muramidase and chitotetrose

An end-modified β-d-galactosyl chitotetraose derivative [4

Topics: Animals; Binding Sites; Chickens; Enzyme Assays; Kinetics; Molecular Docking Simulation; Muramidase; Oligosaccharides; Protein Structure, Tertiary; Spectrophotometry; Substrate Specificity

Protein-carbohydrate interactions play a significant role in biological processes. Presented here is the novel application of amide hydrogen/deuterium exchange mass spectrometry (amide exchange-MS) to the study of the interaction between a protein and its carbohydrate substrate. The degree of deuterium incorporation into hen egg lysozyme was monitored with and without substrate to verify that a carbohydrate can provide sufficiently stable protection of the amide hydrogen atoms in a protein's backbone from exchange with deuterated solvent. The substrate protected a number of amide hydrogens from exchange, implying that protein-carbohydrate binding systems will be compatible with amide exchange-MS. Endopolygalacturonase-II (EPG-II) from Aspergillus niger, a pectin-degrading enzyme, was chosen as the first carbohydrate-binding system to be extensively studied using quenched amide exchange-MS. Monitoring the changes in deuterium incorporation of EPG-II in the presence and absence of an oligomer of galacturonic acid implied the location of substrate binding. This study demonstrates the ability of amide exchange-MS to investigate protein-carbohydrate interactions.

Topics: Amides; Animals; Aspergillus niger; Biology; Carbohydrate Metabolism; Carbohydrates; Chickens; Crystallography, X-Ray; Deuterium; Disaccharides; Hexuronic Acids; Hydrogen; Mass Spectrometry; Muramidase; Oligosaccharides; Ovum; Polygalacturonase; Proteins

The enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis, were studied in detail with a new enzyme assay. In this assay, the enzyme reaction products were derivatized by reductive coupling to a chromophore. Products were separated by HPLC and the amount of product was calculated by peak integration. Penta-N-acetylglucosamine (penta-nag) and hexa-N-acetylglucosamine (hexa-nag) were used as substrates. Hexa-nag was more efficiently converted than penta-nag, which is an indication that hevamine has at least six sugar binding sites in the active site. Tetra-N-acetylglucosamine (tetra-nag) and allosamidin were tested as inhibitors. Allosamidin was found to be a competitive inhibitor with a K(i) of 3.1 microM. Under the conditions tested, tetra-nag did not inhibit hevamine.

Topics: Acetylglucosamine; Binding, Competitive; Chitinases; Chromatography, High Pressure Liquid; Coloring Agents; Euphorbiaceae; Kinetics; Muramidase; Oligosaccharides; Oxidation-Reduction; Plant Proteins; Thermodynamics; Trisaccharides

A major regulatory shift affecting the expression of lysozyme c may have been involved in the origin of two groups of mammals whose nutrition depends on foregut bacteria. A survey of 23 mammalian species reveals that the lysozyme c activity per g of stomach mucosa is many times higher for ruminants and a leaf-eating monkey than for animals lacking a foregut. The implication is that stomach lysozyme c functions as a major digestive enzyme in ruminant-like mammals, helping to make those bacterial which enter the stomach from the foregut available for hydrolysis by conventional digestive enzymes. The high level of stomach lysozyme is due to more enzyme molecules rather than to an increase in the activity of each molecule. This was shown for the cow by purifying the three, non-allelic lysozymes c that account for the lysozyme activity in gastric mucosa and measuring their specific activities and for other foregut fermenters by immunological titration. Lysozyme appears in the stomach mucosa before birth and reaches adult levels before weaning. Other tissues tested from cattle lack lysozyme c and may instead have low levels of another lysozyme that could belong to the g class, the first indication that lysozyme g may be present in mammals. The lysozymes of eight ruminants, four Old World monkeys, and 12 other animals were compared as regards the ability to lyse bacterial cells under various conditions and to resist inactivation by pepsin. There are differences among these species in the dependence of the rate of bacterial lysis on time, pH, and ionic strength. Although not every lysozyme was tested in all of these catalytic respects, there were no exceptions to the following generalizations. First, at ionic strengths above 0.1 and pH values above 5, the rate of lysis by ruminant and monkey lysozymes c rose with the time of reaction, whereas the rate was more nearly constant for the other animal lysozymes. Second, the lytic activity at neutral pH is lower than at pH 5 for the ruminant and monkey lysozymes c when the ionic strength is over 0.1; by contrast, for other lysozymes c under these conditions the activity at neutral pH is about as high as at pH 5. This latter property, which may be viewed as an adaptation for functioning as a digestive enzyme in the stomach, can be explained in part by differences in electrostatic interactions between lysozyme and the substrate due to the relatively non-basic nature of ruminant and monkey lysozymes compared to other lyso

Topics: Animals; Artiodactyla; Gastric Mucosa; Hydrogen-Ion Concentration; Immunodiffusion; Kinetics; Muramidase; Oligosaccharides; Osmolar Concentration; Pepsin A; Stomach; Tetroses; Tissue Distribution

The influence of urea and of guanidine chloride on the binding of the bacterial substrate and of inhibitors such as N-acetylglucosamine or chitotetraose to hen lysozyme were studied at 20 degrees and at 40 degrees C (physiological temperature). The action of urea did not prevent a certain degree of organization of the enzyme compatible with its usual behaviour in the presence of some inhibitors and with its crystallization ; guanidine chloride, already at low concentrations, seemed to have a more severe effect on lysozyme.

Topics: Acetylglucosamine; Animals; Chickens; Chitin; Corynebacterium; Egg White; Guanidines; Kinetics; Muramidase; Oligosaccharides; Temperature; Tetroses; Urea