Is pyridoxal 5'-phosphate an affinity label for phosphate-binding sites in proteins?: The case of bovine glutamate dehydrogenase.

The effects of pyridoxal 5'-phosphate (PalP) on ox liver glutamate dehydrogenase (94% inactivation by 1.8 mM reagent at pH 7 and 25 degrees C) have been compared with those of three analogues, 5'-deoxypyridoxal (96% inactivation), pyridoxal 5'-sulphate (97%) and pyridoxal 5-methylsulphonate (94%), in order to establish whether PalP acts as an affinity label for this enzyme. Like PalP and unlike pyridoxal, which is a much less potent inactivator, none of the analogues has a free 5'-OH group to cyclize with the aldehyde function. The result with 5'-deoxypyridoxal shows that a negative charge, such as that of the phosphate group, is not required for efficient inactivation. With all four reagents, addition of an excess of cysteine or lysine led to 90-100% re-activation over 3-20 h. Dialysis also caused reactivation to a similar extent. A combination of 2.15 mM NADH, 1 mM GTP and 10 mM 2-oxoglutarate gave complete protection against PalP, but only partial protection against the analogues. 5'-Deoxypyridoxal still caused 20-25% inactivation in the presence of the protection mixture. Absorbance measurements after reduction with NaBH4 show the characteristic features of a reduced Schiff's base and allowed estimation of the extent of reaction. With all the reagents the protection mixture decreased incorporation by about 1 mol/mol, but levels of incorporation without protection varied from about 2 mol/mol for PalP up to about 5 mol/mol for 5'-deoxypyridoxal. The labelling at additional sites may explain the residual inactivation in the presence of potent protecting agents.

Determination of foetal calf serum in vaccine preparations by immunoradiometric assay and enzyme-linked immunosorbent assay.

A simple and sensitive immunoradiometric assay (IRMA) for detection of foetal calf serum proteins in vaccine preparations has been developed. In this two-site sandwich assay the same preparation of polyclonal anti-FCS immunoglobulin (IgG) was used for coating the solid phase (as a capture antibody) and in a labelled form (I-125 labelled) for detection. The developed assay allows precise and accurate quantification of FCS proteins (down to 10 ng/ml) in vaccine preparations and has been used for screening of FCS residues (a) during production of measles vaccine, and (b) in various different commercial vaccine preparations. An enzyme-linked immunosorbent assay (ELISA) was also developed based on the same assay two-site sandwich principle, where anti-FCS was directly labelled with horse-radish peroxidase. ELISA was comparable in sensitivity and accuracy with IRMA.

Comparison of immunogenicity of recombinant and plasma derived hepatitis B antigen in guinea-pigs.

Four different preparations of hepatitis B antigen (HBsAg) were tested in parallel with respect to their ability to elicit anti-HBs in guinea-pigs. We compared the effect of low doses (5 micrograms single dose and 30 micrograms total amount) of human plasma derived and recombinant HBsAg, in purified forms or prepared as vaccines, respectively. The highest titre of anti-HBs developed was detected in guinea-pigs immunized with plasma derived HBsAg, followed the by response in animals immunized with plasma derived vaccine. Purified recombinant antigen and recombinant vaccine exhibited poor immunogenicity compared with plasma derived antigen preparations. The increase in the dose of recombinant antigen (150 micrograms total amount) resulted in markedly improved response in two out of ten animals. Anti-HBs immunoglobulin fractions from pooled sera within each group of animals were isolated, partially purified, coupled to polystyrene beads and subsequently used in immunoradiometric assay with monoclonal 125I-anti-HBs. Only the anti-HBs isolated from pooled sera of animals immunized with purified plasma derived antigen met the requirements for high sensitivity and antigen specificity in such assay. Comparable properties were exhibited by the anti-HBs obtained from one guinea-pig immunized with a larger amount of recombinant antigen. With these two anti-HBs immunoglobulin preparations, amounts as low as 1 ng of HBsAg could be detected.

Comparative susceptibility of a peptidoglycan monomer from Brevibacterium divaricatum and its anhydromuramyl analogue to hydrolysis with N-acetylmuramyl-L-alanine amidase. Isolation and characterization of anhydromuramyl-peptidoglycan monomer.

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.

Relationship of metabolism and immunostimulating activity of peptidoglycan monomer in mice after three different routes of administration.

[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.

Partial purification and characterization of N-acetylmuramyl-L-alanine amidase from human and mouse serum.

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.

The metabolic fate of 14C-labeled peptidoglycan monomer in mice. I. Identification of the monomer and the corresponding pentapeptide in urine.

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.

Isolation procedure and properties of monomer unit from lysozyme digest of peptidoglycan complex excreted into the medium by penicillin-treated Brevibacterium divaricatum mutant.

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.

Stimulation of humoral immunity by peptidoglycan monomer from Brevibacterium divaricatum.

Peptidoglycan monomer (PGM), a water soluble and nontoxic disaccharide pentapeptide unit obtained from Brevibacterium divaricatum, was administered intravenously into mice, and the humoral immune response to sheep erythrocytes was assayed by means of Jerne's technique for plaque-forming cells (PFC) in the spleen. The PFC response was evidently stimulated. The counts were increased to practically the same extent over a great range of doses of PGM (from 25 to 1600 microgram per animal), and the effect was present in the mice immunised with optimal, as well as in those immunised with suboptimal, doses of antigen. The magnitude of the immunostimulation depend only on the timing of PGM administration: it was maximal if PGM was injected 1 or 2 days after the antigen. In vitro, in a 4-day culture of spleen cells, PGM did not stimulate PFC formation. We conclude that stimulation of the humoral immune response to sheep red blood cell antigens by PGM probably occurs without cell multiplication and probably involves more than simply a contact of immunocompetent cells with PGM.