The covalent oscillator: A paradigm accounting for the sliding/bending mechanism and wave propagation in cilia and flagella

Summary— In most models of wave propagation in eucaryotic flagella and cilia, a clear distinction is made between the dynein dependent microtubule sliding which represents the oscillatory motor and the bending mechanism which regulates wave propagation. Little is known about the physical elements regulating the latter: in the present model, the bending propagation is postulated to be supported by an open/close cyclic mechanism protease/ligase dependent, which involves transient covalent links between adjacent microtubular doublets; this open/close cycle propagates in register with the powering action of the dynein motor along the exoneme. The implications of the model are discussed in relation to previous data which involve protease/ligase in the axonemal function as well as other data which can be integrated by the proposed model.     

Theozymes and compuzymes: theoretical models for biological catalysis

A theozyme is a theoretical enzyme constructed by computing the optimal geometry for transition-state stabilization by functional groups. It is created in order to permit quantitative assessment of catalytic function. Theozymes have been used to elucidate the role of transition-state stabilization in the mechanisms underlying enzyme- and antibody-catalyzed hydroxyepoxide cyclizations, eliminations and decarboxylations, peptide and ester hydrolyses, and pericyclic and radical reactions. The enzymes studied include orotodine monophosphate decarboxylase, HIV protease and ribonucleotide reductase.     

Reduction of ortho-aminobenzoyl-proline fluorescence and formation of pyrrolobenzodiazepine-5,11-dione

Summary Theortho-aminobenzoic acid (Abz) group is widely employed as a fluorescent marker for peptides used as substrates for the study of proteolytic enzyme activity. In fact, a direct correlation has been observed between fluorescence intensity and enzyme activity. An unusual behavior of the fluorescence properties of this group, which would lead to erroneous evaluation of the enzyme activity, was observed when it is bound directly to proline. Here we report a systematic NMR, fluorescence and X-ray diffraction study of the compounds obtained from Boc-Abz-Pro-NH₂, Boc-Abz-Pro-OH, as well as from various other Boc-Abz-Pro-X derivatives, after treatment with HCl or TFA under anhydrous conditions. We verified that, as recently reported, even under these synthetic conditions, deprotection of Boc-Abz-Pro-NH₂ or Boc-Abz-Pro-OH leads to the formation of the same product: pyrrolobenzodiazepine-5,11-dione. However, the formation of this compound was not detected with Abz-Pro-N(CH₃)₂, Abz-Pro-Leu-Gly-NH₂ or Abz-pyrrolidine. For all these compounds we observed an unusual behavior for the fluorescence quantum yield of Abz that can be explained as the consequence of a non-radiative deactivation process produced, specifically, by the amidation of the Abz carboxyl group with proline or a similar secondary amine such as pyrrolidine. In conclusion, these results indicate that Abz cannot be used as an internal fluorescence marker for proteolytic enzyme activity when bound directly to proline.     

Efficient recovery of secretory recombinant proteins from protease negative mutant Escherichia coli strains

Osmotic shock and lysozyme/EDTA methods were used to recover secreted recombinant proteins from protease negative mutant strains of E. coli. Up to 80% of protein A--lactamase fusion protein was recovered from protease negative mutants by simple osmotic shock. Fractionation by lysozyme/EDTA treatment, increased the recovery of protein A--lactamase fusion protein from the mutant strain up to 93%. Mild fractionation condition allowed efficient recovery of secreted protein from protease negative mutant strains, but not from the parent strain possessing proteases. © Rapid Science Ltd. 1998     

Probing the ligand binding domain of the GluR2 receptor by proteolysis and deletion mutagenesis defines domain boundaries and yields a crystallizable construct.

Ionotropic glutamate receptors constitute an important family of ligand-gated ion channels for which there is little biochemical or structural data. Here we probe the domain structure and boundaries of the ligand binding domain of the AMPA-sensitive GluR2 receptor by limited proteolysis and deletion mutagenesis. To identify the proteolytic fragments, Maldi mass spectrometry and N-terminal amino acid sequencing were employed. Trypsin digestion of HS1S2 (Chen GQ, Gouaux E. 1997. Proc Natl Acad Sci USA 94:13431-13436) in the presence and absence of glutamate showed that the ligand stabilized the S1 and S2 fragments against complete digestion. Using limited proteolysis and multiple sequence alignments of glutamate receptors as guides, nine constructs were made, folded, and screened for ligand binding activity. From this screen, the S1S21 construct proved to be trypsin- and chymotrypsin-resistant, stable to storage at 4 degrees C, and amenable to three-dimensional crystal formation. The HS1S21 variant was readily prepared on a large scale, the His tag was easily removed by trypsin, and crystals were produced that diffracted to beyond 1.5 A resolution. These experiments, for the first time, pave the way to economical overproduction of the ligand binding domains of glutamate receptors and more accurately map the boundaries of the ligand binding domain.     

Continuous culture of Methanococcus jannaschii, an extremely thermophilic methanogen

Methanococcus jannaschii, an extremely thermophilic methanogen isolated from a deep-sea hydrothermal vent, was grown at 80 degrees C in continuous culture on a mineral salts medium gassed with H(2) and CO(2) at three different flow rates. The maximum specific growth rate was 0.56 h(-1), and the maximum specific methane productivity was 0.32 (mol g(-1) h(-1)). Uncoupling of growth and methane production was evidenced by an increase in teh non-growth-associated rate of methane formation, beta, with increasing gaseous input. The specific hydrogenase activity exhibited growth-assiciated behaviour at low growth rates, but showed no dependence on growth at higher growth rates. The growth dependence of hydrogenase activity is consistent with the pressure dependence of hydrogenase activity measured in previous experiments. In contrast, the specific protease activity was independent of the growth rate over the entire range of dilution rates studied. (c) 1994 John Wiley & Sons, Inc.     

Response dynamics of 26-, 34-, 39-, 54-, and 80-kDa proteases in induced cultures of recombinant Escherichia coli

Several researchers have demonstrated that the presence of a heterologous protein in recombinant Escherichia coli elicits a response similar to the heat-shock response, which includes enhanced protease expression. The present work detects, quantifies, and characterizes intracellular protease activity in E. coli that are "shocked" by the induction of a recombinant protein, CAT, which is an endogenous protein in some E. coli strains. A novel, sodium dodecyl sulfate gelatin poly-acrylamide gel electrophoresis (SDS-GPAGE) method is used to detect, quantify, and characterize the presence of these proteases. A hypothesis is proposed which links the amplified protease activity to a temporary depletion of specific amino acid pools, and a stringent-like stress response. (c) 1993 John Wiley & Sons, Inc.     

Influence of larval dietary supply on the food consumption, food utilization efficiency, growth and development of the lacewingChrysoperla carnea

In order to derive quantitative estimates of predation rate from serological gut analysis data, one must have an estimate of the interval during which a meal can be detected after feeding. In practice this has turned out to be ‘D_max,’ defined as ‘...the time from finishing a meal until that meal could just no longer be detected in any individuals.’ However D_max substitutes an absolute limit for what is really a continuous variable with significant variation. We examined this problem in a study of the detectability ofHelicoverpa zea Boddie (Lepidoptera: Noctuidae) fifth instar remains in the guts ofPolistes metricus Say (Hymenoptera: Vespidae). Wasps were maintained onTrichoplusia ni (Hübner) (Lepidoptera: Noctuidae) fifth instars before being fed a singleH. zea fifth instar. They were killed and frozen at 0, 24, 48 and 96 h intervals, with those held for more than 24 h fed a singleT. ni fifth instar at 24 h intervals in order to simulate continued feeding. Wasp abdomens were assayed by immunodot, using a monoclonal antibody toH. zea arylphorin. There was a logarithmic decay in the proportion ofP. metricus positive over time, a singleH. zea fifth instar meal having a detectability half-life of 19.4 h at field temperatures. If prey antigen detectability decays exponentially, then a detectability half-life is a more appropriate unit of detectability than an absolute detectability period.     

桔黄赛多孢霉外泌弹性蛋白酶的生化特性

外泌弹性蛋白酶是桔黄赛多孢霉(Scedosporium aurantiacum)主要毒性蛋白酶之一,本文在前期研究的基础上,对这种蛋白酶的序列、结构和酶学特性进行了研究。首先通过柱层析技术将S. aurantiacum培养上清液中的蛋白进行了分离,然后通过酶谱电泳纯化得到了弹性蛋白酶条带。从凝胶中提取了弹性蛋白酶,通过质谱技术对其序列进行了检测,并对其反应特性、活力和反应动力学参数进行了研究。结果显示,S. aurantiacum外泌弹性蛋白酶对弹性蛋白和牛跟腱胶原蛋白(bovine achilles tendon collagen, BATC)具有较好的水解性能,对鱼皮胶原蛋白(fish skin collagen, FSC)的水解效率高于对鱼鳞明胶的水解效率,对酪蛋白的水解性最差。作用于弹性蛋白时,其催化效率小于猪胰腺弹性蛋白酶。Zn²⁺对酶活力有提升作用,而Ca²⁺、Mg²⁺、Na⁺、(2S)-2-[(4S)-2-氨基-1,4,5,6-四羟基4-嘧啶基]-N-[[[(1S)-1-羰基-3-甲基丁基]氨基]羰基]甘氨酰- N1-[(1S)-1-甲基-2-氧乙基]-l-谷氨酸甲酰胺(elastatinal)和苯甲基磺酰氟(phenylmethanesulfonyl fluoride, PMSF)均对酶活有抑制作用。该蛋白酶和青霉(Paecilomyces lilacinus)外泌丝氨酸蛋白酶(PDB Entry: c3f7oB_)的序列最相似,且有多段保守序列的氨基酸个数多于7个,可以作为PCR反应引物设计的模板。酶学特性实验表明,S. aurantiacum外泌弹性蛋白酶对肺组织中的弹性蛋白具有降解作用,其蛋白表达和毒性机制需要进一步的研究。