一组玉米胚钙沉淀蛋白的初步研究

从玉米胚中分离出一组理化性质相似的可为钙所沉淀的蛋白。该组蛋白可被３％的三氯乙酸和５５％的硫酸铵可逆沉淀,具有较高的热稳定性,在９３￣９４℃下５ｍｉｎ不沉淀。该组钙沉淀蛋白可被等于或大于１ｍｍｏｌ／Ｌ的ＣａＣｌ２可逆地沉淀,但不被ＭｇＣｌ２或ＮａＣｌ沉淀。该组蛋白在在ＥＧＴＡ存在下可与ｐｈｅｎｙｌ－ｓｅｐｈａｒｏｓｅ ４Ｂ结合而被含Ｃａ＾２＋的缓冲液所洗脱。它们由７种蛋白质组成,亚基分子量为１６￣     

盐分和水分胁迫盐地碱蓬幼苗渗透调节效应的研究

利用不同浓度ＮａＣｌ和等渗ＰＥＧ处理４ｄ龄的盐地碱蓬（Ｓｕａｃｄａ ｓａｌｓａ（Ｌ．）Ｐａｌｌ．）幼苗,１０ｄ后测定植株中主要有机溶质和无机离子的含量及叶片涌透势和涌透调节能力。结果表明,ＮａＣｌ处理的无机离子总含量急剧增加,其中Ｎａ＾２＋、Ｃｌ＾－半加最多,占总计算渗透势（ＣＯＰ）的７１％￣８８％,总无机离子占ＣＯＰ的９５％￣９７％。而有机溶质总含量则稍有降低,约上ＣＯＰ的２％￣５％。ＰＥＧ处理     

人前列腺生长因子的分离纯化及生物活性鉴定

从人良性增生前列腺组织中经硫酸铵沉淀和肝素－琼脂糖凝胶层析纯化出人前列腺生长因子（ｈＰＧＦ）,纯化倍数约１０００倍,ＳＤＳ－ＰＡＧＥ和等电聚焦电泳示分子量约为１７ｋＤ、等电点同标准ｈＦＧＦ。利用分离培养的人前列腺间质或纤维细胞进行活性鉴定,发现以１．３￣１．７ｍｏｌ／Ｌ ＮａＣｌ洗脱部分为ｈＰＧＦ,活性最高,对间质成纤维细胞有显著刺激增殖作用。     

抗旱性不同的小麦幼苗对水分和NaCl胁迫的反应

分别测定抗旱小麦的８１３９（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．ｃｖ．８１３９）和干旱敏感品种甘麦８号（Ｔ．ａｅｓｔｉｖｕｍ Ｌ．ｃｖ．Ｇａｎｍａｉ Ｎｏ．８）在２０％ＰＥＧ６０００和１．２％ＮａＣｌ胁迫下的生长、光合作用、蒸腾作用及抗氧化保护系统的变化。结果表明,抗旱小麦８１３９对ＰＥＧ６０００有较强的抗性,但对ＮａＣｌ胁迫的抗性较差。ＮａＣｌ胁迫下,两种小麦根的生长均受到严重抑制,而在ＰＥ     

盐生杜氏藻甘油-3-磷酸脱氢酶的分离纯化及其特性的研究

利用ＰＥＧ分级,ＤＥＡＥ离子交换层析,ＢｌｕｅＳｅｐｈａｒｏｓｅ拟亲和层析,ＭｏｎｏＱ离子交换层析等手段,分离纯化盐生杜氏藻（Ｄｕｎａｌｉｅｌａｓａｌｉｎａ（Ｄｕｎａｌ）Ｔｅｏｄ．）甘油三磷酸（Ｇ３Ｐ）脱氢酶（ＥＣ１．１．１．８）,得到比活为１２．６Ｕ／ｍｇ的电泳纯的酶,并对此酶的生化特性进行了研究。４％～２０％非变性聚丙烯酰胺梯度凝胶电泳测得全酶分子量约为２７０ｋＤ,ＳＤＳＰＡＧＥ表明该酶只有一种分子量约为６５ｋＤ的亚基,据此推测该酶应为同四聚体。酶催化磷酸二羟丙酮（ＤＨＡＰ）还原的最适ｐＨ值为７．５,催化Ｇ３Ｐ脱氢的最适ｐＨ值为１０。该酶对４个底物还原型辅酶Ⅰ（ＮＡＤＨ）,二磷酸吡啶核苷酸（ＤＨＡＰ）,辅酶Ⅰ（ＮＡＤ）,Ｇ３Ｐ的表观Ｋｍ值分别为６３μｍｏｌ／Ｌ,２７２μｍｏｌ／Ｌ,１．５３ｍｍｏｌ／Ｌ,６．５２ｍｍｏｌ／Ｌ。该酶在保存过程中易失活。ＮＡＤＨ能降低酶失活的速度,而ＮＡＤ则不然。低浓度ＮａＣｌ对酶略有保护作用,但高浓度ＮａＣｌ加快酶的失活,且浓度越高效应越明显。     

两相分配法制备玉米根质膜及其纯度鉴定

用Ｄｅｘｔｒａｎ Ｔ５００,ＰＥＧ３３５０两相体系制备玉米根质膜,首先在高盐浓度下选用五种不同的聚合物浓度,研究了玉米根系膜在两相体系中的分配情况,在此基础上研究了ＮａＣｌ浓度对玉米根质膜的纯度及得率的影响。结果表明,制备了玉米根质膜选用６．２％聚合物浓度,７．５ｍｍｏｌ／Ｌ ＮａＣｌ的两相体系比较合适。     

不同培养基对杜氏藻（DunaliellaSalina）生长和无机离子含量的影响

培养在Ｊｏｈｎｓｏｎ培养液、Ｊｏｈｎｓｏｎ＋０．３％ＮａＣｌ培养液、海水和卤水中的杜氏藻,其生长速度有区别,在Ｊｏｈｎｓｏｎ＋０．３％ＮａＣｌ培养液中生长较好,Ｊｏｈｎｓｏｎ培养液和卤水次之,海水中生长较差。杜氏藻生沃的盐度范围为０～１２％,当培养基中ＮａＣｌ浓度超过１２％时,细胞数几乎不增加,甚至略有降低。在不同培养基中藻细胞Ｈ￣＋含量较稳定,而积累ｃａ￣（２＋）,在Ｊｏｈｎｓｏｎ＋０．３％ＮａＣｌ培养液中,杜氏藻细胞Ｎａ￣＋含量增加;而在含高浓度Ｎａ￣＋的海水和卤水中杜氏藻细胞中Ｎａ￣＋的含量低于培养液。     

酿酒酵母3——磷酸甘油脱氢酶的诱导,提纯和性质

在ＹＥＰＤ培养中添加ＮａＣｌ,可以诱导酿酒酵母细胞内３－磷酸甘油脱氢酶的形成,当ＮａＣｌ浓度达５％时,酶比活从０．０５Ｕ／ｍｇ提高到０．５Ｕ／ｍｇ;若再限制培养基中葡萄糖浓度在１００ｍｇ／Ｌ以下,酶比活可达到０．８９Ｕ／ｍｇ。酶比活与培养基中的ＮａＣｌ浓度的函数关系式为：Ｓａ＝０．１２９Ｃ＾３－０．０３８Ｃ＾２＋０．０３４Ｃ＋０．０６３。粗酶液经Ｓｅｐｈａｄｅｘ Ｇ－２５凝胶过滤,Ｂｌｕｅ Ｓｅｐ     

小麦根质膜H^＋—ATPase的部分纯化

以小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）根为材料,采用不连续蔗糖密度梯度离心法制备高纯度质膜微囊。质膜经ＴｒｉｔｏｎＸ１００和ＫＣｌ处理后,再用Ｚｗｉｔｅｒｇｅｎｔ３１４增溶Ｈ＋ＡＴＰａｓｅ,最后用硫酸铵沉淀得到部分纯化的质膜Ｈ＋ＡＴＰａｓｅ。ＳＤＳＰＡＧＥ结果表明,经过上述步骤纯化,分子量为９４ｋＤ的膜蛋白组分得到富集;与质膜相比,其含量提高１５．７倍。部分纯化的质膜Ｈ＋ＡＴＰａｓｅ可以水解ＡＴＰ,受Ｋ＋刺激,并被Ｎ,Ｎ′ｄｉｃｙｃｌｏｈｅｘｙｌｃａｒｂｏｄｉｍｉｄｅ（ＤＣＣＤ）抑制;ＡＴＰ水解活力被Ｎａ３ＶＯ４抑制９５％,但不被ＮａＮ３、ＮａＮＯ３和Ｎａ２ＭｏＯ４抑制。     

盐分和水分胁迫对盐地碱蓬幼苗渗透调节效应的研究

利用不同浓度ＮａＣｌ和等渗ＰＥＧ处理４０ｄ龄的盐地碱蓬（Ｓｕａｅｄａｓａｌｓａ（Ｌ．）Ｐａｌ．）幼苗,１０ｄ后测定植株中主要有机溶质和无机离子的含量及叶片渗透势和渗透调节能力。结果表明,ＮａＣｌ处理的无机离子总含量急剧增加,其中Ｎａ＋、Ｃｌ－增加最多,占总计算渗透势（ＣＯＰ）的７１％～８８％,总无机离子占ＣＯＰ的９５％～９７％。而有机溶质总含量则稍有降低,约占ＣＯＰ的２％～５％。ＰＥＧ处理使有机溶质（氨基酸、糖、有机酸）含量明显增加,特别是氨基酸占ＣＯＰ的９％。不同处理的实测渗透势（ＭＯＰ）均小于ＣＯＰ,说明在这些条件下,还有其它的渗透剂参与盐地碱蓬幼苗的渗透调节。结果还表明,盐地碱蓬幼苗的渗透调节能力随外界盐浓度的增大而增加。     

Epoxy-oxirane activation of PEG for protein ligand coupling

Summary PEG was successfully activated using an epoxy-oxirane based reaction. The coupling of three protein ligands to activated PEG was investigated. Glutathione, Bovine Serum Albumin (BSA) and Protein A were successfully coupled to the activated PEG. Glutathione was coupled to 8% solutions of PEG (8000) and PEG (4000) at concentrations of 1.77 g/l and 1.14 g/l respectively. These values compare favourably with comercially available Sepharose, which gave a Glutathione concentration of 2.22 g/l. Protein A was coupled to a 20% PEG (8000) solution at a concentration of 5.7 g/l and BSA was coupled to a 20% PEG (8000) solution at a concentration of 6.2 g/l.     

α-Amylase production by Bacillus subtilis and B. amyloliquefaciens in different PEG solutions

-Amylase production by Bacillus subtilis and Bacillus amyloliquefaciens was investigated in polyethyleneglycol (PEG)-containing growth medium. Five different molecular weight PEGs (600, 3000, 4000, 8000 and 20,000) were used. Enzyme production with B. subtilis increased 21% in medium containing 5% PEG 3000, but enzyme production with B. amyloliquefaciens increased 31% in medium containing 5% PEG 600 and 21% in medium containing 2% PEG 8000.     

Protective formula and preservation conditions for the endoparasitic fungus, <Emphasis Type="Italic">Esteya vermicola</Emphasis>

The endoparasitic nematophagous fungus, Esteya vermicola, is a bio-control agent with demonstrated ability to attack pinewood nematode (Bursaphelenchus xylophilus). An optimized solution for the protection and preservation of E. vermicola conidia is needed in order to ensure their survival during transportation, preservation, and application. Five protectants, kaolin, arabinose, sorbitol, PEG8000, and Span 80, were selected from 34 agents. These were incorporated into calcium alginate gel capsules at the following concentrations: 10% kaolin, 0.1% Span 80, 1% arabinose, 5% sorbitol, and 5% PEG8000. The improved diffluent formula contained 69.9% soluble starch, 14% wheat flour, 5% PEG8000, 0.1% span 80, 1% arabinose and 10% skim milk. The viability of E. vermicola conidia preserved in the protectant (5% sorbitol and 20% PEG8000) at six temperatures,–70,–20, 4, 26, 37°C, and room temperature (uncontrolled), was also assessed. The highest viability after storage for one month was achieved at–70°C.     

Differential effects of permeating and nonpermeating solutes on the fatty acid composition of Pseudomonas putida

We examined the effect of reduced water availability on the fatty acid composition of Pseudomonas putida strain mt-2 grown in a defined medium in which the water potential was lowered with the permeating solutes NaCl or polyethylene glycol (PEG) with a molecular weight of 200 (PEG 200) or the nonpermeating solute PEG 8000. Transmission electron microscopy showed that -1.0-MPa PEG 8000-treated cells had convoluted outer membranes, whereas -1.0-MPa NaCl-treated or control cells did not. At the range of water potential (-0.25 to -1.5 MPa) that we examined, reduced water availability imposed by PEG 8000, but not by NaCl or PEG 200, significantly altered the amounts of trans and cis isomers of monounsaturated fatty acids that were present in whole-cell fatty acid extracts. Cells grown in basal medium or under the -0.25-MPa water potential imposed by NaCl or PEG 200 had a higher trans:cis ratio than -0.25-MPa PEG 8000-treated cells. As the water potential was lowered further with PEG 8000 amendments, there was an increase in the amount of trans isomers, resulting in a higher trans:cis ratio. Similar results were observed in cells grown physically separated from PEG 8000, indicating that these changes were not due to PEG toxicity. When cells grown in -1.5-MPa PEG 8000 amendments were exposed to a rapid water potential increase of 1.5 MPa or to a thermodynamically equivalent concentration of the permeating solute, NaCl, there was a decrease in the amount of trans fatty acids with a corresponding increase in the cis isomer. The decrease in the trans/cis ratio following hypoosomotic shock did not occur in the presence of the lipid synthesis inhibitor cerulenin or the growth inhibitors chloramphenicol and rifampicin, which indicates a constitutively operating enzyme system. These results indicate that thermodynamically equivalent concentrations of permeating and nonpermeating solutes have unique effects on membrane fatty acid composition.     

Differential Effects of Permeating and Nonpermeating Solutes on the Fatty Acid Composition of Pseudomonas putida

We examined the effect of reduced water availability on the fatty acid composition of Pseudomonas putida strain mt-2 grown in a defined medium in which the water potential was lowered with the permeating solutes NaCl or polyethylene glycol (PEG) with a molecular weight of 200 (PEG 200) or the nonpermeating solute PEG 8000. Transmission electron microscopy showed that −1.0-MPa PEG 8000-treated cells had convoluted outer membranes, whereas −1.0-MPa NaCl-treated or control cells did not. At the range of water potential (−0.25 to −1.5 MPa) that we examined, reduced water availability imposed by PEG 8000, but not by NaCl or PEG 200, significantly altered the amounts of trans and cis isomers of monounsaturated fatty acids that were present in whole-cell fatty acid extracts. Cells grown in basal medium or under the −0.25-MPa water potential imposed by NaCl or PEG 200 had a higher trans:cis ratio than −0.25-MPa PEG 8000-treated cells. As the water potential was lowered further with PEG 8000 amendments, there was an increase in the amount of trans isomers, resulting in a higher trans:cis ratio. Similar results were observed in cells grown physically separated from PEG 8000, indicating that these changes were not due to PEG toxicity. When cells grown in −1.5-MPa PEG 8000 amendments were exposed to a rapid water potential increase of 1.5 MPa or to a thermodynamically equivalent concentration of the permeating solute, NaCl, there was a decrease in the amount of trans fatty acids with a corresponding increase in the cis isomer. The decrease in the trans/cis ratio following hypoosomotic shock did not occur in the presence of the lipid synthesis inhibitor cerulenin or the growth inhibitors chloramphenicol and rifampicin, which indicates a constitutively operating enzyme system. These results indicate that thermodynamically equivalent concentrations of permeating and nonpermeating solutes have unique effects on membrane fatty acid composition.     

Activation in the family of Candida rugosa isolipases by polyethylene glycol

We have investigated activation of two isoenzymes (lip1 and lip3) from Candida rugosa in polyethylene glycol (PEG) media. Aqueous solutions of PEG 8000 and 20,000 activate lip3 but not lip1 from C. rugosa. Maximum activation (260%) of lip3 requires 6 h of pre-incubation with PEG 8000 (4%, w/v). PEG seems to shift the equilibrium between the open and the closed forms of lip3 towards the active conformation. Inhibition experiments demonstrate that ligands have easier access to the lip3 active site than to the lip1 active site, both in the presence and the absence of PEG.

The presence of PEG in the crystallization medium is responsible for reported differences in the crystal structures of lip1 and lip3. A comparative analysis of crystallographic models of lip1 and lip3 suggests a role for PEG in activation of lip3 and further stabilization of the activated/open form via dimerization in aqueous media.     

Thermal features of the bovine serum albumin unfolding by polyethylene glycols.

Albumin showed very poor affinity for polyethylene glycol molecular weight (Mw) 1000 (30 M(-1)) and Mw 8000 (400 M(-1)) (PEG 1000 and PEG 8000). Polyethylene glycol of low Mw favours the ionization of the tyrosine (TYR) residues of albumin. Such variation might be a consequence of the change in dielectric constant at the domain of the protein by PEG binding. PEGs of high Mws stabilize the native compact state of human albumin showing negative preferential interaction with the protein. Interaction between PEGs and albumin is thermodynamically unfavourable, and becomes even more unfavourable for denatured proteins whose surface areas are larger than those of native ones leading to a stabilization of the unfolded state, which is manifested as a lowering of the thermal transition temperature. PEG 8000 perturbs the structure of the protein surface, partially modifying the layer of water and the microenvironment of the superficial aromatic residues (tryptophan, TRP and TYR) which is in agreement with the modifications of the UV spectrum of albumin by PEG 8000 and circular dichroism (CD) spectrum at high temperatures.     

Purification of lipase produced by a new source of Bacillus in submerged fermentation using an aqueous two-phase system

This work discusses the application of an aqueous two-phase system for the purification of lipases produced by Bacillus sp. ITP-001 using polyethylene glycol (PEG) and potassium phosphate. In the first step, the protein content was precipitated with ammonium sulphate (80% saturation). The enzyme remained in the aqueous solution and was dialyzed against ultra-pure water for 18 h and used to prepare an aqueous two-phase system (PEG/potassium phosphate). The use of different molecular weights of PEG to purify the lipase was investigated; the best purification factor (PF) was obtained using PEG 20,000g/mol, however PEG 8000 was used in the next tests due to lower viscosity. The influence of PEG and potassium phosphate concentrations on the enzyme purification was then studied: the highest FP was obtained with 20% of PEG and 18% of potassium phosphate. NaCl was added to increase the hydrophobicity between the phases, and also increased the purification factor. The pH value and temperature affected the enzyme partitioning, with the best purifying conditions achieved at pH 6.0 and 4°C. The molecular mass of the purified enzyme was determined to be approximately 54 kDa by SDS-PAGE. According to the results the best combination for purifying the enzyme is PEG 8000g/mol and potassium phosphate (20/18%) with 6% of NaCl at pH 6.0 and 4°C (201.53 fold). The partitioning process of lipase is governed by the entropy contribution.     

Stimulation of murine granulocyte macrophage-colony stimulating factor production by Pluronic F-68 and polyethylene glycol in transgenic Nicotiana tabacum cell culture

Pluronic F-68, PEG 8000, or PEG 20 000 added to cell suspension cultures of transgenic Nicotiana tabacum promoted cell growth and the production of the recombinant murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in a 5-l stirred tank bioreactor. The specific growth rates were enhanced from 0.27 d^–1 to 0.47 d^–1, 0.37 d^–1 and 0.4 d^–1 when Pluronic F-68, PEG 8000, or PEG 20 000 was added, respectively. The maximum cell density was also increased most to 13.6 g l^–1 when Pluronic F-68 was added (11.3 g l^–1 in the control culture). In terms of mGM-CSF production, PEG 8000 gave the greatest stimulation and with 2 g PEG 8000 l^–1, mGM-CSF increased from 1.6 to 6.6 ng ml^–1.     

Polymer-stimulated ligation: enhanced ligation of oligo- and polynucleotides by T4 RNA ligase in polymer solutions.

The effects of macromolecular crowding were tested on several reactions catalyzed by T4 RNA ligase. The rate of cyclization of oligoriboadenylates was stimulated up to 10-fold by relatively high concentrations of several polymers (polyethylene glycol (PEG) 8000 or 20,000; bovine plasma albumin; Ficoll 70). In addition, higher concentrations of PEG 8000 or PEG 20,000 allowed the novel formation of large linear products from the oligoriboadenylates. Also stimulated by high concentrations of PEG 8000 were the rate at which T4 RNA ligase joined p(dT)10 to oligoriboadenylates and the rate at which the enzyme activated p(dT)n by transfer of an adenylyl moiety from ATP to the oligonucleotides. These results with T4 RNA ligase are compared to earlier studies on the effects of crowding on DNA ligases.