高效液相层析法纯化人胎盘谷胱甘肽硫转移酶

足月分娩的新鲜胎盘组织制成匀浆后,经高速离心、超速离心,谷胱甘肽(GSH)Sepharose 6B亲合层析,Amicon pM-10膜超过滤及高效液相层析,最终经SDS-PAGE鉴定,结果呈现单一亚基区带,其亚基分子量为25000。 根据我们现有高效液相设备条件,用ODS柱代替RadulovicL等报道的特异阴离子柱,用磷酸盐洗脱液代替含谷胱甘肽、二硫苏糖醇及氯化钾的梯度洗脱液,从人胎盘组织成功地制备了谷胱甘肽硫转移酶(GST)纯酶,全过程在15min内完成,保留时间及主峰面积的重复性均较理想,7次实验结果的变异系数为0.2％,最终纯化578.9倍。本研究为各种形式GST的纯化制备提供了一个新的、重复性好、分辨率高及回收理想的简易方法。     

用亲和层析技术分离纯化天冬氨酸酶

<正> 亲和层析技术做为现代生化实验室常用的提纯方法,具有专一性强,活力损失小,分离步骤少等优点。天冬氨酸酶是重要的酶制剂,以往见报的分离纯化工艺步骤繁琐,活力损失大。本文报道的新工艺是用聚乙二醇(PEG)/磷酸钾双水相抽提;DEAE-sophadex A-50柱层析除PEG和部分杂蛋白;环氧氯丙烷活化载体,底物作配基的亲和层析技术获得了电泳纯的天冬氨酸酶。     

Expression of a gene encoding a rabbit sperm membrane protein in mammalian cells.

A general mammalian expression vector designated pSV2-EP was reconstructed by inserting an oligonucleotide fragment into pSV2-dhfr. This vector allowed insertion of cDNAs with EcoRI cohesive ends. The pSV2-EP contains a simian virus 40 (SV40) early promoter, origin for DNA replication, SV40 poly-A site, splicing site, an initiator ATG downstream from the promoter and an EcoRI site for the insertion of cDNA fragment screened from lambda gt11 expression libraries. A recombinant plasmid (pS-VRS-1) was constructed by inserting RSD-1, a cDNA encoding a rabbit sperm tail protein, into the EcoRI site of the pSV2-EP vector. Chinese hamster ovarian (CHO) dhfr-negative cells were cotransformed with pSV2-dhfr and pSVRS-1 by the calcium phosphate method. In selective culture medium without thymidine and hypoxanthine, several cell lines were obtained containing mRNA and DNA that hybridized with RSD-1. One of these transformed cell lines stained intensely with anti-rSMP-B antibodies, demonstrating that the RSD-1 was expressed in the transformed CHO cells.     

Chemical modification of Bacillus thuringiensis subsp. thuringiensis (HD-524) trypsin-activated endotoxin: implication of tyrosine residues in lepidopteran cell lysis

A purified protein fraction from a solubilized and trypsin-digested extract of Bacillus thuringiensis subsp. thuringiensis (HD-524) fermentation powder was lytic to cells from several lepidopteran lines. Maximum yield was obtained by alkaline carbonate-thiocyanate solubilization of washed powder followed by trypsin digestion and Sephacryl (S-300) chromatography. The alkaline carbonate-solubilized fraction consisted predominantly of two bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with MW of 144 +/- 0.9 kDa and 134 +/- 1.4 kDa. After trypsin treatment and column chromatography, the cytolytic fraction consisted of a major band with a MW of 60.0 +/- 1.8 kDa and a minor band of 69 +/- 0.9 kDa. Cells from Trichoplusia ni (TN368) were most susceptible to lysis with 50% of cells lysed at 3 micrograms/ml, followed by Spodoptera frugiperda cells (SF21AE) exhibiting 50% cell lysis at 5 micrograms/ml and Lymantria dispar cells (Ld652Y) showing 40% lysis at 10 micrograms/ml. Chemical modification of the polypeptides was performed to determine the role of certain amino acid residues in the cytolytic activity. The group-specific reagent tetranitromethane was used to nitrate and oxidize tyrosine and cysteine residues, respectively. Cysteine residues alone were also modified with p-hydroxymercuribenzoic acid. Lysine residues were modified with O-methylisourea. Of the three types of amino acid residues, only the modification of tyrosine resulted in reduced cell lysis.     

草鱼免疫应答的初步研究

研究了草鱼在不同水温条件下受抗原刺激后其中和抗体的变化。15℃培养条件下中和抗体上升缓慢,9周内滴度低于1:8;20℃时,3周后抗体可上升到1:256,最高达1:5270,而在25℃时,1周中和抗体即达到1:570,最高可达1:20000以上。并探索了从草鱼血清中提纯抗体的条件,研究其抗体的特性。草鱼血清中的抗体为大分子蛋白,容易解离为抗原性相同,分子量近似于人IgG的较小分子,含有较多的二硫键,具有类似IgM的某些特性。     

多肽的α螺旋结构对多肽与钙调蛋白亲合力的影响

本文设计并采用固相法合成了4种钙调蛋白可结合多肽,这些多肽分成两组,每一组中两个多肽的碱性和疏水性相近,但形成α螺旋结构的倾向(预测)不同。研究了这些多肽与钙调蛋白的相互作用,在Ca~(2+)存在下,这些多肽与丹磺酰钙调蛋白结合,使丹磺酰钙调蛋白的荧光光谱发生显著变化,测定了多肽与钙调蛋白所形成的复合物的解离常数。结果表明,预测形成α螺旋结构倾向较大的多肽与钙调蛋白的亲合力也较大。     

Flavonol glycoside production in callus cultures of Epimedium diphyllum.

Callus cultures of Epimedium diphyllum produced a large amount of epimedoside A in addition to a small amount of diphylloside B, ikarisoside C, epimedoside E, diglycosides of des-O-methylanhydroicaritin (8-gamma, gamma-dimethylallylkaempfero). Icariin, epimedins A-C, which are glycosides of anhydroicaritin, were also produced in the callus cultures. Contents of the flavonol glycosides in callus tissue were higher than those of mother plants, but the composition of each flavonol glycoside mixture in the callus cultures was different from that of the original plants. The time-course experiments showed that an inverse relationship existed between cell growth and flavonol glycoside production. Effects of hormonal factors on cell growth and flavonol glycoside production indicated that 2,4-dichlorophenoxyacetic acid was needed for the production of flavonol glycosides.     

Ultrastructure of the micropyle and its relationship to pollen tube growth and synergid degeneration in sunflower

Summary Ultrastructural studies made on the micropyle of sunflower before and after pollination resulted in the following observations. (1) The micropyle is closed instead of a hole or canal. The inner epidermis of the integument on both sides of the micropyle is in close contact at the apex of the ovule. The boundary between the two sides consists of two layers of epidermal cuticle. (2) The micropyle contains a transmitting tissue. The micropyle is composed of an intercellular matrix produced by the epidermal cells of the integument. (3) The micropyle is asymmetrical, and is much wider on the side proximal to the funicle. On the funicle side the cells adjacent to the micropyle are similar to those of the transmitting tissue: they have large amounts of intercellular matrix and contain abundant dictyosomes, rough ER, and starch grains, and provide an appropriate environment for growth of the pollen tubes. The cells distal to the funicle are rich in rough ER and lipid bodies; they lack large intercellular spaces. (4) The micropyle is variable in the axial direction, i.e., it is much larger and more asymmetric at the level distal to the embryo sac than at a level close to the embryo sac. After pollination, one to four pollen tubes are seen in a micropyle. During their passage through the micropyle, most pollen tubes are restricted to the side proximal to the funicle. There is a greater tendency (81%) for the degenerate synergid to be located toward the funicle, i.e., at the same side as the pollen tube pathway. The data indicate a close relationship between micropyle organization, orientation of pollen tube growth, and synergid degeneration.     

Color regulation in the archaebacterial phototaxis receptor phoborhodopsin (sensory rhodopsin II)

Phoborhodopsin, a repellent phototaxis receptor in Halobacterium halobium, exhibits vibrational fine structure, a feature that has not been identified for any other rhodopsin pigment at physiological temperatures. This conclusion follows form analysis of the absorption properties of the pigment in H. halobium membranes containing native retinal and an array of retinal analogues. The absorption spectrum of the native pigment has a maximum at 487 nm with a pronounced shoulder at 460 nm; however, the bandwidth is that expected for a single retinylidene species. Gaussian band-shape simulation with a spacing corresponding to the vibrational frequencies of polyene stretching modes reproduces the structured absorption spectra of native pigment as well as of analogue phoborhodopsin. Absorption shifts produced by a series of dihydroretinal and other retinal analogues strongly indicate that the dominant factor regulating the color of the pigment is planarization of the retinal ring with respect to the polyene chain.     

Mechanism of adenylate kinase. Structural and functional demonstration of arginine-138 as a key catalytic residue that cannot be replaced by lysine

Replacement of the arginine-138 of adenylate kinase (AK) by lysine or methionine resulted in a decrease in kcat by a factor of 10(4), increases in Km by a factor of 10-20, and relatively little changes in dissociation constants. Proton nuclear magnetic resonance (NMR) studies were then undertaken to obtain structural information for quantitative interpretation of the kinetic data. Since the lysine mutant (R138K) represents a conservative mutation with surprisingly large effects on kinetics, structural studies were focused on the wild type (WT) and R138K. The results and conclusions are summarized as follows: (i) The aromatic spin systems of WT and R138K were assigned from total correlated spectroscopy (TOCSY). Comparison of the chemical shifts of aromatic protons, one-dimensional spectra, TOCSY, and nuclear Overhauser enhanced spectroscopy (NOESY) indicated that the conformation of R138K was almost unperturbed relative to that of WT. Thus Arg-138 is not important for the tertiary structure. (ii) Proton NMR titrations with AMP and MgATP suggested that substrate binding affinities and substrate-induced conformational changes are nearly identical between WT and R138K. Thus arginine-138 should not be involved in stabilizing the first substrate in the binary complex. (iii) Notable differences were observed between the proton NMR spectra of the WT and R138K complexes with the reaction mixture, which agrees with the perturbation in the Km values of R138K. The differences were analyzed in detail by using a "static reaction mixture'--p1, p5-bis(5'-adenosyl)pentaphosphate (MgAP5A). The aromatic spin systems of WT + MgAP5A and R138K + MgAP5A were partially assigned from various two-dimensional spectra.(ABSTRACT TRUNCATED AT 250 WORDS)