No abstract

The sensitivity of a fluorescence labeling method ((1979). J. Biochem. 85, 989--994; 995--1002) for structure analyses of asparagine-linked sugar moieties of glycoproteins was increased by using HPLC with a fluorescence detector. Sugar moieties were separated from polypeptide portions by hydrazinolysis. Free amino groups thus exposed were acetylated and the reducing ends of sugar chains were reductively aminated with a fluorescent reagent, 2-aminopyridine, by the use of sodium cyanoborohydride. The pyridylamino derivatives were purified on a Dowex 1 column to eliminate undesired substances. The separation and identification of the pyridylamino derivatives were carried out by HPLC with a column of C18 reversed phase or gel permeation phase. As little as 0.1 pmol of pyridylamino derivatives can be detected. Ten microgram of Taka-amylase A was easily detected by this system. The method was also applied to some other glycoproteins.

Potential aldehyde groups of several monosaccharides and oligosaccharides were coupled with 2-aminopyridine by reductive amination with sodium cyanoborohydride. The product was isolated by adsorption on a Dowex 50 (H+) column, followed by washing, elution with aqueous ammonia and evaporation. The specimen was analyzed by paper electrophoresis at pH 5.0. Each sugar derivative gave a single spot in addition to one corresponding to excess 2-amino-pyridine when the paper was scanned under a UV lamp. The migration rates of these fluorescent sugars were related to their molecular weights and were independent of their linkage points and anomeric configurations. The reducing end sugar units of oligosaccharide derivatives could be identified by gas-liquid chromatography after hydrolysis of the 2-aminopyridine derivatives of the oligosaccharides.

The log phase cells of autolytic Micrococcus lysodeikticus (luteus) IFO 3333 did not autolyze when grown in the presence of trypsin although the growth curve and morphology of the cells were not influenced.A non-autolytic mutant was obtained by subculture of the wild-type strain IFO 3333 on an agar slant containing 1% glucose. The mutant (strain MT) was arranged in tetrads or in clusters consisting of regular tetrads, in contrast to the wild-type IFO 3333 which occurred singly or in irregular masses. The mutant MT grown in a culture medium containing trypsin caused remarkable alteration in cell morphology : large cell packets consisting of a number of "unit tetrads" arranged regularly in three dimensions were formed by the addition of trypsin to the medium. The findings suggest that inhibition of the separation of divided cells is brought about by inactivation or suppression of a cell wall autolytic enzyme which plays an important role in the separation step and is accessible to externally added trypsin in the mutant cells but not in the wild-type cells.The possibility that there are two kinds or phases of autolytic enzymes, "a physiological autolytic enzyme" and "a useless autolytic enzyme", is discussed.In the previous paper (21), we reported that the log phase cells of Micrococcus lysodeikticus (luteus) IFO 3333 were autolyzed by the action of an N-acetyl-muramyl-L-alanine amidase and this cellular autolysis was depressed by the addition of trypsin to the autolyzing buffer.To investigate the physiological role or significance of the cell wall autolytic enzyme(s), we isolated a non-autolytic mutant of the above strain and compared the autolytic ability, cell morphology and cell wall compositions of this mutant MT with those of the wild-type IFO 3333, particularly by cultivating the mutant and wildtype cells in the presence of externally added proteolytic enzymes.