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Biomedical subjects

M Schindler

Publications and source records attributed to M Schindler.

At least 163 records · Page 9Linked to original sources
PubMed1977

Mechanism of lysozyme catalysis: role of ground-state strain in subsite D in hen egg-white and human lysozymes.

The association constants for the binding of various saccharides to hen egg-white lysozyme and human lysozyme have been measured by fluorescence titration. Among these are the oligosaccharides GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-GlcNAc, GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-N-acetyl-D-xylosamine, and GlcNAc-beta(1 leads to 4-GlcNAc-beta(1 leads to 4)-MurNAc, prepared here for the first time. The binding constants for saccharides which must have N-acetylmuramic acid, N-acetyl-D-glucosamine, or N-acetyl-D-xylosamine bound in subsite D indicate that there is no strain involved in the binding of N-acetyl-D-glycosamine in this site, and that the lactyl group of N-acetylmuramic acid (rather than the hydroxymethyl group) is responsible for the apparent strain previously reported for binding at this subsite. For hen egg-white lysozyme, the dependence of saccharide binding on pH or on a saturating concentration of Gd(III) suggests that the conformation of several of the complexes are different from one another and from that proposed for a productive complex. This is supported by fluorescence difference spectra of the various hen egg-white lysozyme-saccharide complexes. Human lysozyme binds most saccharides studied more weakly than the hen egg-white enzyme, but binds GlcNAc-beta(1 leads to 4)-MurNAc-beta(1leads to 4)-GlcNAc-beta(1 leads to 4)-MurNAc more strongly. It is suggested that subsite C of the human enzyme is "looser" than the equivalent site in the hen egg enzyme, so that the rearrangement of a saccharide in this subsite in response to introduction of an N-acetylmuramic acid residue into subsite D destabilizes the saccharide complexes of human lysozyme less than it does the corresponding hen egg-white lysozyme complexes. This difference and the differences in the fluorescence difference spectra of hen egg-white lysozyme and human lysozyme are ascribed mainly to the replacement of Trp-62 in hen egg-white lysozyme by Tyr-63 in the human enzyme. The implications of our findings for the assumption of superposition and additivity of energies of binding in individual subsites, and for the estimation of the role of strain in lysozyme catalysis, are discussed.

Acetylglucosamine↗
PubMed1976

[Effectiveness and tolerance of the rabies vaccine Lyssavac Berna].

Rabies vaccine derived from human diploid cells (Wyeth, Merieux) and the duck embryo vaccine Lyssavac Berna are prepared from beta-propiolactone-inactivated Pitman-Moore vaccine virus strain. A group of 113 seronegative veterinary students was vaccinated with Lyssavac Berna according to the 4-dose immunization schedule recommended by WHO. Seroconversion was obtained in all test persons. The vaccine was very well tolerated. In agreement with the literature we also found a relatively fast decline in the neutralizing antibody titer after this immunization procedure. However, a booster injection after one year resulted in an antibody rise with titer values comparable with or higher than those obtained after the primary vaccination course. An annual single revaccination dose should be sufficient to protect people at risk.

Adult↗
PubMed1976

A transition state analog of lysozyme catalysis prepared from the bacterial cell wall tetrasaccharide.

Treatment of the cell wall tetrasaccharide GlcNAcbeta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-MurNAc with alkali resulted in the formation of the unsaturated tetrasaccharide GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-delta2,3-2-acetamido-2-deoxy-D-glucoseen. The same compound was also formed by transglycosylation upon incubation of the unmodified tetrasaccharide with the unsaturated disaccharide GlcNAc-beta(1 leads to 4)-delta2,3-2-acetamido-2-deoxy-D-glucoseen (Tipper, D. J. (1968) Biochemistry 7, 1441-1449) and hen egg white lysozyme. The unsaturated tetrasaccharide was further characterized by paper electrophoresis, amino sugar analysis, and NMR. From NMR analysis it is concluded that the delta2,3-2-acetamido-2-deoxy-D-glucoseen at the reducing end of the unsaturated tetrasaccharide has a half-chair conformation. This conformation is similar to the one proposed for the sugar at subsite D in the lysozyme-substrate complex in the transition state. Addition of the unsaturated tetrasaccharide to a solution of hen egg white lysozyme quenched the fluorescence of the enzyme and shifted the fluorescence maximum to the blue, similar to the effect produced by the parent compound. The association constant of the unsaturated tetrasaccharide and lysozyme was measured at pH 6.0 and 24 degrees by spectrofluorimetry and microcalorimetry and found to be 1.45 X 10(5) M-1 and 2.5 X 10(5) M-1, respectively. The average value is 100 times higher than that found for the binding of unmodified tetrasaccharide to the enzyme under the same conditions. The unsaturated tetrasaccharide proved to be a better inhibitor of the lysis of Micrococcus luteus cells than the parent compound by a factor of 35. These results support the hypothesis that the active site of the enzyme is constructed so as to bind the transition state for the reaction it catalyzes more firmly than the substrate itself.

Animals↗
PubMed1976

The binding of glycoconjugates to human-milk D-galactosyltransferase.

Through the use of affinity chromatography, a homogeneous preparation of human beta(1 leads to 4)-D-galactosyltransferase (the A protein of lactose synthase) was obtained. The specificity of this protein for glycoconjugates was studied in the presence and absence of human alpha-lactalbumin. A kinetic analysis of the transfer of D-galactose to N-acetyl-D-glucosamine and the beta(1 leads to 4) linked N-acetylglucosamine oligomers, suggested that the active site region of the enzyme contains more than one binding site for acceptor moleucles. Furthermore, experiments with Na-acetylglucosamine-beta(1 leads to4)-N-acetylmuramic-pentapeptide isolated from Micrococcus luteus indicated that the presence of a peptide chain does not enhance enzymic activity, as compared with the corresponding free disaccharide. Similar results were obtained using ovalbumin and the ovalbumin glycopeptide (which have similar apparent Km values for A protein) as galactose acceptors. In contrast to its ability to inhibit N-acetyllactosamine production, alpha-lactalbumin did not inhibit the transfer of D-galactose to the N-acetylglucosamine oligomers or the glycopeptides. Although alpha-lactalbumin can switch the specificity of A protein from N-acetyl-D-glucosamine to D-glucose resulting in the production of lactose, no transfer of galactose was observed to beta(1 leads to 4)-linked glycose oligomers or to a collagen glycopeptide, D-glycopyranosyl-alpha(1 leads to 2)-D-galactopyranosyloxy-beta(1 leads to 5)-lysine. IT therefore appears that alpha-lactalbumin can only modify human A protein for monosaccharide acceptors.

Acetylglucosamine↗
PubMed1976

The specificity requirements of bacteriophage T4 lysozyme.

A series of bacterial cell wall glycopeptides of low molecular weight and cell wall nucleotide precursors have been tested for their inhibitory action on the digestion by T4 lysozyme of a radioactively labeled linear uncrosslinked peptidoglycan. The disaccharide-peptides GlcNAc-MurNAc-l-Ala-D-Glu(A2pm) (C5) and GlcNAc-MurNAc-L-Ala-D-Glu(A2pm-D-Ala) (C6) as well as the monosaccharide-peptide MurNAc-L-Ala-D-Glu(A2pm) were found to be good competitive inhibitors (with similar Ki values) whereas the disaccharide-pentapeptide GlcNAcMurNAc-L-Ala-DGlu-Gly-L-Lys-D-Ala was a poor inhibitor. T4 lysozyme did not catalyse transglycosylation reactions from Escherichia coli B peptidoglycan to the disaccharide-peptide C6. No changes were seen in the circular dichroism spectra (200-250 nm) or fluorescence emmission spectra upon binding of the good inhibitors. The results obtained indicate that T4 lysozyme has a small active site capable of recognizing a unit consisting of MurNAc-L-Ala-D-Glu(A2pm).

Circular Dichroism↗
PubMed1976

Protein patterns of brush-border fragments in congenital lactose malabsorption and in specific hypolactasia of the adult.

Brush-border membrane proteins of the small-bowel mucosa were separated on polyacrylamide gels from intestinal biopsy specimens obtained from four children with congenital lactose malabsorption and from two adults with specific hypolactasia. In three patients with the congenital type of lactase deficiency the protein band corresponding to brush-border lactase was reduced in intensity, but was never completely absent. No difference in gel patterns was detected when this pattern in congenital deficiency was compared to that obtained from the two patients with adult-type selective hypolactasia. In one patient with congenital lactose malabsorption the protein band corresponding to lactase activity was not detectable. The findings suggest that the mechanisms leading to low lactase activity in the congenital and adult forms of lactose intolerance are similar.

Adult↗
PubMed1976

Quantitative determination of N-acetylglucosamine residues at the non-reducing ends of peptidoglycan chains by enzymic attachment of [14C]-D-galactose.

The ability of human milk galactosyltransferase to attach D-galactose residues quantitatively to the C-4 of N-acetylglucosamine moieties at the ends of oligosaccharides has been utilized for the specific labeling and quantitative determination of the chain length of the glycan moiety of the bacterial cell wall. The average polysaccharide chain length of the soluble, uncrosslinked peptidoglycan secreted by Micrococcus luteus cells on incubation with penicillin G was studied with this technique and found to be approximately 70 hexosamines long. Furthermore, the peptidoglycan chain length of Escherichia coli sacculi of different cell shapes and dimensions was determined both in rod-shaped cells and in filaments induced by temperature shift of a division mutant or by addition of cephalexin or nalidixic acid. The average chain length found in most of these sacculi was between 70 and 100 hexosamines long. Small spherical 'mini' cells had chain lengths similar to those of the isogenic rod-like cells.

Acetylglucosamine↗