P物质对大鼠DRG神经元胞体膜的作用

本文在大鼠ＤＲＧ神经元标本上应用细胞内记录,以确定ＳＰ对ＤＲＧ细胞的膜反应及其可能的离子机制。实验所测ＤＲＧ细胞静息膜电位为－５８．９±８．２ｍＶ（Ｘ±ＳＥ,ｎ＝８１）。传导速度：Ａ_（α／β）细胞为２０．４±４．８ｍ／ｓ（Ｘ±ＳＥ）,范围１４．１－２８．７ｍ／ｓ（４７／６０）;Ａδ及Ｃ类细胞为９．８±５．２ｍ／ｓ,范围１．２－１３．７ｍ／ｓ（１３／６０）。浴槽滴加ＳＰ（１０￣（－７）－３×１０￣（－４）ｍｏｌ／Ｌ）在大多数细胞可引起明显的膜去极化反应（５６／６０）。少数细胞对ＳＰ无反应（４／６０）。在ＳＰ去极化期间膜电导值有所增加,从平均值２．７２×１０￣（－８）ｍｈｏ增加２４．６％（ｎ＝３）。所测逆转电位值在＋４０－＋５０ｍＶ之间（ｎ＝３）。浊流平衡液（ＢＳＳ）中ＮａＣｌ以氯化胆碱置代,或用含ＴＴＸ（１０￣（－５）ｍｏｌ／Ｌ）的ＢＳＳ灌流,可使ＳＰ－去极化幅值大大减小但不能完全消除。而高（２０ｍｍｏｌ／Ｌ）和低（０ｍｍｏｌ／Ｌ）Ｃａ￣（２＋）的ＢＳＳ灌流时,使ＳＰ－去极化幅值相应的增加和降低。用含１０￣（－４）ｍｏｌ／ＬＣｄ￣（２＋）及１０￣（－２）ｍｏｌ／ＬＴＥＡ的ＢＳＳ灌流,均使ＳＰ－去极化明显减小。     

慈菇（Sagittaria safittifolia）α－半乳糖苷酶的纯化与性质

以对硝基苯糖苷基为底物,测定了慈菇的１２种糖苷酶,其中α－甘露糖苷酶、α－和β－半乳糖苷酶活力较高;经硫酸铵分级沉淀,ＳｅｐｈａｄｅｘＧ－１５０分子筛层析,ＣｏｎＡＳｅｐｈａｒｏｓｅ４Ｂ亲和层析,ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ离子交换层析,从慈菇抽提液纯化了α－半乳糖苷酶。纯化酶的比活提高１０７２倍,活力回收１５．６％,在圆盘聚丙烯酰胺凝胶电泳和ＳＤＳ－ＰＡＧＥ上均显示１条蛋白质带,在α－半乳糖苷酶浓度为１５０ｍＵ／ｍｌ的溶液中测不到其他糖苷酶的活力。慈菇α－半乳糖苷酶的分子量用ＳｅｐｈａｄｅｘＧ－１００凝胶过滤柱测定或在ＳＤＳ－ＰＡＧＥ上测定均为６０ｋＤ,酶反应的最适ｐＨ在５．８附近,最适温度为６０℃。该酶分解对硝基苯基－α－半乳糖苷的Ｋ＿ｍ值为３．７×１０￣（－４）ｍｏｌ／Ｌ,Ｖ＿ｍ值为２．１×１０￣（－４）ｍｏｌ／Ｌ。银离子、汞离子显著抑制酶活力,Ｄ－半乳糖和密二糖均竞争性地抑制该酶水解对硝基苯基α－Ｄ－半乳糖苷的活力,根据Ｄｉｘｏｎ作图求得其Ｋ＿ｉ值分别为０．９２×１０￣（－３）ｍｏｌ／Ｌ和１．９８×１０￣（－３）ｍｏｌ／Ｌ。２－脱氧－Ｄ－半乳糖和Ｌ－岩藻糖为酶活力的非竞争性抑制剂。化学修饰     

P物质对GABAA和GABAB受体介导的DRG神经元膜反应的调制作用

实验在幼年大鼠ＤＲＧ标本上进行。应用细胞内记录观察了ＳＰ对ＧＡＢＡ反应的调制作用。结果证明：（１）单独滴加ＳＰ（５×１０（－６）－４×１０（－５）ｍｏｌ／Ｌ）或浴槽灌流ＳＰ（１０（－６）－５×１０（－６）ｍｏｌ／Ｌ）不引起膜电位的改变或仅有轻微的去极化,但却能使ＧＡＢＡ引起的去极化反应减小５０．８±２０．２％（±ＳＤ）（２０／３０）;（２）单独滴加ＳＰ可使多数受检细胞ＡＰＤ５０延长２８．７±９．１％（±ＳＤ）（１０／１８）;（３）在预加ＳＰ后,能使ｂａｃｌｏｆｅｎ所引起的ＡＰＤ５０缩短效应（２０．６±２．９％,±ＳＤ）完全消除（４／１２）或翻转成ＡＰＤ（５０）延长１９．３±８．９％（±ＳＤ）（８／１２）;（４）预加ＧＡＢＡＢ受体激动剂ｂａｃｌｏｆｅｎ（１０（－４）－１０（－３）ｍｏｌ／Ｌ）３０—９０ｓ后明显地抑制ｍｕｓｃｉｍｏｌ（１０－４－１０－３ｍｏｌ／Ｌ）引起的去极化反应,其抑制效应达５４．４±１８．８％（±ＳＤ）（１７／２０）。由于ＤＲＧ神经元的胞体通常可用来作为研究初级传入终末的模型,因而本文实验结果提示：介导伤害性刺激信息的Ｐ物质在背角的释放,可能作用于初级传入终末,从而产生对抗ＧＡＢＡ介导的突触     

泡桐叶片蛋白质提取方法的研究

２４个提取泡桐叶片蛋白质组合的试验结果表明,用ＵＫＣ（９．５ｍｏｌ／Ｌ尿素,５ｍｍｏｌ／ＬＫ２ＣＯ３,１．２５％ＣＴＡＢ,０．５％二硫苏糖醇,２％两性载体电解质ｐＨ３．５～１０,５％ＴｒｉｔｏｎＸ－１００）提取由１０％冷（－４０℃）三氯醋酸（丙酮配制,内含０．０７％的巯基乙醇）处理的泡桐叶片干粉提取出的蛋白质总量和种类最多。这表明该方法可用于泡桐叶片蛋白质的提取。     

胞外青霉素酰化酶的纯化及部分理化性质

巨大芽孢杆菌产胞外青霉素酰化酶发酵液经硫酸铵分级抽提及ＳｅｐｈａｄｅｘＧ－１００、羟基磷灰石、ＤＥＡＥ纤维素ＤＥ５２等层析步骤,提纯了青霉素酰化酶,得到电泳均一的酶制剂。纯酶比活力约为２５Ｕ／ｍｇ蛋白,纯化４９倍,活力回收５８％,经ＰＡＧＥ及ＳＤＳ－ＰＡＧＥ测知该酶不含亚基,其分子量约为１４０ｋＤ。该酶最适ｐＨ为９．０,最适温度４７℃,用底物ＮＩＰＡＢ测活,其Ｋｍ值为６．２×１０~（－４）ｍｏｌ／Ｌ,Ｖｍ值为１．２４×１０４ｍｏｌ／Ｌ。此外还探讨了部分金属离子对该酶的影响。     

鼠伤寒沙门氏菌外膜蛋白诱导BALB／C小鼠免疫保护的实验研究

本实验采用超声破碎、ＴｒｉｔｏｎＸ－１００处理和超速离心技术提取了鼠伤寒沙门氏菌（Ｓａｌｍｏｎｅｌｌａｔｙｈｉｍｕｒｉｕｍ,ＳＴＭ）的外膜蛋白（Ｏｕｔｅｒｍｅｍｂｒａｎｅｐｒｏｔｅｉｎｓ,ＯＭＰｓ）,其中脂多糖（ＬＰＳ）的含量约为５％。ＯＭＰｓ经ＳＤＳ—ＰＡＧＥ显示１０余条蛋白带。对ＯＭＰｓ诱发ＢＡＬＢ／Ｃ小鼠产生典型的迟发型变态反应（ＤＴＨ）进行了检测。经腹腔免疫的ＢＡＬＢ／Ｃ小鼠用５００ＬＤ５０鼠伤寒沙门氏菌（５０１１５）攻击,１００％可得到保护;用５００ＬＤ５０伤寒杆菌（Ｅ６８６）攻击     

缢蛏碱性磷酸酶胍溶液中分子折叠与活力变化研究

报道了缢蛏碱性磷酸酶（简称ＡＬＰ）经不同浓度盐酸胍处理时酶的分子构象所发生的变化以及酶变化和失活的动力学过程。在胍中酶荧光发射峰强度下降,紫外差光谱在２４６ｎｍ和２８５ｎｍ处出现２个负峰,ＣＤ谱中酶的α螺旋度下降,且随浓度增大,变化程度也加大。动力学研究表明,酶在０．５ｍｏｌ／Ｌ、１．０ｍｏｌ／Ｌ、２．０ｍｏｌ／Ｌ３．０ｍｏｌ／Ｌ、４．０ｍｏｌ／Ｌ盐酸胍中的变性速度常数分别为３．２１×１０~（－４）ｓ~（－１）、６．３８×１０~（－４）ｓ~（－１）、２．１７×１０~（－３）ｓ~（－１）、２．３３×１０~（－３）ｓ~９－１）、５．１７×１０~（－３）ｓ~（－１）;而酶在相应盐酸胍中的失活速度常数分别为２．３３×１０~（－４）ｓ~（－１）、３．５７×１０~（－４）ｓ~（－１）、５．８６×１０~（－４）ｓ~（－１）、１．１４×１０~（－３）ｓ~（－１）、３．４５×１０~（－３）ｓ~（－１）;表现为失活与构象伸展变化基本平行。     

南方鲇碱性磷酸酶的分离纯化及部分性质的研究

用正丁醇抽提,硫酸铵分级沉淀,ＤＥＡＥ－纤维素和ＳｅｐｈａｃｒｙｌＳ－２００柱层析,从南方鲇（Ｓｉｌｕｒｕｓ　ｍｅｒｉｄｉｏｎａｌｉｓ　Ｃｈｅｎ）肠粘膜中提取出碱性磷酸酶（ＡＫＰ）。提纯倍数为３９．５０倍,比活为６８．３５μ／ｍｇ蛋白,提取酶液经ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ只呈现一条区带。该酶的分子量为１３２１４０,Ｎ末端氨基酸为门冬氨酸,最适ｐＨ为１０．１０,７．５＞ｐＨ＞１１．５时不稳定,最适温度为４０℃左右,对热不很稳定,以磷酸苯二钠为底物其Ｋ_ｍ值为１．７２×１０~（－３）ｍｏｌ／Ｌ。Ｍｇ~（２＋）、Ｍｎ~（２＋）为该酶的激活剂,ＫＨ_２ＰＯ_４、Ｌ－ＣｙＳ、ＭＥ、ＤＦＰ、ＥＤＴＡ－Ｎａ_２为抑制剂。选用ＫＨ_２ＰＯ_４和ＤＦＰ作抑制类型的判断,结果表明,ＫＨ_２ＰＯ_４属竞争性掏剂,其抑制常数为２．３ｍｍｏｌ／Ｌ;ＤＦＰ为非竞争性抑制剂,抑制常数为１．０５ｍｍｏｌ／Ｌ。     

杜氏盐藻细胞质膜氧化还原系统

杜氏盐藻细胞质膜具的氧化ＮＡＤ（Ｐ）Ｈ,还原Ｆｅ（ＣＮ）＾３－６和Ｏ２的氧化还原系统。当Ｆｅ（ＣＮ）＾３－６浓度为０．６ｍｍｏｌ／Ｌ,氧化ＮＡＤＨ的Ｋｍ为９６μｍｏｌ／Ｌ,Ｖｍａｘ为１５９ｎｍｏｌ１０＾－８ｃｅｌｌｓｍｉｎ＾－１,最适ｐＨ为８．５。ＴｒｉｔｏｎＸ－１００可促进ＮＡＤＨ和Ｆｅ（ＣＮ）＾３－６的氧化还原活性。ＮＡＤＨ能促进藻细胞的氧吸收,最适ｐＨ为８．５。在无外源电子供体存在时,细胞质     

皮质酮快速抑制PC12细胞乙酰胆碱诱发电流影响及机制探讨

目的和方法：本研究采用全细胞膜片箝技术,观察皮质酮（Ｂ）对ＰＣ１２细胞上乙酰胆碱诱发电流（ＩＡＣｈ）的快速作用并初步探讨其可能机制。结果：ＰＣ１２细胞上ＩＡＣｈ是通过烟碱受体（ｎＡＣｈＲ）引起的。箝制电压为－８０ｍＶ时,ＡＣｈ（３０μｍｏｌ／Ｌ）诱发一内向电流;细胞外灌流同时给予ＡＣｈ和Ｂ（１０－５ｍｏｌ／Ｌ）时,Ｂ对ＩＡＣｈ的抑制作用较弱;用Ｂ（１０－５ｍｏｌ／Ｌ）对细胞进行预处理,可提高Ｂ对ＩＡＣｈ峰值的抑制率,作用呈可逆性、浓度依赖性和非电压依赖性;细胞外用ＲＮＡ合成抑制剂放线菌素Ｄ（４×１０－５～４×１０－３ｍｏｌ／Ｌ）或蛋白合成抑制剂放线菌酮（１０－４～１０－３ｍｏｌ／Ｌ）孵育细胞１～２ｈ阻断基因机制,但均不影响Ｂ对ＩＡＣｈ的快速抑制作用。结论：Ｂ对ＰＣ１２细胞上ＩＡＣｈ有快速抑制作用,此作用可能是由非基因组机制介导的。     

Steady-state kinetics, micellar effects, and the mechanism of peroxidase-catalyzed oxidation of n -alkylferrocenes by hydrogen peroxide

 Kinetics of the steady-state oxidation of n–alkylferrocenes (alkyl = H, Me, Et, Bu and C₅H₁₁) by H₂O₂ to form the corresponding ferricenium cations catalyzed by horseradish peroxidase has been studied in micellar systems of Triton X-100, CTAB, and SDS, mostly at pH 6.0 and 25  °C. The rate of oxidation of ferrocenes with longer alkyl radicals is too slow to be measured. The reaction obeying the [RFc]:[H₂O₂] = 2 : 1 stoichiometry is strictly first-order in both HRP and RFc in a wide concentration range. The corresponding observed second-order rate constants k, which refer to the interaction of the peroxidase compound II (HRP-II) with RFc, decrease with the elongation of the alkyl substituent R, and this in turn is accompanied by an increase in the formal redox potentials E°′ in the same medium. Increasing the surfactant concentration lowers the rate constants k, the effect being due to the nonproductive binding of RFc to micelles rather than to enzyme inactivation. The micellar effects are accounted for in terms of the Berezin pseudo-phase model of micellar catalysis applied to the interaction of enzyme with organometallic substrates. The oxidation was found to occur primarily in the aqueous pseudo-phase and the calculated intrinsic second-order rate constants k _w are (1.9 ± 0.5)×10⁵, (2.7 ± 0.1)×10⁴, and (5.9 ± 0.6)×10³ M^–1 s^–1 for HFc, EtFc, and n–BuFc, respectively. The data obtained were used for estimating the self-exchange rate constants for the HRP-II/HRP couple in terms of the Marcus formalism. Received: 15 July 1996 / Accepted: 15 November 1996     

牛小脑肌醇磷脂激酶PI(4)K高产率纯化与特征

对牛小脑膜区肌醇磷脂激酶进行了11 500倍纯化,过程包括：TritonX-100抽提,硫酸铵沉淀,阳离子交换层析(phosphocellulose),亲和层析(Heparin Sepharose CL-6B)和阴离子交换层析(DEAE10,FPLC)等.纯化程度可达95％以上,对SDS-PAGE电泳结果进行扫描分析测其分子质量为56 ku.纯化的肌醇磷脂激酶的特异活性为450 nmol/mg·min, 动力学性质表现为ATP的表观K_m值为7.9×10^-7 mol/L,PI的表观K_m值为6.6×10^-7 mol/L. 腺嘌呤核苷是该酶的有效抑制剂,3.5×10^-7 mol/L腺嘌呤核苷可使该酶活力降低约50％,而TritonX-100对该酶活力具有刺激作用,0.5% TritonX-100可使该酶表现为最高活力.     

Stabilizing hyperactivated lecitase structures through physical treatment with ionic polymers

Lecitase Ultra has been covalently immobilized on cyanogen bromide cross-linked 4% agarose (CNBr) beads, maintaining 70% of the initial activity. The activity of the immobilized enzyme was improved in the presence of Triton X-100, sodium dodecyl sulfate (SDS), and cetyltrimethyl ammonium bromide (CTAB) (e.g., up to 800% when using CTAB). However, CTAB and Triton X-100 presented a negative effect on enzyme stability even at low concentrations, and SDS cannot be used for a long time at 1% concentration. To maintain the hyperactivated conformation of the enzyme in the absence of detergent, ionic polymers were added during incubation of the immobilized enzyme in the presence of detergents. Coating the immobilized enzyme with polyethylenimine in aqueous buffer (PEI) produced a 3-fold increase in enzyme activity. However, in the presence of 0.1% SDS (v/v), this coating produced a 50-fold increase in enzyme activity. Using PEI and 0.01% (v/v) CTAB, the Lecitase activity decreased to 10%. Using irreversible inhibitors, it could be shown that the PEI/SDS-CNBr-Lecitase preparation allowed its catalytic Ser to be more accessible to the reaction medium than the unmodified CNBr-Lecitase.     

糖链结构不同的纤连蛋白与HT1080细胞结合研究

人血浆纤连蛋白（Ｆｉｂｒｏｎｅｃｔｉｎ,Ｆｎ）与人胎盘纤连蛋白两者在肽链结构上基本相同,但人血浆Ｆｎ的Ｎ－糖链结构为二天线结构,而人胎盘Ｆｎ不仅Ｎ－糖链的数量增加,同时还含有多天线结构,分别用~（１２５）Ｉ标记这两种具有不同糖链结构的Ｆｎ,观察两者与ＨＴ１０８０细胞的饱和结合的亲和性,结果发现,在４℃,人血浆Ｆｎ与ＨＴ１０８０细胞的饱和结合为１２９ｎｇ／１０~５细胞,解离常数为２．８３×１０~（－８）ｍｏｌ／Ｌ,人胎盘Ｆｎ与ＨＴ１０８０细胞的饱和结合为１３３ｎｇ／１０~６细胞,解离常数为２．６４×１０~（－８）ｍｏｌ／Ｌ．因而,人血浆Ｆｎ与人胎盘Ｆｎ上Ｎ－糖链的不同并未影响其与受体的结合．     

Adsorption of surfactants on a <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain and the effect on cell surface lypohydrophilic property

The adsorption behavior of five surfactants, cetyltrimethylammonium bromide (CTAB), Triton X-100, Tween 80, sodium dodecyl sulfate (SDS), and rhamnolipid, on a Pseudomonas aeruginosa strain and the effect of temperature and ionic strength (IS) on the adsorption were studied. The change of cell surface lypohydrophilic property caused by surfactant adsorption was also investigated. The results showed that the adsorption kinetics of the surfactants on the cell followed the second-order law. CTAB adsorption was the fastest one under the experimental conditions, and it took longest for SDS adsorption to equilibrate because of electric repulsion. The adsorption of Triton X-100 and Tween 80 was characterized by short equilibration time, and rhamnolipid adsorption reached equilibrium in about 90 min. The adsorption isotherms of all the surfactants on the bacterium fitted Freundlich equation well, but the adsorption capacity and mode were variations for the surfactants as indicated by k and n parameters in the equations. The adsorption mode for all the surfactants except SDS is probably hydrophilic interaction because the adsorption totally turned the cell surface to be more hydrophobic. Neither the temperature nor the IS had significant effect on CTAB adsorption, but higher IS significantly enhanced SDS adsorption and modestly strengthened adsorption of Triton X-100, Tween 80, and rhamnolipid. Higher temperature strengthened adsorption of SDS but weakened the adsorption of Triton X-100, Tween 80, and rhamnolipid.     

Effect of Surfactants on Activity and Stability of Native and Chemically Modified Lipases A and B from Candida Rugosa

The effect of sodium dodecyl sulfate (SDS) and Triton X-100 on the hydrolytic activity of lipases A and B from Candida rugosa has been studied. Lipase B is significantly more affected than lipase A by the presence of both surfactants; Triton X-100 produces a more deleterious effect than SDS with both isoenzymes. In addition, the stability of lipases A and B in the presence of different concentrations of SDS was investigated; lipase A was more stable than isoform B. Both isoenzymes were chemically modified by reaction of their amino groups with octanoyl chloride or activated polyethylene glycol (PEG, mol. wt. 5000). In all cases the modification produced a protective effect against denaturation by SDS. In particular, PEG₅₀₀₀-liPases A and B were significantly more stable (stabilization factor: 3-4) than the native enzymes at the surfactant concentrations tested.     

人乳腺癌cAMP结合蛋白

建立了测定人乳腺癌胞浆ｃＡＭＰ结合蛋白（ｃＡＭＰｂ．ｐ．）方法。综合研究了其温度、保温时间、配体浓度、稳定性等条件。ｃＡＭＰｂ．ｐ．的Ｋ_Ｄ值为２．９０×１０~（－８）ｍｏｌ／Ｌ．并测定了６０例雌激素受体（ＥＲ）Ｆｕ性乳腺癌标本的ｃＡＭＰｂ．ｐ．含量。此组病人术后均接受系统的内分泌治疗,ＥＲ／ｃＡＭＰｂｐ,比值范围为７．７～３６２×１０~（－３）,ＥＲ／ｃＡＭＰｂ．ｐ．比值≥４０×１０~（－３）的五年生存率明显高于比值＜４０×１０~（－３）组,（ｐ＜０．００５）．表明测定ＥＲ／ｃＡＭＰｂ．ｐ．比值对预测患者内分泌治疗疗效,优于单独测定ＥＲ．     

Liposomes as a model for the study of the mechanism of fish toxicity of sodium dodecyl sulfate in sea water.

The mechanism underlying the shark repellency of SDS was studied by comparing it with the shark nonrepelling detergent, Triton X-100. The findings can be summarized as follows: (1) The effective concentration of SDS for termination of shark tonic immobility (an immediate and fast response) was close to its critical micellar concentration in sea water (70 microM). The fish lethal concentrations (LD50) were far below the CMC value for SDS, and at CMC level for Triton X-100. (2) In sea water SDS possesses a strong affinity for lipid membranes, expressed in a lipid sea water partition coefficient (Kp) of about 3000. (3) In liposomal systems examined by assays of turbidity, fluorescence resonance energy transfer and kinetics of carboxyfluorescein (CF) release, the pattern of SDS induced changes in the phospholipid bilayer suggests: (a) absence of vesicle-vesicle fusion; (b) occurrence of vesicle size increase, and (c) nonlytic gradual release of CF above and below its CMC values. In contrast, Triton X-100 above its CMC induces membrane solubilization. (4) Assays coupling CF release from liposomes to potassium diffusion potential induced by valinomycin indicate that SDS related CF release can also be attributed to a specific mechanism such as cation pore formation and not only to membrane solubilization. The hypothesis of pore formation by SDS is discussed.