括搂素的纯化及其免疫毒素的制备

利用凝胶过滤及离子交换层析从Ｔｒｉｃｈｏｓａｎｔｈｅｓ ｋｉｒｉｌｏｗｉｉ Ｍａｘｉｍｏｗｉｃｚ种子中分离出一种核糖体失活蛋白,称之为括楼素。这种毒素在无细胞体系中对蛋白质生物合成具有明显的抑制活性而对完整细胞的毒性很低。与单克隆抗体偶联后,括搂素对靶细胞显示了明显的特异性细胞毒作用。     

苦瓜核糖体失活蛋白的分离纯化及其抑制烟草花叶病毒活性

目的:探讨分离纯化苦瓜种子核糖体失活蛋白(RIP)的方法,并研究利用纯化后的RIP抑制烟草花叶病毒(TMV)的活性。方法:用盐溶液抽提总蛋白后,经30%～90%的(NH4)2SO4分级沉淀,制成粗提液。用CMSepharoseFastFlow离子交换层析结合SephadexG-75凝胶柱分离纯化RIP。结果与结论:经SDS-PAGE检测及RNAN-糖苷酶活性鉴定,确定最终得到的蛋白为苦瓜RIP;所纯化的RIP对感染黄瓜和番茄的TMV有明显的抑制作用,但RIP的浓度并不与其抑制TMV的活性呈正相关。     

苦瓜的核糖体失活蛋白

核糖体失活蛋白是一类专一修饰核糖体的大亚基rRNA从而抑制蛋白质生物合成的蛋白毒素,可分为Ⅰ-型和Ⅱ-型两种类型。苦瓜中含有多种Ⅰ-型核糖体失活蛋白,如α-苦瓜素,β-苦瓜素和MAP30等,这些蛋白成分具有抗肿瘤、抗病毒和抗艾滋病等功能,因而近年来引起人们广泛的关注。对苦瓜核糖体失活蛋白的研究进展和应用前景进行了综述。     

一组丝瓜籽小分子核糖体失活蛋白LuffinS的分离,纯化 …

通过硫酸铵分级沉淀、ＣＭ－５２阳离子交换层析、ＨＲＬＣ分子排阻层析及ＦＰＬＣ Ｍｏｎｏ Ｓ离子交换层析等步骤,从丝瓜籽中分离到一组分子量为８ｋＤ左右的小分子核糖体失活蛋白－ＬｕｆｆｉｎＳ１、ＬｕｆｆｉｎＳ２、ＬｕｆｆｉｎＳ３。末端分析结果表明,它们的Ｎ端氨基酸分别为Ａｌａ、Ｐｒｏ和Ｔｈｒ。氨基酸序列分析确定了ＬｕｆｆｉｎＳ２的Ｎ端９个氨基酸的序列是Ｐｒｏ－Ａｒｇ－Ｇｌｙ－Ｇｌｎ－Ａｌａ－Ｐｈｅ－Ａ     

豆薯种子中两种蛋白质的分离纯化及其性质研究

豆薯（Ｐａｃｈｙｒｒｈｉｚｕｓｅｒｏｓｕｓ）种子经磷酸盐缓冲液抽提,Ｓ－ＳｅｐｈａｒｏｓｅＦａｓｔＦｌｏｗ柱,ＤＥ－５２纤维素柱和ＳｅｐｈａｄｅｘＧ－７５柱层析,提取出两种高纯度的蛋白成分,命名为ＰａｃｈｙｒｉｎＩ和ＩＩ,ＳＤＳ－ＰＡＧＥ测得其分子量分别为３３ｋＤ和１４．５ｋＤ,但ＨＰＬＣ分子筛的结果显示ＰａｃｈｙｒｉｎⅡ的分子量为２８ｋＤ,无论在还原条件下,还是在非还原条件下,ＰａｃｈｙｒｉｎＩ     

豆薯种子中两种蛋白质的分离纯化及其性质研究

豆薯（Ｐａｃｈｙｒｒｈｉｚｕｓｅｒｏｓｕｓ）种子经磷酸盐缓冲液抽提,Ｓ－ＳｅｐｈａｒｏｓｅＦａｓｔＦｌｏｗ柱,ＤＥ－５２纤维素柱和ＳｅｐｈａｄｅｘＧ－７５柱层析,提取出两种高纯度的蛋白成分,命名为ＰａｃｈｙｒｉｎⅠ和Ⅱ．ＳＤＳ－ＰＡＧＥ测得其分子量分别为３３ｋＤ和１４．５ｋＤ,但ＨＰＬＣ分子筛的结果显示ＰａｃｈｙｒｉｎⅡ的分子量为２８ｋＤ,无论在还原条件下,还是在非还原条件下,ＰａｃｈｙｒｉｎⅡ电泳的结果都完全相同,表明该蛋白的亚基不是以二硫键相连。两种蛋白的等电点分别为４．５和６．５．用酸解法测定了它们的氨基酸组成。在无细胞体系中,它们对蛋白合成有较弱的抑制活性,显示它们可能是核糖体失活蛋白（ＲＩＰｓ）家族中的新成员。     

括楼素的纯化及其免疫毒素的制备

利用凝胶过滤及离子交换层析从Trichosanthes kirilowii Maximowicz种子中分离出一种核糖体失活蛋白,称之为括楼素(Trichokirin)。这种毒素在无细胞体系中对蛋白质生物合成具有明显的抑制活性而对完整细胞的毒性很低。与单克隆抗体偶联后,括楼素对靶细胞显示了明显的特异性细胞毒作用。     

苦瓜籽蛋白MAP30的聚乙二醇化学修饰及抗肿瘤活性研究

采用硫酸铵分级沉淀、SP-Sepharose FF离子交换层析和Blue Sepharose CL-6B亲和层析获得苦瓜蛋白MAP30;采用(mPEG)2-Lys-NHS修饰MAP30;对修饰后的MAP30-PEG结合物与MAP30的抗肿瘤活性进行了比较分析。结果发现:MAP30对多数肿瘤细胞株具有明显的抑制作用,而且抑制效果在一定范围内具有剂量和时效依赖关系。但是在相同条件下,MAP30对人正常胚肺二倍体细胞株WI-38的毒性极小。MAP30和MAP30-PEG结合物对小鼠黑色素瘤(B16)、人宫颈癌细胞(Hela)和人表皮癌细胞(A431)细胞均表现出抑制作用,且这种作用在一定范围内呈时效和量效依耐性。但在相同浓度下PEG-MAP30对小鼠黑色素瘤(B16)、人宫颈癌细胞(Hela)和人表皮癌细胞(A431)的抑制作用低于MAP30,约占65%～70%左右。     

核糖体灭活蛋白在植物中的作用

植物核糖体灭活蛋白 (ribosome -inactivatingproteins ,RIPs)能够破坏真核或原核细胞的核糖体大亚基RNA ,使核糖体失活而不能与蛋白质合成过程中的延伸因子相结合 ,从而导致蛋白质合成受到抑制。不同的核糖体对不同RIPs的敏感性不同 ,RIPs对自体或异体核糖体的作用也有很大区别。RIPs对病毒有很强的抑制作用 ,并且有些RIPs表现出对某些真菌和昆虫的抗性 ,因此认为核糖体灭活蛋白在植物的防御反应中扮演重要角色。另外 ,RIPs还可能参与了细胞代谢、细胞死亡等生理调控过程。     

天花粉蛋白基因的克隆及序列分析

本文应用ＤＮＡ多聚酶链式反应（ＰＣＲ）技术,从括楼基因组ＤＮＡ中扩增并克隆了天花粉蛋白（ＴＣＳ）基因。核酸序列分析结果表明,克隆片段包括ＴＣＳ的前原蛋白的编码序列和５＇一侧翼区段。其编码序列与已发表的不同来源的３种ＴＣＳ基因的核苷酸序列的同源性分别为９９．２０％,９８．７４％和９８．６４％。推导出的氨基酸序列与已发表的４种ＴＣＳ的氨基酸序列的同源性分别为９８．６２％、９８．６２％、９７．４１％和９     

Isolation and characterization of ribosome-inactivating proteins from Cucurbitaceae

Due to their RNA-N-glycosidase activity, ribosome-inactivating proteins (RIPs) are attractive candidates as antitumor and antiviral agents in biomedical and agricultural research. We have isolated and characterized two such proteins, foetidissimin II and texanin, from two Cucurbitaceae species. Foetidissimin II, obtained from the roots of Cucurbita foetidissima, was identified as a type-2 RIP, with a molecular weight of 61 kDa, as estimated by gel electrophoresis. It is composed of two chains, a 29-kDa chain A, and a 32-kDa chain B. Texanin, isolated from the fruits of Cucurbita texana, is a type-I RIP, with a single chain of molecular weight 29.7 kDa, as estimated by MALDI-TOF-MS. Both proteins exhibit RNA-N-glycosidase activity, with aniline playing a critical role in rRNA cleavage. The IC50 value of foetidissimin II, determined by cell-free protein-synthesis inhibition, was 0.251 muM. In an in vitro cytotoxicity assay, foetidissimin II exhibited IC50 values of ca. 70 nM to both adenocarcinoma and erythroleukemia cells. Texanin exhibited a weaker anticancer activity against erythroleukemia cells, with an IC50 value of 95 microM, but no activity against adenocarcinoma cells. The N-terminal sequences of both proteins were compared with those of reported RIPs.     

Biosynthesis of ribosome-inactivating proteins from callus and cell suspension cultures of Mirabilis expansa (Ruiz &Pavon)

 Callus and cell suspension cultures from the little known Andean crop Mirabilis expansa were developed and maintained on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (1 mg/l) and kinetin (0.1 mg/l). Callus and cell suspension cultures were screened with antibodies raised against ME1 (27.5 kDa) and ME2 (27 kDa), two ribosome-inactivating proteins (RIPs) previously found in roots of M. expansa. A 29-kDa protein found in callus and cell suspensions reacted strongly with ME1 antibodies. The 29-kDa protein, named MEC, was purified to homogeneity by ammonium sulfate precipitation and cation exchange perfusion chromatography. Amino acid N-terminal sequencing revealed close homology between MEC and ME1. The MEC amino acid sequence examined was highly conserved among RIPs from widely different sources. This new RIP was immunolocalized to the cell walls of callus and cell suspension cultures. Received: 20 August 1999 / Revision received: 10 December 1999 / Accepted: 21 December 1999     

乌珠穆沁牛的染色体研究

用C带、G带、核仁组织者区的银染色(Ag-NOR)技术,研究了内蒙古的4头乌珠穆沁公牛(蒙古牛的一类型)的染色体。乌珠穆沁牛2n=60, X是一大的亚中着丝点染色体,Y为小的亚中着丝点染色体,臂比1.78。乌珠穆沁牛的Ag-NORs定位于2、3、4、11和28号染色体末端,平均每细胞中Ag-NORs出现频率为5.48。     

肥皂草毒蛋白分离纯化及性质研究

从肥皂草里分离到三种核糖体失活蛋白(ribosome inactivating proteins, RIPs): SO3a、SO3b,SO6.并测定SO3a,SO3b的相对分子质量分别约为22 500、19 400, 等电点都为8.4左右. 进行了氨基酸组成分析.用激光拉曼光谱初步预测SO3a和SO3b的二级结构含量.用反相毛细管色谱发现SO6含有两个组分,而Stripe等报道SO6为单一峰.     

17种植物中蛋白质提取物的抗HIV—1活性

以化合物对ＨＩＶ－１诱导Ｃ８１６６细胞形成合胞体的抑制实验和化合物对ＨＩＶ－１感染细胞的保护实验作为初筛方法,筛选了来源７科１７种植物的４７个样品,其中８个样品经测定是核糖体失活蛋白,其余为粗提蛋白。     

Studies on the anti-mitogenic, anti-phage and hypotensive effects of several ribosome inactivating proteins

An investigation was conducted to compare the anti-mitogenic, anti-phage and hypotensive activities of several ribosome inactivating proteins (RIPs) in order to ascertain whether the RIPs differed in their potencies in the various bioassays. Agrostin, luffin and saporin elicited a dose-dependent suppression of the mitogenic response of murine splenocytes to concanavalin A. The three RIPs were approximately equipotent in this regard, with near maximal inhibition attained at a dose of 83 nM and approximately 50% inhibition at 830 pM. Trichosanthin was slightly more potent than the three aforementioned RIPs. All of these RIPs were capable of inhibiting the replication of phage M13 in the bacterium Escherichia coli, the ranking of potencies being luffin>trichosanthin>agrostin when tested at a concentration of 3.5 microM. The RIPs gelonin and saporin did not exert a conspicuous antiviral effect at the same dose. After intravenous administration into normotensive rats via the external jugular vein, the RIPs saporin, trichosanthin, gelonin and momordin evoked a mild hypotensive response while luffin and agrostin were inactive. The hypotensive response, however, lacked dose dependence. The RIPs trichosanthin, momordin and gelonin did not affect the blood pressure response to angiotensin I. Chemical modification of the arginine residues of the RIPs brought about a reduction in their ability to inhibit cell-free translation. It appears that the ranking of potency of RIPs in one bioassay was different from the rankings in other assays.     

Seed reserve-protein glycosylation in an in vitro preparation from developing cotyledons of Phaseolus vulgaris

A particulate preparation from developing cotyledons of Phaseolus vulgaris L. was incubated with uridine-5-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc; [6-³H]glucosamine), and by polyacrylamide gel electrophoretic analysis it was shown that the labeled (N-acetyl)glucosamine (GlcNAc) was incorporated into the principal reserve protein of the cotyledons, vicilin, and also into phytohemagglutinin. Some of the labeled product also reacted with antiserum to vicilin from mature seeds. In contrast it was not possible to detect the incorporation of labeled mannose from guanosine-5-diphospho-D-mannose (GDP-mannose; [U-¹⁴C]mannose) into either of these proteins by gel-electrophoretic analysis of the mannose-labeled products, but we did observe a low incorporation of mannose into material which reacted with antiserum to vicillin. The predominant glycosylation reaction in vitro was therefore probably a transfer of GlcNAc alone, rather than in combination with mannose as preformed oligosaccharide.Abbreviations GlcNAc N-acetyl-D-glucosamine - GDP guanosine 5-diphospho - IEF isoelectric focusing - PHA phytohemagglutinin - SDS sodium dodecylsulfate - UDP uridine-5-diphospho     

Culture senescence and abscisic acid induce saporin production in cultured roots of Saponaria officinalis

The presence and variation of activity of the type 1 ribosome-inactivating protein saporin has been evaluated in cultured roots of the soapwort Saponaria officinalis . Results from western analysis and in vitro protein synthesis inhibition indicate that saporin production is increased in senescing cultures, reaching a maximum value during the late stationary phase. Accordingly, cultures treated with the senescence-related hormone abscisic acid show a significant increase in saporin activity, independently from the culture growth phase. Stress conditions, such as the presence of hydrogen peroxide in the culture medium, had no effect on the modulation of enzymatic activity. The putative regulation of saporin production by abscisic acid and its possible role in accomplishing the ageing programme is discussed.