Deriving Toxicity Values for Organophosphate Nerve Agents: A Position Paper in Support of the Procedures and Rationale for Deriving Oral RfDs for Chemical Warfare Nerve Agents

During the process of deriving oral Reference Dose (RfDs) values for chemical warfare agents, several issues arose regarding the identification of adverse effect levels and the application of uncertainty factors. For those agents that function as cholinesterase inhibitors (e.g., agents VX, GA, GB, and GD), these issues included the following: (1) Is the endpoint of blood cholinesterase inhibition an indicator of toxicity or a biomarker of exposure? (2) Can an experimental animal species be more sensitive than humans, thereby eliminating the need for an animal-to-human uncertainty factor? (3) Can the uncertainty factor that is used to extrapolate from a lowest-observed adverse-effect-level (LOAEL) to a no-observed-adverse-effect-level (NOAEL) be less than the default value of 10? (4) Can an oral RfD be derived from non-oral toxicity data? (5) Can an uncertainty factor of less than 10 be used to extrapolate from subchronic to chronic exposure (e.g., is the critical effect adequately described by the subchronic exposure data)? (6) What constitutes an adequate data base for organophosphate cholinesterase inhibitors, and what uncertainty factor should be used for an incomplete data base? Analysis of relevant data resulted in the following selection and justifications of uncertainty factors. For uncertainty associated with intraspecies extrapolation (UF_H), physiologic and pathologic conditions affecting cholinest-erase activity levels justified maintaining a UF_H of 10 for all of the nerve agents. Because available data indicated that humans tended to be more sensitive than rats regarding anticholinesterase effects, an interspecies variability (UF_A) factor of 10 was retained for agents GA, GB, and GD. For agent VX, however, the available data revealed that the domestic sheep test species exhibited sensitivity equivalent to or greater than that of humans thereby justifying a UF_A of 1. For uncertainties regarding extrapolation from subchronic-to- chronic exposure data, consideration of information on the physiology of cholinergic systems and the available toxicity data for the nerve agents and other cholinest-erase inhibitors indicated that a UF_S of 3 was justified for all four of the nerve agents. For uncertainties regarding LOAEL-to- NOAEL extrapolation (UF_L), the selection of agent GB, GD, and VX doses resulting in cholinesterase inhibition in the absence of clinical signs of toxicity (biomarker of exposure) justified this endpoint as a minimal LOAEL and a UF_L of 3. For agent GA, a NOAEL was used, and therefore no UF_L was required. The uncertainty factor for data base completeness (UF_D), was based upon several considerations. Of primary concern was the fact that chronic toxicity studies are not considered an essential component of the data base requirements for cholinesterase inhibitors because of the unlikelihood that the endpoint will change with an increase in exposure time beyond that defined as a subchronic exposure. Additionally, limited data regarding reproductive and developmental toxicity were not considered to represent critical toxicity endpoints for the nerve agents or cholinesterase inhibitors in general. Although the data base for agents GA, GB, and GD were lacking reproductive and developmental toxicity data to some extent, a UF_D of 3 was justified for the aforementioned reasons. The data base for agent VX was considered complete and a UF_D of 1 was selected for development of the RfD for this agent. A modifying factor (MF) to reflect qualitative assessment of additional uncertainties in the critical study or data base that are not addressed by uncertainty factors was limited to agent GA due to the route-to-route (i.e., intraperitoneal to oral) extrapolation and to insure the equivalent oral NOAEL was not overestimated. This article provides a brief overview of the nerve agents, information on cholinergic systems that is pertinent to deriving toxicity values for nerve agents and other organophosphate cholinesterase inhibitors, and a discussion of key issues regarding the use of uncertainty factors in RfD derivations.     

D-核糖发酵过程中底物抑制及补料的研究

研究了初始葡萄糖浓度对D -核糖发酵的影响 ,证实了较高的葡萄糖浓度对D -核糖发酵的抑制作用 ,并确定了较为适宜的初始葡萄糖浓度为 1 0 0g·L-1 或 1 5 0g·L-1 。前者条件下D -核糖的转化率和生产强度均达最大 ,分别为 32 8g·kg-1 和0 .6 8g·L-1 ·h-1 ;后者条件下D -核糖的产量达最大值 39.4 8g·L-1 。针对底物抑制现象 ,研究了补料工艺对D -核糖发酵的影响 ,确定发酵 2 4h后补加 5 0g·L-1 的葡萄糖为较优的补料工艺 ,在此工艺条件下最终D -核糖产量相对于对照组提高了 4 8.3%。     

A Fortran Program for Evaluation of the Effectiveness Factor of Biocatalysts in the Presence of External and Internal Diffusional Limitations

A Fortran program called SPEFF for evaluation of the effectiveness factor of immobilized enzyme preparations of spherical form in the presence of external and internal mass transfer resistances is described, and a listing of the program is given. Enzyme distribution in the bioparticle may be uniform or nonuniform. In the latter case the enzyme distribution is approximated by fifth-order polynomial. In the program differential equations are replaced by the system of non-linear algebraic equations, and the latter are solved by Newton iteration technique. The program is developed for Michaelis-Menten kinetics with allowance for competitive product inhibition and substrate inhibition. After slight modifications the program can be used for computation of the effectiveness factor of a membrane with an immobilized enzyme, or in the case when the enzyme kinetics are more complex. A typical run on a PDP-11/45 computer took 10-20 seconds. A typical computation time in the case of IBM-compatible TURBO PC was 15-30 seconds.     

Substrate and product inhibition of hydrogen production by the extreme thermophile,Caldicellulosiruptor saccharolyticus

Substrate and product inhibition of hydrogen production during sucrose fermentation by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was studied. The inhibition kinetics were analyzed with a noncompetitive, nonlinear inhibition model. Hydrogen was the most severe inhibitor when allowed to accumulate in the culture. Concentrations of 5-10 mM H(2) in the gas phase (identical with partial hydrogen pressure (pH(2)) of (1-2) x 10(4) Pa) initiated a metabolic shift to lactate formation. The extent of inhibition by hydrogen was dependent on the density of the culture. The highest tolerance for hydrogen was found at low volumetric hydrogen production rates, as occurred in cultures with low cell densities. Under those conditions the critical hydrogen concentration in the gas phase was 27.7 mM H(2) (identical with pH(2) of 5.6 x 10(4) Pa); above this value hydrogen production ceased completely. With an efficient removal of hydrogen sucrose fermentation was mainly inhibited by sodium acetate. The critical concentrations of sucrose and acetate, at which growth and hydrogen production was completely inhibited (at neutral pH and 70 degrees C), were 292 and 365 mM, respectively. Inorganic salts, such as sodium chloride, mimicked the effect of sodium acetate, implying that ionic strength was responsible for inhibition. Undissociated acetate did not contribute to inhibition of cultures at neutral or slightly acidic pH. Exposure of exponentially growing cultures to concentrations of sodium acetate or sodium chloride higher than ca. 175 mM caused cell lysis, probably due to activation of autolysins.     

Inhibition and Substrate Competition Kinetics in Analysis of Porcine Thyroid Alkaline Ribonuclease's Specificity Toward Synthetic Rna's and Trna

Abstract

Inhibition and substrate competition kinetics demonstrated that tRNA is a highly preferred substrate of thyroid alkaline RNase. The pyrimidine-specific RNase cleaved poly(C) 2.8 × 10⁵ faster than poly(U). k_cat: K_M ratios for tRNA and poly(C) based on molecular weights failed to predict preference when both were present. Competition experiments between poly(C) and tRNA revealed tRNA was a tight-binding competing substrate and the cytidylate residues in the 3prime;-CCA terminus of tRNA were preferred about 280: 1 over those in poly(C). Poly(U) was competitive with tRNA. When poly(C) was the substrate, inhibition type by poly(G) depended on poly(G) concentration. Neither tRNA lacking its 3prime; terminal cytidylyl(3prime;-5prime;)adenosine and terminating in a 2prime;:3prime; cCMP residue, tRNA lacking its 3prime; terminal 5prime;AMP residue, guanosine, nor guanylyl(3prime;-5prime;)guanylyl(3prime;-5prime;)guanosine were inhibitors. Product inhibition by adenosine and 2prime;:3prime; cCMP showed the kinetic mechanism for cleavage of tRNA was ordered uni bi.     

Purification and Characterization of Catechol 2,3-Dioxygenase from the Aniline Degradation Pathway of Acinetobacter sp. YAA and Its Mutant Enzyme,Which Resists Substrate Inhibition

Catechol 2,3-dioxygenase (C23O), a key enzyme in the meta-cleavage pathway of catechol metabolism, was purified from cell extract of recombinant Escherichia coli JM109 harboring the C23O gene (atdB) cloned from an aniline-degrading bacterium Acinetobacter sp. YAA. SDS–polyacrylamide gel electrophoresis and gel filtration chromatography analysis suggested that the enzyme (AtdB) has a molecular mass of 35 kDa as a monomer and forms a tetrameric structure. It showed relative meta-cleavage activities for the following catechols tested: catechol (100%), 3-methylcatechol (19%), 4-methylcatechol (57%), 4-chlorocatechol (46%), and 2,3-dihydroxybiphenyl (5%). To elevate the activity, a DNA self-shuffling experiment was carried out using the atdB gene. One mutant enzyme, named AtdBE286K, was obtained. It had one amino acid substitution, E286K, and showed 2.4-fold higher C23O activity than the wild-type enzyme at 100 μM. Kinetic analysis of these enzymes revealed that the wild-type enzyme suffered from substrate inhibition at >2 μM, while the mutant enzyme loosened substrate inhibition.     

Blood-Brain Transfer of Galactose in Experimental Galactosemia, with Special Reference to the Competitive Interaction Between Galactose and Glucose

The interaction between glucose and galactose during transport across the cerebral capillary endothelium was studied in anesthetized rats. Although galactose is present in the diet of suckling mammals and is a potential substrate for brain metabolism in adult mammals, its effect on glucose transport in adult rats is unknown. A kinetic model was formulated to analyze the effect of chronically elevated galactose levels on glucose transport in adult rats. The analysis indicated that galactose and glucose compete for the same transport mechanism in the cerebral capillary endothelium. The Tmax of glucose and galactose were both about 380 mumol 100 g-1 min-1 and the Kt of galactose (30 mM) was about three times that of glucose (10 mM). During prolonged galactosemia in adult rats, neither the Tmax, nor the Kt of either competitor changed substantially when compared with rats subjected to acute galactosemia. At 10 mM galactose in plasma in rats with acute galactosemia, the inhibition of glucose transport, simulated a 25% reduction of plasma glucose, and in rats with chronic galactosemia a 20% reduction. This moderate effect is in contrast to the effect of galactose in suckling rats in which 10 mM galactose in plasma reduced the glucose transport to a level corresponding to a 50% reduction of the plasma glucose concentration.     

Irreversible Inhibition of Transglutaminases by Sulfonium Methylketones: Optimization of Specificity and Potency with w-Aminoacyl Spacers

Abstract

Sulfonium methylketones, of structure Cbz-Phe-NH(CH₂)_nCOCH₂S ⁺ (CH₃)₂, n < 2, are specific and potent inactivators of transglutaminases. The length of the -(CH₂)_n-spacer moiety, n = 1-5, is a critical determinant for both the specificity and potency of the inactivator. The dipeptidyl analog Cbz-Phe-Gly-(CH₂)_nS ⁺ (CH₃)₂, n = 1, is a more powerful inactivator of the thiol proteinase cathepsin B, k/K < 3 × 10⁵ M^?1 min^?1, than of transglutaminases, k_i(appl/K_i(appl < 1.5 × 10⁴ M^?1 min^?1. In contrast, the γ-aminobutyryl analog, n = 3, is a very potent transglutaminase inactivator with k_i(apP/ K_i(appl = 3.1 < 10⁶M^?1min^?1, but does not inactivate cathepsin B. In cell studies, the y-aminobutyryl and w-aminohexyl analogs inhibited the transglutaminase-mediated process of ionophore-induced cross-linked envelope formation by human malignant keratinocytes and the order of potency was related to that found for enzyme inhibition. The sulfonium methylketones, in equilibrium with the resonance stabilized ylides, are chemically inert towards glutathione under ambient conditions demonstrating the potential utility of this novel class of transglutaminase inhibitors for the study of enzyme inhibition in cellular environments.     

Selective Irreversible Inhibition of Neuronal and Inducible Nitric-oxide Synthase in the Combined Presence of Hydrogen Sulfide and Nitric Oxide

Citrulline formation by both human neuronal nitric-oxide synthase (nNOS) and mouse macrophage inducible NOS was inhibited by the hydrogen sulfide (H₂S) donor Na₂S with IC₅₀ values of ∼2.4·10⁻⁵ and ∼7.9·10⁻⁵ m, respectively, whereas human endothelial NOS was hardly affected at all. Inhibition of nNOS was not affected by the concentrations of l-arginine (Arg), NADPH, FAD, FMN, tetrahydrobiopterin (BH4), and calmodulin, indicating that H₂S does not interfere with substrate or cofactor binding. The IC₅₀ decreased to ∼1.5·10⁻⁵ m at pH 6.0 and increased to ∼8.3·10⁻⁵ m at pH 8.0. Preincubation of concentrated nNOS with H₂S under turnover conditions decreased activity after dilution by ∼70%, suggesting irreversible inhibition. However, when calmodulin was omitted during preincubation, activity was not affected, suggesting that irreversible inhibition requires both H₂S and NO. Likewise, NADPH oxidation was inhibited with an IC₅₀ of ∼1.9·10⁻⁵ m in the presence of Arg and BH4 but exhibited much higher IC₅₀ values (∼1.0–6.1·10⁻⁴ m) when Arg and/or BH4 was omitted. Moreover, the relatively weak inhibition of nNOS by Na₂S in the absence of Arg and/or BH4 was markedly potentiated by the NO donor 1-(hydroxy-NNO-azoxy)-l-proline, disodium salt (IC₅₀ ∼ 1.3–2.0·10⁻⁵ m). These results suggest that nNOS and inducible NOS but not endothelial NOS are irreversibly inhibited by H₂S/NO at modest concentrations of H₂S in a reaction that may allow feedback inhibition of NO production under conditions of excessive NO/H₂S formation.     

The Effect of Cations on the Amidase Activity of Human Tissue Kallikrein: 1-Linear Competitive Inhibition by Sodium,Potassium, Calcium and Magnesium. 2-Linear Mixed Inhibition by Aluminium

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide by human tissue kallikrein (hK1) was studied in the absence and in the presence of increasing concentrations of the following chloride salts: sodium, potassium, calcium, magnesium and aluminium. The data indicate that the inhibition of hK1 by sodium, potassium, calcium and magnesium is linear competitive and that divalent cations are more potent inhibitors of hK1 than univalent cations. However the inhibition of hK1 by aluminium cation is linear mixed, with the cation being able to bind to both the free enzyme and the ES complex. This cation was the best hK1 inhibitor. Aluminium is not a physiological cation, but is a known neurotoxicant for animals and humans. The neurotoxic actions of aluminium may relate to neuro-degenerative diseases.     

Antagonism by Local Anesthetics of Sodium Channel Activators in the Presence of Scorpion Toxins: Two Mechanisms for Competitive Inhibition

1. The interaction of veratridine (VTD), a Na+ channel activator, scorpion alpha-toxin (LQ), an open state Na+ channel stabilizer, and the local anesthetic, lidocaine (LID), a channel inhibitor, at the neuronal sodium channel was assessed by measuring VTD-dependent slow depolarizations of frog sciatic nerve using the sucrose-gap method. 2. The slow depolarizing action of veratridine was potentiated more than 10-fold by the peptide LQ toxin, whereas its competitive inhibition by lidocaine was unchanged by LQ. 3. We conclude that the antagonism between VTD and a LID molecule during slow depolarization is allosteric, involving a trapping of the Na+ channel by LID in the inactivated state that has a very low affinity for VTD. 4. The binding of VTD to the open state of the channel, which is stabilized by LQ, may be inhibited by orthosteric competition at overlapping sites since both LID and VTD bind avidly and rapidly to open channels.     

Genetic Factors in Susceptibility: Serum PON1 Variation Between Individuals and Species

In mammals, serum paraoxonase (PON1) is tightly associated with high-density lipoprotein (HDL) particles. In human populations, PON1 exhibits a substrate dependent activity polymorphism determined by an Arg/Gln (R/Q) substitution at amino acid residue 192. The physiological role of this protein appears to be involvement in the metabolism of oxidized lipids. Several studies have suggested that the PON1_R192 allele may be a risk factor in coronary artery disease. PON1 also plays an important role in the metabolism of organophosphates including insecticides and nerve agents. The PON1_R192 isoform hydrolyzes paraoxon rapidly, but diazoxon, soman and sarin slowly compared with the PON1_Q192 isoform. Both PON1 isoforms hydrolyze phenylacetate at approximately the same rate, while PON1_R192 hydrolyzes chlorpyrifos oxon slightly faster than PON_Q192. Animal model studies involving injection of purified rabbit PON1 into mice clearly demonstrated the ability of PON1 to protect cholinesterases from inhibition by OP compounds. The consequence of having low PON1 levels has been addressed with toxicology studies in PON1 knockout mice. These mice showed dramatically increased sensitivity to chlorpyrifos oxon, diazoxon and some increased sensitivity to the respective parent compounds. These observations are consistent with earlier studies that showed a good correlation between high rates of OP hydrolysis by serum PON1 and resistance to specific OP compounds. They are also consistent with the observations that newborns have an increased sensitivity to OP toxicity, due in part to their not expressing adult PON1 levels for weeks to months after birth, depending on the species. Together, these studies point out the importance of considering the genetic variability of PON1₁₉₂ isoforms and levels as well as the developmental time course of PON1 appearance in serum in developing risk assessment models     

Furanocoumarin Content,Antioxidant Activity,and Inhibitory Potential of Heracleum verticillatum,Heracleum sibiricum,Heracleum angustisectum,and Heracleum ternatum Extracts against Enzymes Involved in Alzheimer's Disease and Type II Diabetes

Hexane extracts of Heracleum verticillatum, H. sibiricum, H. angustisectum, and H. ternatum were studied for their furanocoumarin content antioxidant potential and acetylcholinesterase and α‐amylase inhibitory activities. Quantification of the furanocoumarins was performed by ¹H‐NMR. Pimpinellin was found to be the main component in the roots of all studied species. Bergapten and imperatorin were the major compounds in the fruits of H. sibiricum and H. verticillatum, respectively, while byakangelicol dominated in H. angustisectum and H. ternatum fruits. The leaf and fruit extracts of H. angustisectum demonstrated the highest DPPH radical scavenging activity and TEAC (IC₅₀ 0.58 mg/mL and 1.83 mm , respectively). The root extracts of H. verticillatum and H. angustisectum were found to be the most effective against acetylcholinesterase (IC₅₀ 0.30 and 0.34 mg/mL, respectively). The studied extracts were not active or demonstrated a weak inhibitory effect (%Inh. up to 29.7) towards α‐amylase.     

The Mechanisms of Action and Inhibition of Pancreatic Lipase and Acetylcholinesterase: A Comparative Modeling Study

Abstract

Pancreatic lipase and acetylcholinesterase are both serine esterases. Their X-ray structures reveal a similar overall fold, but no sequence homology can be detected. A catalytic triad like in the trypsin family of serine proteases consisting of serine, histidine and aspartate (glutamate in acetylcholinesterase) suggests mechanistic similarities. Models of the transition states of the substrate cleavage have been built and possible catalytic pathways were examined. The model that could produce a consistent pathway throughout the reactions had a transition state of the opposite handedness compared to trypsin. These models could be used to rationalise binding modes of inhibitors of both enzymes. The lipase inhibitor tetrahydrolipstatin (THL) contains a gamma-lactone which is opened by the catalytic serine; the alcohol leaving group prohibits deacylation by locking the pathway for incoming water and thus inactivates the enzyme. Carbamate inhibitors of acetylcholinesterase transfer a carbamoyl group to the serine-OH which deacylates slowly. These observations can be used as a starting point for the discovery of new classes of inhibitors.     

Competitive inhibition analysis of the enzyme-substrate interaction in the carboxy-terminal processing of the precursor D1 protein of photosystem II reaction center using substituted oligopeptides

A clear parallelism was demonstrated between the efficiency as substrate of the substituted oligopeptides corresponding to the carboxy-terminal (C-terminal) sequence of the precursor D1 protein (pD1) in the in vitro enzymatic assay and their competitive inhibitory capacity toward the proteolytic C-terminal processing of the full-length pD1 integrated in the intact photosystem II complex embedded in the thylakoid membrane of Scenedesmus obliquus LF-1 mutant, as shown e.g. by the influence of L343A, A345G and A345V substitutions and the effect of C-terminal fragments. This suggests that the basic mechanism for substrate recognition by the processing protease elucidated in the enzymatic analysis using synthetic oligopeptides is also effective in vivo, although it can sometimes be difficult to detect the consequence of amino acid substitution in the integrated systems.     

Angiotensin III: A Potent Inhibitor of Enkephalin-Degrading Enzymes and an Analgesic Agent

Various angiotensins, bradykinins, and related peptides were examined for their inhibitory activity against several enkephalin-degrading enzymes, including an aminopeptidase and a dipeptidyl aminopeptidase, purified from a membrane-bound fraction of monkey brain, and an endopeptidase, purified from the rabbit kidney membrane fraction. Angiotensin derivatives having a basic or neutral amino acid at the N-terminus showed strong inhibition of the aminopeptidase. Dipeptidyl aminopeptidase was inhibited by angiotensins II and III and their derivatives, whereas the endopeptidase was inhibited by angiotensin I and its derivatives. The most potent inhibitor of aminopeptidase and dipeptidyl aminopeptidase was angiotensin III, which completely inhibited the degradation of enkephalin by enzymes in monkey brain or human CSF. The Ki values for angiotensin III against aminopeptidase, dipeptidyl aminopeptidase, endopeptidase, and angiotensin-converting enzyme, which degraded enkephalin, were 0.66 X 10(-6), 1.03 X 10(-6), 2.3 X 10(-4), and 1.65 X 10(-6) M, respectively. Angiotensin III potentiated the analgesic activity of Met-enkephalin after intracerebroventricular coadministration to mice in the hot plate test. Angiotensin III itself also displayed analgesic activity in that test. These actions were blocked by the specific opiate antagonist naloxone.     

Endogenous ADP-Ribosylation in Brain: Initial Characterization of Substrate Proteins

Cholera and pertussis toxin-mediated ADP-ribosylation has been used extensively to study regulation of guanine nucleotide binding proteins (G proteins) in the nervous system, but much less is known about possible endogenous ADP-ribosylation of G proteins in brain. The present study demonstrates endogenous ADP-ribosylation, in the absence of cholera and pertussis toxins, of four predominate proteins in homogenates of rat cerebral cortex. These proteins showed apparent molecular masses of 20, 42, 45, and 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 42- and 45-kDa proteins comigrated precisely with the major cholera toxin-labeled bands. Furthermore, the endogenous ADP-ribosylated and cholera toxin-ADP-ribosylated bands yielded identical 32P-labeled peptide fragments by one-dimensional peptide mapping, indicating that they are probably the same proteins, presumably the alpha-subunits of Gs. In contrast, peptide maps of the 50-kDa protein, which migrated close to a 48-kDa cholera toxin-labeled band, demonstrated that this protein is distinct from the toxin-labeled band and from Gs alpha. Levels of endogenous ADP-ribosylation activity showed regional heterogeneity in brain, with a nearly threefold variation observed among the brain regions examined. Chronic administration (7 days) of corticosterone significantly increased overall levels of endogenous ADP-ribosylation, indicating that components of this system may be under hormonal control in vivo. Attempts to identify neurotransmitters or second messenger systems that regulate endogenous ADP-ribosylation activity in brain have so far been unsuccessful with one exception.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competitive interactions limiting the number of species in rock pools: experiments with Sigara nigrolineata

Rock-pool corixids (Hemiptera) Arctocorisa carinata and Callicorixa producta were excluded from small rock pools. First-stage larvae of Sigara nigrolineata, separately or together with larvae of either of the rock-pool species, were then introduced daily at a rate simulating reproduction by typical populations. In all three treatments, the survival of S. nigrolineata larvae was high at the beginning of experiment, 0.39–0.83 between the 1st and 5th stages, but later high mortality was observed. C. producta adults tended to invade experimental rock pools and towards the end of the experiment larvae of this species were dominant. Oviposition rates and developmental rates of the three species are comparable. In laboratory tests, young larvae of S. nigrolineata were more vulnerable to interspecific predation than those of the rock-pool species, and older larvae proved to be less efficient predators. In rock pools, there are no environmental factors preventing successful reproduction by S. nigrolineata. However, competition and predation by the two other rock-pool species seem to prevent successful reproduction. Dispersal in S. nigrolineata is too slow for efficient colonization of rapidly changing rock-pool environments.     

Inhibition of CYP2B4 by 2-ethynylnaphthalene: evidence for the co-binding of substrate and inhibitor within the active site

2-Ethynylnaphthalene (2EN) is an effective mechanism-based inhibitor of CYP2B4. There are two inhibitory components: (1) irreversible inactivation of CYP2B4 (a typical time-dependent inactivation), and (2) a reversible component. The reversible component was unusual in that the degree of inhibition was not simply a characteristic of the enzyme-inhibitor interaction, but dependent on the size of the substrate molecule used to monitor residual activity. The effect of 2EN on the metabolism of seven CYP2B4 substrates showed that it was not an effective reversible inhibitor of substrates containing a single aromatic ring; substrates with two fused rings were competitively inhibited by 2EN; and larger substrates were non-competitively inhibited. Energy-based docking studies demonstrated that, with increasing substrate size, the energy of 2EN and substrate co-binding in the active site became unfavorable precisely at the point where 2EN became a competitive inhibitor. Hierarchical docking revealed potential allosteric inhibition sites separate from the substrate binding site.     

固定化脂肪酶有机相中催化己酸乙酯反应动力学研究

有机相中脂肪酶催化反应动力学研究是酶工程领域中一个引起广泛兴趣的研究课题［１］。目前的研究较多地集中于水解反应和转酯反应［２～４］。对于酯化反应的动力学报道不多。酶反应动力学研究对于研究反应机理、确定有效的酶促反应环境、选择合适的酶反应器有着十分重要的作用。因此,对非水相中脂肪酶催化反应动力学的研究无论在理论上还是在实际应用中都有着重要的意义。与自由酶相比,使用固定化酶可提高酶在有机相中的扩散效果和热力学稳定性,是调节控制酶活性常用的手段。但酶固定化以后,影响其动力学的因素增多,反应机制变得更加…