Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A sensitive and reproducible high performance chromatographic procedure is described for the assay of jack bean beta-galactosidase in which the reaction products are separated on a Dionex AS6 ion exchange column under alkaline conditions and detected by triple-pulsed amperometry. Quantition of the enzyme-released galactose is accomplished by using either fucose or lactose, the substrate, as an internal standard. The validity of the procedure as a general method for the assay and kinetic characterization of exoglycosidases was demonstrated by performing parallel measurements of galactose using an established coupled-enzyme assay, and using these values to calculate Km and Vmax values against lactose. Additional data are presented which establish the applicability of using a similar HPLC approach for the assay of glycosyltransferases.
 ...
PMID:The use of HPLC-pulsed amperometry for the characterization and assay of glycosidases and glycosyltransferases. 182 65

Chromatographic methods were developed for the separation and characterization of acidic (sialylated) and neutral (asialo-complex and high-mannose) oligosaccharides released from glycoproteins with peptide N-glycosidase F. endo-beta-N-acetylglucosaminidase F and endo-beta-N-acetylglucosaminidase H using a carbohydrate analyzer (Dionex BioLC). All the carbohydrate separations were carried out on a polymeric pellicular anion-exchange column HPIC-AS6/CarboPac PA-1 (Dionex) using only two eluants namely, 0.5 M NaOH and 3% acetic acid/NaOH pH 5.5, which were mixed with water to generate various gradients. Developed conditions for quantitative detection of carbohydrates with pulsed amperometry were necessary to obtain steady baselines at 0.1-0.3 microA output with suitable sensitivity (less than 5 pmol) in separations employing a variety of acidic and alkaline sodium acetate gradients. Oligosaccharides released from heat-denatured and trypsin-treated glycoproteins were purified initially from large-scale digestion (greater than 0.1 g) by extraction of peptide material into phenol/chloroform and finally by ion-exchange chromatography of the acqueous phase. Oligosaccharides isolated from the peptide N-glycosidase digests of bovine fetuin, human transferrin and alpha 1-acid glycoprotein gave multiple peaks in each charge group in separations based on the charge content at pH 5.5. Alkaline sodium acetate gradients were developed to obtain oligosaccharide maps of the glycoproteins within 60 min, in which separated oligosaccharides eluted in the order of neutral, mono-, di-, tri- and tetra-sialylated species based on both charge, size and structure. Baseline separations were obtained with neutral oligosaccharide types but mixtures of high-mannose and complex types were poorly resolved. The high-mannose peaks were eliminated specifically from complex oligosaccharides by digesting with alpha-mannosidase. Treatment with beta-galactosidase, beta-N-acetylglucosaminidase and alpha-mannosidase resulted in a decrease of the oligosaccharide elution times corresponding to the number of sugar residues lost, the profile of changes was highly reproducible. In contrast, treatment with alpha-L-fucosidase, endo-beta-N-acetylglucosaminidase F and endo-beta-N-acetylglucosaminidase H resulted in an increase in their corresponding oligosaccharide retention times similar to the presence of an additional sugar residue. Conditions developed for separation of the reduced oligosaccharides and also a mixture of monosaccharide to oligosaccharide containing about 15 sugar residues within 30 min were useful in determining the effect of endo- and exo-glycosidases on porcine thyroglobulin oligosaccharides. Changes in elution time of the oligosaccharides following specific glycosidase digestions combined with methylation analysis provided a rapid and sensitive tool for confirmation of the carbohydrate primary structures present in thyroglobulin.
 ...
PMID:Rapid characterization of asparagine-linked oligosaccharides isolated from glycoproteins using a carbohydrate analyzer. 199 74