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.     

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.     

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.     

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.     

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.     

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.     

Binding of N-acetyl-chitotriose to human lysozyme.

The interaction of N-acetyl-chitotriose ((GlcNAc)3) with human lysozyme [EC 3.2.1.17] was studied at various pH values by measuring changes in the circular dichroic (CD) band at 294 or 255 nm and the data were compared with the results for hen and turkey lysozymes reported previously (Kuramitsu et al. (1974) J. Biochem.76, 671-683; Kuramitsu et al. (1975) J. Biochem. 77, 291-301). The pH dependence of the binding constant of (GlcNAc)3 to human lysozyme was different from those for hen and turkey lysozymes. The catalytic carboxyls of human lysozyme, Asp 52 and Glu 35, were not perturbed on binding of (GlcNAc)3. This is consistent with the previous findings that the macroscopic pK values of Asp 52 and Glu 35 of human lysozyme are 3.4 and 6.8 at 0.1 ionic strength and 25 degrees and were unchanged on complexing with (GlcNAc)3. An ionizable group with pK 4.5, which participates in the binding of (GlcNAc)3 to hen lysozyme and was assigned as Asp 101, did not participate in the binding of the saccharide to human lysozyme. Between pH 9 and 11, the binding constants of (GlcNAc)3 to hen lysozyme remained unchanged, whereas perturbation of an ionizable group with pK 10.5 to 10.0 was observed for human lysozyme. This group may be Tyr 62 in the active-site cleft. The binding constants of (GlcNAc)3 to human lysozyme molecules having different microscopic protonation forms, with respect to the catalytic carboxyls, were estimated using the binding constants obtained in the present experiments and the microscopic ionization constants of the catalytic carboxyls obtained previously. All four species of human lysozyme had similar binding constants to (GlcNAc)3. This result is different from those for hen and turkey lysozymes.