肌醇磷脂激酶(PI4-K)单克隆抗体的制备及其对细胞生长的抑制

制备了抗肌醇磷脂激酶 ( PI4- K)单克隆抗体 ( A6D)并测定了抗原 -抗体反应基本特性及功能 .结果表明 ,单克隆抗体与固相及溶液中肌醇磷脂激酶的亲和常数分别为 7.5× 1 0 6和 6× 1 0 8( mol/L) -1.单抗 1 .9× 1 0 -7mol/L可以抑制从细胞提取液的 PI4- K酶活力 50 % .用 FITC标记单抗在蛋白微球引导下进入细胞内 ,主要富集在细胞质膜区 ,并对 He La细胞和小鼠小脑细胞生长有明显抑制作用 .     

肌醇磷脂激酶(PI4-K)单克隆抗体的制备及其对细胞生长的抑制

制备了抗肌醇磷脂激酶 ( PI4- K)单克隆抗体 ( A6D)并测定了抗原 -抗体反应基本特性及功能 .结果表明 ,单克隆抗体与固相及溶液中肌醇磷脂激酶的亲和常数分别为 7.5× 1 0 6和 6× 1 0 8( mol/L) -1.单抗 1 .9× 1 0 -7mol/L可以抑制从细胞提取液的 PI4- K酶活力 50 % .用 FITC标记单抗在蛋白微球引导下进入细胞内 ,主要富集在细胞质膜区 ,并对 He La细胞和小鼠小脑细胞生长有明显抑制作用 .     

磷脂酰肌醇3激酶与胰岛素抵抗

胰岛素(Insulin,INS)通过胰岛素信号转导途径发挥其促进合成代谢、稳定血糖的生理作用,磷脂酰肌醇-3激酶(phos-phatidylinositol-3-kinase,PI-3K)是胰岛素信号转导中的关键分子.PI-3K是由催化和调节亚基构成的异源二聚体.催化和调节亚基在数量上保持平衡,此平衡的紊乱可以改变PI-3K的活性.研究表明调节亚基p85α与胰岛素的敏感性成负相关,动物和人胰岛素抵抗(Insulin resistance,IR)发生调节亚基p85α的过度表达.     

磷脂酰肌醇—4—激酶的免疫分析

在化猪肝微粒体磷脂酰肌醇－４－激酶（ＰＩ－４－Ｋ）的基础上,制备该酶的免疫血清,提纯ＩｇＧ,并建立了ＰＩ４－Ｋ的ＥＬＩＳＡ,进行不同组织中ＰＩ４－Ｋ的免疫沉淀分析和免疫定量。结果证明：猪肝微粒体ＰＩ４－Ｋ的抗血清或抗体ＩｇＧ可沉淀猪肾和猪肺的微粒膜,猪肝细胞膜和人中性粒细胞细胞膜ＴｒｉｔｏｎＸ－１００增溶液（ＴＸＳＭ）中的ＰＩ４－Ｋ表明不同组织,不同亚细胞和不同种属的ＰＩ４－Ｋ有相似的抗原性。猪肝     

酵母菌磷脂酰肌醇(PI)的生物合成及其重要的生理学功能

本文对近年来在酵母菌磷脂酰肌醇 (PI)生物合成 ,PI与酵母菌信号传导的相互关系 ,PI在酵母菌耐浓度酒精中的作用和PI在酵母菌胞外酶分泌解阻遏中的作用等方面最新研究进展进行了较为全面的讨论。     

幽门螺杆菌通过激活磷脂酰肌醇3-激酶信号来调节细胞迁移

目的幽门螺杆菌被认为是诱发胃癌的最强的风险因素。幽门螺旋杆菌的毒性成分是可以增加癌症危险的cag分泌系统,它可以使cagA和肽聚糖易位进入宿主细胞,进而激活信号转导通路。AKT是磷脂酰肌醇3。激酶（PI3K）的目的蛋白,并在胃癌中被激活,但PI3K-AKT和具有潜在致癌性的幽门螺旋杆菌诱导的细胞反应之间的关系尚不清楚。方法我们揭示了介导幽门螺旋杆菌刺激的AKT活化和胃上皮细胞的这些生物学结果之间的分子通路。结果幽门螺旋杆菌以Scr和表皮生长因子受体依赖性方式增加PI3K-AKT的信号,是幽门螺旋杆菌诱导的细胞迁移不可或缺的。结论这些结果表明,PI3K-AKT信号调节幽门螺旋杆菌诱发的病理生理反应,从而降低癌变门槛。     

磷脂酰肌醇－4－激酶的的免疫分析

在纯化猪肝微粒体磷脂酰肌醇－４－激酶（ＰＩ－４－Ｋ）的基础上,制备该酶的免疫血清、提纯ＩｇＧ,并建立了ＰＩ４－Ｋ的ＥＬＩＳＡ,进行不同组织中ＰＩ４－Ｋ的免疫沉淀分析和免疫定量。结果证明：猪肝微粒体ＰＩ４－Ｋ的抗血清或抗体ＩｇＧ可沉淀猪肾和猪肺的微粒体膜、猪肝细胞膜和人中性粒细胞细胞膜ＴｒｉｔｏｎＸ－１００增溶液（ＴＸＳＭ）中的ＰＩ４－Ｋ,表明不同组织、不同亚细胞器和不同种属的ＰＩ４－Ｋ有相似的抗原性。猪肝、肾、肺、胃、小肠和大肠ＴＸＳＭ中ＰＩ４－Ｋ的活力和酶蛋白含量相仿,急性中性粒细胞白血病患者的中性粒细胞膜上ＰＩ４－Ｋ的含量和活力都增加３．５倍左右．加上ＰＩ４－Ｋ抗体沉淀各组织ＰＩ４－Ｋ活力和酶蛋白的百分率基本相同,支持文献报道组织中微粒体或细胞膜中的ＰＩＫ主要是ＰＩ４－Ｋ,而免疫性不同的ＰＩ３－Ｋ含量极少的观点．     

酵母菌磷脂酰肌醇（PI）的生物合成及其重要的生理学功能

本文对近年来在酵母菌磷脂酰肌醇（PI）生物合成,PI与酵母菌信号传导的相互关系,PI在酵母菌耐浓度酒精中的作用和PI在酵母菌胞外酶分泌解阻遏中的作用等方面最新研究进展进行了较为全面的讨论。     

磷脂酰肌醇3-激酶γ/δ与炎症相关疾病

磷脂酰肌醇3-激酶(PI3K)是一类脂质与蛋白激酶家族,其主要通过在磷脂酰肌醇的肌醇环三位进行磷酸化产生胞内重要的第二信使——磷脂酰肌醇-3,4,5-三磷酸(phosphatidyl inositol 3,4,5-trisphosphate,PIP3)而发挥作用.磷脂酰肌醇3-激酶γ/δ(PI3Kγ/δ)是I类PI3K家族中的成员,其主要表达于免疫相关细胞中,这2种PI3K亚型参与先天性与获得性免疫应答.因此,PI3Kγ/PI3Kδ被视为因免疫反应调控异常导致的炎症疾病的治疗药物靶点.目前,利用特异性抑制剂靶向干预PI3Kγ和/或PI3Kδ,成为炎症相关疾病治疗的新策略.本文简介了PI3Kγ与PI3Kδ在不同类型免疫细胞中的功能;并就采用小分子特异性抑制剂,靶向抑制PI3Kγ和/或PI3Kδ在各类炎症相关疾病中的治疗作用和效果进行综述.     

雷公藤对哮喘大鼠气道重构及磷脂酰肌醇3激酶表达的影响

目的:探讨雷公藤内酯醇对哮喘气道重构及磷脂酰肌醇3激酶(PI3K)表达的影响。方法:将40只SD大鼠随机分为5组(n=8):A组(正常对照组);B组(哮喘4周组);C组(哮喘6周组);D组(给药4周组);E组(给药6周组)。测定气道反应性并观察气道壁嗜酸性粒细胞浸润;图像分析软件测定支气管壁厚度、支气管平滑肌厚度及支气管平滑肌细胞核数量;免疫组织化学染色、逆转录聚合酶链式反应(RT-PCR)检测PI3K蛋白及mRNA表达。结果:①B组、C组PI3Kp85α的蛋白及mRNA表达水平显著高于A组(P均<0.01),E组上述指标较B组、C组、D组均显著降低(P<0.01、P<0.01、P<0.05);②B组及C组支气管壁厚度、支气管壁平滑肌厚度、支气管壁平滑肌细胞核数量均较A组明显增加(P均<0.01),而E组上述指标较B组、C组、D组均显著降低(P均<0.01);③B组、C组的气道反应性均高于A组(P均<0.01),E组较B组、C组、D组均显著降低(P<0.01、P<0.01、P<0.05)。结论:气道平滑肌增生是气道重构的一个显著特征,PI3K可能在此起促进作用。雷公藤内酯醇可能通过下调PI3K的表达而减轻哮喘气道高反应性及抑制气道平滑肌增生,对哮喘气道重构有一定治疗作用。     

Identification and Characterization of a Novel Human Phosphatidylinositol 4-kinase

The extensive sequence homology that exists among the catalyticdomains of phosphatidylinositol 3- and 4-kinases allowed usto clone a novel human gene encoding a putative phosphatidylinositolkinase, NPIK. Among other known phosphatidylinositol 3- and4-kinases, NPIK was most closely related to yeast PIK1 phosphatidylinositol4-kinase. Several forms of NPIK cDNAs were isolated, and expressionof NPIK message was detected in a wide variety of tissues. Fluorescencein situ hybridization and radiation hybrid analyses assignedthe NPIK gene to human chromosome 1. Recombinant NPIK proteincatalyzed a conversion from phosphatidylinositol to phosphatidylinositol4-phosphate. The catalytic activity of NPIK was augmented byTriton X-100, and was reduced in the presence of adenosine.Using green .uorescent protein system we determined that NPIKis localized in the cytoplasm. Taken together, the data suggestthat NPIK may play a pivotal role in regulating the synthesisof phosphatidylinositol 4-phosphate at the site(s) accessiblefrom cytoplasm.     

Purification of a Phosphatidylinositol 4-Phosphate Kinase from Bovine Brain Membranes

A phosphatidylinositol 4-phosphate (PIP) kinase (EC 2.7.1.68) was purified from bovine brain membranes in a six-step procedure involving solubilization of the enzyme with 170 mM NaCl followed by chromatography on diethylaminoethyl-cellulose, phosphocellulose, Ultrogel AcA44, hydroxylapatite, and ATP-agarose. The enzyme preparation was nearly homogeneous and was purified 5,600-fold with a final specific activity of 85 nmol/min/mg of protein and a yield of 20%. Its molecular mass was 110 kilodaltons, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was specific for PIP; phosphorylation of phosphatidylinositol and diacylglycerol was not observed.     

Wortmannin Blocks Goldfish Retinal Phosphatidylinositol 3-Kinase and Neurite Outgrowth

The goldfish retina has been used extensively for the study of nerve regeneration. A role for phosphatidylinositol 3-kinase (PI3K) in neurite outgrowth from goldfish retinal explants has been examined by means of wortmannin (WT), a selective inhibitor of the enzyme. The presence of PI3K in retinal extracts was determined by means of immunoprecipitation as well as by an in vitro assay system for catalytic activity. The relative amount of the p85 subunit of PI3K detected by western blot in the retina following optic nerve crush was unchanged. WT inhibited goldfish brain PI3K activity at concentrations as low as 10^–9 M, approximating that reported for inhibition of mammalian PI3K's. Daily addition of 10^–8 M WT to retinal explants, activated by prior crush of the optic nerve, significantly inhibited neurite outgrowth during a 7 day in vitro culture period, while a single addition of WT to freshly explanted retina had no effect on neurite outgrowth. These results suggest that a PI3K-mediated process may be critical for nerve regrowth.     

Purification and Characterization of a Soluble Phosphatidylinositol 4-Kinase from Carrot Suspension Culture Cells

Previously we reported the presence of a soluble phosphatidylinositol 4-kinase (PI 4-Kinase) in carrot (Daucus carota L.) suspension culture cells (C.M. Okpodu, W. Gross, W.F. Boss [1990] Plant Physiol 93: S-63). We have purified the enzyme over 1000-fold using Q-Sepharose ion exchange, hydroxylapatite, and G-100 gel filtration column chromatography. The Mr of the enzyme was estimated to be 83,000 by gel filtration. PI 4-kinase activity was recovered after renaturation of the 80-kD region of polyacrylamide gels, and an 80-kD peptide cross-reacted with antibodies to the yeast 55-kD membrane-associated PI 4-kinase on western blots. The isolated lipid kinase phosphorylated PI but not lysophosphatidylinositol or phosphatidylinositol monophosphate. Maximal PI kinase activity occurred when the substrate was added as Triton X-100/PI mixed micelles at pH 8. The enzyme required divalent cations. At low concentrations (1-5 mM), Mn2+ was more effective than Mg2+ in increasing enzyme activity; however, maximal activity occurred at 25 to 40 mM Mg2+. Calcium from 0.01 [mu]M to 1 mM had no effect on the enzyme activity. The Km of the enzyme for ATP was estimated to be between 400 and 463 [mu]M. The enzyme was inhibited by adenosine (100 [mu]M); however, ADP (up to 100 [mu]M) had no effect on the activity. The biochemical characteristics of the carrot soluble PI 4-kinase are compared with the previously reported PI 4-kinases from animals and yeast.     

Purification and Characterization of Sorbitol Dehydrogenase from Bovine Brain

Sorbitol dehydrogenase (EC 1.1.1.14) was isolated from bovine brain and purified 3,000-fold to apparent homogeneity, as judged by polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 36 units/mg of protein; a molecular weight of 39,000 for each of the four identical subunits and 155,000 for the intact enzyme were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel exclusion chromatography, respectively. The presence of one Zn2+ per subunit was confirmed by atom absorption spectroscopy; inactivation of the enzyme by metal-chelating agents points to the essential role that Zn2+ plays in the catalytically competent enzyme. The enzyme is also inactivated by thiol-blocking reagents; with respect to inactivation by sodium pyrophosphate, sorbitol dehydrogenase is different from closely related alcohol dehydrogenase.     

Cinderella story: PI4P goes from precursor to key signaling molecule

Abstract

Phosphatidylinositol lipids are signaling molecules involved in nearly all aspects of cellular regulation. Production of phosphatidylinositol 4-phosphate (PI4P) has long been recognized as one of the first steps in generating poly-phosphatidylinositol phosphates involved in actin organization, cell migration, and signal transduction. In addition, progress over the last decade has brought to light independent roles for PI4P in membrane trafficking and lipid homeostasis. Here, we describe recent advances that reveal the breadth of processes regulated by PI4P, the spectrum of PI4P effectors, and the mechanisms of spatiotemporal control that coordinate crosstalk between PI4P and cellular signaling pathways.     

一种改进的纯化和鉴定牛脑皮层Gs蛋白的方法

用１％胆酸钠和１５％孢和硫酸铵相结合的方法,从牛脑皮层细胞膜中抽提得到主要含激活型Ｇ－蛋白和腺苷酸环化酶两种蛋白组分的制剂,然后通过Ｓｅｐｈａｒｏｓｅ６Ｂ柱将两者分开,将含Ｇｓ高活力的级分用庚胺－Ｓｅｐｈａｒｏｓｅ４Ｂ柱进一步分离,即可获得高活力的Ｇｓ,ＳＤＳ－ＰＡＧＥ显示为分子量４５０００和３６０００的两条蛋白带,该法具简便,快速,重复性好、产率高等优点,且可同时获得无Ｇｓ污染的ＡＣ。用无Ｇｓ污     

一种改进的纯化和鉴定牛脑皮层Gs蛋白的方法

用1%胆酸钠和15%饱和硫酸铵相结合的方法,从牛脑皮层细胞膜中抽提得到主要含激活型G-蛋白(G_s)和腺苷酸环化酶(AC)两种蛋白组分的制剂,然后通过Sepharose 6B柱将两者分开.将含G_s高活力的级分用庚胺-Sepharose 4B柱进一步分离,即可获得高活力的G_s,SDS-PAGE显示为分子量45 000和36 000的两条蛋白带.该法具有简便、快速、重复性好、产率高等优点,且可同时获得无G_s污染的AC.用无G_s污染的AC脂酶体测定G_s活力亦简便、可靠、灵敏度高.     

Inhibition of Neuroblastoma Cell Phosphatidylinositol 3-Kinase by CDP-Diacylglycerol and Phosphatidate

Abstract: Phosphatidylinositol (PI) 3-kinase is activated by a variety of agents, including various growth factors, and has been proposed to play a role in initiation of cell growth, proliferation, and differentiation. We here investigate the effect of various membrane lipids on PI 3-kinase immunopurified from human SH-SY5Y neuroblastoma cells. CDP-diacylglycerol (CDP-DAG) inhibited PI 3-kinase activity with an IC₅₀ of 6 µ M . Phosphatidate (PA) was also inhibitory (IC₅₀ = 38 µ M ) as was lysophosphatidate. Neither DAG nor any of the other phospholipids examined affected PI 3-kinase activity. The results offer the possibility that CDP-DAG or PA at critical membrane sites may exert functionally significant metabolic regulation at the point of convergence of the PI 3-kinase-directed and the PI 4-kinase-directed phosphoinositide signal transduction pathways.