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Specific Enzyme Activities (specific + enzyme_activity)
Selected AbstractsKinetic studies on aminopeptidase M-mediated degradation of human hemorphin LVV-H7 and its N -terminally truncated productsJOURNAL OF PEPTIDE SCIENCE, Issue 7 2008Harald John Abstract The human hemorphin LVV-H7 belongs to the class of µ-opiod receptor-binding peptides, which also exhibits significant affinity to insulin-regulated aminopeptidase (IRAP) thereby affecting IRAP inhibition. The inhibitory potency towards IRAP is of pharmaceutical interest for the treatment of Alzheimer's disease. Consecutive N -terminal cleavage of the first two amino acid residues of LVV-H7 affects a drastic increase of the binding affinity (V-H7) but ultimately leads to its complete abolition after cleavage of the next amino acid residue (H7). Therefore, we investigated LVV-H7 truncation by aminopeptidase M (AP-M) identified as a LVV-H7 degrading enzyme potentially regulating hemorphin activity towards IRAP in vivo. Using a selective quantitative multi-component capillary zone electrophoretic method (CZE-UV), we analyzed the AP-M-mediated subsequent proteolysis of the hemorphins LVV-H7 (L32 -F41), VV-H7 (V33 -F41), and V-H7 (V34 -F41) in vitro. Incubations were carried out with synthetic hemorphins applied as single substrates or in combination. Maximum velocities (Vmax), catalytic constants (turnover numbers, kcat), and specific enzyme activities (EA) were calculated. L32 cleavage from LVV-H7 happens more than two-times faster (kcat: 140 min,1 ± 9%, EA: 1.0 U/mg ± 9%) than V33 cleavage from VV-H7 (kcat: 61 min,1 ± 10%, EA: 0.43 U/mg ± 10%) or V32 deletion from V-H7 (kcat: 62 min,1 ± 8%, EA: 0.46 U/mg ± 8%). In contrast, we showed that H7 (Y35 -F41) was neither degraded by porcine AP-M nor did it act as an inhibitor for this enzyme. Determined turnover numbers were in the same dimension as those reported for dynorphin degradation. This is the first time that AP-M-mediated truncation of natural underivatized LVV-H7 and its physiological metabolites was analyzed to determine kinetic parameters useful for understanding hemorphin processing and designing hemorphin-derived drug candidates. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. [source] Analysis of Escherichia coli anaplerotic metabolism and its regulation mechanisms from the metabolic responses to altered dilution rates and phosphoenolpyruvate carboxykinase knockoutBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2003Chen Yang Abstract The gluconeogenic phosphoenolpyruvate (PEP) carboxykinase is active in Escherichia coli during its growth on glucose. The present study investigated the influence of growth rates and PEP carboxykinase knockout on the anaplerotic fluxes in E. coli. The intracellular fluxes were determined using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U- 13C6]glucose labeling experiments and 2D nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids and glycerol. Significant activity of PEP carboxykinase was identified in wild-type E. coli, and the ATP dissipation for the futile cycling via this reaction accounted for up to 8.2% of the total energy flux. Flux analysis of pck deletion mutant revealed that abolishment of PEP carboxykinase activity resulted in a remarkably reduced flux through the anaplerotic PEP carboxylase and the activation of the glyoxylate shunt, with 23% of isocitrate found being channeled in the glyoxylate shunt. The changes in intracellular metabolite concentrations and specific enzyme activities associated with different growth rates and pck deletion, were also determined. Combining the measurement data of in vivo fluxes, metabolite concentrations and enzyme activities, the in vivo regulations of PEP carboxykinase flux, PEP carboxylation, and glyoxylate shunt in E. coli are discussed. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 129,144, 2003. [source] Dopamine, Morphine, and Nitric Oxide: An Evolutionary Signaling TriadCNS: NEUROSCIENCE AND THERAPEUTICS, Issue 3 2010George B. Stefano Morphine biosynthesis in relatively simple and complex integrated animal systems has been demonstrated. Key enzymes in the biosynthetic pathway have also been identified, that is, CYP2D6 and COMT. Endogenous morphine appears to exert highly selective actions via novel mu opiate receptor subtypes, that is, mu3,-4, which are coupled to constitutive nitric oxide release, exerting general yet specific down regulatory actions in various animal tissues. The pivotal role of dopamine as a chemical intermediate in the morphine biosynthetic pathway in plants establishes a functional basis for its expansion into an essential role as the progenitor catecholamine signaling molecule underlying neural and neuroendocrine transmission across diverse animal phyla. In invertebrate neural systems, dopamine serves as the preeminent catecholamine signaling molecule, with the emergence and limited utilization of norepinephrine in newly defined adaptational chemical circuits required by a rapidly expanding set of physiological demands, that is, motor and motivational networks. In vertebrates epinephrine, emerges as the major end of the catecholamine synthetic pathway consistent with a newly incorporated regulatory modification. Given the striking similarities between the enzymatic steps in the morphine biosynthetic pathway and those driving the evolutionary adaptation of catecholamine chemical species to accommodate an expansion of interactive but distinct signaling systems, it is our overall contention that the evolutionary emergence of catecholamine systems required conservation and selective "retrofit" of specific enzyme activities, that is, COMT, drawn from cellular morphine expression. Our compelling hypothesis promises to initiate the reexamination of clinical studies, adding new information and treatment modalities in biomedicine. [source] Defining the caspase-containing apoptotic machinery contributing to cornification in human epidermal equivalentsEXPERIMENTAL DERMATOLOGY, Issue 1 2006Vijaya Chaturvedi Abstract:, Whether terminal differentiation/stratum corneum formation of keratinocytes (KCs) represents a form of programmed cell death, utilizing mediators of classical apoptosis, is unclear. Apoptosis, an evolutionarily conserved death process, is comprised of extrinsic and intrinsic pathways, which converge using caspase 3. To define upstream and downstream caspases involved in terminal differentiation, we utilized human epidermal equivalents (EEs). Using submerged cultures comprised of human KCs, EEs were sequentially analyzed before and after being raised to an air/liquid (A/L) interface at 3,24 h intervals. At each time point, EEs were analyzed morphologically and for specific enzyme activity to distinguish different initiator (caspases 1, 2, 8, 9) and effector caspases (3, 6, 7). Terminal differentiation began at 6,8 h, as defined by stratum corneum with loricirin expression and completed at 18,24 h producing an epidermis resembling normal skin. Enzyme activity for caspases 1, 2, 3, 6, 7, 8, and 9 (but not 4, 5) was enhanced (>two-fold nmol/mg/h) at 3,6 h compared with submerged cultures. Processing of caspase 14 occurred at 18 h, and cleaved caspase 14 was increased at 24 h. Activated caspase 3-positive and terminal deoxynucleotidyl transferase-mediated nick end labeling-positive KCs were identified in EEs at 3,6 h corresponding to initiation sites of terminal differentiation. Addition of caspase inhibitors reduced levels of involucrin and loricrin in EEs raised to an A/L interface. We conclude caspases function as important death effectors strategically positioned at intersection of intrinsic and extrinsic pathways in KCs undergoing stratum corneum formation. [source] Characterization of a novel silkworm (Bombyx mori) phenol UDP-glucosyltransferaseFEBS JOURNAL, Issue 3 2002Teresa Luque Sugar conjugation is a major pathway for the inactivation and excretion of both endogenous and exogenous compounds. We report here the molecular cloning and functional characterization of a phenol UDP-glucosyltransferase (UGT) from the silkworm, Bombyx mori, which was named BmUGT1. The complete cDNA clone is 1.6 kb, and the gene is expressed in several tissues of fifth-instar larvae, including fat body, midgut, integument, testis, silk gland and haemocytes. The predicted protein comprises 520 amino acids and has ,,30% overall amino-acid identity with other members of the UGT family. The most conserved region of the protein is the C-terminal half, which has been implicated in binding the UDP-sugar. BmUGT1 was expressed in insect cells using the baculovirus expression system, and a range of compounds belonging to diverse chemical groups were assessed as potential substrates for the enzyme. The expressed enzyme had a wide substrate specificity, showing activity with flavonoids, coumarins, terpenoids and simple phenols. These results support a role for the enzyme in detoxication processes, such as minimizing the harmful effects of ingested plant allelochemicals. This work represents the first instance where an insect ugt gene has been associated with a specific enzyme activity. [source] Characterization of a prolyl endoprotease from Eurygaster integriceps puton (Sunn pest) infested wheatARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2010Charles Darkoh Abstract Sunn pest, Eurygaster integriceps, Puton, infested and uninfested wheat seeds were obtained from the International Center for Agriculture Research in the Dry Areas (ICARDA), Aleppo, Syria, with the primary objective to identify the type of enzyme deposited by the Sunn pest on the wheat responsible for the gluten degradation. Enzyme levels were extremely low due to the enzyme being secreted by the insect in localized areas on the seed. Only extract from the infested wheat contained glutenase activity. Anion exchange, Cu2+ sepharose, and gel filtration chromatography were used to partially purify and enrich protein samples from both infested wheat and uninfested wheat. An SDS-gluten assay was used to show gluten specificity while a commercially available chromogenic proline peptide, benzyloxycarbonyl-Gly-Pro-p-nitroanalide (ZGPpNA), was utilized to identify fractions containing the active proline specific enzyme activity and to determine Michaelis-Menten kinetics. Despite low levels of enzyme on the infested wheat, the enzyme was partially purified and enriched exhibiting a specific activity of 4.5,U/mg of total protein for gluten in a SDS gluten assay (1,U of enzyme activity was defined as the decrease in gel height in millimeters in 1,h) and exhibited a high-affinity Km of 65,µM for ZGPpNA, cleaving at the carboxy terminus of the proline residue. The enzyme exhibited optimal activity between pH 8 and 10.0 at temperatures between 20° and 35°C. The enzyme was identified to be a prolyl endoprotease. © 2010 Wiley Periodicals, Inc. [source] | |||||||||||||