The action of lysozyme on peptidoglycan with N-unsubstituted glucosamine residues. Isolation of glycan fragments and their susceptibility to lysozyme.

1. A peptidoglycan preparation N-acetylated at about 30% of glucosamine residues was obtained by the treatment of the lysozyme-resistant cell wall paptidoglycan of Bacillus cereus with acetic anhydride at pH 7. Fractionation of dialyzable material resulting from lysozyme digestion of the glycan component of this peptidoglycan preparation yielded five oligosaccharides designated as S1 to S5 besides the disaccharide GlcNAc-MurAc. 2. Oligosaccharide S3, which accounted for about 30% of the disaccharide units recovered as disaccharides and oligosaccharides, was identified as GlcN-MurAc-GlcNAc-MurAc. Oligosaccharide S1, accounting for about 20% of the disaccharide units recovered, was characterized as GlcN-MurAc-GlcN-MurAc-GlcNAc-MurAc, while oligosaccharide S2, present in a smaller amount, as GlcNAc-MurAc-GlcN-MurAc-glcNAc-MurAc. Oligosaccharides S4, and S5, present in small amounts, were identified as GlcNAc-MurAc-GlcNAc-MurAc and MurAc-GlcNAc-MurAc, respectively. 3. Oligosaccharides S1, S3 and S5 proved to be completely insusceptible to lysozyme, whereas S2 was digsted by lysozyme to produce GlcNAc-MurAc and S3. S1 was found to act as a more potent inhibitor than S3 in lysozyme-catalyzed digestion of polysaccharides. 4. The results obtained show that the lysozyme-catalyzed hydrolysis of peptidoglycan oligosaccharides had an obligatory requirement for the N-acetyl group on the glucosamine residue located in subsite C in the enzyme-substrate complex.     

Isolation of lysozyme on chitosan.

Lysozyme has been immobilized on chitosan, a polyaminosaccharide, without using any intermediate reagent. The best pH conditions for operating the chitosan columns have been determined and the best eluting agent was found to be a 2% solution of propylamine. The lysozyme activity was determined after reacting lysozyme with the product of glycolchitin and Remazol Brilliant Blue R. The recovery of lysozyme from chicken egg white yields lysozyme with 55% activity.     

Effect of lysozyme or modified lysozyme fragments on DNA and RNA synthesis and membrane permeability of Escherichia coli

Previously we have shown that chicken egg white lysozyme, an efficient bactericidal agent, affects both gram-positive and gram-negative bacteria independently of its muramidase activity. More recently we reported that the digestion of lysozyme by clostripain yielded a pentadecapeptide, IVSDGNGMNAWVAWR (amino acid 98-112 of chicken egg white lysozyme), with moderate bactericidal activity but without muramidase activity. On the basis of this amino acid sequence three polypeptides, in which asparagine 106 was replaced by arginine (IVSDGNGMRAWVAWR, RAWVAWR, RWVAWR), were synthesized which showed to be strongly bactericidal. To elucidate the mechanisms of action of lysozyme and of the modified antimicrobial polypeptides Escherichia coli strain ML-35p was used. It is an ideal organism to study the outer and the inner membrane permeabilization since it is cryptic for periplasmic beta-lactamase and cytoplasmic beta-galactosidase unless the outer or inner membrane becomes damaged. For the first time we present evidence that lysozyme inhibits DNA and RNA synthesis and in contrast to the present view is able to damage the outer membrane of Escherichia coli. Blockage of macromolecular synthesis, outer membrane damage and inner membrane permeabilization bring about bacterial death. Ultrastructural studies indicate that lysozyme does not affect bacterial morphology but impairs stability of the organism. The bactericidal polypeptides derived from lysozyme block at first the synthesis of DNA and RNA which is followed by an increase of the outer membrane permeabilization causing the bacterial death. Inner membrane permeabilization, caused by RAWVAWR and RWVAWR, follows after the blockage of macromolecular synthesis and outer membrane damage, indicating that inner membrane permeabilization is not the deadly event. Escherichia coli bacteria killed by the substituted bactericidal polypeptides appeared, by electron microscopy, with a condensed cytoplasm and undulated bacterial membrane. So the action of lysozyme and its derived peptides is not identical.     

Isolation and characteristics of Klebsiella pneumoniae lysozyme factor

For the first time the preparation of K. pneumoniae antilysozyme factor has been isolated and purified. This factor, having a molecular weight of about 1,000 daltons, is oligopeptide with an oligosaccharide part. It also contains such amino acids as proline, lysine, arginine and tyrosine. The antilysozyme factor is resistant to 30-minute boiling and has no optimal pH value for its action.     

The preparation of protected fragments of lysozyme for semisynthesis.

This paper reports the development of methods for preparing tryptic fragments of hen's-egg lysozyme in an appropriate state of protection for use in the chemical synthesis of modified polypeptides. 1. We describe the cleavage of the disulphide bridges of the enzyme and the simulatneous protection of the liberated thiol groups by S-sulphonation. Lysozyme resisted the usual conditions for this reaction. We have confirmed the stability of the S-sulphonyl group to the conditions met in peptide synthesis. 2. We describe the reversible protection of the amino groups of the enzyme by reaction with various anhydrides of 1,2-dicarboxylic acids. We conclude that 2-methylmaleic anhydride and exo-cis-3,6-endoxo-delta4-tetrahydrophthalic anhydride are unsuitable for our purpose but that maleic anhydride can, in spite of certain drawbacks, be used. 3. We describe the tryptic cleavage of the thiol- and amino-protected protein and the separation of the fragments. 4. We describe the reversible protection of the carboxylic acid groups (including the specific deprotection of the alpha-carboxyl group), the imidazolyl group and the aloph-amino groups of the fragments. Several alternative groups have been evaluated for most of these purposes. The side-chain amides did not present any serious problem of libility, 5. We describe experiments on the stability of the side chain of tryptophan, both protected by formylation and unprotected, to the acid conditions needed for the deprotection of the other functional groups in the peptide. We conclude that protection of tryptophan is unnecessary. We suggest that most of the methods described are of general application in peptide semisynthesis by fragment condensation. An Appendix is included to which points 6-ll appertain...     

Isolation and cloning of Streptomyces terminal fragments

Streptomyces species have a linear chromosome of approximately 8 Mb in size. Many strains also carry linear plasmids. Most of these linear elements contain terminal proteins covalently bound to the 5 ends of the DNA. Using a method for the visualisation of terminal DNA fragments in agarose gels, it was possible to see three fragments in S. rimosus and five fragments in S. avermitilis. The method was also used to clone the 298 bp BamHI fragment carrying the left end of plasmid SLP2. Analysis of the sequence showed that the end resembled other Streptomyces chromosome and plasmid ends, but there were eight palindromes (instead of seven) and a tandem duplication of a 14 bp sequence.     

Isolation of the lysozyme gene of chicken.

The lysozyme gene has been purified by molecular cloning from two chicken gene libraries. Several recombinant phages harbouring sequences homologous to a plasmid carrying a double stranded lysozyme cDNA have been isolated. One recombinant appears to carry an entire lysozyme gene. Electron microscopic studies show that the latter is split by at least three introns. The length of the gene is about 3.9 kb, 6 times longer than lysozyme mRNA.     

Isolation of RNA and DNA fragments using diatomaceous earth

A series of simple and phenol-free silica-based protocols for isolating and cleaning RNA or DNA fragments from different sources were developed. Cytoplasmic RNA isolated from hybridoma cells by this method was used in RT-PCR. DNA fragments obtained using this protocol were suitable for further subcloning, gene transformation and DNA sequencing. © Rapid Science Ltd. 1998     

Antigenic determinants of hen egg-white lysozyme in delayed hypersensitivity. I. Macrophage migration inhibition activities of lysozyme fragments.

Five kinds of fragments of hen egg-white lysozyme (HL) were tested by macrophage migration inhibition (MMI) assay using peritoneal exudate cells (PEC) of guinea pigs immunized with HL in complete Freund's adjuvant. (See article). These four kinds of HL fragments were also shown to be composed of the immunodominant groups of the HL molecule for circulating antibody against HL in guinea pigs. The relationships between the antigenic sites related to circulating antibody and the cellular recognition sites are discussed.     

Isolation and characterization of the bacteriophage T4 tail-associated lysozyme.

Direct evidence has been obtained that the tail-associated lysozyme of bacteriophage T4 (tail-lysozyme) is gp5, which is a protein component of the hub of the baseplate. Tails were treated with 3 M guanidine hydrochloride containing 1% Triton X-100, and the tail-lysozyme was separated from other tail components by preparative isoelectric focusing electrophoresis as a peak with a pI of 8.4. The molecular weight as determined from sodium dodecyl sulfate electrophoresis was 42,000. The tail-lysozyme was unambiguously identified as gp5 when the position of the lysozyme was compared with that of gp5 of tube-baseplates from 5ts1/23amH11/eL1ainfected Escherichia coli cells by two-dimensional gel electrophoresis. The tail-lysozyme has N-acetylmuramidase activity and the same substrate specificity as gene e lysozyme; the optimum pH is around 5.8, about 1 pH unit lower than for the e lysozyme. We assume that the tail-lysozyme plays an essential role in locally digesting the peptidoglycan layer to let the tube penetrate into the periplasmic space. The tail-lysozyme is presumably also responsible for "lysis from without."