纳豆激酶的分离纯化及酶学性质研究

目的　分离纯化纳豆激酶并进行酶学性质研究。方法　从纳豆中分离纯化具有纤溶活性的纳豆激酶 ,采用氯化钠溶液浸提、硫酸铵盐析及 sephadex G- 1 5 0凝胶过滤分离纯化纳豆激酶 ,纯化倍数 5 0倍 ,纤维蛋白平板法测其比活。结果　纳豆激酶比活为 6 0 0 U·mg-1,回收率 40 %。酶学性质研究表明 ,最适反应温度为 5 0℃ ,5 5℃以下稳定 ,最适反应 p H为 8.0 ,在 p H 6～ 1 0溶液中基本稳定 ,分子量约 2 8k D。酶作用机理初探表明 ,纳豆激酶不仅具有纤溶性 ,还有抗凝血性。结论　纳豆激酶有可能成为新型溶栓药物     

用多孔玻璃载体制备固定化青霉素酶的研究

本文就多孔玻璃固定化方法进行了研究。摸索了硅烷化回流时间、γ-氨基丙基三乙氧基硅烷浓度、以及酶与玻璃N羟基琥珀酰亚胺酯共价结合时,pH对固定化青霉素酶活力的影响等。用比活10.2u/mg的青霉素酶粗酶,与150～200目、孔径300～500×10~(-10)m的多孔玻璃所制得的固定化酶活力为248.8u/g,回收率为13.5％。 该固定化酶反应的最适温度为52℃,最适pH为7;其热稳定性较差,pH7条件下65℃及85℃保温30分钟活力分别损失3％及1.2％;其对酸的稳定性优于对碱的稳定性;使用和贮藏稳定性均好。该酶的性能宜用于组装热敏电阻电极。     

牛肝β-D-葡萄糖醛酸苷酶的分离纯化和性质研究

采用自溶、35 %硫酸铵分部沉淀、CM 纤维素柱层析、羟基磷灰石柱层析由动物肝脏中分离纯化β D 葡 萄糖醛酸苷酶 ,获得比活 2 6 70 0U/mg的纯酶 ,活力回收率为 4 2 .8% ,纯化的酶经SDS PAGE呈单一条带。该酶以 β D 酚酞葡萄糖醛酸苷为底物的Km值为 2 .0× 10 -4mol/L ,以对硝基苯酚葡萄糖醛酸苷为底物的Km值为 2 .5× 10 -4mol/L。在 pH值 5～ 6的范围内 ,该酶的活力较高 ,最适 pH值为 5 .5 ,在pH值 5 .5时稳定性最好。在 30～ 5 0℃范围内酶活力均较稳定     

固态发酵黑曲霉产壳聚糖酶的纯化及其性质研究

目的 研究黑曲霉产壳聚糖酶的纯化及其性质.方法 紫外诱变筛选黑曲霉产壳聚糖酶高产菌株,并分离纯化其固态发酵物的粗提液,研究纯化的壳聚糖酶的酶学性质.结果 固态发酵120 h,干培养基的酶活力可达58.12 U·g~(-1).固态培养基提取液通过丙酮分级沉淀,经交联壳聚糖树脂亲和层析、DEAE-Sepharose FF层析得到了一种胞外壳聚糖酶,分子量为63kD,最适反应温度为55℃,最适反应pH6～7,降解壳聚糖反应的米氏常数K_m为2.933 g·L~(-1),V_(max)为0.028 g·L~(-1)·min~(-1).该酶不能降解片状甲壳素和纤维素,能微弱降解CMC和胶体甲壳素,最适底物为脱乙酰度90%的胶体壳聚糖.结论 紫外诱变的成本低、方法简单,利用黑曲霉高产壳聚糖酶菌株发酵能生产一种新型壳聚糖酶.     

心肌黄酶的提纯工艺及其部分性质的研究

通过热变性处理、硫酸铵分级沉淀、DE-AE-Toyopear1650M柱层析及K_2BP-Agarose染料配基层析等方法从猪心肌组织匀浆提取液中分离与纯化了心肌黄酶。应用此工艺,酶活力的总回收率为16.9％,纯化倍数为26.4。用Sephadex G-100凝胶过滤法测得心肌黄酶的分子量为85,000道尔顿;以NADH为底物时其米氏常数为1.0×10~(-3)M;最适作用PH为10.6,在pH5-10时稳定;最适作用温度为65℃,有较好的热稳定性,在75℃保温3小时仍可保留80％的酶活性,提纯的酶溶液在4℃保存六个月未见活性明显损失。     

核酸酶P1的分离纯化及部分酶学性质研究

桔青霉发酵液经离心、超滤、硫酸铵盐析、透析和DE-52型树脂柱色谱分离,纯化得到核酸酶P1.比活为28490u/mg,纯化10.5倍,收率为45%.SDS-PAGE电泳显示为单一条带.酶学性质研究结果表明,以酵母RNA为底物时的Km为3.36mmol/L,最适作用温度70℃,最适pH 5.4.     

猪胰腺羧肽酶B提取工艺中的激活条件与稳定性研究

目的研究猪胰腺羧肽酶B提取工艺中的激活条件以及激活后粗酶液的稳定性,并对酶进行纯化。方法胰腺匀浆、激活,硫酸铵盐析进行初步提取,采用疏水色谱、离子交换色谱和分子筛等方法进行纯化,对胰腺匀浆后的粗酶激活条件进行了研究,考察了不同保存条件下粗酶液的稳定性。结果纯化后的羧肽酶B经SDS-PAGE电泳鉴定为电泳纯。比活为152 u/mg,纯化过程的活性总回收率为51%。粗酶液中羧肽酶B的激活受各种条件影响,特别是激活温度和激活时间,4℃下激活80 h,或37℃下,激活6.5 h,均可获得最大酶活,激活后的粗酶液可直接于35%(NH4)2SO4中保存,保存60 h的酶活损失小于5%。结论通过调整激活温度与激活时间可以获得猪胰腺羧肽酶B粗酶液最大的激活效果,激活后的粗酶液可于35%(NH4)2SO4中短期保存。     

一种具有纤溶活性的蛋白酶的分离纯化及性质

目的分离纯化BacillussubtilisJin2 1发酵产生的具有纤溶活性的蛋白酶 ,并对其性质进行研究。方法通过硫酸铵分级盐析 ,SerineSepharose 2B和SephacrylS— 2 0 0色谱柱进行分离纯化 ,用SDS PAGE测定纯度 ,SDS PAGE和凝胶层析测定相对分子质量 ,纤维蛋白平板法测定酶活力。结果分离得到了电泳纯的酶 ,该酶的相对分子质量为 2 8kD ,等电点约为 8 6 ,最适 pH为 7 5 ,最适温度为 4 0℃ ,在 4 0℃以下较稳定 ,超过 5 0℃时酶活力开始下降 ;金属离子Mn2 + 对酶有明显的激活作用 ,而Zn2 + 对酶有抑制作用 ,二异丙基磷酰氟 (DFP)和苯甲基磺酰氟 (PMSF)完全抑制酶活性。结论该酶是一种丝氨酸蛋白酶 ,与纳豆激酶相似 ,有望开发成为一种新型口服溶栓药物     

固定化胰蛋白酶的性质研究

目的　测定固定化胰蛋白酶的催化特性和部分稳定性。方法　在相同的条件下,测定固定化和可溶性胰蛋白酶的活力,得其最适作用pH值和最适作用温度,再测定活力,得到各自的米氏常数(Km)值;测得两种状态酶的热稳定性和酸碱稳定性。同时测定了固定化胰蛋白酶的低温稳定性。结果　固定化和可溶性胰蛋白酶的最适作用pH值分别为8 .0～9. 0和7. 0～9.0 ,最适作用温度分别为6 0℃和5 5℃,Km值分别为2 . 32 6、1 42. 9mg·ml-1 ;固定化胰蛋白酶在5 5℃条件下,保温5h ,仍有78 92 %的活性;在4℃条件下,8d后,活性才略微下降,两周活性保留达90 %以上;在pH 9.0的活性未下降,在pH 7.0～1 0 .0范围内也比较稳定(相对活性>80 %)。结论　胰蛋白酶经固定化后,最适作用pH值范围变窄,最适作用温度和Km值升高;热稳定性和酸碱稳定性均有所增强     

黑曲霉SL2-111聚半乳糖醛酸酶的纯化和特性

通过硫酸铵分级沉淀、Sephacryl S 200分子筛柱层析、DEAE Sepharose Fast Flow阴离子交换柱层析3个步骤,从黑曲霉SL2 111发酵的果胶酶粗品中纯化出一种电泳纯的聚半乳糖醛酸酶。经SDS PAGE测定其相对分子质量约为71.5 ku。最适pH值和最适温度分别为3.0和65℃。在50℃以下,pH值2.6~5.4之间酶活力相对稳定。浓度为5 mmol/L的Ca2 、Li 、Cu2 、Mg2 、K 对酶活力有激活作用;而Mn2 和Ag 具有抑制作用。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.     

Pharmacological treatment of COVID-19: an opinion paper

The precocity and efficacy of the vaccines developed so far against COVID-19 has been the most significant and saving advance against the pandemic. The development of vaccines has not prevented, during the whole period of the pandemic, the constant search for therapeutic medicines, both among existing drugs with different indications and in the development of new drugs. The Scientific Committee of the COVID-19 of the Illustrious College of Physicians of Madrid wanted to offer an early, simplified and critical approach to these new drugs, to new developments in immunotherapy and to what has been learned from the immune response modulators already known and which have proven effective against the virus, in order to help understand the current situation.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.