两亲分子解除乙醇对内切葡聚糖酶的抑制作用

乙醇对内切葡聚糖酶存在抑制作用,一定浓度的ＢＲＩＪ ３５、Ｔｗｅｅｎ ８０,Ｔｗｅｅｎ ２０可以部分或基本解除乙醇的抑制,而ＳＤＳ不能解除乙醇对酶的抑制,从荧光光谱可见,乙醇使酶分子的生色团埋藏于分子内部,两亲分子由反之。圆二色谱揭示了酶分子α螺旋结构为活性所必需,两亲分子结构不同,解除乙醇对酶抑制的能力也不同。     

Tween类胶束体系中水杨酸铜络合物的SOD样活性

合成了水杨酸铜·乙醇络合物,提纯了Ｔｗｅｅｎ－２０、Ｔｗｅｅｎ－４０,Ｔｗｅｅｎ－６０．Ｔｗｅｅｎ－８０,并测定了它们在ｐＢ７．４的磷酸缓冲液中的ＣＭＣＯ。用Ｃｙｔ,ｃ－ＨＸ－ＸＯ法分别测定了水杨酸铜·乙醇络合物清除超氧阴离子自由基的活性,不同浓度的单纯表面活性剂对Ｏ２的影响,以及表面活性剂与水杨酸铜·乙醇络合物协同清除Ｏ２的作用。观察到在胶束体系中水杨酸铜·乙醇络合物的ＳＯＤ样活性明显高于缓冲液体系无表面活性剂时的ＳＯＤ样活性,表面活性剂本身也具有清除Ｏ２的功能。抗红细胞膜脂质过氧化实验也得到类似结果。     

S/D处理人纤维蛋白原的病毒灭活验证

在人纤维蛋白原制备工艺中增加Ｓ／Ｄ处理灭活病毒步骤,ＴＮＢＰ和Ｔｗｅｅｎ８０终浓度分别为０．３％和１％,在２５℃处理６小时能有效灭活指示病毒ＶＳＶ（〉３．７５Ｌｏｇ）、Ｓｉｎｄｂｉｓ（〉４．４６Ｌｏｇ）、ＨＩＶ（〉３．６７Ｌｏｇ）,盲传三代未检出病毒     

β-葡萄糖苷酶的分离纯化和性质研究

β－葡萄糖苷酶是纤维素酶的重要组分之一,它不仅可水解纤维二糖和寡糖,更可解除纤维二糖对β－１,４－内切葡聚糖酶和外切葡聚糖酶的抑制,提高水解速率和程度．利用ＳｅｐｈａｄｅｘＧ－１５０和ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０层析法从黑曲霉变异株Ｌ－２２中分离提纯了β－葡萄糖苷酶,该酶是由两个分子量相同的亚基组成的二聚体,每个亚基分子量为２０３ｋＤ．该酶最适ｐＨ为４．８,ｐＨ稳定范围在３．６～６．４;最适温度是６０℃,温度稳定范围为４～６０℃;酶分子含糖量为８．３５％．它是一个酸性β－葡萄糖苷水解酶,专一性地水解β－糖苷键．而不水解α－糖苷键,对短链底物表现了相对高的活力．用动力学分析和共价化学修饰方法探讨了与该酶活力有关的必需基团．由ｐＨ对ｌｇＶｍ和ｌｇＶｍ／Ｋｍ的影响,推测出酶活性部位至少有两个可解离基团为酶活性所必需,它们在酶－底物复合物中的ｐＫｅｓ１和ｐＫｅｓ２的值分别为４．０和５．６,在游离酶中的ｐＫ值分别为４．２和５．９．由此可初步判断这两个可解离基团可能为组氨酸和含羧基的氨基酸,它们与酶的催化和底物结合可能有关．     

几种细胞因子对HSV─1感染单核巨噬细胞的影响

以ＨＳＶ─１接种人单核细胞系Ｕ＿（９３７）和小鼠腹腔巨噬细胞,并在接种前后分别以不同的细胞因子处理。经病毒滴定、免疫荧光试验检测病毒抗原及多聚酶链反应技术检测病毒基因,研究了细胞因子对ＨＳＶ─１感染单核巨噬细胞的影响。结果证实,ＴＮＦ─α５０ｕ／ｍＬ　、Ｍ─ＣＳＦ２００ｕ／ｍＬＩＦＮ─γ１０００ｕ／ｍＬ、ＩＦＮ－α１０００ｕ／ｍＬ均能增强单核巨噬细胞对ＨＳＶ─１的抗性,加速细胞对ＨＳＶ─１ＤＮＡ的清除,抑制病毒抗原的表达;并能拮抗ＬＰＳ对ＨＳＶ─１在单核巨噬细胞中表达的促进作用。     

静注丙球低pH孵放灭活病毒效果验证

以水泡性口炎病毒（ＶＳＶ）为指示病毒,考察了低ｐＨ孵放不同时间对低温乙醇法生产的静注丙球（ＩＶＩＧ）中ＶＳＶ的灭活效果,并对不同厂家及不同批号ＩＶＩＧ中病毒灭活情况进行了比较。结果表明,液体ＩＶＩＧ在ＰＨ４．１±０．３,２０－２５℃,孵放２１天可灭活ＶＳＶ达６Ｌｏｇｓ以上（即低于实验检测限）,但不同厂家及不同批号的ＩＶＩＧ在病毒灭活的发生上有所不同     

单纯疱疹病毒的PCR直接基因分型

本文利用ＰＣＲ技术建立一种对ＨＳＶ直接基因分型的方法。在ＨＳＶ－Ⅰ、Ⅱ两型的ＤＮＡ多聚酶基因上设计了一条两型共同的上游引物（ＨＤＰ－Ｂ）和两条型特异的下游引物（ＨＤＰ－１、ＨＤＰ－２）。三条引物共同组成一个扩增反应体系,在ＨＳＶ－Ⅰ产生５４３ｂｐ条带,ＨＳＶ－Ⅱ产生３７２ｂｐ条带,据此在基因水平上对ＨＳＶ进行分型。５株不同来源的ＨＳＶ（２株Ⅰ型,３株Ⅱ型）分型结果与病毒分离及血清学方法完全一致。该     

多聚酶链反应法在检测和研究人类疱疹病毒6型中的应用

人类疱疹病毒６型（ＨＨＶ－６）是８０年代末新发现的一种ＤＮＡ病毒,它对Ｔ淋巴细胞有高度亲嗜性,与婴幼儿急疹（ＥＳ）、淋巴系肿瘤及ＡＩＤＳ等病有密切关系。在近年对ＨＨＶ－６的研究中,多聚酶链反应（ＰＣＲ）被广泛应用。本文就ＰＣＲ法检测ＨＨＶ－６的一般方法、常用引物特点以及此法在研究ＨＨＶ－６中的应用及其结果评价作一综述。     

非洲爪蟾孵化酶的纯化及其部分生化特性研究

本文以ＧＳＴ－ＵＶＳ．２抗体和卵黄膜为检测手段,采用凝胶过滤和离子交换等方法将非洲爪蟾（Ｘｅｎｏｐｕｓｌａｅｖｉｓ）孵化酶纯化了９０倍以上,并研究了其酶活性和生化特性。实验发现,孵化酶分子量为６０ｋＤ,有很强的蛋白酶活性和卵黄膜溶解活性;它很不稳定,在纯化时极易降解为４０ｋＤ分子,４０ｋＤ分子没有卵黄膜溶解活性,但仍有很强的蛋白酶活性。４０ｋＤ分子很可能只代表６０ｋＤ分子中的蛋白酶功能区,而丢失了两个ＣＵＢ重复区。孵化酶对ＥＤＴＡ和金属离子非常敏感、又为ｐ－ＡＰＭＳＦ等胰蛋白酶抑制剂所强烈抑制,表明它是属于胰蛋白酶类型的一种金属蛋白酶。其特异性ＭＣＡ－底物为Ｂｏｃ－Ｌｅｕ－Ｇｌｙ－Ａｒｇ－ＭＣＡ。     

人类疱疹病毒6型的分子生物学研究进展

人类疱疹病毒６型（ＨＨＶ－６）是近几年分离到的、对Ｔ淋巴细胞具有高度亲嗜性的双链ＤＮＡ病毒。ＨＨＶ－６现已分为Ａ、Ｂ两型,它们在生长特性、与单克隆抗体的反应性及限制性酶切图谱等方面存在差异。研究表明ＨＨＶ－６与人类多种疾病有关。随着器官移植的发展和ＡＩＤＳ病人的增多,ＨＨＶ－６的感染变得日益重要。本文主要对其分离培养、流行病学、遗传及分子生物学特性作一综述。     

Tween 80解除乙醇对内切葡聚糖酶(Cx)的抑制作用

通过还原糖量的测定,可知Cx酶在乙醇存在下活力降低。当该体系中加入一定浓度的Tween 80时,Cx酶抗乙醇抑制的能力增强。由紫外光谱及圆二色谱可见,乙醇使Cx酶中α-螺旋含量增加,Tween 80使Cx酶中α-螺旋含量减少,结构变得松散。适量的Tween 80能使Cx酶在乙醇中保持一种具有较高活力的构象。     

The role of lipid phase structure in the interaction of lactate dehydrogenase with phosphatidylserine. Activity studies

Lactate dehydrogenase is one of the enzymes of the glycolytic path. It has been shown to be able to bind in vitro to cellular membranes. The presence of anionic phospholipids induces changes in the catalytic properties of the enzyme similar to those found when the enzyme is bound to natural membranes. In this study, a nonionic detergent (Tween 20), at concentrations not affecting the catalytic activity of LDH, was used to study the role of the lipid supra-molecular structure in the interaction between pig skeletal muscle lactate dehydrogenase and phosphatidylserine. Tween 20 changes the equilibrium of concentrations between the lipid supra-molecular forms. The detergent at the used concentration values did not alter the activity of the enzyme when it was used on its own, but did diminish the level of inhibition induced by the studied phospholipid. The obtained results showed that the interaction is reversible and that the bilayer structure of the lipid is essential for the inhibition.     

Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication.

Gap junction communication in some cells has been shown to be inhibited by pp60v-src, a protein tyrosine kinase encoded by the viral oncogene v-src. The gap junction protein connexin43 (Cx43) has been shown to be phosphorylated on serine in the absence of pp60v-src and on both serine and tyrosine in cells expressing pp60v-src. However, it is not known if the effect of v-src expression on communication results directly from tyrosine phosphorylation of the Cx43 or indirectly, for example, by activation of other second-messenger systems. In addition, the effect of v-src expression on communication based on other connexins has not been examined. We have used a functional expression system consisting of paired Xenopus oocytes to examine the effect of v-src expression on the regulation of communication by gap junctions comprised of different connexins. Expression of pp60v-src completely blocked the communication induced by Cx43 but had only a modest effect on communication induced by connexin32 (Cx32). Phosphoamino acid analysis showed that pp60v-src induced tyrosine phosphorylation of Cx43, but not Cx32. A mutation replacing tyrosine 265 of Cx43 with phenylalanine abolished both the inhibition of communication and the tyrosine phosphorylation induced by pp60v-src without affecting the ability of this protein to form gap junctions. These data show that the effect of pp60v-src on gap junctional communication is connexin specific and that the inhibition of Cx43-mediated junctional communication by pp60v-src requires tyrosine phosphorylation of Cx43.     

Effect of ethanol on the activity and conformation of Penaeus penicillatus acid phosphatase.

The effect of ethanol on the activity of Penaeus penicillatus acid phosphatase has been studied. The results show that ethanol significantly inhibits enzyme activity as a non-competitive inhibitor, with Ki 8.75%. The conformational changes of the enzyme molecule induced by ethanol were followed using fluorescence emission, ultraviolet difference and circular dichroism (CD) spectra. Increasing the ethanol concentration caused the fluorescence emission intensity of the enzyme to increase. The ultraviolet difference spectra of the enzyme denatured with ethanol had two negative peaks at 220 and 278 nm, and a positive peak at 240 nm. Increasing the ethanol concentration produced a small shoulder peak at 287 nm in addition to the increases in the negative magnitudes of the 220 and 278 nm peaks. The changes of the fluorescence and ultraviolet difference spectra reflected the changes of the microenvironments of the tryptophan and tyrosine residues of the enzyme. The CD spectrum changes of the enzyme show that the secondary structure of the enzyme also changed. The results suggest that ethanol is a non-competitive inhibitor and the conformational integrity of the enzyme is essential for its activity.     

Transient kinetic studies of substrate inhibition in the horse liver alcohol dehydrogenase reaction

The rate-limiting step of ethanol oxidation by alcohol dehydrogenase (E) at substrate inhibitory conditions (greater than 500 mM ethanol) is shown to be the dissociation rate of NADH from the abortive E-ethanol-NADH complex. The dissociation rate constant of NADH decreased hyperbolically from 5.2 to 1.4 s-1 in the presence of ethanol causing a decrease in the Kd of NADH binding from 0.3 microM for the binary complex to 0.1 microM for the abortive complex. Correspondingly, ethanol binding to E-NADH (Kd = 37 mM) was tighter than to enzyme (Kd = 109 mM). The binding rate of NAD+ (7 X 10(5) M-1s-1) to enzyme was not affected by the presence of ethanol, further substantiating that substrate inhibition is totally due to a decrease in the dissociation rate constant of NADH from the abortive complex. Substrate inhibition was also observed with the coenzyme analog, APAD+, but a single transient was not found to be rate limiting. Nevertheless, the presence of substrate inhibition with APAD+ is ascribed to a decrease in the dissociation rate of APADH from 120 to 22 s-1 for the abortive complex. Studies to discern the additional limiting transient(s) in turnover with APAD+ and NAD+ were unsuccessful but showed that any isomerization of the enzyme-reduced coenzyme-aldehyde complex is not rate limiting. Chloride increases the rate of ethanol oxidation by hyperbolically increasing the dissociation rate constant of NADH from enzyme and the abortive complex to 12 and 2.8 s-1, respectively. The chloride effect is attributed to the binding of chloride to these complexes, destabilizing the binding of NADH while not affecting the binding of ethanol.     

Effect of surfactants on ethanol fermentation using glucose and cellulosic hydrolyzates

The effect of the non-ionic surfactants on the ethanol fermentation was greatly dependent on the surfactant added. While Tween 20 and Tween 80 slightly enhanced ethanol fermentation, Triton X-100 which exhibited the inghest increase in the enzymatic saccharification had a negative effect on the ethanol fermentation. The negative effect of Triton X-100 on ethanol production was the most pronounced when the cellulosic hydrolyzates were used. Tween 80 showed the best performance for the ethanol production from steam exploded wood hydrolyzate.     

Acetaldehyde Is a Causal Agent Responsible for Ethanol-Induced Ripening Inhibition in Tomato Fruit

Inhibition of tomato (Lycopersicon esculentum Mill.) fruit ripening by exogenously applied ethanol was shown to be caused by elevated endogenous levels of acetaldehyde (AA). Exposure of excised pericarp discs of mature-green tomato fruit to ethanol or AA vapors produced elevated levels of both compounds in the tissue, but only the levels of AA were associated with ripening inhibition. Ripening inhibition was dependent on both the applied concentration and the duration of exposure. Discs treated with inhibitory levels of AA had levels of ethanol that were elevated but below that associated with inhibition of ripening. The in vivo activity of alcohol dehydrogenase was inhibited 40 to 60% by 4-methylpyrazole (4-MP), a competitive inhibitor of this enzyme. The inhibitory effect of ethanol on ripening was reduced by the simultaneous application of 4-MP. Tissue treated with 4-MP plus AA vapors had higher endogenous levels of AA and ripening was inhibited longer than in tissue without 4-MP. The tissue AA level resulting from ethanol or AA application appears to be the critical determinant of ripening inhibition.     

Acetyl CoA mediated specific inhibition of malic enzyme (EC 1.1.1.40) from rat liver and skeletal muscle cytoplasm

Extramitochondrial malic enzyme (EC 1.1.1.40) of rat liver and skeletal muscle is shown for the first time to be subject to significant inhibition by acetyl CoA. The liver enzyme seems to be slightly more sensitive to inhibition than the muscle enzyme, and inhibition can only be observed at malate concentrations below Vmax. Since acetyl CoA is a metabolite of ethanol (acetate) oxidation in liver and muscle, its inhibitory effect on malic enzyme could explain (a) the prolonged disequilibrium of the liver malic enzyme reaction after acute ethanol injection in rats (1), and (b) the inhibition of alanine release from perfused rat hindquarters in the presence of acetate, the major metabolite of ethanol in the peripheral vascular system (2).     

Further evidence for the importance of lipid bilayers in the interaction between lactate dehydrogenase and phosphatidylserine

Lactate dehydrogenase (LDH) is one of the glycolytic enzymes, which have been proved to have the capability to reverse non-specific adsorption on cellular membranous structures in vitro, as well as on the structural proteins of the contractile system of muscle cells. It has been suggested that this binding may play a physiological role, as it alters the enzyme's kinetic properties. Our previous studies on this enzyme showed that its interaction with some anionic phospholipids reveals similar characteristics and similar effect on the activity of the enzyme to those which had been observed for the interaction with membranous structures. Disruption of the lipid bilayers by nonionic detergent (Tween 20) restored the enzyme activity inhibited by the presence of phosphatidylserine (PS) liposomes. In this study, we used the measurement of enzyme tryptophanyl fluorescence spectra to monitor the interaction and possible changes in the enzyme conformation. The investigation provided further evidence of the importance of the bilayer structure in this interaction. Similarly to the effect on the activity of the enzyme, the addition of Tween 20 diminishes the quenching of the LDH tryptophanyl fluorescence, and finally completely restores the fluorescence.     

Cholesterol requirement of mycoplasmas

Cholesterol requirement for growth of mycoplasmas was tested in a serum-free medium supplemented with albumin, l-arginine, palmitic acid, and various concentrations of cholesterol dissolved in Tween 80. In cases in which Tween 80 was shown to inhibit growth, the test medium was supplemented with cholesterol dissolved in ethanol. Of the 31 species examined, all but Mycoplasma laidlawii, M. granularum, and Mycoplasma species strain S-743 exhibited a growth response to cholesterol. No requirement for cholesterol could be shown with the stable L-phase variants of Streptobacillus moniliformis and Proteus species. The results provide experimental support for the view that the large majority of the established Mycoplasma species require cholesterol for growth.