| Popis: |
Tritium-labeled tetrasaccharide, GlcNAc-β(1 → 4)-MurNAc-β(1 → 4)-GlcNAc-β(1 → 4)-MurNAc, and disaccharide, GlcNAcβ(1 → 4)-MurNAc (where MurNAc represents N-acetylmuramyl), were prepared by tritiation of the corresponding unlabeled compounds. The products, obtained in good yield, were found to be homogeneous and radiochemically pure by several chromatographic and electrophoretic criteria and indistinguishable from the starting materials. Chemical and enzymatic studies indicated that they were uniformly labeled. The action of lysozyme on the tetrasaccharide was studied by a combination of paper electrophoresis and paper chromatography. The major product of digestion was the disaccharide. Higher oligosaccharides were formed in the initial stages of the reaction and disappeared after prolonged incubation when only disaccharide together with considerable amounts of tetrasaccharide was present. The rate of disappearance of the tetrasaccharide changed markedly during the course of the reactions; the maximal rate increased with the increase in substrate concentration. Four higher oligosaccharides (hexa-, octa-, deca-, and dodecasaccharides) were isolated from lysozyme digests of the tetrasaccharide-3H. Two of these were found to be identical with the hexa- and octasaccharides isolated from lysozyme digests of Micrococcus lysodeikticus cell walls. The higher oligosaccharides were readily digested by lysozyme to yield the corresponding di- and tetrasaccharides. The hexa- and octasaccharides were digested at a much faster rate than the tetrasaccharide. Addition of small amounts of octasaccharide to the tetrasaccharide increased significantly the rate of digestion of the latter. Upon incubation of unlabeled tetrasaccharide with disaccharide-3H in the presence of lysozyme, the labeled tetrasaccharide formed did not contain significant amounts of radioactivity in the nonreducing half of the molecule. Furthermore, when this newly formed radioactive tetrasaccharide was incubated with GlcNAc-14C and lysozyme, the Glc-NAc-β(1 → 4)-MurNAc-β(1 → 4)-GlcNAc contained insignificant amounts of 3H. These results indicate that in the reactions studied over 90% of the new products must be formed by transglycosylation, and not by reversal of hydrolysis. The results obtained show that hydrolysis of the tetrasaccharide to disaccharide proceeds chiefly via transglycosylation, leading to the formation of high oligosaccharides. This mechanism is consistent with the binding data for the association of various saccharides with lysozyme and can be readily explained on the basis of the three-dimensional model of lysozyme presented by Phillips and co-workers. |
