[Chronic neutrophilic leukemia. Blood muramidase].
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[Serum muramidase assay in patients with refractory anaemia (author's transl)].
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Anemia, Aplastic↗
[Serum activity of muramidase in patients with trichinellosis and some types of helminthiasis].
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Adolescent↗
Interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcal-clostridial cell wall lytic enzyme.
Bacterial autolysins are endogenous enzymes that specifically cleave covalent bonds in the cell wall. These enzymes show both substrate and bond specificities. The former is related to their interaction with the insoluble substrate whereas the latter determine their site of action. The bond specificity allows their classification as muramidases (lysozymes), glucosaminidases, amidases, and endopeptidases. To demonstrate that the autolysin (LYC muramidase) of Clostridium acetobutylicum ATCC824 presents a domainal organization, a chimeric gene (clc) containing the regions coding for the catalytic domain of the LYC muramidase and the choline-binding domain of the pneumococcal phage CPL1 muramidase has been constructed by in vitro recombination of the corresponding gene fragments. This chimeric construction codes for a choline-binding protein (CLC) that has been purified using affinity chromatography on DEAE-cellulose. Several biochemical tests demonstrate that this rearrangement of domains has generated an enzyme with a choline-dependent muramidase activity on pneumococcal cell walls. Since the parental LYC muramidase was choline-independent and unable to degrade pneumococcal cell walls, the formation of this active chimeric enzyme by exchanging protein domains between two enzymes that specifically hydrolyse cell walls of bacteria belonging to different genera shows that a switch on substrate specificity has been achieved. The chimeric CLC muramidase behaved as an autolytic enzyme when it was adsorbed onto a live autolysin-defective mutant of Streptococcus pneumoniae. The construction described here provides experimental support for the theory of modular evolution which assumes that novel proteins have evolved by the assembly of preexisting polypeptide units.
Amino Acid Sequence↗
Factors affecting sensitivity of group B streptococci to an exogenous murein hydrolase.
Group B streptococci treated with cell wall synthesis inhibitors (penicillin or vancomycin) or by a variety of membrane-acting agents are sensitized to the lytic action of exogenous M1 muramidase. Muramidase without a sensitizing agent caused rupture of bacterial chains only, accompanied by the release of a small amount of cell wall peptidoglycan label and an increase of the number of colony-forming units. In combination with sensitizing agents the exogenous muramidase appeared to initiate hydrolysis of biosynthetically new peptidoglycan. Treatment of the cells with chloramphenicol or starvation for nutritionally required amino acids suppressed the rate of cell lysis and peptidoglycan hydrolysis during subsequent sensitization and muramidase treatment of the bacteria. Purified cell walls prepared from the amino acid starved cells were also hydrolyzed with a slower rate by muramidase. It is suggested that agents sensitizing the bacteria to the exogenous muramidase act by perturbing or removing some nonmurein components of the cell envelope which protect the peptidoglycan from the activity of exogenous enzyme. Agents increasing resistance against exogenous muramidase may also cause some alteration in peptidoglycan structure.
Cell Division↗
Structures suggesting cell-wall-deficient forms detected in circulating erythrocytes by fluorochrome staining.
Cell-wall-deficient (CWD) forms of bacteria are associated with certain cases of idiopathic septicemia. In this preliminary study of blood examined immediately after venipuncture, structures with a morphology characteristic of CWD forms were seen parasitizing the erythrocytes. These inclusions were usually circumferential, but in some cases they protruded from the red cells. The CWD forms were detected by staining with Gould's rhodamine-labeled muramidase, which reacted similarly to acridine orange but with greater specificity. A blocking test, employing unlabeled muramidase, indicated the specificity of the reaction between muramidase and the microbial substrate. Reaction of the forms with muramidase indicates their bacterial, rather than mycoplasmal, nature. Thus in vivo CWD forms have a detectable component of muramic acid, at least in certain cases. Sixty-eight individuals with a diagnosis of fever of unknown origin were tested, with 51 nondebilitated individuals serving as controls. More intraerythrocytic forms reacting with muramidase were found in the patients than in the controls. Nearly 40% of the cases had a relatively high incidence of erythrocyte parasitism. In some instances when freshly drawn blood was examined, the structures, which appear to be microbial, extended in rhizoid filaments from the erythrocytes.
Acridines↗
Hydrolysis of soluble, linear, un-cross-linked peptidoglycans by endogenous bacterial N-acetylmuramoylhydrolases.
Soluble, linear, uncross-linked peptidoglycans, prepared from two autolysis-defective mutants of Streptococcus faecium ATCC 9790 and from Micrococcus leuteus, were used as substrates for studies of hydrolysis by an N-acetylmuramoylhydrolase (muramidase). The kinetics of hydrolysis of these substrates and the ability of the muramidases isolated from S. faecium ATCC 9790 and from two autolysis-defective mutants, Lyt-14 and Aut-3, to carry out transglycosylation reactions were compared with the action of hen egg white lysozyme (EC 3.2.1.17). Hydrolysis of these substrates by the endogenous streptococcal muramidases resulted in the production of disaccharide-peptide monomers with the structure (formula; see text) as nearly the sole product. As estimated from increases in reducing groups, hydrolysis proceeded at a linear rate for extended intervals, with consumption of up to 75% of the substrate, even at substrate concentrations well below the Km value. Apparent Km and relative Vmax values for the three streptococcal enzymes were indistinguishable from each other or from those for hen egg white lysozyme. These results indicate that the autolysis-defective phenotype of these mutants cannot be attributed to differences in their muramidases. In contrast to the action of hen egg white lysozyme, the streptococcal muramidase failed to catalyze transglycosylations. The extended periods of hydrolysis at constant rates are consistent with the occurrence of multiple catalytic events after the formation of the enzyme-substrate complex.
Amino Acid Sequence↗
The peptidoglycan-degrading property of lysozyme is not required for bactericidal activity in vivo.
Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in pulmonary host defense. Increased concentration of lysozyme in the airspaces of transgenic mice enhanced bacterial killing whereas lysozyme deficiency resulted in increased bacterial burden and morbidity. Lysozyme degrades peptidoglycan in the bacterial cell wall leading to rapid killing of Gram-positive organisms; however, this mechanism cannot account for the protective effect of lysozyme against Gram-negative bacteria. The current study was therefore designed to test the hypothesis that the catalytic activity (muramidase activity) of lysozyme is not required for bacterial killing in vivo. Substitution of serine for aspartic acid at position 53 (D53S) in mouse lysozyme M completely ablated muramidase activity. Muramidase-deficient recombinant lysozyme (LysM(D53S)) killed both Gram-positive and Gram-negative bacteria in vitro. Targeted expression of LysM(D53S) in the respiratory epithelium of wild-type (LysM(+/+)/LysM(D53S)) or lysozyme M(null) mice (LysM(-/-)/LysM(D53S)) resulted in significantly elevated lysozyme protein in the airspaces without any increase in muramidase activity. Intratracheal challenge of transgenic mice with Gram-positive or Gram-negative bacteria resulted in a significant increase in bacterial burden in LysM(-/-) mice that was completely reversed by targeted expression of LysM(D53S). These results indicate that the muramidase activity of lysozyme is not required for bacterial killing in vitro or in vivo.
Animals↗
Modular organization of the lytic enzymes of Streptococcus pneumoniae and its bacteriophages.
The nucleotide sequences of genes cpl7 and cpl9 of the Streptococcus pneumoniae bacteriophages Cp-7 and Cp-9, encoding the muramidases CPL-7 and CPL-9, respectively, have been determined. The N-terminal domains of CPL-7 and CPL-9 were virtually identical to that previously reported for the CPL-1 muramidase. The C-terminal domain of the CPL-7 muramidase, however, was different from those of the host amidase and the phage Cp-1 and Cp-9 lysozymes. Whereas all enzymes studied are characterized by repeated sequences at their C termini, the repeat-unit lengths are 20 amino acids (aa) in CPL-1, CPL-9 and in the host amidase, but 48 aa in CPL-7. Six repeated sequences represent the C-terminal domains of CPL-1, CPL-9 and the host amidase, and 2.8 perfect tandem repetitions that of CPL-7. The peculiar characteristics of the structure of CPL-7 muramidase correlate with its biochemical and biological properties. Whereas CPL-1, CPL-9 and the pneumococcal amidase strictly depend on the presence of choline-containing cell walls for activity, CPL-7 is able to degrade cell walls containing either choline or ethanolamine. These results support the previously postulated role for the C-terminal domain of these lytic enzymes in substrate recognition and provide further experimental evidence supporting the notion that the proteins have evolved by an exchange of modular units.
Amino Acid Sequence↗
The second peptidoglycan hydrolase of Streptococcus faecium ATCC 9790 covalently binds penicillin.
A second peptidoglycan hydrolase (muramidase-2) of Streptococcus faecium ATCC 9790 (Enterococcus hirae) has been purified to apparent homogeneity. The enzyme has been shown to be a beta-1,4-N-acetylmuramoylhydrolase (muramidase; EC 3.2.1.17) and to differ in substrate specificity from a previously isolated muramidase. Purified enzyme appears as two protein staining bands with molecular masses of 125 and 75 kilodaltons (kDa) on polyacrylamide gels after sodium dodecyl sulfate electrophoresis. Elution and renaturation of protein bands from sodium dodecyl sulfate-polyacrylamide gels showed that both proteins have muramidase-2 activity. Both proteins have been shown to bind radioactive benzylpenicillin and have the same electrophoretic mobilities as penicillin-binding proteins 1 and 5 present in membrane preparations of this organism, respectively. Incubation of a [14C]penicillin G-labeled 125-kDa form of the enzyme with crude alkaline extracts from S. faecium (which did not contain added proteinase inhibitors) showed the endogenous conversion of the radiolabeled 125-kDa form to the radiolabeled 75-kDa form of the enzyme.
Chromatography, Affinity↗
Peptidoglycan-hydrolyzing activity of the FlgJ protein, essential for flagellar rod formation in Salmonella typhimurium.
Because the rod structure of the flagellar basal body crosses the inner membrane, the periplasmic space, and the outer membrane, its formation must involve hydrolysis of the peptidoglycan layer. So far, more than 10 genes have been shown to be required for rod formation in Salmonella typhimurium. Some of them encode the component proteins of the rod structure, and most of the remaining genes are believed to encode proteins involved in the export process of the component proteins. Although FlgJ has also been known to be involved in rod formation, its exact role has not been understood. Recently, it was suggested that the C-terminal half of the FlgJ protein has homology to the active center of some muramidase enzymes from gram-positive bacteria. In this study, we showed that the purified FlgJ protein from S. typhimurium has a peptidoglycan-hydrolyzing activity and that this activity is localized in its C-terminal half. Through oligonucleotide-directed mutagenesis, we constructed flgJ mutants with amino acid substitutions in the putative active center of the muramidase. The resulting mutants produced FlgJ proteins with reduced enzymatic activity and showed poor motility. These results indicate that the muramidase activity of FlgJ is essential for flagellar formation. Immunoblotting analysis with the fractionated cell extracts revealed that FlgJ is exported to the periplasmic space, where the peptidoglycan layer is localized. On the basis of these results, we conclude that FlgJ is the flagellum-specific muramidase which hydrolyzes the peptidoglycan layer to assemble the rod structure in the periplasmic space.
Amino Acid Substitution↗
Cloning, expression and sequence analysis of an endolysin-encoding gene of Lactobacillus bulgaricus bacteriophage mv1.
The lysA gene specifying an endolysin of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv1, was cloned and expressed in Escherichia coli. The 4.05-kb restriction fragment containing this gene was analysed by restriction and deletion mapping, and by subcloning. The nucleotide sequence of a 1150-bp fragment coding for an active lysin was determined. The lysA gene consists of 585 bp and codes for a protein of a deduced Mr of 21,120, which agrees with the size based on in vivo transcription/translation studies. The deduced amino acid sequence of the mv1 lysin (LysA) was compared to that of other known lytic enzymes. Significant homology was observed with the N-terminal portion of the muramidase of the fungus Chalaropsis and that of the muramidase of the Streptococcus pneumoniae phage Cp-1, suggesting that LysA might be a muramidase. In E. coli, the cloned lysA gene was able to complement the muramidase-defective bacteriophage lambda Ram5, proving that the products of these two genes are interchangeable. The lysA gene is preceded by an open reading frame with unknown function and no characteristic prokaryotic promoter sequences could be detected upstream from lysA, suggesting that this gene is part of an operon.
Amino Acid Sequence↗
Properties of cell wall-associated DD-carboxypeptidase of Enterococcus hirae (Streptococcus faecium) ATCC 9790 extracted with alkali.
DD-Carboxypeptidase (DD-CPase) activity of Enterococcus hirae (Streptococcus faecium) ATCC 9790 was extracted from intact bacteria and from the insoluble residue (crude cell wall fraction) of mechanically disrupted bacteria by a brief treatment at pH 10.0 (10 mM glycine-NaOH) at 0 degrees C or by extraction with any of several detergents. Extractions with high salt concentrations failed to remove DD-CPase activity from the crude wall fraction. In contrast to N-acetylmuramoylhydrolase (both muramidase 2 and muramidase 1) activities, DD-CPase activity failed to bind to insoluble cell walls or peptidoglycan matrices. Thus, whereas muramidase 1 and muramidase 2 activities can be considered to be cell wall proteins, the bulk of the data are consistent with the interpretation that the DD-CPase of this species is a membrane protein that is sometimes found in the cell wall fraction, presumably because of hydrophobic interactions with other proteins and cell wall polymers. The binding of [14C]penicillin to penicillin-binding protein 6 (43 kilodaltons) was proportional to DD-CPase activity. Kinetic parameters were also consistent with the presence of only one DD-CPase (penicillin-binding protein 6) in E. hirae.
Cell Membrane↗
"Histiocytic markers" in melanoma.
BACKGROUND: Tumor cells of malignant melanoma, the "great imitator," may morphologically mimic almost any cell, including histiocytes. Immunohistochemical stains for histiocytes are often used to distinguish histiocytic lesions that resemble melanomas, but we have noted and others have reported that these markers may be immunoreactive in melanomas. METHODS: We evaluated 43 primary and metastatic melanomas with traditional markers for melanomas (S100, HMB45, and NKI-C3) and common markers used for histiocytes (alpha-1-antitrypsin or AAT, CD68/KP1, HAM56, Mac387, and Muramidase). The extent (<5%, 5 to 30%, 30 to 60%, 60 to 90%, >90%) and intensity (1+ to 4+) of staining were recorded semi-quantitatively. RESULTS: Melanoma immunoreactivity (>5% of tumor cells) was as follows: S100, 100%; HMB45, 91%; NKI, 91%; AAT, 95%; CD68, 86%; HAM56, 26%; Mac387, 7%; and Muramidase, 30%. Among the histiocytic markers, staining by AAT and CD68 was typically diffuse but weak. Staining by HAM56, Mac387, and Muramidase was usually focal. In contrast, the traditional melanoma markers showed diffuse and strong staining. Interpretation of the histiocytic markers was complicated by scattered atypical histiocytes and pigmented tumor cells. CONCLUSION: Melanomas are commonly immunoreactive for histiocytic markers. AAT and CD68 immunostains are diffusely positive almost as frequently as traditional melanoma markers, although with weaker intensity. HAM56, Mac387, and Muramidase are less commonly positive and exhibit focal staining. Therefore, depending on the context, histiocytic markers may not be helpful in differentiating histiocytes and histiocytic tumors from melanomas.
Adult↗
Multinucleate giant cells in sublabial salivary gland tissue in Sjögren's syndrome. A diagnostic pitfall.
The presence of multinucleate giant cells in the sublabial salivary gland tissue in Sjögren's syndrome is an unusual phenomenon which can give rise to differential diagnostic problems. We found in 4 cases of 55 patients with Sjögren's syndrome multinucleate giant cells. In 2 of these 4 patients epimyoepithelial islands were also present. The combination of both multinucleate giant cells as epimyoepithelial islands can mimic the histological picture of a non- caseating granulomatous disease. To discriminate between an epimyoepithelial island and an epithelioid granuloma the immunoperoxidase technique with antibodies directed against muramidase appeared an useful tool. The epithelioid cells contain muramidase whereas the cells in the epimyoepithelial island do not contain this enzyme. Thus, multinucleate giant cells are a rare phenomenon in Sjögren's syndrome, therefore restricting its diagnostic significance. When they occur in Sjögren's syndrome staining for muramidase can be of help to avoid a false positive diagnosis of diseases in which non- caseating granulomatous inflammation occur, such as in sarcoidosis.
Adult↗
Immunohistochemical study of idiopathic histiocytosis of the mandible and maxilla.
Recent evidence suggests that the proliferative cells of idiopathic histiocytosis may be derived from Langerhans cells. In this study, antisera to S-100 protein, HLA-DR (la-like) antigen, muramidase, and alpha 1-antichymotrypsin were tested on formalin-fixed, paraffin-embedded tissue from nine cases of idiopathic histiocytosis using an immunoperoxidase technique. Tumor cells were positive for S-100 protein and HLA-DR antigen but negative for muramidase and alpha 1-antichymotrypsin. Mononuclear phagocytes were positive for HLA-DR antigen, muramidase, and alpha 1-antichymotrypsin but negative for S-100 protein. The immunohistochemical staining pattern of the tumor cells in these cases of idiopathic histiocytosis is similar to that seen for normal Langerhans cells. When these results are coupled with electron microscopic and histochemical data, it would appear that the origin of cells in idiopathic histiocytosis is from the Langerhans cell or its precursor. Thus, this condition might be better designated "Langerhans cell disease."
Chymotrypsin↗
Peanut agglutinin: a new marker for tissue histiocytes.
The histiocyte (macrophage) is a unique cellular constituent of the immune system which is involved in immune and non-immune cellular reactions, as well as in the genesis of a variety of benign and malignant neoplasms. The ability to distinguish histiocytes from similar appearing cells morphologically, is often difficult, yet may be of considerable practical and theoretical importance. The present study describes a new marker for histiocytes, applicable to routinely processed tissues. An immunoperoxidase procedure to detect binding of peanut agglutinin (PNA) was applied to 58 specimens consisting of lymph nodes, extranodal lymphoid tissues, neoplasms and reactive histiocytic lesions. Results were compared with a currently accepted histiocytic marker--cytoplasmic muramidase. Of the 58 tissues, 51 showed PNA binding to morphologically recognizable histiocytes, whereas muramidase was detected in only 44. PNA binding- appeared at least as sensitive and specific as muramidase, as a marker for histiocytes, and offers the advantage that it does not bind to cells of the granulocytic series.
Cytoplasm↗
Separation and characterization of human neutrophil granules.
Human blood neutrophilic leukocytes were separated and purified by modifications of the Hypaque/Ficoll and dextran separation methods, resulting in a suspension which was greater than 96% neutrophils. Neutrophils were prepared in 0.34 M sucrose containing heparin and were clarified of nongranular debris by sequential passage through polycarbonate filters of pore size 5 mu and 2 mu. Isopycnic sucrose gradients of such filtrates revealed three major bands. The gradient separated fractions were studied by electron microscopy including peroxidase cytochemistry and by enzyme assay for myeloperoxidase (MPO), beta-glucuronidase, muramidase alkaline phosphatase and acid phosphatase utilizing both p-nitrophenylphosphate (pnp) and beta-glycerophosphate as substrates. Peroxidase-positive granules were observed at both density 1.22 (band A) and density 1.20 (band B). Three peroxidase-negative granules were identified: the round or oval peroxidase-negative granule of density 1.22 (band A) and two smaller granules, distinguishable by size and shape at density 1.18 (band C). Band C granules contain crystalloid inclusions. Peaks of muramidase activity coincided with bands A and C, suggesting the presence of muramidase in the peroxidase-negative granules of density 1.22 and in one or both of the peroxidase-negative granules at density 1.18. beta-Glucuronidase was distributed like MPO, with a major peak in band B and a minor peak in band A. Acid beta-glycerophosphatase was largely in band A. Acid pnp phosphatase was nonspecifically associated with soluble nongranular protein which always remained at the origin of sucrose gradients. Alkaline phosphatase was not granule associated and sedimented alone to density 1.145, which is highly suggestive of a cytoplasmic membrane localization for this enzyme.
Acid Phosphatase↗