Interaction of lysozyme with antibiotics-binding of penicillins to lysozyme

Binding of lysozyme with the antibiotics such as penicillin-G, penicillin-V and methicillin at different concentrations and pH was studied by equilibrium dialysis. Co-operative binding isotherms were observed at pH 5.0,7.0 and 9.0 with all the penicillins and the binding ratios decreased slightly with the increase of pH. The Gibbs free energy change calculated on the basis of Wyman’s binding potential concept decreased slightly with the increase of pH indicating slight decrease in the binding strength at higher pH in the case of all penicillins. The ultra-violet difference spectra of lysozyme-penicillin complexes showed a less intense peak in the region of 284–300 nm at pH 5.0. Only penicillin-G complex had a peak at pH 7.0 at these wavelengths with less intensity compared to that at pH 5.0. However, none of the penicillins showed discrete peaks in this region at pH 9.0. The appearance of peaks in the difference spectra of all these complexes at pH 5.0 and with only penicllin-G complex at pH 7.0 in the aromatic region indicated hydrophobic interactions with tryptophan residues as the binding sites. In addition, the ionic interactions with lysine residues in lysozyme were also occurring. The conformational changes induced by the binding of penicillins to lysozyme monitored by circular dichroism showed a slight decrease in the aromatic bands in the 320–250 nm region. However, in the 250–200 nm region, [θ]222nm values obtained at various concentrations of penicillins in the complex indicated an increased α-helical content generating a more ordered structure. These results led to the conclusion that both the hydrophobic and electrostatic interactions prevail in the binding of penicillins to lysozyme.     

Lipopolysaccharide interaction with lysozyme. Binding of lipopolysaccharide to lysozyme and inhibition of lysozyme enzymatic activity

Experiments have been carried out to characterize the binding of lysozyme (LZM) to bacteriol lipopolysaccharide (LPS). The formation of LPS.LZM complexes can be readily demonstrated using either physical-chemical separation techniques or a radiolabeled photoaffinity LPS probe. The binding affinity of LZM for LPS has been estimated to be approximately 10(8) liters/mol. Binding of LPS results in loss of LZM enzymatic activity by a noncompetitive inhibition, as assessed by either particulate or soluble substrates. This interaction of LPS with LZM is dictated primarily by hydrophobic interactions and appears to be a general property of both constituents. Binding can be demonstrated with LZM of both human and avian sources, as well as with LPS isolated from a variety of Gram-negative organisms. The addition of LPS to biologically relevant fluids containing LZM results in dose-dependent inhibition of LZM enzymatic activity suggesting that such interactions may have relevance in Gram-negative infections. Finally LZM has been shown to reduce the endotoxic activity of LPS as assessed by gelation of Limulus amoebocyte lysates.     

Streptomycin and lysozyme

Résumé Les microorganismes sensibles au lysozyme ne subissent pas l'agglutination par la streptomycine. Inversement, la streptomycine empêche la clarification des suspensions microbiennes par le lysozyme. Cet effet est attribuable à la prévention de la dissolution des noyaux. Les mutants résistants à la streptomycine restent sensibles au lysozyme et se comportent, en présence de streptomycine plus lysozyme, comme les souches-mères.

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Lysozyme-sensitive microorganisms are not subject to streptomycin-agglutination. Conversely, streptomycin inhibits the clarification of bacterial suspensions by lysozyme. This effect is mainly due to a prevention of nuclear dissolution. Streptomycin-resistant mutants remain lysozyme-sensitive and behave towards streptomycin plus lysozyme as do parent-cells.

     

Calf rennet lysozyme.

A glycosidase displaying endo-N-acetylmuramoylhydrolase specificity (EC 3.2.1.17) was isolated from calf rennet. This lysozyme was also present in abomasal secretions from calf and adult cattle. Multiple molecular forms revealed by electrofocusing might be artefacts. The main enzyme form had Mr approx. 15 000, pH optimum 5.0, pI7.5, and a remarkable conformation stability. Competitive inhibition was observed with both N-acetylglucosamine and N-acetylmuramic acid, with apparent Ki values of 29 mM and 2.4 mM respectively. The isolated enzyme also displayed significant chitinase activity.     

Surfactant-induced refolding of lysozyme

The surfactant-lysozyme interaction was investigated by circular dichroism, fluorescence, UV, dynamic light scattering, surface tension, turbidity measurements and lysozyme activity assay. A new way of refolding of lysozyme was found. It was shown that the lysozyme unfolded by anionic surfactants could be renatured by adding cationic surfactants. That is, lysozyme formed precipitate with anionic surfactants, the precipitates could be dissolved by adding a cationic surfactant solution, and then the lysozyme was refolded to its native state spontaneously. Different couples of anionic surfactants and cationic surfactants including C10SO3/C10NE, C12SO3/C10NE, C10SO3/C12NE, C10SO3/C12NB, C10SO4/C10NE and C12SO4/C10NE (C(n)SO3, C(n)SO4, C(n)NE and C(n)NB represent sodium alkyl sulfonate/sulfate, alkyl triethyl/butyl ammonium bromide respectively) were investigated, all of them gave similar results. The results were explained in terms of the differences between the interaction of anionic-cationic surfactants and that of surfactant-lysozyme. It was thought that the formation of mixed micelles of anionic-cationic surfactants is a more favorable process than that of lysozyme-surfactant complexes, which induces the dissociation of lysozyme-surfactant complexes when cationic surfactants were added.     

Microbial inhibitors of lysozyme

Lysozyme is one of the most important factors of innate immunity, possessing anti-microbial action against a wide range of microorganisms due to cationic nature of protein and, in a lesser degree, due to muramidase activity. In the process of evolution, different mechanisms of lysozyme inhibition, defined as anti-lysozyme activity (ALA), were formed in microorganisms. The usage of the delayed antagonism principle allowed to reveal anti-lysozyme sign in microorganisms of different phylogenetic groups. In the review, data on the occurrence and level of anti-lysozyme activity in pathogens and representatives of normal microflora were presented; findings on nature and genetic determinants of lysozyme inhibitors are given. questions on drug regulation of ALA of the causative agents in infectious diseases and dysbiotic conditions are discussed.     

溶菌酶的研究进展

对溶菌酶的稳定性的改善、制备、基因工程表达产物的复性处理作了介绍,并且对溶菌酶在医药、食品工业和生物工程上的应用作了概述。