| Description | Product DescriptionEndo H, endo-beta-N-acetylglucosaminidase H, Endoglycosidase HEndo H cleaves Asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, Product DescriptionEndo H, endo-beta-N-acetylglucosaminidase H, Endoglycosidase HEndo H cleaves Asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Detergent and heat denaturation may increase the rate of cleavage for some glycoproteins.Recommended Reagentsincluded with:1 vial: 5x Reaction Buffer - 400 ml250 mM sodium phosphate, pH5.51 vial: Denaturation Solution - 200 ml 2%SDS, 1 M Beta-mercaptoethanol Molecular weightapproximately 29 kDpH optimum:5.5, active over the range 5-6.ApplicationsReleases asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. Endo H cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosac-charide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Detergent and heat denaturation may increase the rate of cleavage for some glycoproteins.Specific ActivityOne unit of Endo H activity is defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 µmole of denatured Ribonuclease B in one minute at 37˚C, pH 5.5. Cleavage is monitored by SDS-PAGE (cleaved Ribonuclease B migrates faster). SpecificityEndo H cleaves Asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Detergent and heat denaturation may increase the rate of cleavage for some glycoproteins.FormulationThe enzyme is provided as a sterile-filtered solution in 20 mM Tris-HCl, 25mM NaCl, 1 mM EDTA (pH 7.5).StabilitySeveral days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly. Quality & PurityEndo H is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37°C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases.Directions for use1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 37.5 µl final volume with de-ionized water.2. Add 10 µl 5x Reaction Buffer 5.5 and 2.5 µl of Denaturation Solution. Heat at 100°C for 5 minutes.NOTE: It is not necessary to add Triton X-100. SDS will not inactivate Endo H.3. Add 2.0 µl of Endo H to the reaction. Incubate 3 hours at 37°C.If SDS or heat denaturation is omitted, increase incubation time to at least 24 hours.Monitor cleavage by SDS-PAGE.The production host strain has been extensively tested and does not produce any detectable glycosidases... Read More | The Leuconostoc GPDH exhibits dual coenzyme specificity, namely NAD and NADP (Olive and Levy, Biochem., 6, 730 730, 1967). When assayed under conditions that are optimal for the particular coenzyme, the ratio of observed catalytic activity is NAD/NADP = 1.8 | Inquire | Inquire | Vabicaserin hydrochloride is a 5-hydroxytryptamine 2C ( 5-HT 2C ) receptor -selective agonist with an EC 50 of 8 nM.In VitroVabicaserin displaces 125 I-(2,5-dimethoxy)phenylisopropylamine binding from human 5-HT 2C receptor sites in Chinese hamster ovary cell membranes with a K i value of 3 nM and Vabicaserin hydrochloride is a 5-hydroxytryptamine 2C ( 5-HT 2C ) receptor -selective agonist with an EC 50 of 8 nM.In VitroVabicaserin displaces 125 I-(2,5-dimethoxy)phenylisopropylamine binding from human 5-HT 2C receptor sites in Chinese hamster ovary cell membranes with a K i value of 3 nM and is >50-fold selective over a number of serotonergic, noradrenergic, and dopaminergic receptors. Binding affinity determined for the human 5-HT 2B receptor subtype using [ 3 H]5HT is 14 nM. Vabicaserin is a potent and full agonist (EC 50, 8 nM; E max, 100%) in stimulating 5-HT 2C receptor-coupled calcium mobilization and exhibits 5-HT 2A receptor antagonism and 5-HT 2B antagonist or partial agonist activity in transfected cells, depending on the level of receptor expression. Vabicaserin exhibits lower affinity at the 5-HT 2C antagonist binding site (22 nM) labeled with [ 3 H]mesulergine. Additional binding studies indicate that Vabicaserin possesses affinity for the 5-HT 2B and 5-HT 1A receptors with K i values of 14 and 112 nM, respectively. MCE has not independently confirmed the accuracy of these methods. They are for reference only.In VivoAfter a single oral dose of [ 14 C]Vabicaserin at 50, 5, and 15 mg/kg, unchanged drug represents less than 19, 20, and 35% of total plasma radioactivity at all the time points examined in mice, rats, and dogs, respectively. The carbamoyl glucuronide (CG) represents approximately 7 to 36% of plasma radioactivity in mice and 2 to 28% of plasma radioactivity in dogs but is not detected in rat plasma after the single [ 14 C]Vabicaserin dose. However, the CG is observed in rat plasma after multiple-dose administration of Vabicaserin at higher doses, and the CG is approximately 20 times less than Vabicaserin based on steady-state AUC 0-24 values. The estimated plasma AUC 0-24 ratios of CG to the parent drug are 1.5 and 1.7 in mice and dogs after the single [ 14 C]Vabicaserin dose, respectively. The plasma AUC 0-24 ratios for the CG to Vabicaserin at steady state with doses used for safety assessment are less for mice (0.2-0.6) and slightly higher for dogs (1.8-4.0) compared with the single dose values. The CG is detected in dog urine in similar amounts to the parent drug, although it is not detected in mouse or rat urine after the single [ 14 C]Vabicaserin dose. Radioactivity in a 0- to 24-h bile collection from rats receiving a 5 mg/kg [ 14 C]Vabicaserin dose accounts for 19 and 24% of the administered dose in males and females, respectively. Although the CG is not detected in urine or feces of rats after a single oral administration, it represents an average of up to 30% of biliary radioactivity in male rats and 15% in female rats. In monkeys after a single oral 25-mg/kg dose of Vabicaserin, the plasma concentrations of the CG exceeded those of Vabicaserin at all the time points (2-24 h) postdose, although the amount of CG relative to Vabicaserin decreased by 24 h postdose, with ratios of 17.5 at 2 h and 1.7 at 24 h. The CG to Vabicaserin AUC 0-24 ratio of 12:1 indicates that the CG is a major metabolite in monkeys. MCE has not independently confirmed the accuracy of these methods. They are for reference only.Animal administrationMice and Rats For metabolism studies in mice, rats, and dogs, radiolabeled doses are used. Male and female CD-1 mice and Sprague-Dawley rats are used. The dose vehicle for mice and rats contained 2% (w/w) Tween 80 and 0.5% methylcellulose in water. Nonfasted male and female mice weighing from 27.8 to 33.8 g at the time of dosing are given a single 50-mg/kg (∼300 µCi/kg) dose of Vabicaserin at a volume of 20 mL/kg via intragastric gavage. Mice are kept in metabolic cages in groups of five. Nonfasted male rats weighing from 318 to 345 g and female rats weighing from 227 to 255 g at the time of dosing are given a single 5-mg/kg (∼300 µCi/kg) dose of Vabicaserin at a volume of 2.5 mL/kg via intragastric gavage. Four bile duct-cannulated male rats weighing from 387 to 411 g and four bile duct-cannulated female rats weighing from 291 to 325 g at the time of dosing are nonfasted and are given a single 5-mg/kg (323 µCi/kg) dose of Vabicaserin at a volume of 5.0 mL/kg via intragastric gavage. Rats are kept individually in metabolism cages. Dogs Four male beagle dogs, weighing from 7.6 to 9.8 kg at the time of dosing, are from an in-house colony. Approximately 11 mg of [ 14 C]Vabicaserin hydrochloride and 940 mg of nonlabeled Vabicaserin hydrochloride are dissolved in methanol and then evaporated under a nitrogen stream to dryness. Capsules (number 2) are filled with accurate amounts (126.7-138.1 mg) of the mixed drug substance according to animal weights to give a dosage of 15 mg/kg (39 µCi/kg). The filled gelatin capsules are then enteric-coated manually. Each dog is given one enteric-coated capsule containing [ 14 C]Vabicaserin as the hydrochloride salt. Animals are fed 2 h before dosing and are housed individually in metabolic cages. Monkey Four male cynomolgus monkeys, weighing from 5.4 to 9.6 kg at the time of dosing, are from an in-house colony. Nonfasted monkeys are given a single 25-mg/kg dose of nonradiolabeled Vabicaserin at a volume of 2 mL/kg via intragastric gavage. The vehicle is the same as used in mice and rats. Animals are housed individually in metabolic cages. aladdin has not independently confirmed the accuracy of these methods. They are for reference only.IC50& Target:5-HT 2C Receptor 8 nM (EC 50 )... Read More |