Lysozyme Substrate Research Articles

Action du lysozyme sur un substrat glycopeptidique isolé de micrococcus lysodeikticus

A soluble and purified glycopeptide substrate of lysozyme was isolated, from which glucose, glucosamine, muramic acid and four amino acids are liberated by acid hydrolysis. The degradation products of this substrate obtained by lysozyme digestion have a strong inhibitory effect on the enzyme activity and are able to form insoluble compounds with lysozyme when the concentration of the latter is high enough. This characteristic allows to understand particularities of the kinetics of the enzyme-catalysed rection, especially the activator effect of neutral salts, and some morphological aspects of the lysis of intact sensible bacteria, i.e. the “ NAKAMURA phenomenon” and the “paradoxical inhibition of lysis” by high lysozyme concentrations. Finally the mechanism of protoplast formation could be explained.

Effect of some lytic agents on Aerobacter aerogenes.

IN the course of a study of the surface structures of Aerobacter aerogenes the action of lysozyme has been investigated. Preliminary experiments indicated that the enzyme produced no observable changes in the optical density of cell suspensions. In an attempt to promote enzymic attack the conditions were varied by altering the concentration of lysozyme, the time and temperature of incubation, the origin and age of the bacteria, and the pH of the suspensions. Cells were also incubated with lysozyme and various concentrations of sodium chloride, magnesium chloride, versene1 and tris-buffer2. No lysis which could be attributed to the action of lysozyme was observed under any of these conditions. Microscopic studies showed the appearance, in solutions containing 20 per cent sucrose, of spherical bodies, which were probably similar to the protoplasts obtained by Weibull3 from B. megatherium. These, however, did not constitute more than 1 per cent of the cell population, except in one case, when bacteria incubated overnight with a 2 per cent solution of lipase were subjected to the action of lysozyme in 20 per cent sucrose. About 5 per cent conversion to spheres was observed. In a test for the presence of lysozyme substrate in the cell walls of Aerobacter aerogenes, washed cells were suspended in saline at pH 3.6 and incubated for 1 hr. at 40° C. with 200 µgm./ml. of lysozyme4. The optical densities of the suspensions were measured before and after incubation. The pH was adjusted to 10.0 by the addition of sodium hydroxide and the optical density measured immediately. A comparison with the results of a similar experiment in the absence of the enzyme showed that there was some lysis due to lysozyme. The specific substrate is thus present in the cell walls of the organism, but it is doubtful whether the amount available to the enzyme is sufficient to permit extensive lysis.

The Pigment of Micrococcus lysodeikticus

WHILE culturing cells of Micrococcus lysodeikticus 1, white cells, resistant to lysozyme, were often produced in liquid media containing relatively high carbohydrate. Resistant cells remained Gram-positive and were not lysed by lysozyme in the presence of ethylenediaminetetraacetic acid2. Optimal concentrations of cultural sodium chloride for growth improved the yellow colour of the organisms3. These observations suggested a relationship of the yellow pigment to the polysaccharide substrate of lysozyme.

Lysozyme in the bacteriolysis of gram-negative bacteria. II. Factors influencing clearing during the Nakamura treatment.

Nakamura conditions are helpful in demonstrating the presence of the lysozyme substrate in Gram-negative bacteria because: (1) the low pH (3.5) pretreatment uncovers or releases the substrate from a "bound" state within the cell wall for lysozyme digestion; (2) the addition of alkali (to pH 10.0 or above) allows for increased hydration and dispersion of the cell proteins once the lysozyme substrate has been removed. Increased hydration and dispersion alter the index of refraction of bacterial suspensions so that it is near the index of water, and clearing of the suspensions results. Irrespective of the acids and/or alkalies used, little difference was noted in their effect on clearing. The need for caution in attributing lack of clearing in bacterial suspensions to inactivation or inhibition of autolytic enzyme systems is discussed.

Lysozyme in the bacteriolysis of gram-negative bacteria. I. Morphological changes during use of Nakamura's technique.

Whole cells or the cell walls of 22 species representing 11 genera of Gram-negative bacteria were exposed to lysozyme using a modified Nakamura technique. The cell walls of all organisms contain the lysozyme substrate in differing amounts (two Brucella spp., Proteus vulgaris X-19, and Vibrio cholerae Chicago are possible exceptions) when evaluated spectrophotometrically and with the electron microscope. Using the latter technique, the sequence of events during bacteriolysis with lysozyme was observed. After exposure to lysozyme in saline, with the modified Nakamura technique and the phase microscope, cells were observed to either swell or shrink depending on the pH of the menstruum. This phenomenon apparently involves reversible hydration of cell proteins with concomitant changes in light transmission.

Growth of Micrococcus lysodeikticus as substrate for lysozyme.

Summary(1) A broth consisting of 1% Bacto-peptone and 0.5% NaCl at pH 7.5 is recommended for growth of M. lysodeikticus as substrate for lysozyme. (2) Cells grown and prepared by the recommended procedure were more susceptible to lysozyme than cells produced by other methods. (3) The rate of lysis of cells of M. lysodeikticus was affected by the concentration of NaCl used in the culture medium.

The effect of lysozyme on the electrophoretic mobility of certain cocci

Small but significant decreases in electrophoretic mobility of Sarcina lutea, Micrococcus lysodeikticus and Micrococcus albus occurred when the cells were subjected to the action of lysozyme. Heat-inactivated enzyme apparently combines with the cells causing a different type of change in the electrophoretic behavior of the cells. These findings are discussed in relation to the location of the substrate of lysozyme in the structure of the cell.

Cell wall of Micrococcus lysodeikticus as the substrate of lysozyme.

Cell wall of Micrococcus lysodeikticus as the substrate of lysozyme.

Polysaccharides in ulcerative colitis and normal human rectum compared by stains of biopsy specimens.

Summary1. The lysozyme substrate contained in Micrococcus lysodeikticus is stained by the Hotchkiss or McManus technic after fixation and sectioning by the same procedure utilized for tissue. After fixation and sectioning, the substrate remains susceptible to lysis by lysozyme. Egg albumin, as used in application of sections to slides, does not lyse the lysozyme substrate.2. Biopsy specimens of rectal mucosa from normal persons and patients with active ulcerative colitis after various methods of fixation show no consistent difference in reaction to the Hotchkiss or McManus stain.3. In vitro application of lysozyme and hyaluronidase to human rectal tissue of normal individuals and patients with ulcerative colitis does not alter the reaction of the tissue to the Hotchkiss or McManus stain.