A comparison of the conversion of 1-amino-2-ethylcyclopropane-1-carboxylic acid stereoisomers to 1-butene by pea epicotyls and by a cell-free system

The characteristics of the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by pea (Pisum sativum L.) epicotyls and by pea epicotyl enzyme are compared. Of the four stereoisomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC), only (1R,2S)-AEC is preferentially converted to 1-butene in pea epicotyls. This conversion is inhibited by ACC, indicating that butene production from (1R,2S)-AEC and ethylene production from ACC are catalyzed by the same enzyme. Furthermore, pea epicotyls efficiently convert ACC to ethylene with a low K _m (66 M) for ACC and do not convert 4-methylthio-2-oxo-butanoic acid (KMB) to ethylene, thus demonstrating high specificity for its substrate. In contrast, the reported pea epicotyl enzyme which catalyzes the conversion of ACC to ethylene had a high K _m (389 mM) for ACC and readily converted KMB to ethylene. We show, moreover, that the pea enzyme catalyzes the conversion of AEC isomers to butene without stereodiscrimination. Because of its lack of stereospecificity, its low affinity for ACC and its utilization of KMB as a substrate, we conclude that the reported pea enzyme system is not related to the in-vivo ethylene-forming enzyme.Abbreviations ACC 1-Amino cyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - KMB 4-methylthio-2-oxobutanoic acid     

A new simple method to determine the diffusion coefficient from Active Enzyme Centrifugation experiments

A simple and accurate procedure to determine the diffusion coefficient from Active Enzyme Centrifugation experiments is presented. Using computer-simulated concentration distributions we demonstrate that the procedure, derived by Vinograd for the conventional band sedimentation, is suitable to exploit Active Enzyme Centrifugation experiments when the Cohen's Difference Curves Method is used. This new empirical method avoids all the difficulties of the rigorous method previously proposed by Cohen et al., without any loss of accuracy. Optimal conditions are described which allow the determination of the enzyme diffusion coefficient with a 5% uncertainty. Such an easy determination of the sedimentation and diffusion coefficients by the AEC technique can provide a good and rapid estimation of the active enzyme molecular weight, either with a low amount of material or in very impure preparations.     

Biochemical and physiological investigations on adenosine 5ʹ monophosphate deaminase from Plasmodium spp.

The interplay between ATP generating and utilizing pathways in a cell is responsible for maintaining cellular ATP/energy homeostasis that is reflected by Adenylate Energy Charge (AEC) ratio. Adenylate kinase (AK), that catalyzes inter‐conversion of ADP, ATP and AMP, plays a major role in maintaining AEC and is regulated by cellular AMP levels. Hence, the enzymes AMP deaminase (AMPD) and nucleotidases, which catabolize AMP, indirectly regulate AK activity and in‐turn affect AEC. Here, we present the first report on AMPD from Plasmodium, the causative agent of malaria. The recombinant enzyme expressed in Saccharomyces cerevisiae was studied using functional complementation assay and residues vital for enzyme activity have been identified. Similarities and differences between Plasmodium falciparum AMPD (PfAMPD) and its homologs from yeast, Arabidopsis and humans are also discussed. The AMPD gene was deleted in the murine malaria parasite P. berghei and was found to be dispensable during all stages of the parasite life cycle. However, when episomal expression was attempted, viable parasites were not obtained, suggesting that perturbing AMP homeostasis by over‐expressing AMPD might be lethal. As AMPD is known to be allosterically modulated by ATP, GTP and phosphate, allosteric activators of PfAMPD could be developed as anti‐parasitic agents.     

Application of standard addition for the determination of carboxypeptidase activity in <Emphasis Type="Italic">Actinomucor elegans</Emphasis> bran koji

Leucine carboxypeptidase (EC 3.4.16) activity in Actinomucor elegans bran koji was investigated via absorbance at 507 nm after stained by Cd-nihydrin solution, with calibration curve A, which was made by a set of known concentration standard leucine, calibration B, which was made by three sets of known concentration standard leucine solutions with the addition of three concentrations inactive crude enzyme extract, and calibration C, which was made by three sets of known concentration standard leucine solutions with the addition of three concentrations crude enzyme extract. The results indicated that application of pure amino acid standard curve was not a suitable way to determine carboxypeptidase in complicated mixture, and it probably led to overestimated carboxypeptidase activity. It was found that addition of crude exact into pure amino acid standard curve had a significant difference from pure amino acid standard curve method (p < 0.05). There was no significant enzyme activity difference (p > 0.05) between addition of active crude exact and addition of inactive crude kind, when the proper dilute multiple was used. It was concluded that the addi-tion of crude enzyme extract to the calibration was needed to eliminate the interference of free amino acids and related compounds presented in crude enzyme extract.     

Mechanisms of liquid flux across pulmonary alveolar epithelial cell monolayers

Summary Active transport of sodium by pulmonary alveolar epithelial cells (AEC) is believed to be an important component of edema clearance in the normal and injured lung. Data supporting this premise have come from measurements of sodium movement across AEC monolayers or from perfused lung model systems. However, direct measurement of fluid flux across AEC monolayers has not been reported. In the present work, AEC were studied with an experimental system for the measurement of fluid flux (Jv) across functionally intact cell monolayers. Primary adult rat type II alveolar epithelial cells were cultured on 0.8 μm nuleopore filters previously coated with gelatin and fibronectin. Intact monolayers were verified by high electrical resistance (> 1000 Θ) at 4–5 d of primary culture. At the same time interval, transmission electron microscopy revealed cells with type I cell-like morphology throughout the monolayer. These were characterized by both adherens and tight junctional attachments. Fluid flux across the monolayers was measured volumetrically over a period of 2 h in the presence of HEPES-buffered DMEM containing 3% fatty acid-free bovine serum albumin. Flux (Jv) was inhibited 39% by 1 × 10⁻⁴ M ouabain (P < 0.01) and 27% by 5 × 10⁻⁴ M amiloride (P < 0.05). These data support the concept that AEC Na⁺/K⁺-ATPase and Na⁺ transport systems are important determinants of AEC transepithelial fluid movement in vitro.     

Stereospecific conversion of 1-aminocyclopropanecarboxylic Acid to ethylene by plant tissues : conversion of stereoisomers of 1-amino-2-ethylcyclopropanecarboxylic Acid to 1-butene

Inasmuch as the molecule of 1-aminocyclopropanecarboxylic acid (ACC) possesses reflective symmetry but lacks rotational symmetry, the two chemically alike methylene groups can be distinguished by a stereospecific enzyme. To determine whether ACC conversion to ethylene by plant tissues proceeds in a stereospecific fashion, the four stereoisomers of 1-amino-2-ethylcyclopropanecarboxylic acid (AEC) were administered to postclimacteric apple (Malus sylvestris Mill., var. Golden Delicious), excised preclimacteric cantaloupe (Cucumis melo L., var. reticulatis Naud cv. PMR-45), and etiolated mung bean (Vigna radiata L., Wilczek, var. Berken) hypocotyls. In each case (1R,2S)-AEC was the preferred substrate yielding 1-butene. In contrast, all AEC isomers were converted equally well to butene by chemical oxidation using NaOCl. Both ACC and AEC appear to be substrates for the same enzyme since both reactions are inhibited in parallel by N₂ or Co²⁺, both reactions are induced in parallel by excision, and when both substrates are present simultaneously each will act as an inhibitor with respect to the other. The aforementioned observations indicate that ACC is stereospecifically converted to ethylene. For AEC to be the most active precursor of 1-butene, the ethyl substituent should be trans to the carboxyl group and the pro-(S) methylene group should be unsubstituted. This observation leads to the suggestion that the enzyme interacts with amino, carboxyl, and pro-(S) methylene groups, a configuration corresponding to a l-amino acid. This view is consistent with the observation that the l-forms of alanine and methionine inhibit the conversion of ACC to ethylene more than the corresponding d-amino acids in the mung bean hypocotyl system.     

Comparison of inactivation and unfolding of green crab (Scylla serrata) alkaline phosphatase during denaturation by guanidinium chloride

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, each active site in which contains a tight cluster of two zinc ions and one magnesium ion. Unfolding and inactivation of the enzyme during denaturation in guanidinium chloride (GuHCl) solutions of different concentrations have been compared. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436] has been applied to a study on the kinetics of the course of inactivation of the enzyme during denaturation by GuHCl. The rate constants of unfolding and inactivation have been determined. The results show that inactivation occurs before noticeable conformational change can be detected. It is suggested that the active site of green crab alkaline phosphatase containing multiple metal ions is also situated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.     

Opinion Practical and ethical considerations for students conducting ecological research involving wildlife

This commentary outlines relevant changes to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes for ecologists and students active in field‐based wildlife research. All field‐based activities that require the approval of an Animal Ethics Committee (AEC) have now been detailed within the revised Code of Practice. Researchers and their students are required to give explicit consideration to the humane principles of replacement, reduction and refinement within their experimental protocols as a part of the AEC permission procedure. University‐based researchers also have obligations to train graduate students in the ethical as well as the practical components of wildlife research. In promoting this educational aim, a summary of ethical considerations in wildlife research is offered as a basis for discussion: the regulatory framework of research animal welfare (including an introduction to the Code of Practice, the AEC process and some thoughts on experimental design); and the humane principles on which the Code of Practice is based. Some practical considerations for field work relevant to students’ research training are also described.     

Lysine overproducer mutants with an altered dihydrodipicolinate synthase from protoplast culture of Nicotiana sylvestris (Spegazzini and Comes)

Summary Two S-(2-aminoethyl)L-cysteine (AEC) resistant lines were isolated by screening mutagenized protoplasts from diploid N. sylvestris plants. Both lines accumulated free lysine at levels 10 to 20-fold higher than in controls. Lysine overproduction and AEC-resistance were also expressed in plants regenerated from the variant cultures. A feedback insensitive form of dihydrodipicolinate synthase (DHPS), the pathway specific control enzyme for lysine synthesis, was detected in callus cultures and leaf extracts from the resistant lines. Aspartate kinase (AK), the other key enzyme in the regulation of lysine biosynthesis, was unaltered in the mutants. Crosses with wild type plants indicated that the mutation conferring insensitivity to feedback in DHPS, with as result overproduction of lysine and resistance to AEC, was inherited as a single dominant nuclear gene.Abbreviations AK aspartate kinase (EC 2.7.2.4) - DHPS dihydrodipicolinate synthase (EC 4.2.1.52) - AEC S-(2-aminoethyl)L-cysteine     

Energy Metabolism in Rhizomes of Acorus calamus (L.) and in Tubers of Solanum tuberosum (L.) With Regard to their Anoxia Tolerance

Rhizomes of the marsh plant Acorus calamus (L.) and tubers of the flooding-intolerant Solanum tuberosum (L.) var. Bintje, both kept under strict anoxia, differ markedly in their fermentation properties. The fermentation capacities as measured by ADH and LDH activities and their respective product concentrations were estimated. While rhizomes of Acorus calamus, having high ADH and low LDH activities, accumulate mainly ethanol, tubers of Solanum tuberosum tend towards lactic acid fermentation. The total amount of adenine nucleotides is quite stable in Acorus calamus, whereas they show a sharp decline in S. tuberosum during the first 6h of anoxia. The adenylate energy charge of A. calamus recovers after a short initial drop (AEC > 0.8). AEC values of S. tuberosum decrease rapidly and remain at very low values (AEC ～ 0.3). Tuber tissues became soft and lost viability after about 48–72 h of anoxia at 25 °C. This might be due to tissue acidification and impaired energy metabolism, but not to the lack of energy reserves. Energy metabolism of A. calamus is well adapted to anoxia.     

Studies on the viscosity of solutions of bovine liver glutamate dehydrogenase and on related hydrodynamic models; effect of toluene on enzyme association

The viscosity of bovine liver glutamate dehydrogenase solutions was studied at 10 and 20° C in 0.2.M sodium phosphate buffer at pH 7, in the concentration range 0.1–8 mg/ml. A method for the study of the viscosity of very dilute solutions of associating enzymes is described. It was found that the reduced specific viscosity η_sp/c of glutamate dehydrogenase continuously increases with increasing enzyme concentration, from about 4 ml/g at the lowest concentrations to about 16 ml/g at 8 mg/ml. In the presence of 10^?3M GTP and 10^?3M NADH the viscosity increase is much smaller and the results can be extrapolated to zero enzyme concentration to yield an intrinsic viscosity [η] = 3.2. The values of η_sp/c in phosphate buffer alone apparently extrapolate to the same value of [η], or to a value close to it. We also observe that, in the presence of toluene η_sp/c increases very much more with enzyme concentration: η_sp/c already equals 16 ml/g at a concentration of 0.75 mg/ml. These observations are in good agreement with the hypothesis that the active oligomer of glutamate dehydrogenase (MW = 312,000) associates with increasing enzyme concentration to form linear rodlike polymers of indefinite length. This association is strongly diminished by the addition of 10^?3M GTP, 10^?3M NADH. Toluene, on the other hand, promotes reversible association to linear polymers of very high molecular weight. The transverse and axial rotary frictional coefficients of macroscopic bodies, similar to a physical model for the structure of glutamate dehydrogenase recently advanced, were determined. Assuming that the viscosity of the model is equal to that of an ellipsoid of rotation with identical frictional coefficients, we calculate [η] = 3.26 ml/g according to Kuhn and 3.20 ml/g according to Simha, for the glutamate dehydrogenase oligomer, in good agreement with the result derived from the study of enzyme solutions.     

The Novel Approach to the Protein Design: Active Truncated Forms of Human 1-CYS Peroxiredoxin

Abstract

The object of the present study is the verification of a new approach to the design of the active truncated forms of enzymes. The method is based on a new way of investigating the protein sequences—the ANalysis of Informational Structure (ANIS). The analysis of informational structure allows to determine the hierarchically organized structures (IDIC-trees) formed by the sites with the Increased Degree of Informational Coordination between residues. The proposed approach involves the consequent removal of the fragments corresponding to the individual IDIC-trees from the wild-type enzyme sequences. The described procedure was applied to the design of the active truncated form of human 1-CYS peroxiredoxin (PrxVI). Two variants of the PrxVI truncated sequences were proposed according to ANIS method. These truncated forms of the enzyme were expressed in E. coli and purified. The respective antioxidant activities were measured. It was shown that one of the truncated recombinant proteins retains more than 90% of the wild-type PrxVI enzymatic activity. According to the results of our study we can assume that ANIS method can be an effective tool for the design of the active truncated forms of the enzymes or the chimeric proteins which combine the enzymatic activities of their wild-type prototypes.     

Purification of long helical capsid of newcastle disease virus from Escherichia coli using anion exchange chromatography

NP_Δc375 is a truncated version of the nucleocapsid protein of Newcastle disease virus (NDV) which self‐assembles into a long helical structure. A packed bed anion exchange chromatography (PB‐AEC), SepFastTM Supor Q pre‐packed column, was used to purify NP_Δc375 from clarified feedstock. This PB‐AEC column adsorbed 76.2% of NP_Δc375 from the clarified feedstock. About 67.5% of the adsorbed NP_Δc375 was successfully eluted from the column by applying 50 mM Tris‐HCl elution buffer supplemented with 0.5 M NaCl at pH 7. Thus, a recovery yield of 51.4% with a purity of 76.7% which corresponds to a purification factor of 6.5 was achieved in this PB‐AEC operation. Electron microscopic analysis revealed that the helical structure of the NP_Δc375 purified by SepFast^TM Supor Q pre‐packed column was as long as 490 nm and 22–24 nm in diameter. The antigenicity of the purified NP_Δc375 was confirmed by enzyme‐linked immunosorbent assay. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 564–567, 2013     

An immunological method to combine the measurement of active and total myeloperoxidase on the same biological fluid,and its application in finding inhibitors which interact directly with the enzyme

Abstract

Myeloperoxidase (MPO) is a pro-oxidant enzyme involved in inflammation, and the measurement of its activity in biological samples has emerged essential for laboratory and clinical investigations. We will describe a new method which combines the SIEFED (specific immunological extraction followed by enzymatic detection) and ELISA (ELISAcb) techniques to measure the active and total amounts of MPO on the same human sample and with the same calibration curve, as well as to define an accurate ratio between both the active and total forms of the enzyme.

The SIEFED/ELISAcb method consists of the MPO extraction from aqueous or biological samples by immobilized anti-MPO antibodies coated onto microplate wells. After a washing step to eliminate unbound material, the activity of MPO is measured in situ by adding a reaction solution (SIEFED). Following aspiration of the reaction solution, a secondary anti-MPO antibody is added into the wells and the ELISAcb test is carried out in order to measure the total MPO content.

To validate the combined method, a comparison was made with SIEFED and ELISA experiments performed separately on plasma samples isolated from human whole blood, after a neutrophil stimulation.

The SIEFED/ELISAcb provides a suitable tool for the measurement of specific MPO activity in biological fluids and for the estimation of the inhibitory potential of a fluid. The method can also be used as a pharmacological tool to make the distinction between a catalytic inhibitor, which binds to MPO and inhibits its activity, and a steric inhibitor, which hinders the enzyme and prevents its immunodetection.     

Primary human airway epithelial cell-dependent inhibition of human lung mast cell degranulation

Introduction

Chronic mast cell activation is a characteristic feature of asthma. BEAS-2B human airway epithelial cells (AEC) profoundly inhibit both constitutive and IgE-dependent human lung mast cell (HLMC) histamine release. The aim of this study was to examine the regulation of HLMC degranulation by primary AEC from healthy and asthmatic subjects, and investigate further the inhibitory mechanism.

Methods

HLMC were co-cultured with both BEAS-2B and primary AEC grown as monolayers or air-liquid interface (ALI) cultures.

Results

Both constitutive and IgE-dependent HLMC histamine release were attenuated by BEAS-2B, primary AEC monolayers and ALI cultures. This occurred in the absence of HLMC-AEC contact indicating the presence of a soluble factor. Unlike healthy ALI AEC, asthmatic ALI-AEC did not significantly reduce constitutive histamine release. AEC inhibitory activity was transferable in primary AEC monolayer supernatant, but less active than with Transwell co-culture, suggesting that the inhibitory factor was labile. The AEC inhibitory effects were attenuated by both AEC wounding and pertussis toxin, indicating the involvement of a G₀/G_i receptor coupled mechanism. Solid phase extraction of lipids (<10 kDa) removed the AEC inhibitory activity. The lipid derivatives resolvin D1 and D2 and lipoxin A₄ attenuated HLMC histamine release in a dose-dependent fashion but were not detectable in co-culture supernatants.

Conclusions

Primary AEC suppress HLMC constitutive and IgE-dependent histamine secretion through the release of a soluble, labile lipid mediator(s) that signals through the G₀/G_i receptor coupled mechanism. Manipulation of this interaction may have a significant therapeutic role in asthma.     

Toward a clinical antifungal peptoid: Investigations into the therapeutic potential of AEC5

Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningitis in immunocompromised individuals. Existing antifungal treatment plans have high mammalian toxicity and increasing drug resistance, demonstrating the dire need for new, nontoxic therapeutics. Antimicrobial peptoids are one alternative to combat this issue. Our lab has recently identified a tripeptoid, AEC5, with promising efficacy and selectivity against C. neoformans. Here, we report studies into the broad-spectrum efficacy, killing kinetics, mechanism of action, in vivo half-life, and subchronic toxicity of this compound. Most notably, these studies have demonstrated that AEC5 rapidly reduces fungal burden, killing all viable fungi within 3 hours. Additionally, AEC5 has an in vivo half-life of 20+ hours and no observable in vivo toxicity following 28 days of daily injections. This research represents an important step in the characterization of AEC5 as a practical treatment option against C. neoformans infections.     

Lysine metabolism in a barley mutant resistant to S(2-aminoethyl)cysteine

Lysine and S(2-aminoethyl)cysteine (AEC) metabolism were investigated in normal barley (Hordeum vulgare L. cv. Bomi) and a hemozygous recessive AEC-resistant mutant (R906). Feedback regulation of lysine and threonine synthesis from [¹⁴C] acetate was unimpaired in plants of the mutant 3 d after germination. Seeds of Bomi and R906 contained similar total amounts of lysine, threonine, methionine and isoleucine. Concentrations of these amino acids in the soluble fraction of plants grown 6 d without AEC were also similar. The concentration of AEC in R906 plants was less than in the parent variety when both were grown in the presence of 0.25 mM AEC for 6 d. The uptake of [³H]AEC and [³H]lysine by roots of R906 was, respectively, 33% and 32% of that by Bomi roots whereas the uptake of these compounds into the scutellum was the same in both the mutant and its parent. The uptake of [³H]leucine and its incorporation into proteins was also the same in Bomi and R906 plants. These results suggest that a transport system specific for lysine and AEC but not leucine is altered or lost in roots of the mutant R906. AEC is incorporated into protein and this could be the reason for inhibition of growth rather than action as a false-feedback inhibitor of lysine biosynthesis.Abbreviations AEC S(2-aminoethyl)cysteine - LYS lysine - THR threonine     

The effect of season and location on phosphoadenylate concentrations and adenylate energy charge in two species of freshwater clams

Summary Concentrations of phosphoadenylate nucleotides and the adenylate energy charge ((ATP+1/2ADP)/(ATP+ADP+AMP)) have been suggested as sensitive integrating measures of the energy state of organisms. This synoptic study investigated the seasonal and spatial variation of phosphoadenylate concentrations and AEC in two freshwater bivalve molluscs, the paper-shell clam, Anodonta imbecillis and the asian clam, Corbicula fluminea. Concentrations of all three adenylates, as well as the total adenylate concentration and adenylate energy charge of both species varied seasonally. These fluctuations were closely related to reproductive periods in both species. Total adenylate concentrations and ATP concentrations were slightly negatively correlated with shell length in A. imbecillis but the ADP and AMP concentrations and AEC were not significantly correlated with shell length. In C. fluminea the AEC was negatively correlated were positively correlated with shell length. Neither species exhibited significant differences in AEC between two collection locations. When C. fluminea collected from the Savannah River were acclimated and fed in the laboratory their AEC increased significantly.     

General method for the derivation and numerical solution of epithelial transport models

Summary A general method is presented for the formulation and numerical evaluation of mathematical models describing epithelial transport. The method is based on the principles of conservation of mass, and maintenance of electroneutrality within the cells and bathing solutions. It is therefore independent of the specific membrane transport mechanisms, and can be used to evaluate different models describing arbitrary transport processes (including passive, active and cotransport processes). Detailed numerical methods are presented that allow computation of steady-state and transient responses under open-circuit, current-clamp and voltage-clamp conditions, using a general-purpose laboratory minicomputer. To evaluate the utility of this approach, a specific model is presented that is consistent with the Koefoed-Johnson and Ussing hypothesis of sodium transport in tight epithelia (Acta Physiol. Scand. 42:298–308, 1958). This model considers passive transport of an arbitrary number of permeant solutes, active transport of sodium and potassium, and osmotically induced water transport across the apical and basolateral membranes. Results of the model are compared to published experimental measurements in rabbit urinary bladder epithelium.