RESUMEN
The enzyme N-acetylmuramyl-L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) has been detected in human, mouse, rabbit, bovine and sheep sera. A method for detection of amidase activity using [14C]peptidoglycan monomer as the substrate has been developed. Partial purification of human and mouse amidase was achieved by gel chromatography on Bio-Gel A-1.5 m, DEAE-Sephadex A-50 and Sephadex G-100. Both amidase preparations exhibited maximal activity at pH 9.0 in Tris-HCl buffer and required Mg2+ for maximal activity. Following digestion of peptidoglycan monomer, GlcNAc-MurNAc-L-Ala-D-isoglutaminyl-meso-diaminopimelyl-D-Ala-D-Ala, the disaccharide GlcNAc-MurNAc and the corresponding pentapeptide L-Ala-D-isoglutaminyl-meso-diaminopimelyl-D-Ala-D-Ala were formed and subsequently isolated and chemically characterized. The enzyme therefore acts as an N-acetylmuramyl-L-alanine amidase by cleaving the bond between N-acetylmuramic acid and L-alanine. The glycan linked, peptide-not-cross-linked peptidoglycan dimer was also shown to be a substrate for human and mouse amidase.
Asunto(s)
Amidohidrolasas/sangre , N-Acetil Muramoil-L-Alanina Amidasa/sangre , Animales , Tampones (Química) , Bovinos , Cromatografía en Gel , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Sustancias Macromoleculares , Magnesio/farmacología , Ratones , N-Acetil Muramoil-L-Alanina Amidasa/aislamiento & purificación , Peptidoglicano/metabolismo , Conejos , Ovinos , Especificidad por SustratoRESUMEN
Peptidoglycan monomer (GlcNAc-MurNAc-L-Ala-D-isoglutamine-meso-diaminopimelic acid-D-Ala-D-Ala), labeled with 14C both in the disaccharide and pentapeptide portions, was incubated with slices of mouse liver, kidney or spleen as well as with mouse and human blood, blood cells plasma and serum. Peptidoglycan monomer was isolated unchanged after incubations with mouse organs and blood cells. However, upon incubation with mouse or human blood, 10-50% of the peptidoglycan monomer underwent hydrolysis to the corresponding disaccharide and pentapeptide. After incubations with plasma and serum more than 90% of the [14C]peptidoglycan monomer was metabolized: about 50% of the administered radioactive dose was recovered in the disaccharide unit and about 35% in the pentapeptide part. These results suggest that in blood, plasma and serum of mouse and man, an N-acetylmuramoyl-L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) exists which splits the amide bond between the lactyl carboxyl group of the muramyl residue and the amino group of the peptide moiety in the peptidoglycan molecule.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/metabolismo , Adyuvantes Inmunológicos/metabolismo , Glicopéptidos/metabolismo , Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Animales , Sangre , Brevibacterium , Humanos , Riñón/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos CBA , N-Acetil Muramoil-L-Alanina Amidasa/sangre , Peptidoglicano , Especificidad de la Especie , Bazo/metabolismoRESUMEN
The distribution of radioactive products in mouse urine following intravenous administration of the 14C-labeled peptidoglycan monomer, GlcNAc-MurNaC-L-Ala-D-isoglutamine-meso-diaminopimelic acid-D-Ala-D-Ala, has been studied. 60--80% of radioactivity was recovered within first 3 h, 4--8% in the next 3 h and 2--7% in the following 18 h. The majority of the label was associated with the unchanged peptidoglycan monomer (42--56% of the dose). 14--21% of the label was incorporated into a compound which was isolated and tentatively identified as L-Ala-D-isoglutamine-meso-diaminopimelic acid-D-Ala-D-Ala. The kinetics of excretion and the distribution of radioactivity did not differ in immunized, when compared to non-immunized, mice.
Asunto(s)
Peptidoglicano/orina , Animales , Cromatografía en Gel , Cinética , Ratones , Oligopéptidos/orina , Peptidoglicano/administración & dosificaciónRESUMEN
1. The peptidoglycan complex excreted in large amounts into the medium by the biotin-requiring mutant Brevibacterium divaricatum NRRL-2311 incubated in the presence of penicillin for 1 h has been investigated. A convenient isolation procedure with high yield for the pure monomeric unit from lysozyme digest of the accumulated polymer is described. 2. It is shown that the released peptidoglycan possesses the linear uncross-linked structure made of repeating disaccharide-pentapeptide unit [GlcNAc-MurNac-Ala-D-Glyn(meso-DAP-D-Ala-D-Ala)] which was isolated by stepwise gel filtration and fractionation of the digestion mixture in 10-mg quantities. Evidence that the minor digestion product of accumulated peptidoglycan possesses the glycan-linked dimer structure is given. Under conditions of beta-elimination, the monomeric unit yielded a lactylpentapeptide which was isolated in pure form by gel filtration. 3. The monomer unit originating from the cultures to which L-[U-14C]glutamic acid was added simultaneously with penicillin incorporated the label exclusively in the peptide chain, whereas that labeled from E11-14C]acetate as the precursor contained radioactivity in both the peptide chain (53%) and N-acetylamino groups (47%) of the glycan portion.
Asunto(s)
Brevibacterium/metabolismo , Penicilina G/farmacología , Aminoácidos/análisis , Brevibacterium/efectos de los fármacos , Hexosaminas/análisis , Sustancias Macromoleculares , Muramidasa , Mutación , Resistencia a las Penicilinas , Peptidoglicano/metabolismoRESUMEN
The preparation and characterization of the essential components to be used in the radioimmunoassay of peptidoglycan monomer (PGM) is described. In order to raise the anti-peptidoglycan monomer antibodies 14C-labelled peptidoglycan monomer-bovine serum albumin conjugate was prepared by the coupling of 14C-peptidoglycan monomer to bovine serum albumin in the presence of glutaraldehyde in 0.1 M NaHCO3 at pH 8.3. The prepared conjugate elicited anti-PGM response in rabbits. A synthetic analog of peptidoglycan monomer, Boc-L-tyrosyl-peptidoglycan monomer was prepared by condensation of unprotected peptidoglycan monomer and N-hydroxysuccinimidester of Boc-L-tyrosine in the presence of triethylamine and the obtained disaccharide-hexapeptide was labelled with Na125I. This compound exhibited the ability of binding to anti-peptidoglycan monomer antibodies. The prepared compounds, namely anti-PGM antibodies and 125I-labelled Boc-L-tyrosyl-peptidoglycan monomer, were used as essential components in competitive radioimmunoassay for peptidoglycan monomer determination in mammalian and human sera and plasma, respectively.
Asunto(s)
Peptidoglicano/análisis , Secuencia de Aminoácidos , Anticuerpos , Brevibacterium , Secuencia de Carbohidratos , Radioisótopos de Carbono , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Cromatografía en Capa Delgada/métodos , Electroforesis en Papel/métodos , Datos de Secuencia Molecular , Peptidoglicano/aislamiento & purificación , Radioinmunoensayo/métodosRESUMEN
The complexes of Fe(III) with small molecules (glycopeptides, amino sugars) related to peptidoglycan monomer (PGM) structure were prepared and characterized by chemical and physicochemical methods. A simple method for preparation using separation of the reaction products on a molecular sieve was introduced. Using this method, monomeric complexes of small molecular weight were obtained. The likely structures were proposed on the basis of analytical results.
Asunto(s)
Amino Azúcares/metabolismo , Compuestos Férricos/metabolismo , Glicopéptidos/metabolismo , Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Acetilmuramil-Alanil-Isoglutamina/química , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Secuencia de Aminoácidos , Amino Azúcares/química , Secuencia de Carbohidratos , Fenómenos Químicos , Química Física , Compuestos Férricos/química , Glicopéptidos/química , Datos de Secuencia Molecular , Nitrógeno/metabolismo , Oxígeno/metabolismo , Peptidoglicano/químicaRESUMEN
A 14C labeled low molecular weight immunomodulator, peptidoglycan monomer (14C-PGM), was injected intravenously (i.v.) into mice. At various time intervals thereafter (15 min-6 h), radioactivity in the urine, whole blood, plasma, kidneys, liver, spleen, lungs, intestines and the brain of the mice was determined. Shortly after injection, 14C-PGM was very rapidly excreted from the organism, so that 1 h following administration, 80% of the radioactivity was found in the urine (62% as unchanged PGM and the rest as the metabolites pentapeptide and disaccharide). At the same time, around 2% of the injected material was found in the blood. Six hours after injection, equal quantities were found in the intestines, liver and blood (0.5%), slightly less in the kidneys, lungs and spleen (0.2-0.3%) and the least quantity in the brain (0.04%). However, the dynamics of retention in the organs was evidently different. In the kidneys, lungs and spleen, radioactivity steadily decreased over the studied period. In the liver following an initial decrease, radioactivity remained the same 3 and 6 h after injection. On the other hand, in the intestines and brain PGM seemed to accumulate rather than disappear following i.v. administration. This fact should be considered when explaining different biological activities of low molecular weight bacterial peptidoglycans.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Adyuvantes Inmunológicos/farmacocinética , Acetilmuramil-Alanil-Isoglutamina/administración & dosificación , Acetilmuramil-Alanil-Isoglutamina/química , Acetilmuramil-Alanil-Isoglutamina/farmacocinética , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/química , Secuencia de Aminoácidos , Animales , Secuencia de Carbohidratos , Femenino , Inyecciones Intravenosas , Ratones , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Peptidoglicano , Distribución TisularRESUMEN
[14C] Peptidoglycan monomer, GlcNAc-MurNAc-L-Ala-D-isoglutamine-meso-diaminopimelic acid (omega NH2)-D-Ala-D-Ala (PGM) was administered to mice by the intravenous (i.v.), subcutaneous (s.c.) or peroral (p.o.) routes. The data on distribution of radioactivity and excretion of radioactive products, as well as the data on immunostimulating effects are presented on the comparative basis for PGM administered by three different routes. When injected i.v. or s.c., the major part of applied radioactivity was found excreted in urine, partly as unchanged original compound and partly as the corresponding pentapeptide, L-Ala-D-isoglutamine-meso-diaminopimelic acid (omega NH2)-D-Ala-D-Ala. If administered p.o., the major part of the radioactivity was retained in the stomach and intestinal tract for several hours. The drop in radioactivity in these organs was followed by exhalation of 14CO2 thus indicating extensive degradation of the original molecule. PGM stimulates the humoral immune response to sheep red blood cells in mice if administered i.v. or s.c., but is completely inactive if administered p.o.. Thus, absence of immunostimulating activity following p.o. administration might be explained by extensive metabolic degradation of peptidoglycan monomer.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Bazo/inmunología , Acetilmuramil-Alanil-Isoglutamina/administración & dosificación , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Administración Oral , Animales , Formación de Anticuerpos , Inyecciones Intravenosas , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Peptidoglicano , Distribución TisularRESUMEN
Peptidoglycan monomer, GlcNAc-beta-(1----4)-MurNAc-L-Ala-D-iGln[ (L)-meso-A2pm-(D)-amide-(L)-D-Ala-D-Ala] (PGM), from Brevibacterium divaricatum is composed of the disaccharide pentapeptide containing muramic acid with a reducing end (ca. 90-95%) and of the anhydromuramyl analogue (anhydromuranyl-PGM; ca. 5-10%), according to analysis by high-performance liquid chromatography (HPLC) and fast atom bombardment mass spectrometry (FAB-MS). The two peptidoglycan analogues cannot be separated by simple physico-chemical procedures. The enzyme N-acetylmuramyl-L-alanine amidase (mucopeptide amidohydrolase, E.C. 3.5.1.28) cleaves the bond between N-acetylmuramic acid and L-alanine in the PGM molecule. It is shown that anhydromuramyl-PGM is also a substrate for the amidase. In a preparation containing both analogues, the amidase hydrolyses preferentially PGM rather than anhydromuramyl-PGM. The experimental conditions for treatment with the amidase were adjusted with respect to time and enzyme concentration to allow hydrolysis to proceed for several hours. The course of hydrolysis was followed by analysis of the unhydrolyzed substrate by HPLC, and FAB-MS at predetermined time intervals; after 6 h, the amount of anhydromuramyl-PGM in the unhydrolyzed substrate increased to 25% as compared to the starting material containing only 6%. Such a mixture was suitable for separation of components by preparative thin-layer chromatography and for isolation of completely purified PGM and the corresponding anhydromuramyl analogue containing an intramolecular 1,6-anhydromuramyl end. The separated purified compounds were characterized by HPLC and their structure confirmed by FAB-MS-MS.