Pressure affects enzyme function in organic media

Pressure affects enzyme function in nonaqueous media. Activation volumes have been determined and provide evidence that the primary effect of pressure is to enhance the stripping of water off an enzyme in polar organic solvents and leads to decreased enzymatic activity. Activation volumes of subtilisin Carlsberg in organic solvents, particularly with the enzyme hydrated, have a larger magnitude than activation volumes determined in aqueous solutions. This study provides further evidence that enzymatic activity in polar organic solvents is dominated by the interaction of enzyme-bound water with the solvent. From a practical standpoint, however, the results of this study suggest that enzymatic catalysis in organic solvents may be controlled by the combined effects of pressure and enzyme hydration. (c) 1993 John Wiley & Sons, Inc.     

遮阴对石笔木苗木生长和保护酶活性的影响

为探讨石笔木Tutcheria championi苗木对遮阴环境的适应性,以全光照(无遮阴覆盖)为对照,研究遮阴30%、50%和70%对2年生石笔木苗生长和生理特性的影响。结果表明,全光照下石笔木苗木生长较为健壮,其苗高、地径生长优于遮阴处理;全光照下石笔木总生物量、根生物量比以及根冠比显著大于遮阴处理(P<0.05);50%遮阴处理苗木叶片叶绿素含量显著高于全光照和70%遮阴处理(P<0.05),叶绿素a/b值在各处理间无显著差异。丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性在各处理间无显著差异(P>0.05);70%遮阴处理的苗木叶片过氧化氢酶(CAT)活性最高,显著高于全光照和50%遮阴。由此表明,充足的光照环境有利于石笔木苗木生长。     

Suppression of mitochondrial alternative oxidase can result in upregulation of the ROS scavenging network: some possible mechanisms underlying the compensation effect

Mitochondrial alternative oxidase is an important protein involved in maintaining cellular metabolic and energy balance, especially under stress conditions. AOX genes knockout is aimed at revealing the functions of AOX genes. Under unfavourable conditions, AOX-suppressed plants (mainly based on Arabidopsis AOX1a-knockout lines) usually experience strong oxidative stress. However, a compensation effect, which consists of the absence of AOX1a leading to an increase in defence response mechanisms, concomitant with a decrease in ROS content, has also been demonstrated. This review briefly describes the possible mechanisms underlying the compensation effect upon the suppression of AOX1a. Information about mitochondrial retrograde regulation of AOX is given. The importance of ROS and mitochondrial membrane potential in triggering the signal transmission from mitochondria in the absence of AOX or disturbance of mitochondrial electron transport chain functions is indicated. The few available data on the response of the cell to the absence of AOX at the level of changes in the hormonal balance and the reactions of chloroplasts are presented. The decrease in the relative amount of reduced ascorbate at stable ROS levels as a result of compensation in AOX1a-suppressed plants is proposed as a sign of stress development. Obtaining direct evidence on the mechanisms and signalling pathways involved in AOX modulation in the genome should facilitate a deeper understanding of the role of AOX in the integration of cellular signalling pathways.     

核酸去甲基酶AlkB在毕赤酵母中的表达纯化及在tRNA相关研究中的应用

[目的]核酸的甲基化修饰是一种常见的化学修饰形式,具有重要的生物学功能,却也在一定程度上给一些核酸研究过程带来了技术难度。tRNA上具有的大量甲基化修饰会阻碍逆转录进程,从而降低荧光定量PCR (real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)和高通量测序对其的检测效率。来自大肠杆菌(Escherichia coli)的AlkB蛋白是一种多功能的脱烷基化酶,可以去除DNA和RNA上多种甲基化为代表的修饰,有望解决以上问题。[方法]针对大肠杆菌来源的AlkB,分别尝试在大肠杆菌和毕赤酵母(Pichia pastoris)表达系统中进行诱导表达和纯化,对纯化获得的AlkB进行酶学性质测定。最后以tRNA_UAU^Ile等两种tRNA为代表,研究AlkB的处理对于荧光定量PCR法检测tRNA表达水平的影响。[结果]AlkB在大肠杆菌中表达时多以包涵体形式存在,但是在毕赤酵母中可以成功分泌表达。使用镍柱分离纯化后获得了纯度高于95%的AlkB蛋白,其酶学性质参数如...     

Soluble Sema4D cleaved from osteoclast precursors by TACE suppresses osteoblastogenesis

Bone remodelling is mediated by orchestrated communication between osteoclasts and osteoblasts which, in part, is regulated by coupling and anti-coupling factors. Amongst formally known anti-coupling factors, Semaphorin 4D (Sema4D), produced by osteoclasts, plays a key role in downmodulating osteoblastogenesis. Sema4D is produced in both membrane-bound and soluble forms; however, the mechanism responsible for producing sSema4D from osteoclasts is unknown. Sema4D, TACE and MT1-MMP are all expressed on the surface of RANKL-primed osteoclast precursors. However, only Sema4D and TACE were colocalized, not Sema4D and MT1-MMP. When TACE and MT1-MMP were either chemically inhibited or suppressed by siRNA, TACE was found to be more engaged in shedding Sema4D. Anti-TACE-mAb inhibited sSema4D release from osteoclast precursors by ~90%. Supernatant collected from osteoclast precursors (OC-sup) suppressed osteoblastogenesis from MC3T3-E1 cells, as measured by alkaline phosphatase activity, but OC-sup harvested from the osteoclast precursors treated with anti-TACE-mAb restored osteoblastogenesis activity in a manner that compensates for diminished sSema4D. Finally, systemic administration of anti-TACE-mAb downregulated the generation of sSema4D in the mouse model of critical-sized bone defect, whereas local injection of recombinant sSema4D to anti-TACE-mAb-treated defect upregulated local osteoblastogenesis. Therefore, a novel pathway is proposed whereby TACE-mediated shedding of Sema4D expressed on the osteoclast precursors generates functionally active sSema4D to suppress osteoblastogenesis.     

Synthetic chimeras of mouse growth factor-associated glandular kallikreins. I. Kinetic properties.

A series of six chimeric proteins, composed of fragments corresponding to either one or the other of the growth factor-associated mouse glandular kallikreins-epidermal growth factor binding protein (EGF-BP) and the gamma-subunit of nerve growth factor (gamma-NGF)--were expressed in Escherichia coli and isolated, and their kinetic properties were characterized. The assembly of these synthetic proteases involved the substitution of regions of the proteins containing four specific surface loops that have been postulated to influence both kinetic specificity and the formation of growth factor complexes. The substrates utilized in the kinetic characterization of these chimeric kallikreins were tripeptide nitroanilides representing carboxyl termini of both the EGF and beta-NGF mature hormones, putative processing sites for these kallikreins in the precursors. Characterization of these hybrid enzymes demonstrates that Km and kcat kinetic constants may be independently affected by the regions utilized in construction of these chimeric kallikreins. Specifically, loop 1, located in the amino terminal region (Bode, W., et al., J. Mol. Biol. 164, 237-282, 1983), in gamma-NGF enhanced the kcat for substrates containing threonine in the P2 position, as is the case during the processing of the carboxy terminus of the beta-NGF precursor. Also, the central regions of the kallikreins containing loop 2 and the kallikrein loop dictated the generally inverted Km and kcat kinetic constants observed between EGF-BP and gamma-NGF. Finally, in gamma-NGF the autolysis loop, found in the carboxyl terminal region, functions to lower the Km kinetic constant for a variety of substrates. The results allow previously characterized kinetic differences between EGF-BP and gamma-NGF to be interpreted in terms of specific regions of the proteins and identify a subset of amino acid positions responsible for these functional characteristics.     

Comparison of the crystal structures of genetically engineered human manganese superoxide dismutase and manganese superoxide dismutase from Thermus thermophilus: differences in dimer-dimer interaction.

The three-dimensional X-ray structure of a recombinant human mitochondrial manganese superoxide dismutase (MnSOD) (chain length 198 residues) was determined by the method of molecular replacement using the related structure of MnSOD from Thermus thermophilus as a search model. This tetrameric human MnSOD crystallizes in space group P2(1)2(1)2 with a dimer in the asymmetric unit (Wagner, U.G., Werber, M.M., Beck, Y., Hartman, J.R., Frolow, F., & Sussman, J.L., 1989, J. Mol. Biol. 206, 787-788). Refinement of the protein structure (3,148 atoms with Mn and no solvents), with restraints maintaining noncrystallographic symmetry, converged at an R-factor of 0.207 using all data from 8.0 to 3.2 A resolution and group thermal parameters. The monomer-monomer interactions typical of bacterial Fe- and Mn-containing SODs are retained in the human enzyme, but the dimer-dimer interactions that form the tetramer are very different from those found in the structure of MnSOD from T. thermophilus. In human MnSOD one of the dimers is rotated by 84 degrees relative to its equivalent in the thermophile enzyme. As a result the monomers are arranged in an approximately tetrahedral array, the dimer-dimer packing is more intimate than observed in the bacterial MnSOD from T. thermophilus, and the dimers interdigitate. The metal-ligand interactions, determined by refinement and verified by computation of omit maps, are identical to those observed in T. thermophilus MnSOD.     

Dietary protein requirement for captive juvenile green turtles (Chelonia mydas)

Head-starting programs are extremely important for restoring the population of sea turtles in wild whereas husbandry conditions and feeding regimens of captive turtles are still limited. In the current study, the optimal dietary protein requirement for green turtle (Chelonia mydas) was investigated to support rearing in head-starting programs. Twenty-five-day-old turtles (44.5–46.2 g body weight, n = 45) were randomly distributed into 15 experimental plastic tanks, comprising three treatment replications of 3 turtles each. They were fed fishmeal-based feeds containing different levels of protein (30%, 35%, 40%, 45%, and 50%) for 8 weeks. At the end of feeding trial, growth performance (specific growth rate = 1.86% body weight/day) and feed utilization (protein efficiency ratio = 3.30 g gain/g protein) were highest in turtles fed with 40% protein in feed (p < .05). These nutritional responses were significantly supported by specific activities of fecal digestive enzymes, especially trypsin, chymotrypsin, amylase, and the amylase/trypsin ratio. Also, this dietary level improved the deposition of calcium and phosphorus in carapace, supporting a hard carapace and strong healthy bones. There were no negative effects in general health status of reared turtles, as indicated by hematological parameters. Based on a broken-line analysis between dietary protein levels and specific growth rate, the optimal protein level for green turtles was estimated as 40.6%. Findings from the current study support the use of artificial diets of specific protein levels to rear captive green turtle before release to natural habitats.     

Production of pectin lyase by Penicillium griseoroseum as a function of the inoculum and culture conditions

Optimum activity of an extracellular pectin lyase produced by Penicillium griseoroseum in submerged culture was after 120 h using 0.1% (w/v) citrus pectin as substrate. Sucrose at 0.1% (w/v) stimulated enzyme production and citrus pectin gave the highest activity of enzyme per unit growth.