳和Sephadex G-100凝胶过滤法测得其天然酶的分子量为273000,又用SDS聚丙烯酰胺凝胶电泳测得它的亚基分子量为34500,故该酶有8个分子量相同的亚基。用凝胶电聚焦法测得其pI_(22 c)=9.0。     

慈菇中两种结晶的多功能蛋白酶抑制剂的研究

由于蛋白酶抑制剂在生理、生化、病理、药理上都占有很重要的地位,特别是具有多功能的蛋白酶抑制剂被广泛应用于临床,近年来愈来愈受到各方面的重视。本文报道了从慈菇中提取两种结晶的多功能蛋白酶抑制剂,它们都具有两个活力相等的活性中心,这与其他已知的同类型植物蛋白酶抑制剂不同,有它的特殊性。(1)慈菇蛋白酶抑制剂A、B,除能抑制胰蛋白酶外,还能抑制胰凝乳蛋白酶及猪颌下腺的舒缓激肽释放酶。用酰胺、酯及蛋白等不同底物分别求出抑制剂A、B对胰蛋白酶的当量抑制比值及对胰凝乳蛋白酶的半抑制比值。两者对胰蛋白酶的抑制常数在10~(-9)～10~(-10)的范围内,抑制剂B对胰蛋白酶较之A有更大的结合力,但抑制剂A对胰凝乳蛋白酶及舒缓激肽释放酶较之B却有更明显的抑制活力。(2)用葡聚糖凝胶过滤及聚丙烯酰胺凝胶电泳分别测定抑制剂A、B的分子量均在17000左右。从抑制剂A、B对胰蛋白酶的当量抑制比值求得分子量为8500。这说明每一抑制剂分子中具有两个活力相等的活性中心。(3)测定了抑制剂A、B的氨基酸组成,二者除碱性氨基酸及天冬氨酸含量略有差异外其余皆相同,各含有两对二硫键,非极性氨基酸含量较高约占60%左右。由氨基酸组成求得最小分子量约16500。(4)用二硝基氟苯,二甲基氨基萘磺酰氯及氨肽酶M测定抑制剂A、B的N末端,都证实是天冬氨酸。     

海枣曲霉β-葡萄糖苷酶的提纯与性质

通过聚乙二醇6000一磷酸钾缓冲液双相分离,Sephadex G—100凝胶过滤、DEAE-Sephadcx A-50及SE-Se phadex C-50离子交换柱层析等提纯步骤,从海枣曲霉(Aspe rgillutphoenlcis)麦麸培养物抽提液中提纯到凝胶电泳均一的β-葡萄糖苷酶。该酶的最适pH5．0,最适温度60℃,在pH 4．0--7．5之间及55℃以下稳定。Ag+及Hg2+对该酶有强烈的抑制作用。用SDS-凝胶电泳法及梯度凝胶电泳法测得该酶均分子量分别为118000及195000薄层凝胶等电聚焦法测得其等电点为pH 3.95。     

枯草杆菌产弹性酶及提纯工艺研究

对枯草杆菌产酶弹性蛋白酶和酶提纯生产工艺进行了系统研究,得到了含弹性蛋白酶活力为3万u/g的纯酶粉。     

康氏木霉(Trichoderma koningii)纤维素酶系中β-葡萄糖苷酶的提纯与性质

从康氏木霉培养抽提液扣分离提纯了β-葡萄糖苷酶。提纯步骤通过DEAE SephadexA-50、SE Sephadex C-50和Sephadex G-75柱层析三步纯化。提纯后比潘提高766倍,回收率为25.8%。经硷性和酸性Disc 电泳以及SDS 凝胶电泳鉴定均为单一带。用SDS 凝胶电泳测得该酶的分子量为77000。用加温、改变pH 或化学变性剂(SDS)处理均能使酶失活并且改变该酶的凝胶电泳行为,揭示该酶有亚基存在的可能性。用底物(纤维二糖)或竞争性抑制剂(葡萄糖酸δ-内酯)能保护该酶使其酶活和电泳行为基本上不改变或变化较小。δ-内酯的K(?)比K_m 要小二到三个数量级,它很可能是该酶底物过渡态中间物的类似物。     

Purification and properties of streptococcal nicotinamide adenine dinucleotide glycohydrolase.

Highly purified streptococcal nicotinamide adenine dinucleotide glycohydrolase (NADase) was obtained by utilizing disodium tetrathionate to protect the enzyme by blocking the sulfhydryl groups of streptococcal proteinase. This was followed by two-step ion-exchange chromatography. The pure enzyme, demonstrated as a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, had a specific activity of 11,200 NADase units per mg of protein and was devoid of hemolytic activity. NADase had a molecular weight of about 55,000 as determined by gel filtration, by summation of amino acid residues, and by sodium dodecyl sulfate/gel electrophoresis. The purified enzyme had optimal activity at pH 7.3 and at 40 C and a calculated Km of 5.1 times 10- minus 4 mM. It was inhibited by alpha-iodoacetamide.     

Characterization of fructose 1,6-bisphosphatase from bakers' yeast

Active nonphosphorylated fructose bisphosphatase (EC 3.1.3.11) was purified from bakers' yeast. After chromatography on phosphocellulose, the enzyme appeared as a homogeneous protein as deduced from polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A Stokes radius of 44.5 A and molecular weight of 116,000 was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate resulted in three protein bands of Mr = 57,000, 40,000, and 31,000. Only one band of Mr = 57,000 was observed, when the single band of the enzyme obtained after polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate was eluted and then resubmitted to electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated 1030 residues/mol of enzyme including 12 cysteine moieties. The isoelectric point of the enzyme was estimated by gel electrofocusing to be around pH 5.5. The catalytic activity showed a maximum at pH 8.0; the specific activity at the standard pH of 7.0 was 46 units/mg of protein. Fructose 1,6-bisphosphatase b, the less active phosphorylated form of the enzyme, was purified from glucose inactivated yeast. This enzyme exhibited maximal activity at pH greater than or equal to 9.5; the specific activity measured at pH 7.0 was 25 units/mg of protein. The activity ratio, with 10 mM Mg2+ relative to 2 mM Mn2+, was 4.3 and 1.8 for fructose 1,6-bisphosphatase a and fructose 1,6-bisphosphatase b, respectively. Activity of fructose 1,6-bisphosphatase a was 50% inhibited by 0.2 microM fructose 2,6-bisphosphate or 50 microM AMP. Inhibition by fructose 2,6-bisphosphate as well as by AMP decreased with a more alkaline pH in a range between pH 6.5 and 9.0. The inhibition exerted by combinations of the two metabolites at pH 7.0 was synergistic.     

Purification and characterisation of a carboxylesterase from the latex ofSynadenium grantii Hook, ‘f’

The latex ofSynadenium grantii was found to contain esterolytic activity. Polyacrylamide gel electrophoretic study coupled with substrate and inhibitor specificity studies revealed the presence of multiple forms of carboxylesterases and cholinesterases in the latex. One of the carboxylesterases of the latex was purified by acetone fractionation, carboxymethyl-Sephadex chromatography and Sepharose-6B gel filtration. The homogeneity of the enzyme was established by polyacrylamide gel electrophoresis, isoelectric focussing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme consists of a single polypeptide chain with a molecular weight of 14,000. The amino acid analysis of the purified enzyme revealed that it contained a greater number of neutral and acidic, compared to basic amino acid residues. The isoelectric pH of the enzyme was found to be 4.0. The enzyme was a glycoprotein as revealed by periodic acid Schiff-staining technique. Studies with different organophosphate and carbamate inhibitors showed that this enzyme was sensitive to organophosphates. The product inhibition studies with this enzyme showed linear competitive inhibition with acetate and linear non-competitive inhibition with 1-naphthol.     

黑曲霉(Aspergillus niger)β-D-葡萄糖苷酶的纯化及其性质

利用垂直板凝胶制备电泳从黑曲霉(Aspergillus niger,AS 3.316)中分离提纯了β-D-葡萄糖苷酶(EC3.2.1.21),经凝胶电泳鉴定为单一带。酶作用的最适pH为4.4,在pH4.0—6.2稳定;最适温度65℃,热稳定性较好,于60℃保温4小时,活力保留80％。此酶作用于纤维二糖的Km值为6.09mM。聚丙烯酰胺薄层等电聚焦测得其pI值为5.5;用SDS凝胶电泳测得其分子量为77000。此酶不仅能水解纤维二糖和对硝基苯-β-D-葡萄糖苷,还能微弱地水解对硝基苯β-D-半乳糖苷和β-D-木糖苷。金属离子Fe~(2+)、Hg~(2+)、Cu~(2+)、Al~(3+)、Hg~+和Ag~+等对此酶有不同程度的抑制作用,蛋白质侧链修饰剂N-溴代琥珀酰亚胺对此酶有较强的抑制作用,2-羟基-5-硝基溴苯对酶也有一定的抑制作用,推测色氨酸残基对β-D-葡萄糖苷酶的活力是非常必要的。     

Ribonuclease isolated from yeast (Saccharomyces carlsbergensis): Characterization and properties

The homogeneity of a purified ribonuclease from brewers' yeast was determined by velocity sedimentation and polyacrylamide gel electrophoresis techniques. The velocity sedimentation pattern gave a single peak with a S_app 3.46 and polyacrylamide gel electrophoresis showed one major band. The absorption spectrum of the enzyme showed maximum absorption at 277–278 nm and minimum at 252 nm. The enzyme was relatively stable to extreme pH values and high temperature. Both NaCl and KCl increased the enzyme activity whereas enzyme was inhibited by divalent metal ions. The inhibition of the enzyme was increased in the order of Ca²⁺ > Mg²⁺ > Fe²⁺Cu²⁺. Chemical modification studies of the enzyme showed that tryptophan residues and disulfide bonds were required for enzyme activity.     

Partial purification and characterization of an alkaline proteinase from the rabbit testis

A proteolytic enzyme capable of cleaving intact proteins and synthetic substrates α?N?benzoyl?DL?arginine β?naphthylamide (Bz-Arg-NNap), α-N-benzoyl-L-arginine p-nitroanilde (Bz-Arg-NPhNO₂), and α-N-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt) was purified 92– fold from the rabbit testes. The enzyme exhibited optimal activity at pH 9.0 and 50°C. The polyacrylamide gel electrophoresis and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified enzyme demonstrated multiple forms; the major band in the SDS-polyacrylamide gel electrophoresis corresponded to a M_t 48,000. The same value was established by the gel filtration over Sephadex G-75. The rabbit testicular alkaline proteinase (TAP) resembled acrosin in the hydrolysis of Bz-Arg-OEt. However, CaCl₂, a potential stimulator of acrosin activity, inhibited the alkaline proteinase. The strong inhibitors of acrosin, eg pheny methyl sulphonyl fluoride (PMSF), tosyl lysine chloromethyl ketone (TLCK), and benzamidine did not inhibit the alkaline proteinase. TAP was activated by an acrosin inhibitor isolated from the rabbit testes. Since 0.5 M KCl was necessary for complete extraction of the enzyme and the bulk of the activity was present in 9,000g pellet of the testicular homogenate. The alkaline proteinase appeared to be associated with the membranous structures.     

Purification and characterization of cathepsin D-like proteinase from the tadpole tail of bullfrog, Rana catesbeiana

1. An acid aspartic proteinase in the regressing tadpole tail was purified about 800-fold with a 36% recovery. 2. The mol. wt of the enzyme was found to be 42,000 on gel filtration and 38,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively. 3. The purified enzyme had a maximum activity at pH 3.5 and an apparent Km of 0.084% with acid-denatured hemoglobin as substrate. 4. The enzyme activity was strongly inhibited by pepstatin. In addition, diazoacetylnorleucine methyl ester inactivated the enzyme in the presence of cupric ions. 5. The enzyme was identified as a cathepsin D (EC. 3.4.23.5)-like proteinase.     

Isolation,Purification, and Properties of Cysteine Proteinase from <Emphasis Type="Italic">Bombyx mori</Emphasis> L. Eggs

Silkworm moth (bombyx) egg cysteine proteinase with maximal activity at pH 3.0 was purified by chromatography and isoelectrofocusing. On SDS-electrophoresis in polyacrylamide gel the purified enzyme showed a single band of molecular mass 50 kD. Isoelectrofocusing revealed that the bombyx egg cysteine proteinase exists in two forms with pI values of 5.95 and 6.43, respectively. The enzyme activity was sensitive to inhibition by iodoacetamide and p-chloromercuribenzoate but resistant to EDTA, pepstatin, and phenylmethylsulfonyl fluoride. The cysteine proteinase hydrolyzes storage proteins of bombyx eggs but it was inactive with respect to N-benzoyl-D,L-arginine-p-nitroanilide (BAPNA). It is a cathepsin L-like enzyme.     

Purification and characterization of an extracellular serine proteinase from Acanthamoeba castellanii

An extracellular proteinase of Acanthamoeba castellanii was purified and its biochemical and pathological properties were characterized. The molecular mass of the purified enzyme was approximately 42 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 HR gel-filtration chromatography. Therefore, its structure seemed to be monomeric with a single polypeptide. Its activity was inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride. Its activity was optimum at 30 to 50 degrees C with a maximum at 50 degrees C; optimal pH was 8.0. As much as 70% of the enzyme activity was maintained at 50 degrees C for at least 12 h but was rapidly inactivated thereafter. The purified enzyme degraded collagen and rabbit corneal extract. Furthermore, it exhibited strong cytopathic effects on human corneal epithelial cells and fibroblast cells. These suggest the possible role of this enzyme in the pathogenesis of Acanthamoeba.     

东亚钳蝎毒透明质酸酶的纯化和部分性质的研究

用CM-SephadexC50,CM-SephadexC25和SephadexG-75凝胶过滤,从东亚钳蝎毒中提纯蝎毒透明质酸酶,应用低pH系统不连续聚丙烯酰胺凝胶圆盘电泳,SDS-不连续聚丙烯酰胺凝胶垂直板电泳鉴定均为单一条带,活力提高34倍,产率为12％,纯品无出血活性,无神经毒性。用凝胶过滤法和SDS电泳法测得分子量为54000,PAS染色证实为糖蛋白。 纯化的透明质酸酶的最适pH为4.5～6.5,最适温度为37℃,该酶对热的稳定性比蛇毒透明质酸酶高一些,但在碱性环境中也易失活。0.15MNaCl对酶活性有明显稳定作用,Fe~(2+)、Fe~(3+)及肝素对酶活性有明显的抑制作用,Cu~(2+)对酶活力也有一定影响。