ALKALINE PHOSPHATASE ACTIVITIES AMONG POPULATIONS OF THE COLONY-FORMING DIAZOTROPHIC CYANOBACTERIUM TRICHODESMIUM SPP. (CYANOBACTERIA) IN THE RED SEA

Alkaline phosphatase activities of the diazotrophic marine cyanobacterium Trichodesmium were studied among natural populations in the northern Red Sea and in laboratory cultures of Trichodesmium sp. strain WH9601. Open-water tuft-shaped colonies of Trichodesmium showed high alkaline phosphatase activities with 2.4–11.7 μmol p-nitrophenylphosphate (PNPP) hydrolyzed·μg chl a ^{− 1}·h ^{− 1}, irrespective of date or origin of the sample. Coastal populations of the Trichodesmium tuft colonies had low alkaline phosphatase activities with 0.2–0.5 μmol PNPP·μg chl a ^{− 1}·h ^{− 1}. An exception was the Trichodesmium fall maximum, when both tuft colonies and the plankton community (<100 μm) had alkaline phosphatase activities of 0.6–7.4 μmol PNPP·μg chl a ^{− 1}·h ^{− 1}. Likewise, the more rare puff and bow-tie colonies of Trichodesmium spp. in coastal waters had elevated alkaline phosphatase activities (0.8–1.6 μmol PNPP·μg chl a ^{− 1}·h ^{− 1}) as compared with tuft colonies coinhabiting the same waters. Intact filaments of tuft-forming Trichodesmium sp. strain WH9601 from phosphate-replete cultures had a base alkaline phosphatase activity of 0.5 μmol PNPP·μg chl a ^{− 1}·h ^{− 1}. This activity underwent a 10-fold increase in phosphate-deplete cultures and in cultures supplied with glycerophosphate as the sole P source. The elevated level of alkaline phosphatase activity was sustained in P-deplete cultures, but it declined in cultures with glycerophosphate. The decline is suggested to result from feedback repression of alkaline phosphatase synthesis by the phosphate generated in the glycerophosphate hydrolysis. The enhanced alkaline phosphatase activities of Trichodesmium spp. populations provide evidence that P stress is an important factor in the ecology of Trichodesmium in the northern Red Sea.     

长吻鮠碱性磷酸酶的动力学研究

采用生化手段,从长吻鮠(Leiocassis longirostris Günther)肝中提取出碱性磷酸酶(AKP).提纯倍数为62.08倍,比活为66.43单位/mg蛋白,提取酶液经PAGE和SDS-PAGE只呈现一条区带.该酶的最适pH为10.05,7.0>pH>11.0时不稳定;最适温度为40℃,;对热不很稳定;以磷酸苯二钠为底物其Km值为1.82×10-3mol/L.Mg2+为该酶的激活剂,L-Cys、KH2PO4、DFP、ME、EDTA-Na2为抑制剂.选用KH2PO4,和DFP作抑制类型的判断,结果表明,KH2PO4,属竞争性抑制剂,其抑制常数为2.41mmol/L,DFP为非竞争性抑制剂,抑制常数为1.01mmol/L.       

RETENTION OF P-NITROPHENOL AND 4-METHYLUMBELLIFERONE BY MARINE MACROALGAE AND IMPLICATIONS FOR MEASUREMENT OF ALKALINE PHOSPHATASE ACTIVITY1

During standardization of the methodology for estimating “cell-bound” alkaline phosphatase activity (APA: phosphomonoesterase) in Fucus spiralis L. (Phaeophyta), some of the nonphosphate moiety of the original phosphomonoester was found to be released to the medium subsequent to completion of the routine assays. This occurred with the two substrates generally employed in APA measurements: p-nitrophenyl phosphate (pNPP) and 4-methylumbelliferyl phosphate (MUP). Other marine macrophytes tested showed the same phenomenon. The conditions influencing retention were investigated to establish the simplest procedure for measuring APA. When using pNPP, the release of the product (p-nitrophenol) after the assays was maximum when assays were run for longer than 20 min and at slightly acid pH or at high pNPP concentrations. When using MUP, the leakage of the product (4-methylumbelliferone) after the assays was maximum when APA measurements were run for longer than 40 min and at neutral pH or at high MUP concentrations. The significance of the leakage of the nonphosphate moiety after APA assays is discussed.     

小牛碱性磷酸酶同工酶的一些特性

我们用正丁醇抽提,经ＳｅｐｈａｄｅｘＧ－１００凝胶过滤和ＤＥＡＥ－纤维素柱层析两步纯化,得到了比活性增大４０倍和ＰＡＧＥ纯的牛ＡＫＰ同工酶。对各ＡＫＰ同工酶分别进行酶活性、电泳迁移率（Ｒｆ值）、热抑制、尿素抑制、组织特异性抑制实验,以鉴别每种同工酶的特性。实验结果表明：①各同工酶Ｒｆ值,肝同工酶＞骨同工酶＞小肠同工酶;②经５６℃加热１０ｍｉｎ,只有骨同工酶完全失活,而肠同工酶对热是稳定的;③尿素强烈地抑制骨同工酶活性;肠同工酶对尿素是稳定的;④组织特异性抑制剂苯丙氨酸强烈抑制肠ＡＫＰ活性。     

THE IDENTIFICATION AND PURIFICATION OF A CELL-SURFACE ALKALINE PHOSPHATASE FROM THE DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)1

Two cell-surface proteins were identtjied in the dinoflagellate Prorocentrum minimum (Pavillard) Schilkr strain CCMP 1329 that are evident in phosphate-limited cultures, but not in nitrate-limited cultures or cultures growing exponentially in complete media. These proteins were detected by labeling cell-surface proteins with the biotinylating reagent succinimidyl 6-(biotinamido) hexanoate. One protein, of appoximately 200,000 daltons was purified using differential centrifugation, detergent extraction, and gel filtration chromatography. This purified protein was able to hydrolyze orthophosphate groups from p-nitrophenylphosphate at pH 8, indicating it is an alkaline phosphatase, although it is larger than other alkaline phosphatases isolated to date tom most microorganisms. This protein may be induced to help P. minimum cleave orthophosphate groups from organic forms of phosphate in marine environments. Ultimately, this protein could represent a unique antigen for developing an antibody probe for examining the relationships between phosphate stress and bloom formation in P. minimum, and perhaps other dinoflagellates, in the field.     

ALKALINE PHOSPHATASE ACTIVITY AS A FUNCTION OF INTERNAL PHOSPHORUS CONCENTRATION IN FRESHWATER PHYTOPLANKTON1

Single‐cell alkaline phosphatase (AP) activity is being increasingly used to characterize phosphorus (P) status of individual species of phytoplankton. As phytoplankton growth rates depend more directly on the internal rather than external P concentrations, we determine the AP activity in the two species of freshwater phytoplankton, Scenedesmus quadricauda (Turpin) Bréb. and Asterionella formosa Hassall, as a function of internal P concentration. AP activity strongly correlated with cellular P, increasing almost linearly with decreasing cellular P in both species. The dynamics of initial responses of AP activity to P limitation, as well as the final levels of AP activity, when cellular P approached minimum quotas, differed in two species. After P addition, cellular P concentrations increased rapidly, but AP activity remained high for several days. The lag in AP activity down‐regulation following an increase in cellular P made it difficult to infer current P status of cells under dynamic P conditions.     

电镜图像定量分析肺毛细血管碱性磷酸酶在高压氧研究中的应用

采用碱性磷酸酶（ＡＬＰ）的电镜细胞化学方法,观察在高压氧条件下小鼠肺毛细血管ＡＬＰ活性的分布、活性强度与超微结构改变之间的联系,并用计算机图像定量测量方法对酶活性变化进行了２１个参数的定量分析。文中介绍了多参数图像定量分析方法及计算公式。     

野黄芩苷对内毒素抑制人牙周膜细胞碱性磷酸酶活性的影响

目的观察野黄芩苷对内毒素(LPS)抑制人牙周膜细胞的碱性磷酸酶活性的影响。方法原代培养人牙周膜细胞,采用酶动力学方法观察野黄芩苷对LPS抑制人牙周-膜细胞碱性磷酸酶活性的影响。结果100μg/mL LPS可显著抑制体外培养的人牙周膜细胞碱性磷酸酶活性。加入0.001-10μg/ml野黄芩苷干预后,对LPS抑制碱性磷酸酶活性有一定的拮抗作用,在1μg/ml时达到高峰。结果 野黄芩苷可能通过拮抗LPS抑制牙周膜细胞碱性磷酸酶的活性,促使牙周膜细胞向成骨细胞分化而利于牙周组织再生修复。     

碱性磷酸酶标A蛋白加强的PAP技术

本文介绍一种碱性磷酸酶标Ａ蛋白加强的ＰＡＰ技术。采用ＰＡＰ技术、碱性磷酸酶标Ａ蛋白（ＰＡＡＰ）技术ＰＡＡＰ和加强的ＰＡＰ（ＰＡＰ－ＰＡＡＰ）技术显示下丘脑室旁核催产素（ＯＴ）能神经元。结果发现,其中使用ＰＡＰ－ＰＡＡＰ技术免疫反应产物的显色最深。此技术的原理可能是,由于Ａ蛋白分子至少有四个位点能与ＩｇＧ分子的Ｆｃ段高亲合性地结合,故在该技术中,先经过ＰＡＰ程序的三步免疫反应并显色后,每个与一抗结合的二抗分子上和每个与二抗结合的ＰＡＰ复合物分子上各暴露一个能与Ａ蛋白分子结合的Ｆｃ段,在随后经过ＰＡＡＰ技术处理时,部分ＰＡＡＰ复合物分子就结合在这些Ｆｃ段上,经显色后,ＰＡＡＰ技术显示的浅紫兰色与ＰＡＰ技术显示的浅棕褐色重叠,变成更深的反差明显的深棕褐色。     

COMPARATIVE ALKALINE PHOSPHATASE CHARACTERISTICS OF THE ALGAL BLOOM DINOFLAGELLATES PROROCENTRUM DONGHAIENSE AND ALEXANDRIUM CATENELLA,AND THE DIATOM SKELETONEMA COSTATUM1

The alkaline phosphatase (AP) characteristics of three algal bloom species in the coastal waters of China [Prorocentrum donghaiense D. Lu, Alexandrium catenella (Whedon et Kof.) Balech, and Skeletonema costatum (Grev.) Cleve] were analyzed in a laboratory batch culture experiment using bulk assay and the single‐cell enzyme‐labeled fluorescence (ELF) method. Results showed that the AP of these three test species shared some common characteristics: AP was inducible in all three species and was expressed by algae under phosphorus (P)–stress conditions; no constitutive AP enzyme was detected in the three test species. Once AP was produced, all three test species gradually released the enzymes into the water, and the algae would reinduce AP production. There were also different specific AP characteristics among the three test species under severe P‐stressed conditions. In P. donghaiense, AP covered most of the cell, and the AP production sites were mainly on the cell surface, although some could be observed inside cells. AP also covered the whole cell of A. catenella, but the AP sites were mainly inside the cell with only some on the cell surface. Only one or two AP sites could be detected in S. costatum, and they were all on the cell surface.     

妊娠小鼠子宫腺细胞区的碱性磷酸酶与酸性磷酸酶的研究

小鼠子宫系膜三角区在妊娠后出现上皮样细胞群,群内的细胞称颗粒子宫腺细胞(granu-lated metriial gland cells,GMG细胞),该区改称子宫腺细胞区(metriial gland cell area,MGCA).取孕12～19天MGCA,液氮速冻,恒冷箱切片,偶氮偶联法显示碱性磷酸酶(ALP)与酸性磷酸酶(ACP).结果为,ALP主要分布于GMG细胞群间的疏松结缔组织中;GMG细胞为阴性反应.ACP主要位于GMG细胞内,群间结缔组织含量较少.两种酶的活性随胎龄增加而减弱.ALP与ACP的定位与活性变化特性显示它们与GMG细胞功能关系密切.     

背角无齿蚌碱性磷酸酶的功能基团研究

在一定条件下分别采用PMSF、DTT、PCMB、NBS、TNBS、SUAN、BrAc及IBr等化学修饰剂选择修饰背角无齿蚌碱性磷酸酶的多种氨基酸残基,并测定其酶活力变化。结果表明,PMSF、NBS、TNBS、SUAN、DTT的修饰能显著抑制酶的活力,活力的降低与修饰剂的浓度相关。BrAc、IAc、PCMB的修饰不表现对酶的抑制作用。作者初步认为,Ser、Lys和Trp残基是背角无齿蚌碱性磷酸酶的必需功能基团,部分二硫键时保护酶的催化功能也是必需的。     

ALKALINE PHOSPHATASE IN FRESHWATER CLADOPHORA‐EPIPHYTE ASSEMBLAGES: REGULATION IN RESPONSE TO PHOSPHORUS SUPPLY AND LOCALIZATION1

Extracellular alkaline phosphatase enzyme activity (APA) is important for algal phosphorus (P) acquisition in P‐limited freshwater ecosystems and is often used as an indicator of P deficiency. APA allows access to organic P (monophosphate esters), but the regulation of APA in response to availability of both PO₄³⁻ and organic P is poorly characterized. This study aimed to examine the regulation of APA in freshwater Cladophora‐epiphyte assemblages in response to PO₄³⁻ and a hydrolyzable organic P source, and for the first time to apply enzyme linked fluorescence (ELF) to localize APA within freshwater macroalgal‐epiphyte assemblages. In response to elevated PO₄³⁻ concentrations, a component of net APA was suppressed, but there was also a constitutive APA, which was maintained even after prolonged exposure to nearly 1,000 μM PO₄³⁻ and saturation of internal P pools. When supplied with organic glycerol P as the sole P source, the algae maintained APA in excess of needs for supplying PO₄³⁻ for uptake, resulting in PO₄³⁻ release into the medium. Constitutive APA may be adaptive to growth under chronic P limitation in oligotrophic freshwater habitats. Excess APA and release of PO₄³⁻ could benefit different algal and bacterial partners within assemblages. APA in both Cladophora sp. and epiphytic algae was localized with ELF only when ethanol fixation was omitted. In algal subsamples exposed to different P treatments, there was no correlation between bulk APA (using 4‐methylumbelliferyl phosphate [MUP] substrate) and % cell labeling with ELF, suggesting that ELF labeling of APA was at best semiquantitative in the algal assemblages.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF A CALCIUM‐DEPENDENT ALKALINE PHOSPHATASE FROM THE CYANOBACTERIUM ARTHROSPIRA PLATENSIS 1

In the present study, Triton X‐114 (TX‐114) is used to extract and partially purify alkaline phosphatase (ALP) from a membranous fraction of Arthrospira platensis Gomont containing cell wall, plasma membrane, thylakoids, and sheath. TX‐114 has a double effect: solubilizing cell components to liberate the enzyme and, after phase partitioning, removing chl and other pigments present in the crude extract. The recovery of the enzyme in the aqueous phase suggests the overall hydrophilic character of this enzyme. ALP was kinetically characterized at pH 11.0 using p‐nitrophenyl phosphate as substrate, giving a K_m value of 1.7 mM. Orthovanadate was seen to be a competitive inhibitor of ALP, with a K_i of 0.8 mM. The enzyme was almost completely inactivated in the presence of 70 μM EDTA, although the addition of Ca²⁺ reverted this inactivation; these results indicate that ALP from A. platensis is a calcium‐dependent metalloenzyme. When the effect of Ca²⁺ was investigated in detail, a value of 0.067 μM^?1 for the affinity constant was obtained. The enzyme was histochemically localized in the cytoplasm, cell wall, and sheath using the enzyme‐labeled fluorescent substrate (ELF) method. It is assumed that the same enzyme is either soluble in the cytoplasm and in some way “trapped” in the cell wall or in the sheath. ALP localization within the sheath and the subsequent release of phosphorus (P) may benefit the neighboring cells surrounding this layer.     

C-myc反义RNA抑制血管平滑肌细胞增生的实验研究

观察血管内膜损伤后C－myc反义RNA在抑制血管平滑肌细胞的增生、迁移及防止动脉粥样硬化形成和血管再狭窄中的作用。选雄性日本大白兔36只,经球囊造成腹主动脉内膜损伤的同时,局部给予C－myc反义RNA、正义RNA及盐水,高胆固醇喂养12周处死,测量内膜厚度,计数增生细胞核抗原PCNA阳生细胞数。结果显示：盐水及正义对照组内膜明显增厚（317μm）,PCNA核阳性细胞数明显增多（63％）,可见明显的粥样斑块灶形成,而反义RNA治疗组内膜增厚仅为217μm,PCNA阳性细胞为30.5%,明显低于对照组,P＜0.01。结果表明：C－myc反义RNA具有显抑制VSMC增生,减轻粥样斑块形成的作用。     

蜜环菌侵染天麻皮层过程中酸性磷酸酶的细胞化学研究

被蜜环菌(Armillaria mellea)侵染的天麻(Gastrodia elata B1.)皮层中,由外至内形成三种类型的染菌细胞:菌丝结细胞、空腔细胞和消化细胞。外部两类细胞中的酸性磷酸酶定位显示,一些位于空腔细胞或衰老的菌丝结细胞中的菌丝内部逐渐产生大量酸性磷酸酶,随后菌丝发生自溶。这两类细胞中未发现明显的释放水解酶消化菌丝的现象。当菌丝进入消化细胞以后,情况与此不同,大量包含酸性磷酸酶的微小颗粒出现在菌丝周围,随后这些酶颗粒相互融合,形成包围菌丝的消化泡,菌丝被溶酶体水解酶所消化。最后消化泡变为包含代谢废物的残体。     

小鼠胸腺哺育细胞内淋巴细胞碱性磷酸酶电镜观察

碱性磷酸酶（ａｌｋａｌｉｎｅｐｈｏｓｐｈａｔａｓｅ,ＡＬＰ）是研究Ｔ淋巴细胞分裂分化的标记之一。胸腺内淋巴母细胞具有膜性ＡＬＰ。本文采用ＡＬＰ超微结构细胞化学方法,探讨小鼠胸腺哺育细胞（Ｔｈｙｍｉｃｎｕｒｓｅｃｅｌ,ＴＮＣ）内淋巴细胞（ＴＮＣ－Ｌ）ＡＬＰ的活性分布。观察结果,２０％的ＴＮＣ内有少数淋巴细胞呈阳性反应,而ＴＮＣ外游离的淋巴细胞均为ＡＬＰ阴性反应。结果表明,部分ＴＮＣ内含有少量淋巴母细胞亚群,ＴＮＣ是Ｔ淋巴细胞分裂分化的微环境之一     

背角无齿蚌碱性碳酸酶的分离、纯化及其动力学研究

作者对背角无齿蚌外套膜的碱性磷酸酶（ＡＫＰ）进行了分离纯化,并对其动力学性质进行了初步研究。外套膜匀浆,经正丁醇抽提、盐析、ＳｅｐｈａｄｅｘＧ—１００凝胶过滤等步骤,得到了比活力为１４９．６单位／ｍｇ蛋白的酶制品。通过动力学方法测得其最适ｐＨ值为９．５,最适温度为４０℃,以磷酸苯二钠作底物的Ｋｍ值为０．５７ｍｍｏｌ／Ｌ。Ｍｇ２＋、Ｃａ２＋对酶有激活作用,而Ｃｕ２＋、Ｚｎ２＋、ＫＨ２ＰＯ４、ＥＤＴＡ和巯基乙醇有抑制作用。