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.     

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.     

The lysozyme of the starfish Asterias rubens. A paradygmatic type i lysozyme.

On the basis of a partial N-terminal sequence, Jollès and Jollès previously proposed that the lysozyme from the starfish Asterias rubens represents a new form of lysozyme, called type i (invertebrate) lysozyme. Indeed, it differed from both the types c (chicken) and g (goose) known in other animals, as well as from plant and phage lysozymes. Recently, several proteins belonging to the same family have been isolated from protostomes. Here we report the complete mature protein sequence and cDNA sequence of the lysozyme from Asterias. These sequences vindicate the previously proposed homology between the starfish, a deuterostome, and protostome lysozymes. In addition, we present a structural analysis that allows us to postulate upon the function of several conserved residues.     

Frog lysozyme III. The effects of metamorphic inhibition on Rana pipiens lysozyme ontogeny.

The inhibition of Rana pipiens metamorphosis by thiouracil altered the ontogeny of lysozyme. Certain isozymes of the enzyme remained absent. There was, nevertheless, an increase in tissue lysozyme concentration.     

Proton pathways in lysozyme.

Protonic conduction studies are reported for lysozyme as a function of the number of bound water molecules. Lysozyme samples employing proton-injecting palladium black electrodes exhibited conductivities up to eight orders of magnitude greater than those retained between control (copper) electrodes. The results indicate that water involved in multiple hydrogen bond contact with the enzyme together with hydrogen bonded segments of the enzyme structure provide a hydrogen bond network which is capable of supporting considerable protonic conduction.     

The circular dichroism of lysozyme.

The circular dichroism spectra of hen egg white lysozyme, and of lysozyme derivatives in which tryptophan residues 62 or 108, or both, are selectively oxidized, have been measured as a function of pH over the range of 200 to 310 nm. Neither Trp-62 nor Trp-108 is principally responsible for the positive rotational strength in the 280 to 300 nm region. The spectrum in the 200 to 230 nm region is nearly the same in the native protein and in the derivatives, and is little affected by binding of saccharide. These results are used to reinterpret the circular dichroism spectra of the lysozymes and alpha-lactalbumins.     

Titration study of acetylated lysozyme.

To study the interaction between carboxyl groups and amino groups in native lysozyme [EC 3.2.1.17], and to identify the positions and the pK values of the abnormal carboxyl groups, N-acetylated lysozyme was prepared. The acetylation did not affect the molecular shape of the enzyme, but changed six amino groups to a non-ionizable form, leaving one amino group free; this was determined to be Lys 33. In addition, pH titration of the acetylated lysozyme in 0.2 or 0.02 M KCl aqueous solution indicated fewer titratable groups with pK(int) of 7.8 or 10.4 compared with the native protein, though the number of titratable carboxyl groups was not affected by the acetylation. From the pH titration results and structural considerations, the unititratable carboxyl groups were suggested to be Asp 48, Asp 66, and Asp 87. On the other hand, spectrophotometric titration in 0.2 M KCl showed that all three tyrosine residues are titratable in the acetylated protein, although an abnormal tyrosine residue exists in the native state. Tyr 20 was suggested to be untitratable in the pH range of 8-12.6.     

Spin-label assay for lysozyme.

A spin-label assay for lysozyme, which is based on the enzymatic hydrolysis of spin-labeled peptidoglycan, is described. Hydrolysis of this polymer by lysozyme results in sharpening of the esr spectrum. The rate of spectral sharpening is a function of enzyme concentration. When the activities of hen egg-white and human lysozymes are compared by this method, human lysozyme is 3.5 times as active as the hen enzyme. The pH optima for both enzymes are pH 5.0. At this pH, the maximal activity for the hen egg-white lysozyme is observed at an ionic strength of 0.09. This assay is suitable for measuring lysozyme levels in biological fluids. It is a sensitive, continuous assay that measures muramidase activity on a defined substrate.     

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.     

Low-frequency Raman spectra of lysozyme.

New techniques in laser Raman spectroscopy are used to obtain spectra of aqueous solutions of lysozylme for frequency shifts as small as 5 cm^?1. In addition, Raman measurements are made on two crystalline forms of hen egg white lysozyme. The spectra obtained from the solution and from the crystal are found to be similar for frequencies above 100 cm^?1. However, a low-frequency band at 25 cm^?1 observed in crystalline lysozyme is not found in the solution, indicating that this band cannot be attributed to an internal molecular vibration.     

Binding with lysozyme of antibodies against surface-simulation peptides representing the lysozyme antigenic sites.

Previously it had been shown that native lysozyme has three discontinuous antigenic sites (comprising spatially adjacent residues that may be distant in sequence) that were mimicked by surface-simulation synthetic peptides that had the capacity to bind the bulk (97-99%) of the antibody response against native lysozyme. In the present work these three surface-simulation synthetic peptides were coupled to succinoylated bovine serum albumin, and the conjugates were injected into rabbits. Antibodies against each peptide reacted, as expected, only with that peptide, but it was also found that the antibodies could bind with lysozyme, and the complete specificity of this binding was rigorously established. The advantages of these findings in conformational and immunological investigations are outlined.     

Evolution of cow nonstomach lysozyme genes.

Expansion of the lysozyme gene family is associated with the evolution of the ruminant lifestyle in ruminant artiodactyls such as the cow. Gene duplications allowed recombination between stomach lysozyme genes that may have assisted in the evolution of an enzyme adapted to survive and function in the stomach environment. Despite amplification of lysozyme genes, cow tears, milk, and blood are considered to be lysozyme deficient. Here we have identified 2 new cow lysozyme cDNA sequences and show that at least 4 different lysozymes are expressed in cows in nonstomach tissues and probably function as antibacterial defence enzymes. These 4 lysozyme genes are in addition to the 4 digestive lysozyme genes expressed in the stomach, yielding a number of expressed lysozyme genes in the cow larger than that found in most nonlysozyme-deficient mammals. In contrast to expectations, evidence for recombination between stomach and nonstomach lysozyme genes was found. Recombination, through concerted evolution, may have allowed some lysozymes to acquire the ability to survive in occasional acidic environments.     

Biosynthesis of glycosylated human lysozyme mutants.

Complementary DNA encoding human lysozyme was subjected to oligonucleotide-directed mutagenesis. At one of three selected positions, amino acid residues 22, 68, or 118, the signal for N-linked glycosylation was created. The mutant DNAs were inserted into a eucaryotic vector and transfected into cultured hamster cells. The three mutant cDNAs directed synthesis of lysozyme mutants, which were named LI, LII, and LIII. The mutant lysozymes LI and LII comprised mixtures of glycosylated and nonglycosylated forms. The glycosylated and nonglycosylated forms of mutant LI were found to have an enzymatic activity similar to normal human milk lysozyme. The usage of the glycosylation sites in the mutants was similar in Chinese hamster ovary (CHO) and baby hamster kidney cells. Approximately two of every three molecules in mutant LI, approximately one of every eight molecules in mutant LII, and practically no molecules in mutant LIII became glycosylated. In CHO cells, the processing of the oligosaccharide side chains yielded several larger products than in baby hamster kidney cells. This size variability of glycosylated lysozyme from CHO cells may be explained by the presence of biantennary and triantennary endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides with N-acetyllactosamine repeats of variable length and by the presence of hybrid oligosaccharides, as suggested by affinity to several lectins and sensitivity to endo-beta-galactosidase. In both cell types, the majority of the glycosylated forms were secreted and thus behaved similarly to nonglycosylated lysozyme. A small proportion of mutant LI lysozyme remained associated with the cells. The retained lysozyme was recruited predominantly from the molecules bearing high mannose oligosaccharides. These molecules were targeted to lysosomes, and their carbohydrate was trimmed to an endo-beta-N-acetylglucosaminidase H-resistant form. Owing to the small size of mutant LI lysozyme, minor changes in the size of its carbohydrate moiety result in detectable changes in the electrophoretic mobility of the whole glycoprotein. We suggest that this novel glycoprotein could be used as a reporter in studies on processing and segregation of glycoproteins.