百合巯基蛋白酶抑制剂的纯化及部分性质研究

百合的鳞茎中含有一种对木瓜蛋白酶有强抑制作用的巯基蛋白酶抑制剂．百合的鳞茎经浸取加热处理,木瓜蛋白酶偶联的Ｓｅｐｈａｒｏｓｅ４Ｂ柱亲和层析和ＳｅｐｈａｄｅｘＧ－１００分子筛层析,可获得在ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ均为单一蛋白带的百合巯基蛋白酶抑制剂（ＣＰＩ）．此ＣＰＩ为单链蛋白,含有０．３０７％的中性糖;Ｎ端氨基酸为Ｉｌｅ;ＳＤＳ－ＰＡＧＥ测得亚基分子量为１２０００;ＳｅｐｈａｄｅｘＧ－１００测得分子量为１２５００．百合ＣＰＩ在１００℃内和ｐＨ２～１２范围内非常稳定;对木瓜蛋白酶的抑制属竞争性抑制类型,其Ｋｉ值为１．１５×１０~（－９）ｍｏｌ／Ｌ,对木瓜蛋白酶的抑制摩尔比为８．５：１．     

水稻巯基蛋白酶抑制剂的纯化及其性质研究

水稻的糠皮和胚经生理盐水浸取、离心后的上清液加热至８０℃处理１０ｍｉｎ,离心获得的上清液调ｐＨ至８．０,得到沉淀。沉淀溶解于０．０１ｍｏｌ／ＬＨＣｌ,经透析冷冻干燥得水稻巯基蛋白酶抑制剂（ＣＰＩ）粗品;粗品再经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱线性离子梯度洗脱和ＳｅｐｈａｄｅｘＧ－１００柱分子筛层析,即可获得在ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和ＨＰＬＣ上均为单一蛋白带的ＣＰＩ样品。经上述步骤,ＣＰＩ可被纯化５８倍。经ＳｅｐｈａｄｅｘＧ－１００和ＳＤＳ－ＰＡＧＥ测定其分子量均为１２０００,Ｎ末端氨基酸为Ｐｒｏ,等电点５．６．水稻ＣＰＩ经１００℃处理１０ｍｉｎ后,其抑制活性无任何变化,在ｐＨ２．０～９．０之间,活性也不发生改变,但ｐＨ在９．０以上,其活性逐渐下降,水稻ＣＰＩ对木瓜蛋白酶是一种高亲和性的抑制剂,它对木瓜蛋白酶和无花果蛋白酶有强抑制作用,对菠萝蛋白酶仅有弱抑制作用,但对胰蛋白酶则全无抑制作用;其抑制类型属竞争性抑制剂类型,Ｋ＿ｉ值约３．５×１０￣（－８）ｍｏｌ／Ｌ对木瓜蛋白酶的抑制摩尔比约为１：１。     

冬瓜蛋白酶的分离纯化及其部分性质研究(英文)

冬瓜的果肉中发现了丰富的蛋白水解酶.用硫酸铵将冬瓜果肉汁分级盐析,得到粗酶液.再经DEAE-Sepharose FF离子交换层析和Superdex-75柱层析等步骤得到一种电泳纯的冬瓜蛋白酶.SDS-PAGE测得其分子量为64 kDa.以酪蛋白做底物时,该酶的最适反应温度为70℃,最适作用pH为6.5,在pH 4.5～10.5,40～70℃范围内较稳定.PMSF强烈抑制该蛋白酶的活性.另外,Hg~(2+)对该酶有强烈的抑制作用,Mn~(2+)离子对其有保护作用,Zn~(2+)、Ca~(2+)和Cu~(2+)等离子对其活性没有影响.     

假菠萝麻蛋白酶分离纯化及特性研究

从假菠萝麻叶汁中用乙醇分部沉淀法得到一种蛋白酶,对酪蛋白有强烈的水解活性,经Ｓｅｐｈａｄｅｘ Ｇ－１００柱层析和ＳＰ－Ｓｅｐｈａｄｅｘ Ｃ－５０离子交换柱层析等步骤纯化,可得到六角形结晶。结晶酶液经ＰＡＧＥ圆盘电泳,每条胶柱上只显示一条蛋白带,其活性在ｐＨ４。５－１０、５５℃范围内较稳定,最适ｐＨ８．５最适温度５０℃,Ｋｍ（酪蛋白）值为０．１８５％（Ｗ／Ｖ）。用ＳＤＳ－ＰＡＧＥ和Ｓｅｐｈａｄｅｘ     

元宝枫蛋白酶的分离纯化及其生化性质

采用有机溶剂沉淀和柱层析方法,从元宝枫(Acer truncatum Bunge)叶片中提取并纯化了叶蛋白酶.该蛋白酶的比活性为1 408.04 U·mg-1,纯化倍数50.77.以酪蛋白为底物时,该蛋白酶最适pH 7.5,最适温度60℃;该蛋白酶在pH 5.0～pH 10.0范围内以及在60℃以下较为稳定.该酶的活力能被半胱氨酸和EDTA激活,但受HgCl2抑制,具有巯基蛋白酶的特性.     

ISOLATION AND PARTIAL CHARACTERIZATION OF MACRO- AND MICRONUCLEI FROM PARAMECIUM AURELIA

A method was developed for the isolation of macro- and micronuclei from Paramecium aurelia. This method utilized ionic and nonionic detergents to rupture the intact cells, calcium ions and spermidine were employed to protect the nuclei, and the nuclei were purified by centrifugation. Macronuclei consisted of 22% DNA, 10% RNA, and 68% protein. Micronuclei were composed of 9% DNA, 11% RNA, and 80% protein. DNA from both macro- and micronuclei had a density of 1.687 g/cc in CsCl and 1.417 g/cc in Cs₂SO₄. These values corresponded to G + C content of about 23%. The RNA of macronuclei was examined by gel electrophoresis, and two high molecular weight species were identified having molecular Weights of 1.3 x 10⁶ and 2.8 x 10⁶ daltons. Three syngens were studied, and in each case the conditions for isolation of the nuclei were the same and no differences were observed in the properties of the nuclei.     

天麻球茎中一种抗真菌蛋白的分离和部分特性

白天麻(Gastrodia elata)顶生球茎中分离并纯化了一种抗真菌蛋白(Gastrodia Antifungal Protein),简称GAFP。在马铃薯葡糖琼脂培养基上,4μg GAFP滴加在直径0.6 cm圆纸片上可明显抑制木霉(Trichoderma reesei)菌丝生长。从每千克鲜球茎中可分离得GAFP约20 mg。用SDS-PAGE和凝胶过滤层析测得该蛋白为单多肽链,分子量14.0kD;用离子交换结合法测得电点为8.1;富含Asn,Ala,Gly和Leu,但无Met,Cys和Pro。未经热变性或SDS处理的GAFP与考马氏亮蓝试剂不发生显色反应。GAFP不具有几丁质酶和β-1,3-葡聚糖酶活性。球茎外层组织富含这种蛋白,内层薄壁组织无此蛋白。认为GAFP在阻止真菌侵染当年生顶生和侧生球茎的防卫机制中起重要作用。     

ISOLATION AND PARTIAL CHARACTERIZATION OF FRACTIONS ENRICHED IN SYNAPTOSOMES FROM CHICK BRAIN

–From a pool of hemispheres, optic lobes and cerebellum of chick 3 fractions containing synaptosomes have been prepared. They were obtained by subcellular fractionation of a homogenate and centrifugation of a crude mitochondrial suspension on a discontinuous Ficoll density gradient in iso-osmoticsucrose. The synaptosomal fractions were isolated from bands at the interface of 5–9, 9–12 and 12–16% Ficoll. The characterization of these fractions by marker enzymes, such as lactate dehydrogenase, acetyl-cholinesterase, monoamine oxidase, acid phosphatase and rotenone-sensitive and -insensitive NADH: cytochrome c reductase is reported. Electron microscopic analyses showed that the first fraction (AB) at the 5–9% Ficoll interface contained myelin and other membrane fragments as well as synaptosomes, the second fraction (C) at the 9–12% Ficoll interface contained mainly synaptosomes, and the third fraction (D) at the 12–16% Ficoll interface contained synaptosomes and free mitochondria. A fourth fraction (E) was obtained as a pellet, and was enriched in free mitochondria. There was fair agreement between the distribution pattern of the marker enzyme activities and the particles of the fractions seen by electron microscopy. The content of glycoprotein-bound N-acetylneuraminic acid and total phospholipid of these fractions has been determined. Relative to the mitochondrial fraction (E) the synaptosome fraction contained on basis of particulate protein, respectively, 2–3 times as much protein-bound N-acetylneuraminic acid and 10–20 per cent more total phospholipid.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF A GUT TRYPSIN-LIKE PROTEASE FROM LARVAE OF FLESH FLY BOETTCHERISCA PEREGRIN A (DIPTERA: SARCOPHAGAE)

Abstract A 16kD protease was purified from the gut extract of larvae of Boettcherisca peregrina , after ammonium sulfate precipitation, DEAE-Sephadex A-25 ion-exchange chromatography and SBBI-Sepharose 4B affinity chromatography. The results of substrate and inhibitor specificity indicated that the protease behaved as a trypsin-like protease. It possesses high activity against non-specific substrate casein and Hide powder azure, and against trypsin-specific substrates Bz-Phe-Val-Arg NA, Bz-Pro-Phe-Arg NA and Bz-Val-Gly-Arg NA. It can be strongly inhibited by PMSF, phenymethysulfonyl fluoride (serine protease inhibitor), SBBI, soybean Bowman-Birk inhibitor and Leupeptin (trypsin-specific inhibitor). Activity of this protease was found to be maximal at the alkaline range of pH 8. 5–9. 5.     

类产碱假单胞菌杀虫物质的分离纯化和鉴定

类产碱假单胞菌是一株昆虫病原菌,该菌对草地蝗虫、竹蝗等具有良好的致死作用,经鉴定,该菌对蝗虫具有毒杀作用的物质为其代谢分泌到胞外的一种蛋白质。类产碱假单胞菌培养物经硫酸按沉淀,SephadexG-100凝胶过滤及DEAE-SephadexA-50阴离子交换柱层析,纯化获得的杀虫蛋白只含一种亚基,分子量25100,等电点5．16,含17种氨基酸,其中谷氨酸含量最高,胱氨酸含量最低,最大吸收峰为278．3nm。     

海芋胰蛋白酶抑制剂的分离纯化及性质研究

利用亲和层析和分子筛凝胶过滤等技术,从海芋根茎中分离纯化到一种胰蛋白酶抑制剂,简称ＡＭＴＩ。经ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔ鉴定均显示单一条带,经ＳＤＳ－ＰＡＧＥ测定,其分子量为２２０００,经等电聚焦（ＩＥＦ）测定,其等电点为６．２。根据对胰蛋白酶的抑制比可知该抑制剂为单头抑制剂,其抑制活性在６０℃和ｐＨ５￣１１范围内保持稳定。     

四棱豆根瘤菌的分离及特性

从四棱豆根瘤上分离出一株慢生型根瘤菌(Rhizolia Ps)。在显微镜下,该菌株为革兰氏阴性,有一根亚极生鞭毛,大小为0.59×1.49微米。其增代时间为15.52小时,在39℃和含1.5％NaCl培养基中均不能生长。回接时能使四棱豆幼苗根部结瘤,固氮酶活性为2.81微摩尔乙烯·克鲜瘤~(-1)·小时~(-1)。本文对该菌株的含碳化合物利用、B.T.B.反应、在肉膏蛋白胨上的生长情况、石蕊牛奶反应、淀粉和明胶水解等生理生化特征进行了试验。     

CHARACTERIZATION AND PURIFICATION OF GUT FIBRINOLYTIC PROTEASE FROM TABANUS AMAENUS

Abstract After ammonium sulphate precipitation, Sephadex G-75 gel filtration, Lys-Sepharose 4B affinity chromatography and elution from electrophoresis, the fibrinolytic protease (TAFP) was isolated and purified from the extract of T. amaenus Walker gut. It appeared a single band corresponding to molecular weight of approximately 67kD on SDS-PAGE and an probably pI of 7.2 on IEF. On fibrin plate and plasminogen-free fibrin plate (heated at 85°C for 30 minutes to eliminate plasminogen), TAFP showed same fibrinolytic activity. The result might indicate that TAFP is a fibrinolytic enzyme degrading fibrin, as well as a plasminogen activator degrading fibrin via activating plasminogen. The result of chromogenic substrates indicated that TAFP possesses trypsin-like activity specifically degrading argininyl amide bond or peptide bond, but has no chymotrypsin activity. TAFP was almost inhibited powerfully by antipain, PMSF, soybean trypsin inhibitor and soybean Bowman-Birk inhibitor. However, leupeptin, antitrypsin and TLCK was more powerful effective inhibitors of TAFP. Optimal reaction pH of TAFP was 7.5, and it was stable in 5.5–7.0 of pH range.     

华广虻肠道溶纤活性蛋白的纯化和性质(英文)

经过 75 %饱和度硫酸铵沉淀、SephadexG 75凝胶过滤层析、Lys Sepharose 4B亲和层析和电泳制备洗脱 ,从华广虻 (TabanusamaenusWalker)腹部组织匀浆液中分离纯化出分子量约为 6 7KD的溶纤活性蛋白TAFP。经纤维蛋白平板测定表明 ,TAFP不仅具有纤溶酶作用 ,还具有激活纤溶酶原的作用 ;通过三肽生色底物测定发现 ,TAFP能分解纤溶酶原激活剂的生色底物—ChromozymUK及S 2 2 88。还能水解胰蛋白酶专一底物Bz Phe Val Arg NA及CBZ Gly Pro Arg NA ,表明TAFP具有类胰蛋白酶活性 ,专一水解精氨酸形成的酰胺键 (或肽键 )。TAFP无胰凝乳蛋白酶活性     

ISOLATION AND CHARACTERIZATION OF CHROMATIN FROM NEUROSPORA CRASSA

Different preparations of chromatin isolated from mycelia of Neurospora crassa were analyzed for DNA-associated RNA and proteins. The UV absorption spectra, the ultrastructure of chromatin, and the amino acid composition of the acid-extractable proteins were studied. The protein:DNA ratios range from 1.5 to 2.8; the RNA:DNA ratios range from 0.5 to 1.24. UV absorption shows a macimum at 259 mµ and a minimum at 238–239 mµ. The E280/E260 ranges from 0.59 to 0.70. Electron microscopy reveals a fibrous structure with individual fibers of 120–150 A average diameter. Attempts were made to study the protein by polyacrylamide gel electrophoresis and amino acid analysis. The results indicate that Neurospora chromatin does not contain basic proteins comparable to calf thymus histone. The ratios of basic to acidic amino acids range from 0.93 to 1.19. On electrophoresis, no bands are seen whose positions correspond to those of histones. Staining for basic proteins with fast green or eosin Y at pH 8.2 also shows a negative reaction, suggesting the absence of histones.