黄鳝碱性磷酸酶的分离纯化及其部分性质研究

经Tris-HCl缓冲液(pH8.6)抽提,正丁醇处理,30%-75%硫酸铵分级沉淀分离,DEAE-Sepharose离子交换柱层析,Sephacryl S-200凝胶过滤纯化,从黄鳝内脏组织中分离纯化出电泳纯的碱性磷酸酶。该酶提纯倍数为564倍,比活力达到3015U/mg。酶学性质和动力学性质研究表明,该酶催化磷酸苯二钠的水解反应,最适pH值为10.2,pH小于7和大于12均不稳定;最适温度为40℃,温度高于50℃不稳定;米氏常数Km值为1.17mmo1/L。金属离子对该酶的催化活力有不同的影响,K+对该酶活力无影响,Mg2+对该酶有激活作用,Zn2+对该酶有抑制作用。       

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Ｓｅｐｈａｃｒｙｌ Ｓ－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１３２ １４０,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     

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。     

大瓶螺碱性磷酸酶的分离纯化及部分性质研究

大瓶螺碱性磷酸酶的分离纯化及部分性质研究李清漪,曾和期（西南师范大学生物系,重庆６３０７１５）碱性磷酸酶（ＥＣ３．１．３．１,简称ＡＫＰ）是广泛存在于动物组织中的水解酶。对软体动物中ＡＫＰ的研究仅有少数报道［１,２］,对属于单壳贝类的水生食用螺──大...     

大凉疣螈碱性磷酸酶的分离纯化及部分性质

碱性磷酸酶 (alkaline phosphatase,AKP)在生物界的分布很广 ,动物、植物、微生物中均广泛存在 .提纯的 AKP常被应用于对核酸等的研究 ,是基因工程常用的工具酶 ,也是酶标免疫测定技术的常用工具酶之一 .人类血清中的 AKP在不同疾病状态下有显著变化 ,临床上将血清 AKP变化指标作为诊断某些疾病的依据 .对于细菌和高等动物的 AKP已有广泛的研究 ,但国内外对两栖爬行类动物 AKP的研究报导却很少 ,仅有蛇毒中 AKP的研究报导 [1,2 ] .本文对大凉疣螈皮肤的 AKP进行了分离纯化 ,并对其部分性质进行了初步研究 .1　材料和方法1 .1　材…     

人胎盘碱性磷酸酶的纯化及其抗血清的制备

人胎盘匀浆经过正丁醇抽提,丙酮沉淀以及ＤＥＡＥ－纤维素,ＳｅｐｈａｃｒｙｌＳ－２００,羟基磷灰石３种柱层析等步骤,从中分离出电泳纯的胎盘碱性磷酸酶,纯化系数达１０００倍以上,以此酶作为抗原,免疫新西兰纯种白兔,制备出１：５０００以上滴度的抗血清,该抗血清已用于科研和临床诊断工作中。     

硫酸粘菌素对碱性磷酸酶部分性质的影响

硫酸粘菌素对碱性磷酸酶有抑制作用,用Dixon作图求出抑制常数为Ki=4.88×10-6mol/L,抑制类型为非竞争性抑制.通过紫外吸收光谱,发现硫酸粘菌素引起碱性磷酸酶的空间结构发生改变;由荧光光谱变化,发现硫酸粘菌素对碱性磷酸酶有荧光淬灭作用,淬灭机理是因能量转移而引起的静态淬灭.     

Design and synthesis of selective inhibitors of Placental Alkaline Phosphatase

Placental Alkaline Phosphatase (PLAP) is a tissue-restricted isozyme of the Alkaline Phosphatase (AP) superfamily. PLAP is an oncodevelopmental enzyme expressed during pregnancy and in a variety of human cancers, but its biological function remains unknown. We report here a series of catechol compounds with great affinity for the PLAP isozyme and significant selectivity over other members of the AP superfamily. These selective PLAP inhibitors will provide small molecule probes for the study of the pathophysiological role of PLAP.     

五氯酚对人胎盘碱性磷酸酶抑制的研究

监测人胎盘碱性磷酸酶在不同浓度五氯酚溶液中活力与荧光光谱的变化;测定五氯酚对其抑制的类型及pH对其抑制的影响.结果显示：低浓度的五氯酚(＜1.0 mmol/L)使酶活力及其荧光强度迅速下降,发射峰位明显红移;继续提高五氯酚浓度,其活力与荧光强度逐渐下降,发射峰位仍在红移;五氯酚浓度为5.0 mmol/L时,荧光淬灭,但酶仍保留51.4%的活力;五氯酚浓度高达10.0 mmol/L时,酶剩余活力为30.0%.进一步实验表明五氯酚使L-色氨酸的荧光强度降低,并伴有发射峰位的红移,当五氯酚浓度为5.0 mmol/L时,L-色氨酸的荧光淬灭.五氯酚是人胎盘碱性磷酸酶的反竞争性抑制剂,其抑制常数(K_i)为3.86 mmol/L.其抑制也受溶液pH影响,pH＜7.5时,对酶无抑制;pH为7.5～10.5时,随pH的增加,抑制作用逐渐增强.提示五氯酚能够进入酶分子内部使其荧光淬灭,并抑制了酶活力;表明酶活力抑制与五氯酚的解离状态有关.     

人胎盘碱性磷酸酯酶基因在毕赤酵母中的表达和活性检测

将人胎盘碱性磷酸酯酶 (hPLAP)基因克隆到质粒ppICZαA中并在巴斯德毕赤酵母Pichiapastoris蛋白酶缺陷菌株SMD1 1 6 8中诱导表达。结果表明 :2拷贝子的重组酵母诱导表达产物酶活性最高 ,拷贝Mut 和Muts 表型不同对hPLAP酶活性没有显著影响     

苦瓜素类似物的分离纯化及其性质研究

本文采用硫酸铵分级沉淀、亲和层析、凝胶过滤等方法,从苦瓜(Momordicacharantia)种仁分得苦瓜素Ⅰ、Ⅱ。它们在SDS-聚丙烯酰胺凝胶电泳和等电聚焦电泳上均呈现单一区带,其分子量分别为26 000和28 000,等电点8.3,含糖量1.6％和2.0％。苦瓜素Ⅰ对小鼠腹腔给药LD_(50)值为2.54mg/kg,苦瓜素Ⅱ的致死剂量范围是9-14mg/kg。它们对无细胞体系蛋白合成都有强烈的抑制活性,其IC_(50)均小于0.1ng/mL。我们将苦瓜素Ⅰ、Ⅱ与文献报道的苦瓜抑制剂(Momordica chanantiainhibitor,MCI)和α、β-momorcharin进行了比较,发现它们分别与MCI和α-momorcharin相似。由于苦瓜素Ⅱ的毒性较低,更适合于制备免疫毒素。     

Purification and Characterization of an Extracellular Alkaline Phosphatase from Penicillium Chrysogenum

Abstract

An extracellular alkaline phosphatase from Penidllium chrysogenum was purified to homogeneity using DEAE ion-exchange chromatography and size exclusion chromatography. SDS-PAGE of the purified enzyme indicated a molecular weight of 58,000. The mobility of the native enzyme on a Superose 12 column suggests that the active form of the enzyme is a monomer. The enzyme catalyzes the hydrolysis of phosphate from a variety of substrates including p-Miitrophenyl phosphate, α-naphthyl phosphate and the anti-tumor compound etoposide phosphate. The apparent K_m for the substrate p-nitrophenyl phosphate is 1.3 mM and the enzyme is inhibited by inorganic phosphate. The pH optimum of the enzyme is 9.0 with a broad optimal temperature range between 40 and 50 °C. The isoelectric point of the enzyme is approximately 5.5. The enzyme is a glycoprotein; digestion with endoglycosidase H indicates that the protein consists primarily of N-inked carbohydrates. Enzymatic activity is enhanced by the addition of divalent cations such as Mg⁺⁺ and Mn⁺⁺ and inhibited by addition of a chelator such as EDTA suggesting a metal ion requirement. The enzyme was found to be an inexpensive catalyst for the conversion of etoposide phosphate to etoposide in the manufacture of this anti-tumor compound.     

Carcinoplacental Alkaline Phosphatase

Using HeLa TCRC-1, a cell line which is monophenotypic with respect to the Regan isoenzyme of alkaline phosphatase, we have examined the factors which influence its expression in relation to events of the cell cycle.
DNA synthesis is not required for hormone induction of the Regan isoenzyme as in the presence of hydroxyurea, a specific inhibitor of DNA synthesis, we found induction to occur. Additionally, when partially synchronised cells were allowed to leave the S period prior to hormone treatment, and hydroxyurea was added to prevent cells from entering the next S period, hormone induction of the Regan isoenzyme was still observed. This indicates that initiation of expression of hormone-induced carcinoplacental alkaline phosphatase occur prior to the DNA synthetic phase of the cell cycle.
We propose a hypothetical two-step mechanism of hormone induction to interpret the present findings in relation to previous results.     

Identification and Purification of a Derepressible Alkaline Phosphatase from Anacystis nidulans R2

We have examined the increase in alkaline phosphatase activity in the cyanobacterium Anacystis nidulans R2 upon phosphate deprivation. Much of the activity is released into the medium when A. nidulans is osmotically shocked, indicating that the enzyme is located either in the periplasmic space or is loosely bound to the cell wall. The polypeptide associated with phosphatase activity has been identified as a single species of M_r 160,000. Several lines of evidence demonstrate that this polypeptide is responsible for alkaline phosphatase activity: (a) It is absent when cells are grown in the presence of phosphate and specifically accumulates during phosphate deprivation. (b) It is the major periplasmic polypeptide extracted by osmotic shock. (c) It represents over 90% of the protein in a fraction of periplasmic polypeptides enriched for phosphatase activity. (d) Antibodies raised against the purified species of M_r 160,000 inhibit phosphatase activity by approximately 70%.