底物浓度选择在酶Km值测定中的重要性

以胰蛋白酶对酪蛋白的Ｋｍ值测定为例，讨论了底物浓度选择在酶Ｋｍ值测定中的重要性。底物酪蛋白的浓度分为三组：高浓度（１～５ｍｇ／ｍｌ），低浓度（０．１～１ｍｇ／ｍｌ）和极低浓度（０．０２～０．０８ｍｇ／ｍｌ），高底物浓度（＞８Ｋｍ）的测定结果不能有效地区分多酶（作用于同一底物的多种酶）的存在，也不符合作图法要求的准确度，所得到的Ｋｍ值可能是不准确的或错误的。因此，测定Ｋｍ值时所用的底物浓度必须在０．２～５Ｋｍ范围内，必要时应包括更广泛的浓度范围（包括高底物浓度）。     

郑州地区汉族健康人群和糖尿病合并冠心病患者血清芳香酯酶活性频数分布态性与192位基因的多态性

目的:研究郑州地区汉族健康人群和糖尿病合并冠心病 (DM CAD)患者血清芳香酯酶 (ArE/PON1 )活性频数分布态性与 192位基因多态性的分布。方法:郑州地区汉族健康人群 321例,DM CAD90例。用终点法测定血清ArE/PON1活性。PCR检测年龄相近的 182例健康人和 90例DM CAD患者ArE/PON1 192位基因多态性。结果:郑州市汉族健康人群与DM CAD患者血清ArE/PON1活性频数均呈单峰正态分布;ArE/PON1 192存在基因多态性,等位基因为Q和R。DM CAD患者ArE/PON1酶活性 (0. 198±0. 015)u/ml,低于正常对照组的 (0. 258±0. 020)u/ml(t=37. 92,P<0. 01);DM CAD患者R基因频率和RR基因型分布频率分别为 62%和 31%,高于正常对照组的 46%和 12% (P< 0. 01)。2个观察组各基因型间血清ArE/PON1酶活性差异无统计学意义。结论:郑州地区汉族人群血清ArE/PON1活性频数分布为单峰正态分布,并存在有ArE/PON1 192位基因多态性,R基因可能是该地区DM CAD的独立危险因素。     

Measurement of mouse liver glutathione S-transferase activity by the integrated method

Objective: The integrated method was investigated to measure Vm/Km of mouse liver glutathione S-transfer-ase (GST) activity on GSH and 7-Cl-4-nitrobenzofurazozan. Methods: Presetting concentration of one substrate twenty-fold above the other's and taking maximum product absorbance Am as parameter while Km as constant, Vm/Km was obtained by nonlinear fitting of GST reaction curve to the integrated Michaelis-Menten equation In [Am/(Am -Ai)] + Ai/ ( ξ× Km ) = ( Vm/Km )×ti (1). Results: Vm/Km for GST showed slight dependence on initial substrate concentration and data range, but it was resistant to background absorbance, error in reaction origin and small deviation in presetting Km. Vm/Km was proportional to the amount of GST with upper limit higher than that by initial rate. There was close correlation between Vm/Km and initial rate of the same GST. Consistent results were obtained by this integrated method and classical initial rate method for the measurement of mouse liver GST. Conclusion: With the co     

2型糖尿病合并冠心病患者血清芳香酯酶、黄嘌呤氧化酶及脂蛋白检测

目的:检测2型糖尿病并发冠心病(DM-CHD)患者血清芳香酯酶(ArE/PON1)和黄嘌呤氧化酶(XO)活性及脂蛋白含量.方法:测定78例2型糖尿病(DM)、80例DM-CHD患者与81例健康人血清ArE/PON1、XO、丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、高密度脂蛋白胆固醇(HDL-C)和氧化型低密度脂蛋白(ox-LDL)等有关指标,分析上述指标在DM-CHD发病中的意义.结果:与健康组相比,DM与DM-CHD组患者血清ArE/PON1活性降低、XO活性升高;MDA、CAT等水平升高,SOD活性降低;oxLDL水平升高,HDL-C降低,且DM-CHD组的改变较DM组明显.结论:血清ArE/PON1、OX、SOD及脂蛋白的测定可反映DM与DM-CHD患者机体氧化应激与脂代谢紊乱状况,是监测DM并发CHD的有用指标.     

10-23 DNA enzyme对乙型肝炎病毒C基因体外转录产物的切割活性

目的:探讨10-23 DNA enzym e对乙型肝炎病毒C基因体外转录产物的特异切割活性。方法:设计并合成3种能针对乙型肝炎病毒C基因开放阅读框A1816UG的特异性脱氧核酶,分别命名为D rzBC-7、D rzBC-8和D rzBC-9。应用体外转录的方法获得相应的底物RNA,观察D rzBC-7、D rzBC-8和D rzBC-9对其的体外切割效应。以D rzBC-9为例,观察不同浓度的M gC l2对体外切割效率的影响,根据L inew eaver Burk作图法,计算相关的酶动力学参数Km、K cat和K cat/Km。结果:通过体外转录获得用于切割反应的底物RNA,其大小为300 nt。在特定的切割条件下,D rzBC-7、D rzBC-8和D rzBC-9均能对相应的底物RNA进行有效的切割,切割产物分别为109 nt和191 n。t以D rzBC-9为例,在切割反应体系中缺乏M gC l2时,未见有切割反应。M gC l2浓度在150 mm o.lL-1时达到最高切割效率,再提高M gC l2的浓度,切割效率未见明显增大。酶动力学参数Km、K cat和K cat/Km分别为1.4×10-9m o.lL-1,1.6 m in-1和1.1×109m o.lL-1.m in-1。结论:针对乙型肝炎病毒C基因的10-23 DNA enzym e在体外对相应的转录产物有特异切割作用。     

Estimation of population pharmacokinetic parameters of free-phenytoin in adult epileptic patients

BACKGROUND: Serum concentrations of free-phenytoin (F-PHT) obtained in adult epileptic patients receiving PHT in monotherapy were analyzed to estimate the Michaelis-Menten pharmacokinetic parameters. METHODS: Steady-state F-PHT serum concentrations, PHT dosing history, and associated information were collected prospectively. The maximum metabolic rate (Vm) and Michaelis-Menten constant (Km) of F-PHT and their interindividual variability data were estimated using nonlinear mixed effects modeling (NONMEM). RESULTS: Twenty-nine patients with two or more available steady-state F-PHT serum concentrations (total of 63 dose/serum concentration pairs) met the inclusion criteria. Patients were taking PHT (100-500 mg/day) in monotherapy. The population estimates of F-PHT for Vm and Km were 9.1 mg/kg/day and 7.3 mg/L, respectively. The model was prospectively evaluated in a small group (seven) of additional patients. CONCLUSIONS: The recommended daily dose in this population to achieve a F-PHT concentration of 1.5 mg/L is 6.1 mg/kg.     

用4-硝基-1-萘基磷酸酯为底物测定碱性磷酸酶活性

目的：合成4-硝基-1-萘基磷酸酯 (4-nitro-1-naphthyl phosphate,NNPP)为显色底物测定牛小肠粘膜碱性磷酸酶 (alkaline phosphatase, ALP)。方法：4-硝基-1-萘酚(4-nitronaphthol, 4-NNP)与三氯氧磷反应,经硅胶柱纯化制得NNPP;检测产物吸收跟踪水解过程测定初速度,双倒数法测定米氏常数(Michaelis-Menten constant, Km)。结果：ALP水解NNPP产物最大差吸收峰接近460 nm,等吸收波长接近405 nm。与p-硝基苯酚相比,在pH 6.0~7.0间4-NNP消光系数为其3倍以上,在pH 7.0以上为其2倍以上。ALP对NNPP的Km约12?mol/L而p-硝基苯基磷酸酯的Km约35?mol/L,产物磷酸相对NNPP的竞争性抑制常数接近20?mol/L。ALP催化NNPP水解效率接近水解p-硝基苯基磷酸酯的40％。结论：NNPP可用于测定ALP活性, 且有望与作用于p-硝基苯酚类显色底物的其它酶联用实现单通道两种酶同步测定。     

郑州地区汉族正常人群、糖尿病和糖尿病并发冠心病患者芳香酯酶Q192R基因多态性

目的：研究郑州地区汉族正常人群、2型糖尿病（DM）及DM并发冠心病（DM－CAD）患者芳香酯酶（Arylesterase/Paraoxonase,ArE/PON1)192位基因多态性。方法：用比色法测定郑州地区汉族正常人群（61例）、DM（60例）和DM－CAD（67例）患者血清Ar/PON1活性，用聚合酶链式反应分析技术检测ArE/PON1基因192位多态性，用酶法和酶联免疫法分别测定血清胆固醇、高密度脂蛋白胆固醇（HDLc)、氧化型低密度脂蛋白（oxLDL)等指标。结果：郑州地区汉族人群中存在有ArE/PON1192位基因多态性，Q、R基因频率健康人群中分别为0.56与0.44；DM组为0.53与0．47；DM－CAD组为0．38和0．62。DM－CAD组R基因频率高于DM组和对照组。基因型百分比DM－CAD组RR（31％）高于DM组（13％）和对照组（11％）（P＜0．05）。DM与DM－CAD组血清ArE/PON1活性水平低于对照组，以DM－CAD组为最低（P＜0．05）。酶活性的降低与HDLc、HDL2C呈正相关，与oxLDL呈负相关。结论：郑州地区汉族人群存在ArE/PON1 192位基因多态性，并提示R基因是郑州地区汉族人群DM－CAD的危险因素。     

Specific and reversible inhibition of Entamoeba histolytica cysteine-proteinase activities by Zn2+: implications for adhesion and cell damage.

BACKGROUND: Cysteine-proteinases are thought to play an important role in E. histolytica pathogenicity. Although effective blockage of proteolytic activities can be obtained with several known inhibitors, the high cellular toxicity of most of the inhibitors precludes experimentation with live cells or animal models. Specific cysteine-proteinase inhibitors that could be utilized in studies of virulence are of great need in the field of amebiasis. METHODS: Cysteine-proteinase activities were determined in trophozoite lysates by azocasein degradation and after PAGE and gelatin zymograms. Inhibition of the activities was assessed in the presence of 0.01-2.5 mM concentrations of divalent cations of the IIB and VIII series such as Zn, Cd, Hg, Ni, and Co. Reversibility was induced with 25 mM L-cysteine or 50 mM L-histidine and by metal chelation with 5 mM phenantroline. The inhibitory effect of ZnCl2 was tested with live cells in fibronectin-binding and cytotoxicity assays. RESULTS: ZnCl2 specifically inhibited cysteine-proteinase activities in trophozoite lysates in a concentration-dependent manner. Additionally, 1.0-2.5 mM ZnCl2 blocked proteolysis in more than 70%. This inhibition was completely reverted by L-cysteine, L-histidine, or phenantroline. Similar results were obtained by analyzing individual cysteine-proteinase activities separated in gelatin zymograms. At these concentrations, ZnCl2 significantly interfered with trophozoite adhesion, thus making amebas deficient in substrate degradation and cell damage. CONCLUSIONS: ZnCl2 is an effective inhibitor of amebic cysteine-proteinases. Its low toxicity at relatively high concentrations, high solubility, and low cost make it adequate for live cell experimentation and animal models of amebic virulence.     

肾移植患者血清芳香酯酶活性与其基因192位多态性的研究

目的 研究郑州地区汉族居民肾移植患者血清芳香酯酶（ArE／PONl）活性变化与其基因192位多态性和脂代谢紊乱的关系。方法 通过测定肾移植（100例）、慢性肾小球肾炎的引起的尿毒症患者（101例）和对照组（105例）血清ArE／PON1活性、胆固醇（TC）、甘油三酯）（TG）、高密度脂蛋白胆固醇（HDL2-C）、高密度脂蛋白2胆固醇（HDL2-C）、高密度脂蛋白3胆固醇（HDL3-C）和氧修饰低密度脂蛋白（oxLDL）等的含量。并用聚合酶链式反应（PCR）分析技术检测了ArE／PON1基因192多态性，进行分析研究。结果 肾移植后60d患者血清ArE／PON1活性、ArE／TC、ArE／HDL3-C水平高于尿毒症组，低于对照组，血清HDL-C、HDL2-C和HDL2-C／HDL3-C水平的变化和ArE／PON1活性相似，HDL3-C、NAG／Cr、BUN和SCr水平降低。仍高于对照组水平，血清LDL-C、TC和TG水平反有升高；肾移植和尿毒症组患者血清HDL—C／TC低于对照组。但两组间的差异无统计学意义。ArE／PON1基因192位等住基因Q、R频率健康人基因分别为0．54与0．46。肾移植组为0．51和0．49，尿毒症组0．53与0．47，3组间Q和R基因频率差异无显著性。三组中各组内基因型RR患者血清ArE／PON1与TC、TG、HDL-C、oxLDL等的水平和基因型QR、QQ患者差异无显著性。结论 提示原发病为慢性肾小球肾炎的尿毒症肾移植后合并冠心病和基因R无明显关系。血清ArE／PON1活性降低对观察肾移植受者脂代谢紊乱合并心血管疾病则具有临床意义。     

兔乳酸脱氢酶C4的分离纯化及动力学研究

建立兔乳酸脱氢酶Ｃ４（ＬＤＨ－Ｃ４）分离纯化的新方法，研究该酶的动力学特性。采用Ｂｌｕｅ Ｄｅｘｔｅｒａｎ和Ｓｅｐｈａｒｏｓｅ ４Ｂ交联制成Ｂｌｕｅ Ｄｅｘｔｒａｎ－Ｓｅｐｈａｑｒｏｓｅ ４Ｂ染料配体亲和层析柱，结合ＱＡＥ－Ｓｅｐｈａｄｅｘ Ａ４５０离子交换层析分离兔ＬＤＨ－Ｃ４，以作图法测定该酶动力学数据。     

尼索地平控释包心片释药机理的研究

目的：研究尼索地平(NS)控释包心片体外释放机理。方法：本片为包心片，外层以羟丙甲纤维素(HPMC)作为骨架材料，内层采用速释片心制备而成。采用零级、一级、Higuchi及Peppas方程对释药过程进行整体和分段的处理。结果：0～6h释药符合零级方程，6～10h释药符合一级过程。结论：NS包心片两段释放机制均为非Fickian扩散即药物扩散与骨架溶蚀的协同作用，但扩散与溶蚀各自所占比例有很大差别。     

钩吻素子在人和猪、大鼠、猴、犬肝微粒体的酶促动力学及CYP450酶亚型分析

目的 研究和比较钩吻素子(KM)在人与各种实验动物肝微粒体体外代谢的酶促动力学及选择性CYP450酶抑制剂对其代谢的影响.方法 采用优化的反应体系和UPLC检测方法,测定系列浓度的KM与各种属肝微粒体孵育的降解曲线,以底物消除法计算酶动力学参数;共孵育方法考察选择性CYP450酶抑制剂对KM在各种属肝微粒体代谢的影响.结果 在人肝微粒体,KM的米氏常数(Km)、最大反应速率(Vmax)、半哀期(T1/2)、固有清除率(CLint)分别为(0.135±0.067)mmol/L、(1.89±0.19)μmol·min-1·g-1pro、(712±23)min和(15.7±5.2)mL·min-1·g-1pro.与人相比,犬、猪的Km值较大,大鼠及猴的CLint较高,4种实验动物的Vmax值均较大、T1/2均较短.酮康唑在各种属均可显著抑制KM代谢,在人、猪、犬IC50<1 μmol/L,在大鼠、猴IC50为1~10 μmol/L,可见CYP3A为主要代谢酶.噻氯匹定、奎尼丁和磺胺苯吡唑仅对大鼠,噻氯匹定仅对猴和犬的KM代谢为弱抑制(抑制率20.43%~44.31%),说明在体情况下,CYP2B、CYP2D和CYP2C与KM的代谢可能无关.结论 KM在人与猪、大鼠、犬、猴肝微粒体中的主要代谢途径相同,均由CYP3A酶催化,但酶活性和代谢动力学特征有一定差异.     

野菊花提取物对牙龈卟啉单胞菌胰酶样蛋白酶活性的抑制作用

目的:探讨野菊花提取物对牙龈卟啉单胞菌(P.g)胰酶样蛋白酶(TLP)活性的影响,为野菊花治疗牙周炎提供理论依据。方法:配制牛心脑浸液培养基(BHI),低、中、高浓度对倍稀释的野菊花提取物(0.1563、0.3125和0.6250 g·L^-1)和一定浊度的P.g菌悬液,实验分为低、中、高浓度野菊花提取物组、阳性对照组(不加野菊花提取物)和阴性对照组(加入低浓度野菊花提取物,不加P.g)。各组含溶液的EP管在37℃恒温条件下厌氧培养48 h,测定培养物上清液和菌细胞破碎后上清液中TLP活性和蛋白水平,计算TLP比活。结果:与阳性对照组比较,中、高浓度野菊花提取物组上清液中TLP活性(游离态和结合态)均降低(P<0.05);与低浓度野菊花提取物组比较,中、高浓度野菊花提取物组上清液中TLP活性(游离态和结合态)均降低(P<0.05);与中浓度野菊花提取物组比较,高浓度野菊花提取物组上清液中TLP活性(游离态和结合态)降低(P<0.05),各组上清液中蛋白水平和TLP比活无明显变化(P<0.05)。结论:野菊花提取物对TLP活性有抑制作用。     

ENZYME KINETIC STUJDIES WITH BETA-LACTAMASES AGAINST CEFOTAXIME AND OTHER CEPHALOSPORINS*

Enzyme stability and enzyme kinetic studies with plasmid.mediated and chromosome-mediated beta- lactamases against cefotaxime, cefoperazone and other cephalosporins were performed using spectro- photometry. Beta-lactamases were extracted from granvnegative organisms and identified by isoelectric focusing with the special reagent nitrocefin. The relative hydrolysis rate (%), Michaelis-Menten constant (Km), maximum velocity (Vmax) and inhibition constant (Ki) were obtained and used as indicators for comparative study of enzyme stability in six cephalosporins. The affinity of compounds bindinp; to target sites of beta-lactamases was also determined. The results show that cefotaxime is the most enzyme stable drug among the cephalosporins studied. The Vmax of TEM-1, SHV-I and P99 enzyme hydro lyzing cefotaxime was less than 0.10% as compared with cephaloridine. Cefotaxime demonstrates high affinity to the chromosmm>mediated enzyme P99, and its Ki value is 0.036 µm. Cefoperazonc exhibits less enzyme inhibitory activity and is unstable with most of the beta-lactamases used.     

基因重组人碱性成纤维细胞生长因子药物动力学实验研究──家兔静脉快推给药室分析

目的：研究家兔静脉快推基因重组人碱性成纤维细胞生长因子（ｒｈｂＦＧＦ）后血药浓度随时间的变化关系,并对其代谢动力学参数进行估计。方法：家兔静脉一次快推ｒｈｂＦＧＦ４０００ＩＵ（１０μｇ）,给药后１０,２０,３０ｍｉｎ及１．０,１．５,２．０,３．０,４．０,６．０,８．０ｈ耳缘静脉采血,放免分析法检测血中各个时间点的药物浓度。用数学方法拟合血药浓度－时间的函数关系,并估计药物动力学参数。结果：家兔静脉一次快推给药后血中ｒｈｂＦＧＦ浓度属双室模型一级动力学特征,其血药浓度－时间函数的解析表达式为：Ｃｔ＝９０．１１５６ｅ－２．０１０７ｔ＋１３．０７０７ｅ－０．１７８３ｔ。结论：本研究为指导ｒｈｂＦＧＦ的临床应用提供药物动力学的基本依据。     

氯化聚丙烯超声降解的动力学

测定了不同浓度氯化聚丙烯(CPP)甲苯溶液超声降解的分子量,并用数据拟合法求出了聚合物超声降解极限分子量。由于常用的超声降解动力学方程对实验数据拟合的精度不能令人满意,为了准确建立CPP在甲苯溶液中的超声降解动力学方程,提出了一个改进的动力学方程式:1Mt-Mlim-M0-1Mlim=kt。它能准确地描述CPP超声降解过程。用该方程拟合的聚合物超声降解数据,效果比文献提供的方程好。在超声降解过程中,随CPP浓度的增大极限分子量增大而降解速率常数则减小。     

人胎盘谷胱甘肽S-转移酶的纯化及其部分性质的研究

从人胎盘中分离得到谷胱甘肽S-转移酶纯化倍数470倍.比活力为37.897μmol/min·mg.经PAGE和SDS-PAGE证实为一条区带,亚基分子量为23000 Dalton;等电聚焦电泳结果,此酶等电点为4.6.底物GSH和CDNB的Km值分别为0.259mmol/L(r=0.97)和0.350mmol/L(r=0.99).不同浓度的巯基乙醇对GST-π活性有保护作用,1mmol/L浓度的保护作用较强.DEN,Triton X—100、苯巴比妥钠和AAF对GST-π活性起抑制作用,抑制作用随浓度增高而加强.     

颈动脉粥样硬化患者血纤溶活性与同型半胱氨酸含量的变化及相关性分析

目的:研究颈动脉粥样硬化(AS)患者血液纤溶活性与同型半胱氨酸(Hcy)含量的变化及其之间的相关性。方法:收集98例颈AS患者与94例无颈AS的对照组,分别采用产色法测定纤溶酶原激活物(t-PA)、纤溶酶原激活抑制物-1(PAI-1)活性,荧光偏振光法测定Hcy含量。结果:颈AS患者血PAI1活性和Hcy含量显著高于对照组患者(P<0.05),t PA活性显著低于对照组患者(P<0.05)。多元Logistic回归分析显示:吸烟、高血压、PAI-1活性升高、t-PA活性降低、Hcy含量升高、高胆固醇与颈AS相关。结论:血液纤溶活性降低和高同型半胱氨酸血症可能是颈AS的独立危险因素。     

Dipeptide transport: an active process with energy- and proton-dependence in the human intestinal cell line, Caco-2

Transport of protein in the form of small peptides(di/tripeptides)across the small intestinal wall is amajor route of dietary protein absorption.The dipeptidetransporter,PepT1,is known to be located in the in-testine and the kidney,and plays an important …