甘蓝型油菜β碳酸酐酶的结构预测

根据已经克隆到的甘蓝型油菜β碳酸酐酶基因序列,概念地翻译成蛋白质的氨基酸序列。利用Vector NTISuite、SOPMA、Swiss-Model和NCBI-VAST等软件和服务器对甘蓝型油菜β碳酸酐酶的一级结构、二级结构、三维结构进行分子结构模型预测,并进行三维结构的比对。预测结果显示,甘蓝型油菜β碳酸酐酶是定位于叶绿体基质的蛋白质,具有β类碳酸酐酶所特有的保守性基序Cys-Xn-His-X2-Cys;SOPMA预测二级结构显示α螺旋(39.88%)、随机卷曲(39.27%)、β折叠(16.31%)和β转角(4.53%);用同源建模法构建了三维结构图;通过VAST矢量比对工具将甘蓝型油菜β碳酸酐酶与模板(1ekjG)进行三维结构比对,显示甘蓝型油菜β碳酸酐酶与豌豆β碳酸酐酶同型八聚体中的一个单体(1ekjG)很好的匹配,推测甘蓝型油菜β碳酸酐酶全酶也是同型八聚体。     

小黑麦碳酸酐酶蛋白质三维结构预测

根据已经克隆到的小黑麦碳酸酐酶基因序列,将其概念地翻译成蛋白质的氨基酸序列。利用MEGA4.1、DNAStar5.02、SOPMA、Swiss-Model Workspace和NCBI-VAST等在线软件和服务器对该小黑麦碳酸酐酶(CA)的一级结构、二级结构及三维结构进行了分子结构模型预测,并对其三维结构进行了比对。结果显示,小黑麦碳酸酐酶定位于线粒体内膜和叶绿体类囊体膜上,具有β类碳酸酐酶所特有的保守性基序C-[SA]-D-S-R-[LIVM]-x-[AP];SOPMA预测的二级结构显示,该酶含有α-螺旋(38.61%)、随机卷曲(54.44%)和β-折叠(6.95%)。通过VAST矢量比对工具将小黑麦碳酸酐酶与模板(lekjA)三维结构进行比对,结果显示小黑麦碳酸酐酶与豌豆β碳酸酐酶同型八聚体中的一个单体(lekjA)具有很好的匹配,故推测小黑麦碳酸酐酶全酶也可能是同型八聚体。     

不同植物的碳酸酐酶活力差异研究

碳酸酐酶是催化二氧化碳的可逆水合反应的一种含锌金属酶。测定不同植物、同一植物不同部位、同一植物同一部位不同时间的碳酸酐酶的活力,研究诸葛菜和油菜碳酸酐酶及其胞外酶活力的差异,初步探讨碳酸酐酶活力与植物抗干旱能力之间的关系。研究结果为诸葛菜的喀斯特适生性的研究提供依据。     

碳酸酐酶在红细胞分化发育过程中的功能研究

目前红系分化调控相关的研究主要集中在细胞因子、转录因子、lncRNA及表观遗传方面,为了对红系分化调控机制进行更加深入的解析,研究了碳酸酐酶在红系分化中的功能。碳酸酐酶可以高效催化二氧化碳的水合,但它在红细胞发育过程中的功能尚不清楚。利用脐带血来源的CD34⁺细胞在体外进行红细胞诱导分化,在分化过程中通过慢病毒介导的基因敲降的方法能够降低碳酸酐酶1和碳酸酐酶2的表达,并使用流式细胞仪检测红细胞的生成和分化效率。研究结果表明,与对照组相比,碳酸酐酶1的表达缺陷使红细胞的晚期分化明显受阻,而碳酸酐酶2的表达缺陷则将红细胞的分化阻滞在早期阶段。研究结果表明,虽然作用窗口不同,但碳酸酐酶1和碳酸酐酶2在红系分化的过程中均发挥着重要的调控作用,这一发现对将来在体外红细胞生成具有指导意义。     

嗜热蓝细菌碳酸酐酶基因的异源表达及酶学性质

【背景】碳酸酐酶(carbonic anhydrase,CAH)因其高效催化CO2转化为HCO3–的能力成为当今碳减排工艺中的研究热点,但因工厂烟道气的温度较高,因此寻求热稳定性高的嗜热碳酸酐酶是碳酸酐酶仿生学固碳的关键所在。【目的】克隆嗜热蓝细菌Thermosynechococcuselongatus PKUAC-SCTE542和SynechococcuslividusPCC6715的碳酸酐酶基因Ecah、Pcah,实现其在大肠杆菌细胞中异源高效表达,并进行初步酶学性质研究。【方法】利用PCR技术获得碳酸酐酶基因cah,构建重组基因工程菌BL21_pETM11_CAH,利用IPTG诱导方法高效表达蛋白,表达产物(CAH)经Ni-Agarose亲和层析柱纯化后,进行酶学性质研究。【结果】从E542、PCC6715中克隆得到大小均为534 bp的碳酸酐酶基因,以CO2为底物,酶催化CO2水合的活性分别为42.6 WAU/mg-protein、47.6 WAU/mg-protein。碳酸酐酶ECAH 50°C处理30 min后,酶活提高了8%,而PCAH却下降了10%。Zn2+、磺胺对两种碳酸酐酶有显著抑制作用,Ca2+对ECAH有轻微激活作用,对PCAH无显著抑制作用。【结论】E542嗜热蓝细菌表达的碳酸酐酶比PCC6715的热稳定性强,符合处理工业高温点源烟道气的CO2的基本要求,丰富了碳减排的嗜热碳酸酐酶基因库。     

植物的碳酸酐酶

本文介绍了植物碳酸酐酶的化学特性,在植物中的分布、定位以及环境条件对酶活性的影响。对植物碳酸酐酶的可能生理功能进行了论述,同时对近年来藻类碳酸酐酶的研究成果也做了较详细的介绍。     

碳酸酐酶与临床医学

碳酸酐酶是一类含锌的蛋白酶,共发现了10种同工酶及三种碳酸酐酶相关蛋白,广布于人体各组织,能可逆性地催化C02的水合反应,参与调节pH、离子运输等多种生理过程,碳酸酐酶的缺乏和异常将可能会导致一系列的疾病。     

莱茵藻胞外碳酸酐酶分子定位与活性诱导

胞外碳酸酐酶是藻类CCM机制和光合作用的一个重要组分 ,藻类从高CO2 转入低CO2 浓度培养时可诱导出胞外碳酸酐酶。应用金标免疫分子定位和pH调节对胞外碳酸酐酶分子定位和CO2 诱导机制进行研究 ,结果表明 :胞外碳酸酐酶主要分布于胞壁空间 (细胞质膜与细胞壁之间 ) ,且细胞壁上也有较多分布 ,细胞壁外分布较少。说明胞外碳酸酐酶能从胞壁空间穿过细胞壁。通过CO2 诱导和pH调节(升高 ) ,均可提高碳酸酐酶活性 ,且pH提高幅度越大 ,胞外碳酸酐酶活性也越大 ,说明胞外碳酸酐酶的CO2 诱导与pH调节有一定关系     

莱茵藻胞外碳酸酐酶分子定位与活性诱导

胞外碳酸酐酶是藻类CCM机制和光合作用的一个重要组分,藻类从高CO2转入低CO2浓度培养时可诱导出胞外碳酸酐酶。应用金标免疫分子定位和pH调节对胞外碳酸酐酶分子定位和CO2诱导机制进行研究,结果表明：胞外碳酸酐酶主要分布于胞壁空间（细胞质膜与细胞壁之间）,且细胞壁上也有较多分布,细胞壁外分布较少。说明胞外碳酸酐酶能从胞壁空间穿过细胞壁。通过CO2诱导和pH调节（升高）,均可提高碳酸酐酶活性,且pH提高幅度越大,胞外碳酸酐酶活性也越大,说明胞外碳酸酐酶的CO2诱导与pH调节有一定关系。     

碳酸酐酶Ⅵ的生理功能与酶缺陷性疾病

碳酸酐酶(carbonic anhydrase,CA)催化可逆的水合反应CO2+H2O?ΗCO3?+H+,参与维持pH值平衡、CO2与离子的转运、细胞凋亡等生理过程。碳酸酐酶VI(CA-VI)作为该类含锌酶中惟一的细胞分泌型碳酸酐酶,在哺乳动物及人的唾液腺、乳腺、泪腺、支气管等腺体中表达,对维持口腔、上消化道和呼吸道的生理功能起重要作用。     

Composition and activity of external carbonic anhydrase of microalgae from karst lakes in China

The species composition, the net photosynthetic O₂ evolution rate and the activity of external carbonic anhydrase (CA) of microalgae from three reservoirs were studied. Carbonic anhydrase activity had a significant positive correlation with the density of Cyanobacteria in Lake Aha. Microalgae's carbonic anhydrase activity in Lakes Baihua and Hongfeng was related to the density of Chlorophyceae. The species abundances of microalgae in Lake Aha, Lake Baihua, and Lake Hongfeng were different. A relationship between carbonic anhydrase activity and net photosynthetic O₂ evolution rate had also been established. Algae with external CA influenced the algal productivity. These results demonstrate the role of external CA in facilitating the availability of CO₂ that limits the photosynthesis of microalgae in karst lakes in China.     

Carbonic anhydrases in higher plants and aquatic microorganisms

At physiological pH-values CO₂ and HCO⁻₃are the dominant inorganic carbon species and the interconversion between both is catalyzed by carbonic anhydrase (EC 4.2.1.1). This enzyme is widely distributed among photosynthetic organisms. In the first part of the review, the similarities and the differences of carbonic anhydrases from plants and animals are briefly described. In the second part recent advances in molecular biology to understand the structure of carbonic anhydrase from higher terrestrial plants as well as its involvement in photosynthetic CO₂ fixation are summarized. Lastly, the review deals with the presence of carbonic anhydrase in aquatic organisms including cyanobacteria, microalgae, macroalgae and angiosperms. Evidence for the presence of extracellular and intracellular isozymes in these organisms are discussed. The properties and function(s) of carbonic anhydrase during the operation of the inorganic carbon concentrating mechanism are also described.     

An investigation of carbonic anhydrase activity in the gills and blood plasma of brown bullhead (Ameiurus nebulosus), longnose skate (Raja rhina), and spotted ratfish (Hydrolagus colliei)

Separated plasma and whole blood non-bicarbonate buffering capacities, together with plasma and gill carbonic anhydrase activities and endogenous plasma carbonic anhydrase inhibitor activity were investigated in three species of fish: the brown bullhead (Ameirus nebulosus), a teleost; the longnose skate (Raja rhina), an elasmobranch; and the spotted ratfish (Hydrolagus colliei), a chimaeran. The objective was to test the hypothesis that species possessing gill membrane-bound carbonic anhydrase and/or plasma carbonic anhydrase activity would also exhibit high plasma nonbicarbonate buffering capacity relative to whole blood non-bicarbonate buffering capacity and would lack an endogenous plasma carbonic anhydrase inhibitor. Separated plasma non-bicarbonate buffering capacity constituted > or = 40% of whole-blood buffering in all three species. In addition, all species lacked an endogenous plasma carbonic anhydrase inhibitor. Separated plasma from skate and ratfish contained carbonic anhydrase activity, whereas bullhead plasma did not. Examination of the subcellular distribution and characteristics of branchial carbonic anhydrase activity revealed that the majority of branchial carbonic anhydrase activity originated from the cytoplasmic fraction in all species, with only 3-5% being associated with a microsomal fraction. The microsomal carbonic anhydrase activity of bullhead and ratfish was significantly reduced by washing, indicating the presence of carbonic anhydrase activity that was not integrally associated with the membrane pellet, microsomal carbonic anhydrase activity in skate was unaffected by washing. In addition, microsomal carbonic anhydrase activity from skate and ratfish but not bullhead gills was released to a significant extent from its membrane association by treatment with phosphatidylinositol-specific phospholipase C. The results obtained for skate are consistent with published data for dogfish, suggesting that the possession of branchial membrane-bound carbonic anhydrase activity may be a generalised elasmobranch characteristic. Ratfish, which also belong to the class Chondrichthyes, exhibited a similar pattern. Unlike skate and ratfish, bullhead exhibited high plasma non-bicarbonate buffering capacity and lacked an endogenous carbonic anhydrase inhibitor in the absence of plasma and gill membrane-bound carbonic anhydrase activities.     

How to optimise photosynthetic biogas upgrading: a perspective on system design and microalgae selection

Photosynthetic biogas upgrading using microalgae provides a promising alternative to commercial upgrading processes as it allows for carbon capture and re-use, improving the sustainability of the process in a circular economy system. A two-step absorption column-photobioreactor system employing alkaline carbonate solution and flat plate photobioreactors is proposed. Together with process optimisation, the choice of microalgae species is vital to ensure continuous performance with optimal efficiency. In this paper, in addition to critically assessing the system design and operation conditions for optimisation, five criteria are selected for choosing optimal microalgae species for biogas upgrading. These include: ability for mixotrophic growth; high pH tolerance; external carbonic anhydrase activity; high CO₂ tolerance; and ease of harvesting. Based on such criteria, five common microalgae species were identified as potential candidates. Of these, Spirulina platensis is deemed the most favourable species. An industrial perspective of the technology further reveals the significant challenges for successful commercial application of microalgal upgrading of biogas, including: a significant land footprint; need for decreasing microalgae solution recirculation rate; and selecting preferable microalgae utilisation pathway.     

Zinc and carbonic anhydrase III distribution in mammalian muscle

Zinc and carbonic anhydrase III measurement in human and rat muscle extracts indicate that: 1. About one fifth of zinc in human soleus is associated with carbonic anhydrase III isozyme, and even higher levels of zinc and carbonic anhydrase III are found in rat soleus, where about one half of the zinc is in carbonic anhydrase III. Other muscle was also analysed in a similar way, (see text). Heart is notable in containing lower levels of zinc but negligible carbonic anhydrase III. 2. Treatment of muscle with water or phosphate solutions showed that all the carbonic anhydrase III was water extractable, whereas significant zinc remained bound, but was partially extractable by phosphate solutions. 3. Dialysis of muscle extracts showed that whilst some zinc was dialysable, there was no significant contribution from the carbonic anhydrase III in the dialysed extract. EDTA enhanced the release of dialysable zinc from muscle extract. These findings are discussed in relation to muscle disease.     

Immunohistochemical localization of carbonic anhydrase isoenzymes I and II in human bone,cartilage and giant cell tumor

Summary Carbonic anhydrase isoenzymes I and II have been localized in human bone and cartilage. Osteoclasts are strongly positive for carbonic anhydrase II but very little if any reaction is observed for carbonic anhydrase I. In tendon giant cell tumor osteoclastlike-giant cells contained high amounts of carbonic anhydrase II suggesting the close relation of these cells to normal osteoclasts. In growth plate cartilage strong staining was obtained in late proliferative and hypertrophic chondroxytes as well as in extracellular matrix of hypertrophic zone also only with anti-human carbonic anhydrase II.     

The 30 kDa abnormal protein in avian dystrophic muscle is indistinguishable from carbonic anhydrase III by physicochemical, immunological, and enzymological criteria

In the accompanying paper, we described the existence, molecular characterization, and ontogeny of a 30 kDa abnormal protein in chicken dystrophic muscles. In this study, we have purified chicken carbonic anhydrase III and the 30 kDa protein and directly compared them. In terms of its enzymological features, the 30 kDa protein is a typical carbonic anhydrase III. Like carbonic anhydrases, it contains one mole zinc per mole of protein. The protein selectively cross-reacted with a chicken carbonic anhydrase III antibody. Antibody to the 30 kDa protein cross-reacted with chicken skeletal muscle carbonic anhydrase III. Moreover, the distribution of the abnormal protein is exactly identical to that of carbonic anhydrase III; however, there is a possibility that the 30 kDa protein is a variant of carbonic anhydrase III. Slight differences were found in antigenicities and in the apparent molecular weights of the two proteins. We have compared the two proteins by 125I-labeled two-dimensional peptide mapping. Tryptic maps have shown that the two proteins are highly homologous. Combined, these results strongly indicate that the 30 kDa protein and carbonic anhydrase III are similar, if not identical.     

Distribution of carbonic anhydrase, K+-ATPase and K+-phosphatase in subcellular fractions of gastric mucosa]

The distribution of carbonic anhydrase, K+-ATPase and K+-phosphatase in the subcellular fractions of gastric mucosa was studied. It was found that 90% of carbonic anhydrase are localized in the hyaloplasm, whereas K+-ATPase and K+-phosphatase are predominantly localized in the microsomal fraction. Subfractionation of the microsomal fraction in a sucrose density gradient showed that the membrane-bound carbonic anhydrase (5% of total content) and K+-ATPase are bound to various cell organelles. It is concluded that carbonic anhydrase functions as an intracellular pH-stat and is not directly involved in proton generation by the cell.     

Minireview plant carbonic anhydrase.

This article reviews the literature concerning plant carbonic anhydrase. The following topics are discussed: discovery, molecular weight and structure, zinc content, amino acid composition, mechanism and kinetics of catalytic activity, and function and localization in the plant. Where deficiencies exist in the literature on plant carbonic anhydrase reference is made to articles dealing with mammalian carbonic anhydrase. The theories of carbonic anhydrase function in plants are examined critically and evaluated in the light of existing evidence.     

Sexual differentiation of rat liver carbonic anhydrase III

Using radioimmunoassay, the concentration of carbonic anhydrase III in the livers of adult male rats was found to be approx. 30-times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver carbonic anhydrase III concentrations which could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomised female rats induced about a 5-fold increase in liver carbonic anhydrase III concentration. Immunoprecipitation analysis of the products of liver mRNA translation in vitro with antiserum specific for carbonic anhydrase III showed that hormonal control of the levels of carbonic anhydrase III in liver is mediated by changes in the amount of translatable carbonic anhydrase III mRNA. Marked changes in liver carbonic anhydrase III concentrations were also observed in developing and ageing male rats.