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Hydrolytic Enzymes (hydrolytic + enzyme)
Terms modified by Hydrolytic Enzymes Selected AbstractsThermotropic Lipid Phase Transition and the Behavior of Hydrolytic Enzymes in the Kidney Cortex Brush Border MembraneCHEMISTRY & BIODIVERSITY, Issue 10 2006Sankar Abstract Functional interactions of lipids and proteins were examined in brush-border membranes isolated from the kidney cortex by studying the temperature dependence of the hydrolytic enzyme activities. A close relationship was observed for the membrane proteins and the thermotropic lipid phase transitions. Three lines of evidences were provided for such dependence: a) Arrhenius relationship of the membrane-bound enzyme activities, and the effect of temperature in native and partially delipidated membranes, b) differential scanning calorimetric study of the membrane lipid phase transitions in the native and delipidated membranes, multilamellar vesicles prepared from the membrane extracted lipids, and in vesicles from dimyristoyl phosphatidylcholine, and c) the excimer (dimer)-formation studies of the membrane extrinsic fluorescent probe, pyrene, and the resultant membrane microviscosity. The brush-border membranes were partially delipidated with BuOH and 2,2,2-trifluoroethanol. The functional interactions of the delipidated membranes, which were greatly lost on lipid removal, were largely restored by the addition of exogenous lipids in the reconstitution process, which indicate the critical dependence of the membrane integral proteins on the neighboring lipid molecules in the bulk lipid phase. [source] Hydrolytic enzymes as virulence factors of Candida albicansMYCOSES, Issue 6 2005Martin Schaller Summary Candida albicans is a facultative pathogenic micro-organism that has developed several virulence traits enabling invasion of host tissues and avoidance of host defence mechanisms. Virulence factors that contribute to this process are the hydrolytic enzymes. Most of them are extracellularly secreted by the fungus. The most discussed hydrolytic enzymes produced by C. albicans are secreted aspartic proteinases (Saps). The role of these Saps for C. albicans infections was carefully evaluated in numerous studies, whereas only little is known about the physiological role of the secreted phospholipases (PL) and almost nothing about the involvement of lipases (Lip) in virulence. They may play an important role in the pathogenicity of candidosis and their hydrolytic activity probably has a number of possible functions in addition to the simple role of digesting molecules for nutrition. Saps as the best-studied member of this group of hydrolytic enzymes contribute to host tissue invasion by digesting or destroying cell membranes and by degrading host surface molecules. There is also some evidence that hydrolytic enzymes are able to attack cells and molecules of the host immune system to avoid or resist antimicrobial activity. High hydrolytic activity with broad substrate specificity has been found in several Candida species, most notably in C. albicans. This activity is attributed to multigene families with at least 10 members for Saps and Lips and several members for PL B. Distinct members of these gene families are differentially regulated in various Candida infections. In future, prevention and control of Candida infections might be achieved by pharmacological or immunological tools specifically modulated to inhibit virulence factors, e.g. the family of Saps. [source] Novel role for aspartoacylase in regulation of BDNF and timing of postnatal oligodendrogenesisJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2006Jeremy S. Francis Abstract Neuronal growth factors are thought to exert a significant degree of control over postnatal oligodendrogenesis, but mechanisms by which these factors coordinateoligodendrocyte development with the maturation of neural networks are poorly characterized. We present here a developmental analysis of aspartoacylase (Aspa)-null tremor rats and show a potential role for this hydrolytic enzyme in the regulation of a postnatal neurotrophic stimulus that impacts on early stages of oligodendrocyte differentiation. Abnormally high levels of brain-derived neurotrophic factor (BDNF) expression in the Aspa -null Tremor brain are associated with dysregulated oligodendrogenesis at a stage in development normally characterized by high levels of Aspa expression. BDNF promotes the survival of proliferating cells during the early stages of oligodendrocyte maturation in vitro, but seems to compromise the ability of these cells to populate the cortex in vivo. Aspartoacylase activity in oligodendrocytes is shown to provide for the negative regulation of BDNF in neurons, thereby determining the availability of a developmental stimulus via a mechanism that links oligodendroglial differentiation with neuronal maturation. © 2006 Wiley-Liss, Inc. [source] Assessing the use of a dietary probiotic/prebiotic as an enhancer of spinefoot rabbitfish Siganus rivulatus survival and growthAQUACULTURE NUTRITION, Issue 6 2007A.Y. EL-DAKAR Abstract The use of prebiotics and probiotics as feed supplements that improve efficiency of intestinal bacteria is becoming de rigueur in animal husbandry in many regions worldwide. We tested the effects of a commercial probiotic (Biogen®) containing allicin, high unit hydrolytic enzyme, Bacillus subtilis spores and ginseng extracts on survival, growth, carcass composition and feed cost/benefit in rabbitfish Siganus rivulatus. Fifteen net cages (100 × 100 × 40 cm; L × W × H) were stocked with 10 juvenile rabbitfish (10.3 g per fish) each and placed in a large rectangular tank and offered feed at 4% body weight daily. Cages were offered one of five isonitrogenous and isocaloric diets containing 0, 1, 2, 3 and 4 g kg,1 probiotic at three replicates per treatment for 98 days. Fish in all cages were weighed at 2-week intervals and feed regimen was adjusted accordingly. Rabbitfish offered the control diet exhibited lower growth and feed utilization than all experimental treatments. There was no effect of probiotic inclusion level on survival but growth was better at all inclusion levels than in the control. No significant differences (P > 0.05) in growth were observed among fish groups fed various levels of the probiotic. Carcass composition was not affected by dietary probiotic inclusion. Ultimately, when all variables are considered, Biogen® inclusion to diets appears to reduce feed cost per unit growth of rabbitfish. [source] Xylanases, xylanase families and extremophilic xylanasesFEMS MICROBIOLOGY REVIEWS, Issue 1 2005Tony Collins Abstract Xylanases are hydrolytic enzymes which randomly cleave the , 1,4 backbone of the complex plant cell wall polysaccharide xylan. Diverse forms of these enzymes exist, displaying varying folds, mechanisms of action, substrate specificities, hydrolytic activities (yields, rates and products) and physicochemical characteristics. Research has mainly focused on only two of the xylanase containing glycoside hydrolase families, namely families 10 and 11, yet enzymes with xylanase activity belonging to families 5, 7, 8 and 43 have also been identified and studied, albeit to a lesser extent. Driven by industrial demands for enzymes that can operate under process conditions, a number of extremophilic xylanases have been isolated, in particular those from thermophiles, alkaliphiles and acidiphiles, while little attention has been paid to cold-adapted xylanases. Here, the diverse physicochemical and functional characteristics, as well as the folds and mechanisms of action of all six xylanase containing families will be discussed. The adaptation strategies of the extremophilic xylanases isolated to date and the potential industrial applications of these enzymes will also be presented. [source] Geranyl acetate esterase is commonly present but linalyl acetate esterase occurrence is highly limited in plantsFLAVOUR AND FRAGRANCE JOURNAL, Issue 3 2007Neelam S. Sangwan Abstract Esterases are a group of hydrolytic enzymes that split ester bonds by addition of water and are ubiquitously present in diverse biosystems. Although animal esterases are well studied and are catalytically and functionally classified into different groups, plant enzymes have been viewed rather generally and are casually recruited as biochemical markers in morphogenesis, genetic characterization of plants, etc., without functional emphasis. Some volatile oil plants constitutively synthesize their characteristic monoterpene esters, geranyl acetate and linalyl acetate being the most common among them in the acyclic monoterpene class, whereas other plants also synthesize some volatile hemi- to sesquiterpene esters but inductively under certain ecological situations, such as herbivory, wounding, etc. This study concerns screening relative distribution of geranyl acetate esterase and linalyl aceate esterase activities in selected medicinal and aromatic plants, and reveals that in plants geranyl acetate (a primary alcohol ester) esterase is commonly present, while linalyl acetate (a tertiary alcohol ester) esterase seems to be highly limited to those plants (e.g. Lippia alba, Mentha citrata) that biosynthesize the tertiary monoterpene alcohol linalool and its ester. Such contrasting distribution of the two discrete types of esterases has been discussed in light of scenario of their microbial counterparts and structure,function relationships established thereon. This study makes it obvious that the GGG(A)-X motif esterases (acting on tertiary alcohol esters) are rare entities in plants too, similar to microbes. Furthermore, their presence in some volatile oil plants renders such plants novel phytoresources of the GGGX/GGAX motif hydrolases. Detailed characterization of the motif-specific plant esterases would have an immense impact on understanding of their structure,function relationships in plants. Copyright © 2007 John Wiley & Sons, Ltd. [source] Drug metabolism and disposition in childrenFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 3 2003M. Strolin Benedetti Abstract Key factors undergoing maturational changes accounting for differences in drug metabolism and disposition in the pediatric population compared with adults are reviewed. Gastric and duodenal pH, gastric emptying time, intestinal transit time, bacterial colonization and probably P-glycoprotein are important factors for drug absorption, whereas key factors explaining differences in drug distribution between the pediatric population and adults are membrane permeability, plasma protein concentration and plasma protein characteristics, endogenous substances in plasma, total body and extracellular water, fat content, regional blood flow and probably P-glycoprotein, mainly that present in the gut, liver and brain. As far as drug metabolism is concerned, important differences have been found in the pediatric population compared with adults both for phase I enzymes [oxidative (e.g. cytochrome CYP3A7 vs. CYP3A4 and CYP1A2), reductive and hydrolytic enzymes] and phase II enzymes (e.g. N -methyltransferases and glucuronosyltransferases). Finally, key factors undergoing maturational changes accounting for differences in renal excretion in the pediatric population compared with adults are glomerular filtration and tubular secretion. It would be important to generate information on the developmental aspects of renal P-glycoprotein and of other renal transporters as done and still being done with the different isozymes involved in drug metabolism. [source] Seasonal variation in enzyme activities and temperature sensitivities in Arctic tundra soilsGLOBAL CHANGE BIOLOGY, Issue 7 2009MATTHEW D. WALLENSTEIN Abstract Arctic soils contain large amounts of organic matter due to very slow rates of detritus decomposition. The first step in decomposition results from the activity of extracellular enzymes produced by soil microbes. We hypothesized that potential enzyme activities are low relative to the large stocks of organic matter in Arctic tundra soils, and that enzyme activity is low at in situ temperatures. We measured the potential activity of six hydrolytic enzymes at 4 and 20 °C on four sampling dates in tussock, intertussock, shrub organic, and shrub mineral soils at Toolik Lake, Alaska. Potential activities of N -acetyl glucosaminidase, ,-glucosidase, and peptidase tended to be greatest at the end of winter, suggesting that microbes produced enzymes while soils were frozen. In general, enzyme activities did not increase during the Arctic summer, suggesting that enzyme production is N-limited during the period when temperatures would otherwise drive higher enzyme activity in situ. We also detected seasonal variations in the temperature sensitivity (Q10) of soil enzymes. In general, soil enzyme pools were more sensitive to temperature at the end of the winter than during the summer. We modeled potential in situ,-glucosidase activities for tussock and shrub organic soils based on measured enzyme activities, temperature sensitivities, and daily soil temperature data. Modeled in situ enzyme activity in tussock soils increased briefly during the spring, then declined through the summer. In shrub soils, modeled enzyme activities increased through the spring thaw into early August, and then declined through the late summer and into winter. Overall, temperature is the strongest factor driving low in situ enzyme activities in the Arctic. However, enzyme activity was low during the summer, possibly due to N-limitation of enzyme production, which would constrain enzyme activity during the brief period when temperatures would otherwise drive higher rates of decomposition. [source] Enzymes involved in flavour formation by bacteria isolated from the smear population of surface-ripened cheeseINTERNATIONAL JOURNAL OF DAIRY TECHNOLOGY, Issue 1 2004A G Williams Twenty-five bacterial isolates recovered from the surface population of smear-ripened cheese were assigned phenotypically as Brevibacterium spp., Corynebacterium spp. and Aureobacterium spp. using the Biolog GP2 microplate system and database. The range and activity of hydrolytic enzymes involved in the formation of cheese flavour constituents were monitored in cell-free lysates of the isolates. Esterase activity and the presence of a range of enzymes involved in amino acid release and breakdown was confirmed in all strains examined although there were pronounced interspecies and strain differences in the level of activity detected. Peptidolytic activities present in the smear bacteria included dipeptidyl peptidase and aminopeptidases that cleaved various N-terminal amino acids including proline. Subsequent breakdown of the released aromatic and branched-chain amino acids was mediated by ,-keto acid dependent aminotransferase action and several of the isolates were able to form thiols from sulphur-containing amino acid precursors. It was confirmed that the enzymic activity of the smear population could be manipulated by the use of defined starter cultures comprising selected combinations of smear isolates. The hydrolytic activities of the smear bacteria are involved in the generation of cheese flavour compounds and the enzyme profile is thus an important selection criterion for strains to be evaluated for use in defined surface smear preparations. [source] Enzymes involved in the bioconversion of ester-based prodrugsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 6 2006Bianca M. Liederer Abstract Enzymes are essential for the activation of many prodrugs. In this review, the most important enzymes (e.g., paraoxonase, carboxylesterase, acetylcholinesterase, cholinesterase) involved in the bioconversion of ester-based prodrugs will be discussed in terms of their biology and biochemistry. Most of these enzymes fall into the category of hydrolytic enzymes. However, nonhydrolytic enzymes, including cytochrome P450s, can also catalyze the bioconversion of ester prodrugs and thus will be discussed here. Other factors influencing the ability of these enzymes to catalyze the bioconversion of ester-based prodrugs, particularly species and interindividual differences and stereochemical and structural features of the prodrugs, will be discussed. © 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:1177,1195, 2006 [source] Development of a Sensitive Serological Method for Specific Detection of Latent Infection of Macrophomina phaseolina in CowpeaJOURNAL OF PHYTOPATHOLOGY, Issue 1 2009Leonard Afouda Abstract A double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific detection and quantification of Macrophomina phaseolina in plant tissue. Both polyclonal antisera produced against immunogens from mycelium and culture filtrate of M. phaseolina detected the fungus in mycelial and plant extracts, although the antibodies raised against mycelium were more sensitive. No cross-reaction occurred with Rhizopus stolonifer, Pythium ultimum, Mucor hiemalis, Fusarium oxysporum, Septoria nodorum, Rhizoctonia solani, Sclerotinia sclerotiorum, Phytophthora infestans and Aspergillus niger. In enzyme assays, activity of the endo-acting hydrolytic enzymes 1,3-,-glucanase and, less, cellulase, but not xylanase was detected in infected plants. DAS-ELISA was more sensitive than the 1,3-,-glucanase assay. In polyacrylamide gel electrophoresis (PAGE) up to 18 protein bands were observed, with four bands occurring in the 12 tested isolates deriving from various geographical origin in Niger and Nigeria. The enzyme assays and protein patterns were considered not suitable for specific M. phaseolina detection. Macrophomina phaseolina was essentially located in the roots and hypocotyls, and less in epicotyls and leaves of infected plants. The antibodies were also useful to detect latent infection and the infection of cowpea seeds. [source] Trichoderma enzymes promote Fibrobacter succinogenes S85 adhesion to, and degradation of, complex substrates but not pure cellulose,JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 10 2004Diego P Morgavi Abstract The effects of an enzyme preparation from Trichoderma longibrachiatum (TE) on adhesion and growth of the fibrolytic rumen bacterium Fibrobacter succinogenes S85 was studied to gain a better understanding of the action of feed enzyme additives on fibre digestion by ruminants. Adhesion experiments were performed on crystalline cellulose, corn silage and alfalfa hay. Adhesion of F succinogenes to cellulose was negatively related to the concentration of TE (p < 0.05). At the highest concentration used, TE reduced adhesion to cellulose from 65 to 39%. For corn silage and alfalfa hay, TE stimulated adhesion at low levels (p < 0.05) but this effect was lost at higher levels. Culture experiments were performed on crystalline cellulose and corn silage. The presence of TE in media containing cellulose failed to increase substrate disappearance or gas production although it increased numbers of non-adherent bacteria (p < 0.05). When corn silage was used, the addition of TE increased NDF disappearance (p < 0.05) at 24 and 48 h (33 and 52% in controls versus 53 and 65% in TE treatments). Growth rate and gas production were also stimulated (p < 0.05). We conclude that, for cellulose, the hydrolytic enzymes in TE obstructed available binding sites decreasing bacterial adherence. Fibrobacter succinogenes digested cellulose efficiently and addition of exogenous cellulases did not further increase substrate disappearance. However, for complex plant substrates, low concentration of TE increased bacterial adhesion and plant (corn) fiber degradation. For the Department of Agriculture and Agri-Food, Government of Canada, © Minister of Public Works and Government Services Canada 2004. Published for SCI by John Wiley & Sons, Ltd. [source] Starch,poly(,-caprolactone) and starch,poly(lactic acid) fibre-mesh scaffolds for bone tissue engineering applications: structure, mechanical properties and degradation behaviourJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 5 2008M. E. Gomes Abstract In scaffold-based tissue engineering strategies, the successful regeneration of tissues from matrix-producing connective tissue cells or anchorage-dependent cells (e.g. osteoblasts) relies on the use of a suitable scaffold. This study describes the development and characterization of SPCL (starch with ,-polycaprolactone, 30:70%) and SPLA [starch with poly(lactic acid), 30:70%] fibre-meshes, aimed at application in bone tissue-engineering strategies. Scaffolds based on SPCL and SPLA were prepared from fibres obtained by melt-spinning by a fibre-bonding process. The porosity of the scaffolds was characterized by microcomputerized tomography (µCT) and scanning electron microscopy (SEM). Scaffold degradation behaviour was assessed in solutions containing hydrolytic enzymes (,-amylase and lipase) in physiological concentrations, in order to simulate in vivo conditions. Mechanical properties were also evaluated in compression tests. The results show that these scaffolds exhibit adequate porosity and mechanical properties to support cell adhesion and proliferation and also tissue ingrowth upon implantation of the construct. The results of the degradation studies showed that these starch-based scaffolds are susceptible to enzymatic degradation, as detected by increased weight loss (within 2 weeks, weight loss in the SPCL samples reached 20%). With increasing degradation time, the diameter of the SPCL and SPLA fibres decreases significantly, increasing the porosity and consequently the available space for cells and tissue ingrowth during implantation time. These results, in combination with previous cell culture studies showing the ability of these scaffolds to induce cell adhesion and proliferation, clearly demonstrate the potential of these scaffolds to be used in tissue engineering strategies to regenerate bone tissue defects. Copyright © 2008 John Wiley & Sons, Ltd. [source] Experimental Models To Investigate Inflammatory Processes in Chronic Venous InsufficiencyMICROCIRCULATION, Issue S1 2000RONALD J. KORTHUIS ABSTRACT Chronic venous insufficiency (CVI) is characterized by leukocyte adhesion and infiltration, venous hypertension and dilatation, and valvular dysfunction. The fact that activated white cells can direct a powerful cytotoxic arsenal at parenchymal cells following their extravasation into the tissues led to the original proposal that leukocytes may play a causative role in the pathogenesis of venous disease. A large body of subsequent work indicates that white blood cells are indeed activated in CVI. However, identification of the factors responsible for initiating leukosequestration and activation in such disorders and determination of whether these activated cells then contribute to the progression of venous disease have been hampered by the lack of appropriate animal models that accurately mimic the human condition. Tantalizing evidence suggesting that cyclical periods of ischemia and reperfusion (I/R) may occur in diseased regions of the skin is beginning to accumulate. As is the case with CVI, leukocyte infiltration is a prominent feature in I/R and activated neutrophils play a causative role in the reperfusion component of tissue injury via the targeted release of reactive oxygen metabolites and hydrolytic enzymes. In light of these considerations, many investigators have suggested that examining the mechanisms of I/R injury in skin and skeletal muscle, where ischemia is produced by arterial occlusion, may provide a relevant model for studying the pathogenesis of CVI. Others have suggested that venous occlusion may represent a more appropriate model, as this approach also produces the venous hypertension that is characteristic of the disease. The purpose of this review is to summarize the evidence pointing to the involvement of I/R and venous hypertension as causative factors in CVI-induced leukocyte recruitment. In addition, we will describe the evidence in favor of the view that white blood cells contribute to the pathogenesis of CVI. Finally, we will describe several different experimental models that have been used to examine the role of I/R-induced microvascular dysfunction as it may pertain to the development of CVI, together with a discussion of the relative advantages and limitations of the various models. [source] The protein secretory pathway of Candida albicansMYCOSES, Issue 4 2009William A. Fonzi Summary Virulence of the opportunistic pathogen, Candida albicans, relies on an assemblage of attributes. These include the secretion of hydrolytic enzymes, cell surface adhesins, morphological transition between yeast and hyphae, phenotypic switching and biofilm formation. These diverse features are united by their dependence on the protein secretory apparatus for expression. Although the secretory apparatus of C. albicans has been studied limitedly, it appears to conform to the well-conserved eukaryotic system of vesicle-mediated transport between intracellular compartments and the cell surface. Genome comparison with Saccharomyces cerevisiae, however, shows multiple differences whose functional significance is yet unstudied. A unique aspect of the secretory pathway of C. albicans is its structural, and perhaps functional, rearrangement in hyphal vs. yeast cells. This, and evidence of non-conserved secretion mechanism(s), suggest that there is much fundamental knowledge to be derived from the analysis of secretion in C. albicans, which will be relevant to its ability to cause disease. [source] Hydrolytic enzymes as virulence factors of Candida albicansMYCOSES, Issue 6 2005Martin Schaller Summary Candida albicans is a facultative pathogenic micro-organism that has developed several virulence traits enabling invasion of host tissues and avoidance of host defence mechanisms. Virulence factors that contribute to this process are the hydrolytic enzymes. Most of them are extracellularly secreted by the fungus. The most discussed hydrolytic enzymes produced by C. albicans are secreted aspartic proteinases (Saps). The role of these Saps for C. albicans infections was carefully evaluated in numerous studies, whereas only little is known about the physiological role of the secreted phospholipases (PL) and almost nothing about the involvement of lipases (Lip) in virulence. They may play an important role in the pathogenicity of candidosis and their hydrolytic activity probably has a number of possible functions in addition to the simple role of digesting molecules for nutrition. Saps as the best-studied member of this group of hydrolytic enzymes contribute to host tissue invasion by digesting or destroying cell membranes and by degrading host surface molecules. There is also some evidence that hydrolytic enzymes are able to attack cells and molecules of the host immune system to avoid or resist antimicrobial activity. High hydrolytic activity with broad substrate specificity has been found in several Candida species, most notably in C. albicans. This activity is attributed to multigene families with at least 10 members for Saps and Lips and several members for PL B. Distinct members of these gene families are differentially regulated in various Candida infections. In future, prevention and control of Candida infections might be achieved by pharmacological or immunological tools specifically modulated to inhibit virulence factors, e.g. the family of Saps. [source] Enzymatic stability of 2,-ethylcarbonate-linked paclitaxel in serum and conversion to paclitaxel by rabbit liver carboxylesterase for use in prodrug/enzyme therapyBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 5 2008Tadatoshi Tanino Abstract In prodrug/enzyme therapy for cancer, information on the sensitivity of hydrolytic enzymes to prodrug is required to reduce adverse effects of the parental drug and to find the activating enzyme. The aim of this study was to characterize the enzymatic stability of 2,-ethylcarbonate-linked paclitaxel (TAX-2,-Et) in the sera of several different species including humans. TAX-2,-Et disposition in serum was kinetically analysed using models with hydrolytic and/or degradation processes. To further evaluate the capability of liver carboxylesterases (CESs) in TAX-2,-Et hydrolysis, a CES isolated from rabbit liver (Ra-CES) was utilized as a model enzyme. Rat serum provided rapid enzymatic hydrolysis of TAX-2,-Et with a half-life of 4 min. The degradation of paclitaxel (TAX) (degradation rate constant, 0.16,h,1) was accompanied by the formation of an unknown compound. The conversion to TAX was almost completely inhibited by phenylmethyl sulfonylfluoride (PMSF) and bis(p-nitrophenyl) phosphate (BNPP). In human and rabbit sera, the degradation rate constant of TAX-2,-Et was 5.1,×,10,2 and 0.15,h,1, respectively, when excepting hydrolysis. The degradation products had the same molecular weight as TAX-2,-Et. The amount of TAX produced accounted for only 8,11% of the decrease in TAX-2,-Et after a 9 h exposure to rabbit or human serum. PMSF, but not BNPP, inhibited more than 90% of the TAX production in a 1.5,h incubation with human or rabbit serum. Ra-CES enzyme converted TAX-2,-Et to TAX with Vmax and Km of 74.7±13.8 nmol/min/mg protein and 8.8±2.8 µM, respectively. These results indicate that TAX-2,-Et is sensitive to serum CESs, but not cholinesterases. However, serum CESs show species-dependent hydrolysis of TAX-2,-Et. Although human serum allows the slow release of TAX, TAX-2,-Et is expected to reduce the side-effects of TAX. The Ra-CES enzyme is capable of hydrolysing TAX-2,-Et, which may be beneficial for the development of a TAX-2,-Et/enzyme therapy strategy for ovarian cancer. Copyright © 2008 John Wiley & Sons, Ltd. [source] Modeling the Inhibitor Activity and Relative Binding Affinities in Enzyme-Inhibitor-Protein Systems: Application to Developmental Regulation in a PG-PGIP SystemBIOTECHNOLOGY PROGRESS, Issue 3 2004Wayne W. Fish Within a number of classes of hydrolytic enzymes are certain enzymes whose activity is modulated by a specific inhibitor-protein that binds to the enzyme and forms an inactive complex. One unit of a specific inhibitor-protein activity is often defined as the amount necessary to inhibit one unit of its target enzyme by 50 %. No objective quantitative means is available to determine this point of 50 % inhibition in crude systems such as those encountered during purification. Two models were derived: the first model is based on an irreversible binding approximation, and the second, or equilibrium, model is based on reversible binding. The two models were validated using the inhibition data for the polygalacturonase-polygalacturonase-inhibiting protein (PG-PGIP) system. Theory and experimental results indicate that the first model can be used for inhibitor protein activity determination and the second model can be used for inhibitor protein activity determination as well as for comparison of association constants among enzymes and their inhibitor-proteins from multiple sources. The models were used to identify and further clarify the nature of a differential regulation of expression of polygalacturonase-inhibiting protein in developing cantaloupe fruit. These are the first relations that provide for an objective and quantitative determination of inhibitor-protein activity in both pure and crude systems. Application of these models should prove valuable in gaining insights into regulatory mechanisms and enzyme-inhibitor-protein interactions. [source] N -Linked Glycosylated , -Peptides Are Resistant to Degradation by Glycoamidase ACHEMISTRY & BIODIVERSITY, Issue 12 2005Matthew , -Peptides are resistant to degradation by a variety of proteolytic enzymes that rapidly degrade natural , -peptides. This is one of many characteristics that make , -peptides an attractive class of compounds for drug discovery efforts. To further understand the molecular recognition properties of , -peptides and the ability of enzymes to degrade them, we have synthesized a series of N- linked glycosylated , - and , -peptides, and tested their stability towards a glycosidase. We found that glyco- , -peptides that contain N- acetylglucosamine (1) or N,N -diacetylchitobiose (2) are completely stable to degradation by glycoamidase A. In comparison, the glyco- , -peptides 3 and 4 containing N -acetylglucosamine or N,N -diacetylchitobiose are degraded. Inhibition experiments using increasing concentrations of a glyco- , -peptide fail to inhibit degradation of the corresponding glyco- , -peptide, even when the glyco- , -peptide is at a 128-fold higher concentration than the glyco- , -peptide. Evidently, the glyco- , -peptides have a much weaker affinity for the active site of the glycosidase than the corresponding glyco- , -peptide. These and the results with proteolytic enzymes suggest that the additional CH2 group introduced into the , -amino acid residues causes , -peptides not to be recognized by hydrolytic enzymes. The results described herein suggest the potential of , -peptides that are functionalized with carbohydrates for biological and biomedical investigations, without having to be concerned about the carbohydrate being removed. [source] | ||||||||||||||||