Purification of ornithine carbamoyltransferase from kidney bean (Phaseolus vulgaris L.) leaves and comparison of the properties of the enzyme from canavanine-containing and -deficient plants

Kidney bean (Phaseolus vulgaris L.) ornithine carbamoyltransferase (OCT; EC 2.1.3.3) was purified to homogeneity from leaf homogenates in a single-step procedure, using δ-N-(phosphonoacetyl)-l-ornithine-Sepharose 6B affinity chromatography. The 8540-fold-purified OCT exhibited a specific activity of 526 micromoles citrulline per minute per milligram of protein at 35 °C and pH 8.0. The enzyme represents approximately 0.01% of the total soluble protein in the leaf. The molecular mass of the native enzyme was approximately 109 kDa as estimated by Sephacryl S-200 gel filtration chromatography. The purified protein ran as a single band of molecular mass 36 kDa when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at a single isoelectric point of 6.6 when subjected to denaturing isoelectric focusing. These results suggest that the enzyme is a trimer of identical subunits. Among the tested amino acids, l-cysteine and S-carbamoyl-l-cysteine were the most effective inhibitors of the enzyme. The OCT of kidney bean showed a very low activity towards canaline. The OCTs of canavanine-deficient plants have very low canaline-dependent activities, but the OCTs of canavanine-containing plants showed high canaline-dependent activities. It was assumed that the substrate specificity of this enzyme determines the canavanine synthetic activity of the urea cycle. Received: 22 July 1997 / Accepted: 4 December 1997     

番茄抗病基因Cf9的3′UTR区含有内含子序列

从番茄（ＬｙｃｏｐｅｒｓｉｃｏｎｅｓｃｕｌｅｎｔｕｍＭｉｌ．）抗病品种“中杂９号”基因组ＤＮＡ中扩增并克隆了番茄抗叶霉病基因Ｃｆ９。序列分析结果表明,该基因全长２７５１ｂｐ,含有一个编码８６３个氨基酸的开放阅读框架。在该基因的３′ＵＴＲ区发现了一段未曾报道的内含子序列,长１１５ｂｐ,它所处的位置与番茄另一个抗叶霉病基因Ｃｆ２内含子的位置相似,其５′和３′边界序列为一重复序列,ＴＣＣＡＧＧ（Ｔ）ＡＴＴＣ,并与Ｃｆ２基因内含子边界序列高度同源。与已报道的Ｃｆ９的ｃＤＮＡ序列相比,它的第３７１位的核苷酸Ｔ突变成了Ｃ,从而使其编码蛋白的ＬＲＲ区第１２１位的亮氨酸突变为脯氨酸。     

The use of neamine as a molecular template: Identification of active site residues in the bacterial antibiotic resistance enzyme aminoglycoside 3′-phosphotransferase type IIa by mass spectroscopy

Four novel aminoglycoside-based affinity inactivators were shown to covalently modify the active site of aminoglycoside 3′-phosphotransferase type IIa (APH(3′)-IIa), an important resistance factor in bacteria for aminoglycoside antibiotics. Standard peptide mapping techniques failed with this enzyme. A novel mass spectroscopic analysis which combines protease digestion on the instrument probe, followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described which permitted rapid identification of the sites of protein modification. By this new technique, Glu-3 and Asp-23 were identified as active-site residues, the side chains of which potentially may serve as counter ions for the ammonium functionalities at positions 6′, and 1 and 3 of the antibiotic substrates, respectively. These findings contradict previous assertions that the C-terminal third of the enzyme should form the active site, by placing the active site clearly in the N-terminal portion of the enzyme.     

近金线属的分类地位及金线属的鉴别特征(鲤形目:鲤科:亚科)

本文根据系统学原理,提出了建立新类元的依据;并据此从分类和系统发育上论证了近金线属ＡｎｃｈｉｃｙｃｌｏｃｈｅｉｌｕｓＬｉｅｔＬａｎ独立成属的可能性,认为应将近金线属归于金线属;修订了金线属ＳｉｎｏｃｙｃｌｏｃｈｅｉｌｕｓＦａｎｇ的鉴别特征。     

不同理化因子对雪莲培养细胞中黄酮类形成的影响

研究了不同理化因子对水母雪莲(Saussurea medusa Maxim)愈伤组织生长及黄酮类化合物生物合成的影响。结果表明,有利于细胞生长及黄酮形成的合适温度为25℃。白光对愈伤组织生长无促进作用,但有利于黄酮的形成。培养基中添加1mg／L NAA和O．2mg／L的KT组合对细胞的生长较有促进作用。5％蔗糖和1％葡萄糖的组合有利于细胞的生长和黄酮的形成。用⁶⁰C0-γ射线辐照愈伤组织,在剂量为4000Gy的条件下,获得一个合成黄酮能力高于原愈伤组织70％的细胞系。用高效液相和紫外分光光度法,测定离体培养光照条件下干细胞总黄酮的含量为3．2％,是暗培养的4．4倍。培养温度25℃时干细胞黄酮的含量为2．02％,分别为20℃,35℃时的5倍和3．2倍。     

发展城郊游憩活动的效益：———以抚顺市高湾经济特区为例

１前言随着我国经济的高速发展,特别是实行双休日以来,城镇居民的假期游憩活动呈现大幅度上升的趋势。各大城市利用城郊风光,如山地、水库、森林、草地以及人文古迹等开辟了多种形式的游憩地,供人们游乐活动、旅游度假。抚顺市高湾经济特区位于抚顺市西部,距市中心１...     

甜菜坏死黄脉病毒内蒙分离物RNA3序列分析及编码25kD蛋白基因在大肠杆菌中的表达

以甜菜坏死黄脉病毒（ＢＮＹＶＶ）内蒙分离物的总ＲＮＡ为模板,通过反转录－ＰＣＲ扩增获得ＢＮＹＶＶＲＮＡ３全长ｃＤＮＡ。将其克隆到ｐＧＥＭ－７Ｚｆ（＋）上,得到重组质粒ｐＧＢＹ５６。序列分析结果表明,内蒙分离物ＲＮＡ３基因组全长为１７７５ｎｔ,其中包含３个开放阅读框架,分别编码２５ｋＤ蛋白、４．６ｋＤ蛋白和一种由５９个氨基酸组成的Ｎ蛋白。与法国Ｆ２分离物、德国Ｇ１分离物和日本Ｓ分离物相比,其核苷酸序列的同源性分别为９６．４％、９６．８％和９７．３％。将２５ｋＤ蛋白编码基因克隆到ｐＪＷ２上,构建了该基因的原核表达载体。ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ分析结果表明,２５ｋＤ蛋白基因在Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）中经温度（４２℃）诱导后,可特异地表达２５ｋＤ蛋白     

重组含有Epstein-Barr病毒潜伏膜蛋白1(LMP1)基因的杆状病毒在昆虫细胞中的表达

用杆状病毒表达系统重组病毒,在昆虫细胞中表达了完整的含有ＥＢＶ－ＬＭＰ１基因３个外显子开放读码框架的长２．３ｋｂ的ｃＤＮＡ片段。用重组病毒感染Ｓｆ９细胞,用免疫荧光染色,结果表明：４８小时表达重组蛋白,７２小时细胞较完整,免疫荧光染色强阳性,９６小时后细胞出现破碎。我们采集７２小时的组织培养上清和细胞破碎裂解液,分别采用ＳＤＳ－ＰＡＧＥ、ＨＰＬＣ分子筛法,用免疫蛋白印迹法实验证明,表达的蛋白能被抗ＬＭＰ１的单克隆抗体所识别,测定表达蛋白的分子量为６０ｋＤ。经蛋白含量扫描图分析,采用Ｓｅｐｈａｄｅｘ－７５柱初步纯化表达的ＬＭＰ１蛋白,将后者进行裸鼠体内致瘤实验,未见肿瘤生长。     

流行性感冒灭活疫苗的研制

将流感病毒接种于鸡胚尿囊腔培养,收集鸡胚尿囊液及羊水,经甲醛灭活后,采用蔗糖速率区带超离心的方法进行提纯经脱糖、紫外照射后,按照《ＷＨＯ生物制品规程》的要求,试制出含有Ａ１型、Ａ３型和Ｂ型流感病毒的流行性感冒Ⅲ价灭活疫苗