棕色固氮菌缺失nifZ基因的突变种固氮酶MoFe蛋白的纯化和性质

采用 52℃下加热 6 min,后经 DEAE- 52、Sephacryls S- 2 0 0和 Q- Sepharose等柱层析方法 ,分离纯化了棕色固氮菌 (Azotobacter vinelandii)缺失 nif Z基因突变种固氮酶 Mo Fe(Δnif Z Mo Fe)蛋白 ,其纯度达到电泳纯。Δnif Z Mo Fe蛋白的固氮活性为 2 83nmol C2 H2 还原 / (min·mg蛋白 ) ,远低于野生种 Mo Fe蛋白。Δnif Z Mo Fe蛋白对氧更敏感 ;热稳定性略低于野生种。Δnif Z Mo Fe蛋白的可见光吸收光谱与野生种 Mo Fe蛋白极为相似。其圆二色谱和磁圆二色谱在 450～ 550 nm与野生种 Mo Fe蛋白显著不同 ,表明其 P- cluster及其周围环境与野生种 Mo Fe蛋白有所差异。这亦可能是造成缺失 nif Z突变种 Mo Fe蛋白固氮活性低的原因。     

Purification and some properties of a novel microbial lactate oxidase

 Geotrichum candidum was found to produce a lactate oxidase. The enzyme was purified by gel filtration and ion-exchange chromatography. The purified lactate oxidase showed a molecular mass of 50 kDa under denaturing and about 400 kDa under non-denaturing conditions. Transmission electron micro-scopy analysis confirmed an octameric structure. FMN was found to be a cofactor for this enzyme. Polarographic studies confirmed an oxygen uptake by the lactate oxidase. The enzyme showed specificity towards the L isomer of lactate and did not oxidise pyruvate, fumarate, succinate, maleate and ascorbate. It was stable at alkaline pH and also for 15 min at 45°C. The addition of glycerol and dextran 500 000 to the enzyme sample enhanced storage stability. Received: 28 September 1995/Received revision: 10 January 1996/Accepted: 15 January 1996     

活菌制剂活菌数急剧衰降的原因与减缓衰降的方法

市售活菌制剂口服液活菌数急剧衰降的原因是营养缺乏,特别是缺乏部分氨基酸及某些维生素。增加动物性蛋白胨及强化酵母膏,添加适量维生素可使活菌数一年后保持在１０^６／ｍｌ以上。     

荧光假单胞菌抗噬菌体菌株的选育

本实验从荧光假单胞菌（Ｐｓｅｕｄｏｍｏｎａｓｆｌｕｏｒｅｓｃｅｎｓ）ＡＳ—３菌株的不正常发酵液中分离到一种噬菌体,将其命名为ＰＦＡＳ。ＡＳ—３菌株能利用葡萄糖发酵产生Ｄ－异维生素Ｃ的前体物质２－酮基－Ｄ－葡萄糖酸。电镜观察表明ＰＦＡＳ噬菌体呈蝌蚪形,具有直径为６６ｎｍ的六角形头部及长１１７ｎｍ的尾部。通过紫外线诱变及自然选育两种途径,配合简便有效的初筛方法,经多次分离、纯化、复筛最终在摇并发酵试验中获得６株产量稳定地高于对照敏感菌的抗噬菌体菌株,可望用于生产。     

Effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic acetylcholine receptors on transmembrane potentials and currents in guinea pig ventricular myocytes

The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K⁺ current and decreased the inward basal I Ca significantly. The reversal potential of both carbachol-and antibody-induced extra currents were close to –80 mV, being in proximity to the calculated E_k of –90 mV. A -adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward I_ca and outward I_k simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated I_Ca but not the isoprenaline-stimulated I_k. The antibody- or carbachol-induced outward K⁺ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated I_ca were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events.     

Fluorescence and Fourier-transform infrared spectroscopic studies on the role of disulfide bond in the calcium binding in the 33 kDa protein of Photosystem II

The 33 kDa protein of Photosystem II has one intrachain disulfide bond. Fluorescence spectroscopy shows that the major groups in the protein that bind to Ca²⁺ should be the carboxylic side groups of glutamic acid and/or aspartic acid. Fluorescence and Fourier-transform infrared (FTIR) spectroscopic studies indicate that the conformation of the 33 kDa protein is altered upon reduction, while the reduced protein still retains the secondary structure. FTIR spectroscopy also shows that the metal ions induce a relative decrease of unordered structure and -sheet, and a substantial increase of -helix in both the intact and the reduced 33 kDa protein. This indicates that the addition of cations results in a much more compact structure and that both the intact and the reduced 33 kDa proteins have the ability to bind calcium. The above results may suggest that the disulfide bridge is not essential for calcium binding.Abbreviations CD circular dichroism - FTIR Fourier transform infrared - La lanthanum - PS photosystem - Tb terbium     

Structure and analysis of phospholipase D gene from Ricinus communis L

Phospholipase D (PLD; EC 3.1.4.4) has been proposed to play a pivotal role in various cellular processes, but molecular understanding of this enzyme is rather limited. This report describes the nucleotide sequence, structure, and genomic organization of a PLD gene from castor bean (Ricinus communis L. cv. Hale). The PLD gene was isolated from a castor bean genomic library using the PLD cDNA as a hybridization probe. Sequence comparison with the PLD cDNA revealed that the PLD gene consisted of four exons and three introns, one of which interrupts the 5-untranslated region. Southern blot analysis indicated that the cloned PLD gene was present as a single-copy gene, and yet there were other PLD or PLD-related sequences in the castor bean genome.     

懒猴属的核糖体DNA变异及其种间分化关系

用１５种限制性内切酶和人２８Ｓ、１８ＳｒＤＮＡ探针构建了懒猴属各物种核糖体ＤＮＡ重复单位的限制性内切酶图谱。在进化速率较高的非转录间隔区,在大、中、小懒猴中分别定位了２３、２４、２４个酶切位点。大懒猴与中懒猴有１２个位点不同,与小懒猴有１４个位点不同,而中、小懒猴间则只有一个位点的差异。经过计算,大懒猴与中懒猴的遗传距离值为１２．６５％,与小懒猴的差异为１４．２４％,中、小懒猴间的差异则仅为０．７     

云南姬鼠的蛋白多态性及其遗传分化关系

本文采用蛋白电泳技术对来源于云南省若干地区的姬鼠属（Ａｐｏｄｅｍｕｓ）的３种姬鼠──高山姬鼠（Ａ．ｃｈｅｖｒｉｅｒｉ）８只,中华姬鼠（Ａ．ｄｒａｃｏ）３只和大耳姬鼠（Ａ．ｌａｔｒｏｎｕｍ）１只,以及作为外群的同科的绒鼠属的大绒鼠（Ｈａｐａｌｏｍｙｓｄｅｌａｌｏｒｉ）３只进行了分析。共检测遗传座位２７个,发现２１个座位存在多态性。根据蛋白多态的数据对研究对象进行遗传分化关系的探讨,用系统分析软件ＰＨＹＬＩＰ计算它们之间的分化关系,得到了一棵无根系统树。结果表明,作为外群的大绒鼠明显不同于其它３种姬鼠而聚在最外面。８只高山姬鼠个体汇聚成独立的一支,中华姬鼠的３个个体也聚成一支,但大耳姬鼠却聚在中华姬鼠一支中,因此我们认为大耳姬鼠同中华姬鼠的分化时间可能比较晚近。