Crystal structure of cobra-venom phospholipase A2 in a complex with a transition-state analogue

The crystal structure of a complex between a phosphonate transition-state analogue and the phospholipase A2 (PLA2) from Naja naja atra venom has been solved and refined to a resolution of 2.0 angstroms. The identical stereochemistry of the two complexes that comprise the crystal's asymmetric unit indicates both the manner in which the transition state is stabilized and how the hydrophobic fatty acyl chains of the substrate are accommodated by the enzyme during interfacial catalysis. The critical features that suggest the chemistry of binding and catalysis are the same as those seen in the crystal structure of a similar complex formed with the evolutionarily distant bee-venom PLA2.     

Structures of free and inhibited human secretory phospholipase A2 from inflammatory exudate

Phospholipase A2 (PLA2) participates in a wide range of cellular processes including inflammation and transmembrane signaling. A human nonpancreatic secretory PLA2 (hnps-PLA2) has been identified that is found in high concentrations in the synovial fluid of patients with rheumatoid arthritis and in the plasma of patients with septic shock. This enzyme is secreted from certain cell types in response to the proinflammatory cytokines, tumor necrosis factor or interleukin-1. The crystal structures of the calcium-bound form of this enzyme have been determined at physiological pH both in the presence [2.1 angstrom (A) resolution] and absence (2.2 A resolution) of a transition-state analogue. Although the critical features that suggest the chemistry of catalysis are identical to those inferred from the crystal structures of other extracellular PLA2s, the shape of the hydrophobic channel of hnps-PLA2 is uniquely modulated by substrate binding.     

内毒素对体外培养仓鼠肾细胞膜酶PLA2和ATPase活性影响及阳离子A拮抗效应研究

为研究内毒素(Endotoxin,ET)对体外培养的仓鼠肾细胞膜活性影响及阳离子A(Cation A,CA)对其的保护效应。随机将体外培养的仓鼠肾细胞(BHK-21)分为:对照组(Ⅰ组)、ET处理组(Ⅱ组)和ET+CA组(Ⅲ组),采用微量滴定法和定磷法,第3、6、12、24小时测定细胞膜上磷脂酶A2(PLA2)和ATP酶(ATPase)的活性。结果表明,II组中PLA2活性在整个实验过程中均显著高于I组(P<0.01或P<0.05),Na+-K+-ATPase、Ca2+-Mg2+-ATPase活性呈现先增高后降低的趋势(3、6、12 h增高,24 h以后降低)。III组与II组相比,PLA2的活性显著降低(P<0.05),Na+-K+-ATPase、Ca2+-Mg2+-ATPase活性在整个实验过程中均显著增高(P<0.01或P<0.05)。细胞膜上的PLA2活性增高,ATPase的活性先增高后降低,提示细胞膜受到损伤;CA能明显改善内毒素所致的体外细胞膜损伤。     

鸭肉肌内磷脂水解酶的提取及相关酶系的酶活测定

为了优化肌内磷脂水解酶的提取过程,建立各种鸭肉肌内磷脂水解酶活的测定方法。采用pH 7.5、8.0、8.5的0.1 mol.L-1的Tris-HCl缓冲液提取鸭肉肌内磷脂水解酶,用饱和度范围为0～90%的硫酸铵进行盐析,研究新鲜鸭肉及板鸭中酸性脂肪酶、中性脂肪酶、总磷脂酶、磷脂酶A2(PLA2)、磷脂酶C(PLC)和磷脂酶D(PLD)的活力。结果表明,肌内磷脂水解酶的最佳pH为8.0,最佳盐析条件为70%饱和度的硫酸铵溶液。透析过后的粗酶液中酸性脂肪酶活略大于中性脂肪酶,磷脂酶活次之。     

辛夷油抗炎机理探讨

通过测定小鼠炎症足组织中前列腺素E2 (PGE2 )和组胺的含量 ,以及胸膜炎大鼠胸腔渗出液中白细胞介素 1(IL - 1)、肿瘤坏死因子 (TNF)和磷脂酶A2 (PLA2 )的活性 ,发现给动物灌服辛夷挥发油对动物体内这四种炎症介质及磷脂酶A2 均有抑制作用 ,为辛夷油的抗炎效应提供了实验依据。     

How do enzymes work?

The principle of transition-state stabilization asserts that the occurrence of enzymic catalysis is equivalent to saying that an enzyme binds the transition state much more strongly than it binds the ground-state reactants. An outline of the origin and gradual acceptance of this idea is presented, and elementary transition-state theory is reviewed. It is pointed out that a misconception about the theory has led to oversimplification of the accepted expression relating catalysis and binding, and an amended expression is given. Some implications of the transition-state binding principle are then explored. The amended expression suggests that internal molecular dynamics may also play a role in enzymic catalysis. Although such effects probably do not make a major contribution, their magnitude is completely unknown. Two examples of recent advances due to application of the transition-state binding principle are reviewed, one pertaining to the zinc protease mechanism and the other to the generation of catalytic antibodies.     

伤害对巴西橡胶树胶乳中磷脂酶A2的影响

以巴西橡胶树（Hevea brasiliensis Mull.Arg.）无性系PR107和热研7—33—97为试验对象,研究机械伤害、外源莱莉酸及乙烯（利）等条件对胶乳C-乳清中磷脂酶A2的影响。结果表明,伤害和外源莱莉酸及乙烯（利）对磷脂酶A2均有诱导作用。其中机械伤害的诱导效应最为明显,外源茉莉酸最适诱导浓度为0.08％,磷脂酶A2活性随着外源乙烯刺激剂量的增加而增强。伤害信号能在伤害早期较明显地诱导磷脂酶A2的活性。     

Interference of quantized transition-state pathways in the H + D2 -> D + HD chemical reaction

The collision-energy dependence of the state-resolved differential cross section at a specific backward-scattering angle for the reaction H + D2 --> D + HD is measured with the D-atom Rydberg "tagging" time-of-flight technique. The reaction was modeled theoretically with converged quantum scattering calculations that provided physical interpretation of the observations. Oscillations in the differential cross sections in the backward-scattering direction are clearly observed and are attributed to the transition-state structures that originate from the interferences of different quantized transition-state pathways.     

新型生物可降解PLA沙障与传统草方格沙障防风效益

为了解新型材料生物可降解聚乳酸纤维(PLA)沙障防风效益,通过测定PLA沙障样地内不同高度风速,分别研究1 m×1 m、2 m×2 m、3 m×3 m规格PLA沙障防风效能、地表粗糙度与风速廓线特征,同时以相同坡位同种规格的传统半隐蔽式麦草沙障样地和流动沙丘为对照,对比研究了PLA沙障与传统麦草沙障的防风效益。结果表明,新型生物可降解PLA沙障防风效能显著大于麦草沙障,2种类型沙障最大差值可达10.3%,2种材料沙障不同规格防护效果为1 m×1 m规格较其他2种规格更好;2种材料沙障增加地表粗糙度表现为PLA沙障麦草沙障流动沙丘,且均随规格增大呈逐渐下降趋势,在相同地形条件下,PLA沙障地表粗糙度均值为麦草沙障的1.4倍;2种材料沙障在迎风坡坡底、坡中、坡顶及背风坡4种地形下地表粗糙度差异不明显,其地表粗糙度均值为0.7 cm;1 m×1 m规格的PLA方格沙障和麦草沙障内的风速廓线曲线呈现“S”型曲线特征,而在2 m×2 m和3 m×3 m规格的2种材料沙障内,风速廓线与对照裸沙丘相似,其风速廓线均呈指数函数分布。随着沙障规格的增大,降低风速作用减弱,其风速廓线逐渐由“S”型趋向于指数函数曲线。      

Transition-state models and hydrogen-isotope effects. Kinetic isotope effects provide a sensitive test for detailed models of reacting systems

The transition-state theory of chemical kinetics, coupled with relatively crude methods of constructing potential-energy surfaces for reacting systems, has great utility in the forecasting of kinetic properties. In particular, it permits prediction of the effect of isotopic substitution on rate constants, and comparison of these predictions with experimental data provides a particularly sensitive test for the combination of potential-energy surface and transition-state theory. More rigorous tests of each of these factors depend on future developments in quantum chemistry, in studies of chemical reactions in molecular beams, and in detailed trajectory calculations of scattering processes.     

Receptor-coupled activation of phosphoinositide-specific phospholipase C by an N protein

Cleavage of phosphatidylinositol 4,5-bisphosphate by phospholipase C results in the production of two important second messengers: inositol-1,4,5-trisphosphate and 1,2-diacylglycerol. Although several receptors promote this cleavage, the molecular details of phospholipase C activation have remained unresolved. In this study, occupancy of a Ca2+-mobilizing receptor, the oligopeptide chemoattractant receptor on human polymorphonuclear leukocyte plasma membranes, was found to lead to the activation of a guanine nucleotide regulatory (N) protein by guanosine 5'-triphosphate. The activated N protein then stimulated a polyphosphoinositide-specific phospholipase C by reducing the Ca2+ requirement for expression of this activity from superphysiological to normal intracellular concentrations. Therefore, the N protein-mediated activation of phospholipase C may be a key step in the pathway of cellular activation by chemoattractants and certain other hormones.     

Major enhancement of the affinity of an enzyme for a transition-state analog by a single hydroxyl group

The compound 1,6-dihydropurine ribonucleoside, prepared by reduction of nebularine in the presence of ultraviolet light, is bound by adenosine deaminase approximately 10(8)-fold less tightly than 6-hydroxy-1,6-dihydropurine ribonucleoside, a nearly ideal transition-state analog. This difference in affinities, which is associated with the presence of a single hydroxyl group in the second compound, suggests the degree to which one or a few hydrogen bonds may stabilize the transition state in an enzyme reaction of this type.     

Observation of transition-state vibrational thresholds in the rate of dissociation of ketene

Rate constants for the dissociation of highly vibrationally excited ketene (CH(2)CO) have been measured at the threshold for the production of CH(2)((3)B(1)) and CO((1)Sigma(+)). The rate constant increases in a stepwise manner with increasing energy, consistent with the long-standing premise that the rate of a unimolecular reaction is controlled by flux through quantized transition-state thresholds. The data give the energies of the torsional and C-C-O bending vibrations of the transition state.     

Atomic structure of adenosine deaminase complexed with a transition-state analog: understanding catalysis and immunodeficiency mutations

The crystal structure of a murine adenosine deaminase complexed with 6-hydroxyl-1,6-dihydropurine ribonucleoside, a nearly ideal transition-state analog, has been determined and refined at 2.4 angstrom resolution. The structure is folded as an eight-stranded parallel alpha/beta barrel with a deep pocket at the beta-barrel COOH-terminal end wherein the inhibitor and a zinc are bound and completely sequestered. The presence of the zinc cofactor and the precise structure of the bound analog were not previously known. The 6R isomer of the analog is very tightly held in place by the coordination of the 6-hydroxyl to the zinc and the formation of nine hydrogen bonds. On the basis of the structure of the complex a stereoselective addition-elimination or SN2 mechanism of the enzyme is proposed with the zinc atom and the Glu and Asp residues playing key roles. A molecular explanation of a hereditary disease caused by several point mutations of an enzyme is also presented.     

A G protein mutant that inhibits thrombin and purinergic receptor activation of phospholipase A2

The stimulation of phospholipase A2 by thrombin and type 2 (P2)-purinergic receptor agonists in Chinese hamster ovary cells is mediated by the G protein Gi. To delineate alpha chain regulatory regions responsible for control of phospholipase A2, chimeric cDNAs were constructed in which different lengths of the alpha subunit of Gs (alpha s) were replaced with the corresponding sequence of the Gi alpha subunit (alpha i2). When a carboxyl-terminal chimera alpha s-i(38), which has the last 38 amino acids of alpha s substituted with the last 36 residues of alpha i2, was expressed in Chinese hamster ovary cells, the receptor-stimulated phospholipase A2 activity was inhibited, although the chimera could still activate adenylyl cyclase. Thus, alpha s-i(38) is an active alpha s, but also a dominant negative alpha i molecule, indicating that the last 36 amino acids of alpha i2 are a critical domain for G protein regulation of phospholipase A2 activity.     

聚乳酸薄膜处理对黄花菜保鲜效果分析

聚乳酸(PLA)是一种完全生物降解材料,为研究PLA薄膜对黄花菜的保鲜效果,以聚乙烯(PE)薄膜为对照,用不同厚度PLA薄膜对黄花菜进行了气调保鲜处理,在(25±1)℃贮藏期间对黄花菜的相关品质指标进行了测定。结果表明,PLA薄膜O_2和CO_2透过率低于PE薄膜,贮藏期间形成低氧高二氧化碳气体环境,抑制黄花菜呼吸作用,可明显减缓可溶性蛋白质、可溶性糖、叶绿素和抗坏血酸(Vc)含量的下降,抑制细胞膜渗透率、过氧化物酶(POD)活性的上升,延长黄花菜的贮藏保鲜期至10 d,而PE薄膜仅能贮藏6 d,厚度25μm的PLA薄膜保鲜效果最好。     

Direct Br?nsted analysis of the restoration of activity to a mutant enzyme by exogenous amines

A true Br?nsted analysis of proton transfer in an enzyme mechanism is made possible by the chemical rescue of an inactive mutant of aspartate aminotransferase, where the endogenous general base, Lys258, is replaced with Ala by site-directed mutagenesis. Catalytic activity is restored to this inactive mutant by exogenous amines. The eleven amines studied generate a Br?nsted correlation with beta of 0.4 for the transamination of cysteine sulfinate, when steric effects are included in the regression analysis. Localized mutagenesis thus allows the classical Br?nsted analysis of transition-state structure to be applied to enzyme-catalyzed reactions.     

亚临界R134a体系中的绿色酶促反应研究(英文)

以环境友好的亚临界1,1,1,2-四氟乙烷(R134a)作为反应介质,用磷脂酶D催化卵磷脂(PC)的酰基转移反应制备磷脂酰丝氨酸(PS)。探讨了压力、温度、水分、酶量、底物对酶促反应的影响。结果表明,磷脂酶D在亚临界R134a中(303～323 K、2～8MPa)表现出较高的催化活性。与传统的有机溶剂反应体系相比,亚临界R134a体系能够改善酶促反应动力学,即需要较少的酶量即可迅速达到较高的转化率;在亚临界R134a体系压力6 MPa、体系温度313 K的条件下,9.6 U磷脂酶D催化反应240 min,磷脂酰丝氨酸的含量达到84.88%。     

康氏木霉PLA_2基因的生物学功能及诱导玉米抗弯孢霉叶斑病作用

本研究从康氏木霉T30的cDNA中克隆了PLA2基因,然后用PLA2敲除突变株为材料在玉米接弯孢霉叶斑病菌的条件下研究此基因的生物学功能。敲除突变株P82（ΔPLA2）所分泌的胞外几丁质酶和β-1,3-半乳糖苷酶的酶活要比野生型低。玉米接种弯孢霉叶斑病菌条件下,突变株的生防实验表明其具有较好的诱抗作用。此基因大肠杆菌表达产物在离体玉米叶片上同对照相比产生更大的叶斑。PLA2基因在木霉对抗弯孢霉叶斑病菌同玉米互作可能是一种负调控方式,要对此基因与玉米的非亲和互作还需对此基因互补进行功能验证。