Multiple Forms Of Enzyme Research Articles

Chemical Composition of Seeds in Soybean Glycine soja (Fabaceae) of Amur Oblast.

The wild soybean Glycine soja Sieb. et Zucc. is an ancestor of the cultivated soybean Glycine max (L.) Merr. and a source of many valuable genes missing in the G. max genome, including genes that determine stress resistance to adverse environmental factors. Biochemical parameters (protein, oil, ascorbic acid, carotene, higher fatty acids, and specific activities and multiple forms of enzymes of the oxidoreductase and hydrolase classes) were studied in five G. soja accessions from the collection of the All-Russian Institute of Soybean (КА-1413, КА-342, КBl-29, КBl-24, and Kеl-72). The accessions provide unique natural gene banks. Wild seeds were collected in three districts (Arkharinskii, Blagoveshchensk, and Belogorskii) of Amur Oblast. Based on superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO), ribonuclease (RNase), acid phosphatase, esterase, and amylase (AML) activities and biochemical parameters of seeds, the G. soja accession KA-1413 was found to have higher contents of protein, oleic acid, and linolenic acid; a lower polyphenol oxidase specific activity; and higher activities of SODs, esterases, and RNases. The accession KA-1413 was therefore recommended to use as a source of dominant genes in breeding to increase the adaptive potential of new soybean varieties. A higher heterogeneity of multiple forms was observed for SOD, AML, RNase, and esterase, which can provide markers of adaptation to environmental conditions.

Oxidoreductase activity of soybeen seedlings at the early stage of ontogenesis

The results of analysis of specific activity and multiple forms of oxidoreductase class enzymes are presented: antioxidant complex (catalase and peroxidase) and dehydrogenases (alcohol dehydrogenase, NAD+-malate dehydrogenase, glucose-6-phosphate dehydrogenase). Seven varieties of soybean (Glycine max (L.) Merrill) served as the object of the study. Dormant seeds and 3- and 7-day-old soybean seedlings were used for the analysis. Protein content was determined by the Lowry method, peroxidase activity was determined by colorimetric, catalase and the studied dehydrogenases – by spectrophotometric methods, electrophoretic spectra of enzymes – by electrophoresis on 7.5% polyacrylamide gel columns. Identification of the zones with enzymatic activity on the gel was performed by appropriate histochemical methods. Analysis of specific activity of antioxidant enzymes in dormant soybean seeds revealed increased activity of peroxidase and low activity of catalase. During seed germination, the inverse relationship of the specific activity of these enzymes is observed. On the 7th day the specific activity of catalase increases, that of peroxidase decreases to a minimum. In soybean germination, 5 forms of catalase were detected, indicating low polymorphism and stability of the enzyme, and 18 forms of peroxidase, which confirm high polymorphism and the possibility of using this enzyme as a marker of biochemical processes. Alcohol dehydrogenase and glucose-6-phosphate dehydrogenase were found to be of low heterogeneity, with the specific activity of these enzymes decreasing during germination compared to the dormant period of soybean seeds. The specific activity of NAD+-malate dehydrogenase increases slightly during germination. Eight forms of this enzyme were detected in the soybean varieties studied indicating increased polymorphism. In dormant soybean seeds, the electrophoretic spectra of NAD+-malate dehydrogenase exhibited varietal diversity allowing the enzyme to be used as a marker for further studies.

Chemical Composition of <i>Glycine soja</i> (Fabaceae) Seeds from the Amur Region Territory

Abstract—Wild soybean Glycine soja Sieb. Zucc., the wild ancestor of the cultivated soybean Glycine max (L.) Merr., is the source of many valuable genes missing in the genotype of cultivated soybean, including stress resistance to adverse environmental factors. The biochemical parameters (protein, oil, ascorbic acid, carotene, higher fatty acids, specific activity and multiple forms of enzymes of the oxidoreductase and hydrolase classes) of five forms of wild soybeans from the collection of the All-Russian Research Institute of Soybean, which are unique natural gene banks, were studied. The wild seeds were collected for in three districts of the Amur Region (Arkharinsky, Blagoveshchensk, Belogorsky) and grown on the crop rotation field. The obtained results of enzymatic activity (superoxide dismutase, catalase, peroxidase, polyphenol oxidase, ribonuclease, acid phosphatase, esterase and amylase) and biochemical parameters of the studied seeds of wild soybean forms allowed us to identify the form KA-1413 with high biochemical parameters (protein, oleic and linolenic acids), a low specific polyphenol oxidase activity, and an increased activity of superoxide dismutases, esterases and ribonucleases. Thus, the wild soybean form KA-1413 can be recommended as a source of dominant genes, which will help to increase the adaptive potential of new soybean varieties. The increased heterogeneity of multiple forms of SOD, AML, RNase and esterase in wild soybean seeds can be used as adaptation markers to environmental conditions.

ROLE OF THE ANTIOXIDANT IN FUNCTIONING OF THE AGING MECHANISMS ON THE EXAMPLE OF BARLEY

Introduction. Despite close attention to the process of seed aging, there is still no unambiguous answer to questions about the mechanisms of aging. The solution of these issues is due to both economic needs and the need to store plant genetic resources. Aim. The aim was to study the effect of long-term storage of seeds of different barley varieties, previously differentiated by tolerance to seed aging, on the state of the antioxidant system enzymes. Methods. The studies were carried out on three varieties of barley (Hordeum vulgare L.) Rosava, Selena Star and Trudivnik which had been stored under non-specialized laboratory conditions in paper envelopes for 8 years. The seed viability was assessed by germination energy and germinating ability according to the state standards. The direct test was supplemented with a cytogenetic study: an anatelophase test. The condition of the antioxidant system was characterized by an electrophoretic method for studying nonspecific peroxidase and superoxide dismutase in germinated shoots. The number of multiple forms of enzymes, their relative electrophoretic mobility and specific gravity (share) in percent in the total spectrum were determined. Enzymatic activity was assessed by the area of the peaks of the corresponding multiple forms on the densitograms. Results. In terms of tolerance to long-term storage in direct and cytogenetic tests, the varieties were arranged as follows: Trudivnik > Rosava > Selena Star. It has been shown that the viability of seeds after storage is closely related to the activity of peroxidase and superoxide dismutase: more tolerant varieties are characterized by the stability of the total activity of peroxidase and superoxide dismutase. Conclusion. The specific activity of the medium-mobile peroxidase fraction and low- and medium-mobile SOD fractions plays a leading role in determining the viability of seeds during long-term storage.

Influence of Septoria glycines Hemmi on the enzymes’ activity of hydrolytic Glycines max (L.) Merr seed complex

The Septoria leaf spot (Septoria glycines Hemmi), is a harmful pathogen of soybean. To create a resistant variety to this pathogen, a detailed study of physiological processes at the biochemical level is necessary. Enzymes play a leading role in adapting to stressors. It was shown that the specific activity of esterases and RNases of soybean seeds infected with septoria decreased, relative to the control. Multiple forms of enzymes with different electrophoretic mobility were identified, which indicates a decrease in metabolism under the influence of fungal disease. At the same time, the specific activity of amylases and acid phosphatases of soybean seeds was not strongly affected by S. glycine, as evidenced by the relatively stable multiple forms of enzymes.

Biochemical adaptation of wild and cultivated soybean against toxicity of lead salts

Agricultural production is becoming increasingly dependent on the environmental factors that alter soil properties, plant productivity, and product quality. Environment pollution caused by heavy metals because of human activities are among the most dangerous pollutants on the biosphere. Here, we have studied the biochemical adaptation of wild and cultivated soybeans to the simulated effects of lead nitrate and lead acetate. Lead in the form of acetate had a relevant toxic effect, as evidenced by a significant increase in the concentration of malonic dialdehyde in the treated samples relative to control samples. Catalase and peroxidase, possibly performing a signaling function, are involved in the adaptation to the toxicity of Pb salts. The studied Pb salts showed a predominant stimulating effect on the specific activity of acid phosphatases in cultivated soybean, while the ribonuclease activity changed in both Glycine species. Moreover, in wild soybean, it was mostly suppressive, except for the first day. We found that the electrophoretic spectra of acid phosphatases of soybean seedlings was highly stabile, while that of ribonucleases varied depending on the salt. On the seventh day of exposure, lead nitrate caused a decrease in the specific activity of the studied hydrolases of seedlings of cultivated and wild soybeans. A change in the number or electrophoretic mobility of multiple forms of enzymes during treatment with Pb salts was revealed, which indicates the adaptation of the plants at the molecular genetic level. These results imply that the observed enzymes can be used as sensitive indicators for predicting the effects of heavy metals on soybean.

GENETIC AND BIOCHEMICAL ASPECTS OF THE ASSOCIATION OF THE PPD-D1 LOCUS WITH THE RESISTANCE OF WINTER SOFT WHEAT TO LOW TEMPERATURE

The aim of this work was to study the expression of some enzymes under the influence of unfavorable temperature conditions on plants that differ in allelic composition of Ppd genes. To accomplish this goal, the following tasks were set: 1) to carry out qualitative and quantitative analysis of electrophoregrams of multiple forms of antioxidant enzymes (catalase, peroxidase, superoxide dismutase); 2) to establish whether there is a relationship between the spectra of the enzyme under study and the tolerance to low temperatures and allelic composition of the Ppd-D1 gene.Material. Winter wheat varieties: Mironovskaya 808 (recessive alleles of the Ppd-D1 gene) and Tira (the dominant allele of the Ppd-D1 gene). Methods. Electrophoresis. Complex comparative analysis. Methods of descriptive statistics. Computer data processing.Results. It was shown that in varieties that differ in the composition of Ppd-D1 alleles and resistance to hypothermia gene-enzyme systems function in a different, often opposite way. Moreover, the difference was observed both in control conditions and under stress. This indicates that for the formation of adaptation, not only the peculiarities of the reaction of the metabolic reaction to adverse conditions, but also their initial state are important.In Mironovskaya 808 plants, unlike Tire, peroxidase plays a major role in neutralizing peroxide. This is manifested both in higher activity and in features of changes in enzyme expression during hypothermia, as well as in the ratio of peroxidase activity to the activity of other antioxidant enzymes. The leading role of peroxidase, due to its versatility, leads to a finer regulation of cellular metabolism during adaptation. In addition, in Mironovskaya 808, in response to the action of low temperature, the functioning of specific peroxidases, ascorbate peroxidase and ferulic-specific peroxidase, changes in the opposite way, compared to the variety Tira. The expression of such an important antioxidant enzyme as SOD also changes in different ways.Based on the data obtained, it can be assumed that Tira's system of protection against oxidative explosion in stress does not cope with the excess of superoxide radicals formed.

Inulinase immobilization on polyethylene glycol/polypyrrole multiwall carbon nanotubes producing a catalyst with enhanced thermal and operational stability.

This paper describes the development of a simple method for mixed non-covalent and covalent bonding of partially purified inulinase on functionalized multiwall carbon nanotubes (f-MWCNTs) with polypyrrole (PPy). The pyrrole (Py) was electrochemically polymerized on MWCNTs in order to fabricate MWCNTs/PPy nanocomposite. Two multiple forms of enzyme were bound to N-H functional groups from PPy and -COO- from activated MWCNTs to yield a stable MWCNTs/PPy/PEG immobilized preparation with increased thermal stability. Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) were used to confirm functionalization of nanoparticles and immobilization of the enzyme. The immobilization yield of 85% and optimal enzyme load of 345μg protein onto MWCNTs was obtained. The optimum reaction conditions and kinetic parameters were established using the UV-Vis analytical assay. The best functional performance for prepared heterogeneous catalyst has been observed at pH 3.6 and 10, and at the temperatures of 60 and 80ºC. The half-life (t 1/2) of the immobilized inulinase at 60 and 80ºC was found to be 231 and 99min, respectively. The reusability of the immobilized formulation was evaluated based on a method in which the enzyme retained 50% of its initial activity, which occurred after the eighteenth operation cycle.

Survey of Drug Oxidation Activities in Hepatic and Intestinal Microsomes of Individual Common Marmosets, a New Nonhuman Primate Animal Model.

Background: Common marmosets (Callithrix jacchus) are potentially useful non-human primate models for preclinical studies. Information for major drug-metabolizing cyto-chrome P450 (P450) enzymes is now available that supports the use of this primate species as an animal model for drug development. Here, we collect and provide an overview of infor-mation on the activities of common marmoset hepatic and intestinal microsomes with respect to 28 typical human P450 probe oxidations.Results: Marmoset P450 2D6/8-dependent R-metoprolol O-demethylation activities in hepatic microsomes were significantly correlated with those of midazolam 1′- and 4-hydroxylations, testosterone 6β-hydroxylation, and progesterone 6β-hydroxylation, which are probe reactions for marmoset P450 3A4/5/90. In marmosets, the oxidation activities of hepatic microsomes and intestinal microsomes were roughly comparable for midazolam and terfenadine. Overall, multiple forms of marmoset P450 enzymes in livers and intestines had generally similar sub-strate recognition functionalities to those of human and/or cynomolgus monkey P450 enzymes.Conclusion: The marmoset could be a model animal for humans with respect to the first-pass extraction of terfenadine and related substrates. These findings provide a foundation for un-derstanding individual pharmacokinetic and toxicological results in nonhuman primates as pre-clinical models and will help to further support understanding of the molecular mechanisms of human P450 function.

Molecular dynamics simulations of viral neuraminidase inhibitors with the human neuraminidase enzymes: Insights into isoenzyme selectivity

Inhibitors of viral neuraminidase enzymes have been previously developed as therapeutics. Humans can express multiple forms of neuraminidase enzymes (NEU1, NEU2, NEU3, NEU4) that share a similar active site and enzymatic mechanism with their viral counterparts. Using a panel of purified human neuraminidase enzymes, we tested the inhibitory activity of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir, oseltamivir, and peramivir against each of the human isoenzymes. We find that, with the exceptions of DANA and zanamivir, these compounds show generally poor activity against the human neuraminidase enzymes. To provide insight into the interactions of viral inhibitors with human neuraminidases, we conducted molecular dynamics simulations using homology models based on coordinates reported for NEU2. Simulations revealed that an organized water is displaced by zanamivir in binding to NEU2 and NEU3 and confirmed the critical importance of engaging the binding pocket of the C7–C9 glycerol sidechain. Our results suggest that compounds designed to target the human neuraminidases should provide more selective tools for interrogating these enzymes. Furthermore, they emphasize a need for additional structural data to enable structure-based drug design in these systems.

Serum Biochemical Profile of Nile Tilapias (Oreochromis niloticus) Bred in Net Cages during Summer and Winter

Background: The growth of fish farming in Brazil has been noticeable over the past few years, and the Nile tilapia (Oreochromis niloticus) is the most bred species in the country. Studies on the physiology of these animals are necessary so that the production continues to grow and to improve its rates. However, very discrepant data are still found in the area of blood biochemistry. The aim of this study was to evaluate the physiological variations in serum concentrations of proteins, metabolites, electrolytes and serum enzymes in Nile tilapias bred in net cages in Cachoeira Dourada, state of Minas Gerais, Brazil, during summer and winter.Materials, Methods & Results: During the year of 2016, samples from 100 Nile tilapias were collected during summer (January and February) and samples from 100 Nile tilapias were also collected during winter (July and August). Only one blood sample was collected by caudal venipuncture of each fish, just before the slaughter, always in the morning. The serum samples obtained were then analyzed for serum concentrations of proteins, metabolites, electrolytes, lipids and serum enzymes. Serum concentrations of total proteins, globulins, triglycerides, high density lipoprotein (HDL), alkaline phosphatase, sodium and chloride were significantly higher during winter in comparison to summer, while the serum concentration of uric acid was significantly lower during winter in comparison to summer.Discussion: Due to the lower temperature of water during winter, tilapias reduced food intake and became more susceptible to diseases, which led to an increase in circulating globulins. In that same season, there was an increase in serum concentrations of sodium and chloride as a result of adding sodium chloride to the water in order to stimulate mucus secretion on the fishes’ skin and gills to prevent pathologies. Triglycerides, HDL, and uric acid are considered indicators of nutritional status of animals. The increase in serum concentrations of triglycerides and HDL indicates that tilapias are mobilizing energy reserves, however, when the increase in serum concentrations of uric acid is observed, it is fair to assume there has been an increase in food intake, raising protein catabolism. The increase of serum concentration in alkaline phosphatase is caused by the presence of multiple forms of enzymes that are induced by the action of endogenous corticosteroids, which are produced as a result of the stress suffered by tilapias during the fishing process and during winter, when the water temperature is lower than the ideal for this species. It can be concluded that the season has influence on some serum biochemical constituents of Nile tilapias bred in net cages, and that the predominant variations happen during winter due to immunity decline and during summer due to the increase in food intake and, consequently, in the metabolism of tilapias.

Identification and analysis of OsttaDSP, a phosphoglucan phosphatase from Ostreococcus tauri.

Ostreococcus tauri, the smallest free-living (non-symbiotic) eukaryote yet described, is a unicellular green alga of the Prasinophyceae family. It has a very simple cellular organization and presents a unique starch granule and chloroplast. However, its starch metabolism exhibits a complexity comparable to higher plants, with multiple enzyme forms for each metabolic reaction. Glucan phosphatases, a family of enzymes functionally conserved in animals and plants, are essential for normal starch or glycogen degradation in plants and mammals, respectively. Despite the importance of O. tauri microalgae in evolution, there is no information available concerning the enzymes involved in reversible phosphorylation of starch. Here, we report the molecular cloning and heterologous expression of the gene coding for a dual specific phosphatase from O. tauri (OsttaDSP), homologous to Arabidopsis thaliana LSF2. The recombinant enzyme was purified to electrophoretic homogeneity to characterize its oligomeric and kinetic properties accurately. OsttaDSP is a homodimer of 54.5 kDa that binds and dephosphorylates amylopectin. Also, we also determined that residue C162 is involved in catalysis and possibly also in structural stability of the enzyme. Our results could contribute to better understand the role of glucan phosphatases in the metabolism of starch in green algae.

Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology

Millions of individuals are infected with and die from tuberculosis (TB) each year, and multidrug-resistant (MDR) strains of TB are increasingly prevalent. As such, there is an urgent need to identify novel drugs to treat TB infections. Current frontline therapies include the drug isoniazid, which inhibits the essential NADH-dependent enoyl-acyl-carrier protein (ACP) reductase, InhA. To inhibit InhA, isoniazid must be activated by the catalase-peroxidase KatG. Isoniazid resistance is linked primarily to mutations in the katG gene. Discovery of InhA inhibitors that do not require KatG activation is crucial to combat MDR TB. Multiple discovery efforts have been made against InhA in recent years. Until recently, despite achieving high potency against the enzyme, these efforts have been thwarted by lack of cellular activity. We describe here the use of DNA-encoded X-Chem (DEX) screening, combined with selection of appropriate physical properties, to identify multiple classes of InhA inhibitors with cell-based activity. The utilization of DEX screening allowed the interrogation of very large compound libraries (1011 unique small molecules) against multiple forms of the InhA enzyme in a multiplexed format. Comparison of the enriched library members across various screening conditions allowed the identification of cofactor-specific inhibitors of InhA that do not require activation by KatG, many of which had bactericidal activity in cell-based assays.

Apparent cooperativity and apparent hyperbolic behavior of enzyme mixtures acting on the same substrate

It is well known that a negative cooperative behavior displayed by a monomeric enzyme may be associated with the simultaneous presence of two enzymes acting on the same substrate. In this paper, emphasis is given to the effect exerted by a rapid equilibrium between the enzyme forms in leading to a hyperbolic behavior, thus masking the presence of multiple enzyme forms.

Regulation of human skin mast cell histamine release by PDE inhibitors

BackgroundMast cell and basophiles are thought to be central to inflammation that has an allergic basis as allergens activate these cells in an IgE-dependent manner to generate mediators such as histamine, eicosanoids and cytokines. Phosphodiesterase (PDE) is known to exist as multiple molecular forms of enzyme that metabolise the second messengers. Studies of our own have shown that, of a variety of isoform-selective drugs, the PDE4-selective inhibitors, such as rolipram, attenuate the IgE-mediated release of histamine from human basophiles but not from human lung mast cells (HLMC).The main aim of the present study was to characterise the type and role of PDEs regulating human skin mast cells by using selective and non-selective PDE inhibitors. MethodsCells were pre-treated for 15min with these agents and then challenged with an optimal releasing concentration of anti IgE (1:300) for a further 25min for the release of histamine. ResultsThe data show that all the selective PDE-inhibitor compounds (10−5M) were ineffective whereas the non-selective PDE inhibitor, theophylline (10−3M), inhibited histamine release from HSMC (74±4% inhibition; p<0.05). None of the selective PDE inhibitors had any effect on histamine release from HLMC whereas, in basophiles, compounds with activity at PDE 4 (rolipram, denbufylline, Ro-2017, Org 30029) were effective inhibitors of histamine release. ConclusionThe data suggest that unlike most inflammatory cells, PDE-selective inhibitors are ineffective stabilisers of HSMC activity which is similar to HLMC.

Gag-Pol Processing during HIV-1 Virion Maturation: A Systems Biology Approach

Proteolytic processing of Gag and Gag-Pol polyproteins by the viral protease (PR) is crucial for the production of infectious HIV-1, and inhibitors of the viral PR are an integral part of current antiretroviral therapy. The process has several layers of complexity (multiple cleavage sites and substrates; multiple enzyme forms; PR auto-processing), which calls for a systems level approach to identify key vulnerabilities and optimal treatment strategies. Here we present the first full reaction kinetics model of proteolytic processing by HIV-1 PR, taking into account all canonical cleavage sites within Gag and Gag-Pol, intermediate products and enzyme forms, enzyme dimerization, the initial auto-cleavage of full-length Gag-Pol as well as self-cleavage of PR. The model allows us to identify the rate limiting step of virion maturation and the parameters with the strongest effect on maturation kinetics. Using the modelling framework, we predict interactions and compensatory potential between individual cleavage rates and drugs, characterize the time course of the process, explain the steep dose response curves associated with PR inhibitors and gain new insights into drug action. While the results of the model are subject to limitations arising from the simplifying assumptions used and from the uncertainties in the parameter estimates, the developed framework provides an extendable open-access platform to incorporate new data and hypotheses in the future.

Isoliquiritigenin induces growth inhibition and apoptosis through downregulating arachidonic acid metabolic network and the deactivation of PI3K/Akt in human breast cancer

Arachidonic acid (AA)-derived eicosanoids and its downstream pathways have been demonstrated to play crucial roles in growth control of breast cancer. Here, we demonstrate that isoliquiritigenin, a flavonoid phytoestrogen from licorice, induces growth inhibition and apoptosis through downregulating multiple key enzymes in AA metabolic network and the deactivation of PI3K/Akt in human breast cancer. Isoliquiritigenin diminished cell viability, 5-bromo-2′-deoxyuridine (BrdU) incorporation, and clonogenic ability in both MCF-7 and MDA-MB-231cells, and induced apoptosis as evidenced by an analysis of cytoplasmic histone-associated DNA fragmentation, flow cytometry and hoechst staining. Furthermore, isoliquiritigenin inhibited mRNA expression of multiple forms of AA-metabolizing enzymes, including phospholipase A2 (PLA2), cyclooxygenases (COX)-2 and cytochrome P450 (CYP) 4A, and decreased secretion of their products, including prostaglandin E2 (PGE2) and 20-hydroxyeicosatetraenoic acid (20-HETE), without affecting COX-1, 5-lipoxygenase (5-LOX), 5-lipoxygenase activating protein (FLAP), and leukotriene B4 (LTB4). In addition, it downregulated the levels of phospho-PI3K, phospho-PDK (Ser241), phospho-Akt (Thr308), phospho-Bad (Ser136), and Bcl-xL expression, thereby activating caspase cascades and eventually cleaving poly(ADP-ribose) polymerase (PARP). Conversely, the addition of exogenous eicosanoids, including PGE2, LTB4 and a 20-HETE analog (WIT003), and caspase inhibitors, or overexpression of constitutively active Akt reversed isoliquiritigenin-induced apoptosis. Notably, isoliquiritigenin induced growth inhibition and apoptosis of MDA-MB-231 human breast cancer xenografts in nude mice, together with decreased intratumoral levels of eicosanoids and phospho-Akt (Thr308). Collectively, these data suggest that isoliquiritigenin induces growth inhibition and apoptosis through downregulating AA metabolic network and the deactivation of PI3K/Akt in human breast cancer.

Biochemical Analyses of Multiple Endoxylanases from the Rumen Bacterium Ruminococcus albus 8 and Their Synergistic Activities with Accessory Hemicellulose-Degrading Enzymes

Ruminococcus albus 8 is a ruminal bacterium capable of metabolizing hemicellulose and cellulose, the major components of the plant cell wall. The enzymes that allow this bacterium to capture energy from the two polysaccharides, therefore, have potential application in plant cell wall depolymerization, a process critical to biofuel production. For this purpose, a partial genome sequence of R. albus 8 was generated. The genomic data depicted a bacterium endowed with multiple forms of plant cell wall-degrading enzymes. The endoxylanases of R. albus 8 exhibited diverse modular architectures, including incorporation of a catalytic module, a carbohydrate binding module, and a carbohydrate esterase module in a single polypeptide. The accessory enzymes of xylan degradation were a β-xylosidase, an α-l-arabinofuranosidase, and an α-glucuronidase. We hypothesized that due to the chemical complexity of the hemicellulose encountered in the rumen, the bacterium uses multiple endoxylanases, with subtle differences in substrate specificities, to attack the substrate, while the accessory enzymes hydrolyze the products to simple sugars for metabolism. To test this hypothesis, the genes encoding the predicted endoxylanases were expressed, and the proteins were biochemically characterized either alone or in combination with accessory enzymes. The different endoxylanase families exhibited different patterns of product release, with the family 11 endoxylanases releasing more products in synergy with the accessory enzymes from the more complex substrates. Aside from the insights into hemicellulose degradation by R. albus 8, this report should enhance our knowledge on designing effective enzyme cocktails for release of fermentable sugars in the biofuel industry.

Redox homeostasis and respiratory metabolism in camels (Camelus dromedaries): comparisons with domestic goats and laboratory rats and mice

We have previously reported the occurrence of multiple forms of drug-metabolizing enzymes in camel tissues. Here, we investigate glutathione (GSH)-dependent redox homeostasis, reactive oxygen species (ROS) production and mitochondrial respiratory functions in camel tissues and compare them with imported domestic goats and laboratory rats and mice. Cytochrome P450 2E1 (CYP 2E1) and GSH-metabolizing enzymes were differentially expressed in the liver and kidney of these animals. Camel liver has significantly lower GSH pool than that in goats, rats and mice. Mitochondria isolated from the tissues of these animals showed a comparable ability to metabolize specific substrates for respiratory enzyme complexes I, II/III and IV. These complexes were metabolically more active in the kidney than in the liver of all the species. Furthermore, the activity of complex IV in camel tissues was significantly lower than in other species. On the other hand, complex II/III activity in camel kidney was higher compared to the other species. In addition, as expected, we observed that inhibitors of these enzyme complexes augment the production of mitochondrial ROS in camel and goat tissues. These results help to better understand the metabolic ability and adaptation in desert camels in comparison with domestic goats and laboratory rats and mice since they are exposed to different environmental and dietary conditions. Our study may also have implications in the pharmacology and toxicology of drugs and pollutants in these species.

Estimation of spectra of multiple molecular forms of enzymes using the index of system internal diversity level

Comparative analysis of the most widely used indexes of system diversity is carried out using model electrophoretic spectra. The basic requirements for the diversity index of electrophoretin spectra are formulated. A formula for estimating the level of spectra diversity of multiple forms on the basis of synthesis of the Ashby system complexity index and Simpson index is offered.