呋塞米对碳酸酐酶的抑制效应再研究

呋塞米是一种高效利尿剂,本实验主要探究其对碳酸酐酶的抑制效应.相比较乙酰唑胺而言,呋塞米在其浓度接近碳酸酐酶浓度时能使该酶基本失活.研究发现,呋塞米对碳酸酐酶的抑制效应表现为非竞争性抑制,其IC50为0.759μM,KI为0.61μM,乙酰唑胺的IC50为0.199μM,KI为0.099μM,表现为竞争性抑制.     

Structure and metal exchange in the cadmium carbonic anhydrase of marine diatoms

Carbonic anhydrase, a zinc enzyme found in organisms from all kingdoms, catalyses the reversible hydration of carbon dioxide and is used for inorganic carbon acquisition by phytoplankton. In the oceans, where zinc is nearly depleted, diatoms use cadmium as a catalytic metal atom in cadmium carbonic anhydrase (CDCA). Here we report the crystal structures of CDCA in four distinct forms: cadmium-bound, zinc-bound, metal-free and acetate-bound. Despite lack of sequence homology, CDCA is a structural mimic of a functional beta-carbonic anhydrase dimer, with striking similarity in the spatial organization of the active site residues. CDCA readily exchanges cadmium and zinc at its active site--an apparently unique adaptation to oceanic life that is explained by a stable opening of the metal coordinating site in the absence of metal. Given the central role of diatoms in exporting carbon to the deep sea, their use of cadmium in an enzyme critical for carbon acquisition establishes a remarkable link between the global cycles of cadmium and carbon.     

Models for metal ion function in carbonic anhydrase.

A model catalyst is described which has properties in common with carbonic anhydrase. The model demonstrates the availability of a mechanism, previously only hypothetical, for the action of the enzyme. It also shows, however, that this mechanism alone is not adequate to produce the high activity of the enzyme.     

Thermodynamic study on conformation change of carbonic anhydrase

The effect of pHs on structural stability of the zinc-containing metalloenzyme carbonic anhydrase has been studied by means of differential scanning calorimetry. The rule of the conformational change for the enzyme with pH change has been found by thermodynamic analysis. The experimental results also provide valuable information: in the pH range from 5.26 to 9.04, two independent endothermal peaks were observed on DSC thermogram. It shows the existence of two structural domains in the molecule. The transition temperature and enthalpy of the two domains are different.     

Density functional theory study of proton transfer in carbonic anhydrase

Proton transfer in carbonic anhydrase Ⅱ has been studied at the B3LYP/6-31G（D） level. The active site model consists of the zinc ion, four histidine residues, two threonine residues, and three water molecules. Our calculations showed that the proton of the zinc-bound water molecule could be transferred to the nearest water molecule and an intermediate containing H3O^＋ is then formed. The intermediate is only 1.3 kJ·mol^-1 above the reactant complex, whereas the barrier height for the proton transfer is about 8.1 kJ·mol^-1.     

Relationship among Photosys-tem Ⅱ carbonic anhydrase,extrinsic polypeptides and manganese cluster

Effects of Photosystem Ⅱ (PS Ⅱ) extrinsic polypeptides of oxygen-evolving complex and manganese clusters on PSⅡ carbonic anhydrase (CA) were studied with spinach PSⅡ membranes. The result supported that membrane-bound CA is located in the donor side of PSⅡ. The extrinsic polypeptides played an important role of maintaining CA activity. After removing manganese clusters, oxygen evolution activity was inhibited, but PSⅡ-CA activity was unchanged. It was concluded that CA activity is independent of the presence of manganese clusters, and was not directly correlated with oxygen evolution activity.     

Determination of sulfonamides with fluorescent inclusion complex of carbonic anhydrase and dansylamide

A new fluorimetric method for determination of sulfonamides was described based on the formation of a fluorescent inclusion complex of carbonic anhydrase(CA) with dansylamide(DNSA). The binding of DNSA to CA resulted in an enhancement in the fluorescence emission at 460 nm with excitation at 280 nm. Dissociation constants were determined for the carbonic anhydrase sulfonamide complexes. Linear calibration graphs of sulfonamides were obtained within a concentration range of 0- 0.058 μg/mL for DNSA; 0-0.344 μg/mL for sulfanilamide (SAN) and 0-0.286 μg/mL for P toluenesulfonamide (PTSN). The relative standard deviations were within 1.8%-4.2%. Limits of detection for DNSA, SAN and PTSN were 0.84, 19.5 and 6.1 ng/mL, respectively. The method was applied to the determination of sulfonamides in cow milk at ng/mL level.     

虹鳟假鳃组织中的碳酸酐酶免疫细胞化学定位

研究目的：假腮的功能早已引起科学家兴趣,但还有待阐明。本文通过研究硬骨鱼类品种虹鳟鱼（Oncorhynchusmykiss）的假腮碳酸酐酶的免疫定位,来探讨假腮碳酸酐酶的生理功能。研究方法：免疫组织化学染色技术。重要结论：免疫组化结果显示碳酸酐酶分布在假腮细胞中,更精确地说是在其细胞顶端分布。细胞基底端、管状系统、毛细血管和柱细胞均无免疫染色。免疫细胞化学定位进一步验证了这些结果,并显示一部分是细胞质碳酸酐酶,其余的与细胞膜结构连接。此外,腔隙层未显示出免疫过氧化物酶的活性。本研究揭示了假腮碳酸酐酶的功能与细胞外介质有关,碳酸酐酶能干预传入神经纤维刺激机制。     

不同环境条件下水稻叶片中叶绿体内碳酸酐酶活性的变化

以水稻(Oryza．Sativa L．)为材料,在不同光照、高盐、高温及缺N、K培养等条件下研究碳酸酐酶活性的变化。结果表明;在16μmolES^-2-64μmolES^-2。光强范围内,随着光照的增加,碳酸酐酶活性并无明显的变化．在高盐逆境条件下碳酸酐酶活性降低2-3倍,在高温逆境条件下碳酸酐酶活性降低了2～4倍,在缺N培养条件下碳酸酐酶活性降低了7-8倍,在缺K的培养条件下碳酸酐酶活性降低了2—3倍。     

杜氏盐藻碳酸酐酶N/C结构域耐盐性的研究

为研究杜氏盐藻碳酸酐酶的两个结构域的耐盐性,将杜氏盐藻碳酸酐酶的两个串联结构域分别表达与纯化,利用酶学方法和光谱学方法分别研究这两个结构域各自的活性及其对盐的耐受性.研究发现,N端结构域不具有催化活性,仅有C端结构域具有活性,而且其活性随着盐浓度的增加而增加,这同杜氏盐藻碳酸酐酶在广泛的盐浓度内的活性变化趋势一致.光谱学方法发现,随着盐浓度的增加,C端结构域的二级结构和三级结构并不发生显著变化,活性中心结构逐渐松弛,从而增加了C端结构域活性中心的活力,提高了盐藻碳酸酐酶的活性.