关于酶促反应速度与酶反应的初速度的教学

酶促反应速度是酶促反应动力学中的一个基本概念。一定条件下 ,酶催化反应的活力大小要由该反应条件下酶促反应速度来反映和确定 :酶促反应速度越小 ,酶活力越小 ;反之 ,酶促反应速度越大 ,酶活力也越大。在研究底物浓度、酶浓度、温度、ｐＨ、激活剂、抑制剂等因素对酶活力的影响时 ,都必须测定酶促反应的速度。为了排除实验中存在的一些干扰 ,一个酶的酶促反应的速度要在酶促反应进行的初期测定 ,即测定所谓的酶促反应的初速度。在教学过程中 ,学生不易弄清楚酶促反应速度与酶促反应的初速度之间的关系。我们发现 :当一般地问起酶反应速度…     

苯丙氨酸解氨酶

苯丙氨酸解氨酶由四个亚基组成,含两个脱氢丙氨酸残基。植物酶具有内在不稳性,由多种同工酶组成。酶催化过程中发生构象变化,底物经过负碳离子中间体完成反应。该酶并非是二单体负协同变构酶。一级结构表明,酶以无规则卷曲结构为主。酵母基因约2.7kb,有六个内含子,编码75kD_a肽。植物酶由多基因编码,有一个内含子,编码78kD_a肽。启动子部位有两个富含A、C碱基的序列,为胁迫作用基因活化因子结合部位。     

放线菌果胶酶产酶条件及酶促反应条件的研究

从埋麻土壤中分离到放线菌２９８株,经初筛和复筛得到产酶活性较高的一株放线菌５－７１。最适产酶条件是：果胶０．５％,葡萄糖０．５％,蛋白胨０．５％,酵母粉０．１％,Ｋ２ＨＰＯ４ ０．１％,ＫＨ２ＰＯ４ ０．１％,ＮａＣｌ０．１％,ＭｇＳＯ４·７Ｈ２Ｏ０．０５％,ｐＨ８．０。２５℃培养３天达产酶高峰。通气量对产酶影响不大。酶促反应最适条件是：ｐＨ９．６,４５℃,底物果胶浓度０．７５％,作用时间９０ｍｉ     

大鼠甘氨肽酰化单氨酶基因的克隆与表达

本文采用原位杂交及ＰＣＲ方法,从大鼠脑ｃＤＮＡ库中,筛选到３个大鼠甘氨肽酰化单氧酶基因片段。经ＤＮＡ全序列分析表明,它们跨越ｒＰＡＭ－２全部编码区。通过点突变,ＰＣＲ重组技术等,分别拼接出此双功能酶的ｒＰＨＭ,ｒＰＳＡＬ及其ｒＰＡＭ全构建了多种大肠杆菌表达质粒。     

香蕉低温酶促褐变

7℃低温导致香蕉果皮变褐,褐变程度可用提取液的OD_(450)值表示。随低温处理时间的延长,OD_(450)增大;还原性物质抗坏血酸、谷胱甘肽含量迅速下降;多酚氧化酶底物多巴胺先有增加,接着降低。 低温下,多酚氧化酶(PPO)、过氧化物酶(POD)和过氧化氢酶(CAT)活性较常温低,PPO和POD的游离态酶活性增高。此外,H_2O_2处理加速果皮变褐,刺激PPO及POD活性;棓酸丙酯处理起相反的效应。故认为香蕉低温褐变是低温损伤、酶促褐变引起的一个复杂过程。     

血浆激肽释放酶新底物PK—120的研究

血浆激肽释放酶新底物ＰＫ┐１２０的研究蒲小平（军事医学科学院基础医学研究所,北京１００８５０）关键词血浆激肽释放酶底物１．发现补体系统由２０余种血浆蛋白组成,在机体防御和炎症应答反应中起重要作用。１９８９年Ｈａｍｍｅｒ等在用Ｃ４ｂ－Ｓｅｐｈａｒｏｓｅ...     

A spectrophotometric, enzymatic assay for D-3-hydroxybutyrate that is not dependent on hydrazine

Because of the potential carcinogenic properties of hydrazine and because of other health hazards associated with its use in the laboratory, an enzymatic assay has been developed for D-3-hydroxybutyrate that is not dependent on hydrazine to drive the reaction toward completion. The use of a high concentration of NAD+ and a buffer at pH 9.5 resulted in a favorable conversion of D-3-hydroxybutyrate to acetoacetate by D-3-hydroxybutyrate dehydrogenase even though the reaction favors D-3-hydroxybutyrate formation under physiological conditions. The assay was also completed faster than previous assays using hydrazine so that the amount of enzyme used for the assay could be reduced. The recovery of D-3-hydroxybutyrate added to liver samples was 98 +/- 1% (mean +/- SEM, n = 6). The assay was found to be suitable for the measurement of D-3-hydroxybutyrate in samples such as perchloric acid extracts of isolated hepatocytes even when the acetoacetate to D-3-hydroxybutyrate ratio was 4 to 1. This assay presents a reliable alternative to the use of hydrazine and may be used for the assay of D-3-hydroxybutyrate in a variety of physiological and experimental samples.     

Lipid-protein ratios by infrared spectroscopy

A method for measuring the ratio of diacyl phospholipid to protein in lipid-protein mixtures and membranes by infrared spectroscopy is described. Samples made of diacyl phospholipid and proteins mixed in known ratios were analyzed for lipid-protein ratio by the infrared (ir) method. Results had a standard deviation of less than +/- 4% over the lipid-protein molar ratio range of 9:1 to 320:1. Calculations of the ratio of total lipid to protein require that the diacyl phospholipid-to-protein ratio be divided by the mole fraction of diacyl phospholipid in the total lipid. Phospholipid-protein ratios for various sarcoplasmic reticulum membrane preparations (R1-washed, octylglucoside purified, deoxycholate treated) were determined by the ir method and compared to literature values. Also, phospholipid-protein ratios were determined for R1-washed sarcoplasmic reticulum by three chemical analyses using different protein assays and were compared with ratios obtained by the infrared method. The infrared results were closest to those of a chemical method designed specifically for membrane proteins.     

The application of triazine dye affinity chromatography to the large-scale purification of glycerokinase from Bacillus stearothermophilus

Gram quantities of homogeneous glycerokinase have been prepared from the thermophilic bacterium, Bacillus stearothermophilus, using three major steps: precipitation of debris at pH 5.1, ion-exchange chromatography on DEAE-Sephadex, and affinity chromatography on Procion Blue MX-3G-Sepharose. This method is a considerable improvement over conventional techniques; the purified enzyme was obtained with a 40% recovery and a specific activity of 120 units (mumol/min)/mg protein. A modified culture medium enabled yields of 3.4 X 10(6) units of enzyme to be obtained from 400-liter production cultures.     

游动放线菌Actinoplanes sp.SE50/110中阿卡波糖脱氧氨基糖单元的生物合成

【目的】解析Actinoplanes sp.SE50/110(简称SE50/110)中阿卡波糖脱氧氨基糖单元的生物合成机制。【方法】经过BLASTp分析,推测了Acb A、Acb B和Acb V负责阿卡波糖脱氧氨基糖单元的生物合成。首先,本研究在SE50/110中分别构建了acb A、acb B和acb V的同框缺失和回补突变株。然后,利用大肠杆菌BL21(DE3)/p Gro7分别对Acb A、Acb B和Acb V成功实现了可溶性表达。最后,以D-葡萄糖-1-磷酸为起始底物,通过体外催化反应,研究脱氧氨基糖单元的生物合成过程和相关蛋白的酶学性质。【结果】在SE50/110中分别缺失acb A、acb B和acb V基因后,相应突变株均丧失了阿卡波糖的合成能力,将acb A、acb B和acb V基因分别回补后,各菌株又恢复了阿卡波糖的合成能力,证明了它们均为阿卡波糖生物合成的必需基因。在体外酶促反应中,D-葡萄糖-1-磷酸-胸腺嘧啶转移酶Acb A催化D-葡萄糖-1-磷酸和d TTP合成d TDP-D-葡萄糖,对D-葡萄糖-1-磷酸的Km值为(0.185±0.053)mmol/L,Vmax为(2.366±0.217)μmol/(min·mg);对d TTP的Km值为(4.964±1.089)mmol/L,Vmax为(60.310±5.419)μmol/(min·mg)。d TDP-D-葡萄糖-4,6-脱水酶Acb B催化d TDP-D-葡萄糖转化为d TDP-4-酮基-6-脱氧-D-葡萄糖,Km值和Vmax分别为(0.353±0.089)mmol/L和(306.401±28.740)μmol/(min·mg)。氨基转移酶Acb V催化d TDP-4-酮基-6-脱氧-D-葡萄糖生成d TDP-4-氨基-4,6-双脱氧-D-葡萄糖,Km值和Vmax分别为(1.411±0.293)mmol/L和(3.447±0.279)μmol/(min·mg)。【结论】本研究阐明了阿卡波糖脱氧氨基糖单元的生物合成过程,为全面解析阿卡波糖生物合成途径奠定了基础。同时,测定了相关酶的动力学参数,为代谢工程改造SE50/110,提高阿卡波糖产量提供了重要的理论依据。     

An Enzymatic Fluorometric Assay for Pyridoxal with High Specificity and Sensitivity

An enzymatic fluorometric assay for pyridoxal with pyridoxal dehydrogenase was developed. The detection limit was about 10 pmol: the calibration curve of pyridoxal showed high linearity (r=0.993). The values obtained by this method correlated well with those by the HPLC method. The enzyme had a high specificity for pyridoxal, and thus animal samples could be directly analyzed without separation of pyridoxal 5′-phosphate by column chromatography.     

真菌漆酶的酶活测定方法评价

目前真菌漆酶酶活的测定方法多样,没有统一的标准,致使不同研究之间的漆酶酶活无法进行比较分析,也造成漆酶产品在酶活质量意义上的混乱。因此,对测定真菌漆酶酶活的各种不同的分光光度法进行了综述和比较分析,认为采用ABTS法作为测定漆酶酶活的方法较具合理性和科学性,建议作为漆酶酶活测定的统一方法。     

Quantification of various phosphatidylcholines in liposomes by enzymatic assay

The purpose of this research was to adapt a colorimetric, phospholipase D-based serum-phospholipid assay for the quantification of phosphatidylcholine (PC) in liposomes using a microtitre plate reader. PC from natural egg PC liposomes was quantified reliably. In contrast, poor sensitivity was found for liposomes composed of saturated PCs (dipalmitoyl-phosphatidylcholine [DPPC], hydrogenated egg PC). Triton X-100 was then added to the liposomes followed by heating above the phase transition temperature. This modified sample preparation resulted in recoveries of 102.6%±1.0%, 104.4%±7.6%, and 109.4%±3.2% for E80, E80-3/cholesterol, and DPPC liposomes, respectively. Absolute quantification of unknown PCs against a choline chloride standard is feasible, but relative measurements against the very same PC are recommended wheneve possible. Validation experiments revealed an absolute quantification limit of 1.25 μg per assay, a good linearity in the range of 25 to 1000μg/mL PC (r²≥0.9990) and a quite high accuracy (99.8%–101.4% of theory) and precision (relative standard deviation ≤3.2%) for all 3 PCs studied. The method is thus regarded as suitable for sensitive, rapid, and reliable routine quantification of PCs in liposomes.     

Multi-step reactions on microchip platform using nitrocellulose membrane reactor

A straightforward and effective method is presented for immobilizing enzymes on a microchip platform without chemically modifying a micro-channel or technically microfabricating a column reactor and fluid channel network. The proposed method consists of three steps: the reconstitution of a nitrocellulose (NC) membrane on a plane substrate without a channel network, enzyme immobilization on the NC membrane, and the assembly of another substrate with a fabricated channel network. As a result, enzymes can be stably and efficiently immobilized on a microchip. To evaluate the proposed method, two kinds of enzymatic reaction are applied: a sequential two-step reaction by one enzyme, alkaline phosphatase, and a coupled reaction by two enzymes, glucose oxidase and peroxidase, for a glucose assay.