Biological activity of (lipo)polysaccharides of the exopolysaccharide-deficient mutant Rt120 derived from Rhizobium leguminosarum bv. trifolii strain TA1.

Lipopolysaccharides (LPS) from Rhizobium leguminosarum biovar trifolii TA1 (RtTA1) and its mutant Rt120 in the pssBpssA intergenic region as well as degraded polysaccharides (DPS) derived from the LPS were elucidated in terms of their chemical composition and biological activities. The polysaccharide portions were examined by methylation analysis, MALDI-TOF mass spectrometry, and (1)H NMR spectroscopy. A high molecular mass carbohydrate fraction obtained from Rt120 DPS by Sephadex G-50 gel chromatography was composed mainly of L-rhamnose, 6-deoxy-L-talose, D-galactose, and D-galacturonic acid, whereas that from RtTA1 DPS contained L-fucose, 2-acetamido-2,6-dideoxy-D-glucose, D-galacturonic acid, 3-deoxy-3-methylaminofucose, D-glucose, D-glucuronic acid, and heptose. Relative intensities of the major (1)H NMR signals for O-acetyl and N-acetyl groups were 1 : 0.8 and 1 : 1.24 in DPS of Rt120 and RtTA1, respectively. The intact mutant LPS exhibited a twice higher lethal toxicity than the wild type LPS. A higher in vivo production of TNFα and IL-6 after induction of mice with Rt120 LPS correlated with the toxicity, although the mutant LPS induced the secretion of IL-1ß and IFNγ more weakly than RtTA1 LPS. A polysaccharide obtained by gel chromatography on Bio-Gel P-4 of the high molecular mass material from Rt120 had a toxic effect on tumor HeLa cells but was inactive against the normal human skin fibroblast cell line. The polysaccharide from RtTA1 was inactive against either cell line. The potent inhibitory effect of the mutant DPS on tumor HeLa cells seems to be related with the differences in sugar composition.

Purification of cobalt-activated acylase by affinity chromatography.

alpha-Hydroxyisocaproyltyrosine (HyIc-Tyr-OH), a potent competitive inhibitor of the cobalt-activated acylase form 2, was synthesized. Its derivative, alpha-aminopentyl-HyIc-Tyr-OEt was coupled to cyanogen bromide-activated Sepharose 4B and was used for about 100-fold purification of the acylase from human liver by affinity chromatography. The preparation obtained did not show aminoacylase, aspartyl acylase or alanylarylamidase activities. The same chromatographic method was also applied to isolate form 2 of the serum acylase from patients with viral hepatitis and guinea pig placenta.

Penicillin amidase from Proteus rettgeri.

1. Penicillin amidase from Proteus rettgeri was purified 580-fold by a four-step chromatographic procedure. Titration with phenylmethanesulphonyl fluoride showed that the purified preparation contains 53% of the enzyme. 2. The molecular weight of the amidase was found to be 65.000. The enzyme is strongly inhibited by N-bromosuccinimide and zinc ions. It hydrolyses penicillins, cephalosporins and some synthetic substrates, and in addition it catalyses synthesis of ampicillin from methyl ester of phenylglycine and 6-aminopenicillanic acid. 3. The immobilized amidase obtained by copolymerization of the chemically modified enzyme with acrylamide was applied for preparative hydrolysis of benzylpenicillin.

Polymorphism of the cobalt-activated acylase in human tissues.

A method was elaborated for obtaining polyacrylamide gel zymograms of the cobalt-activated acylase after electrophoresis. Two fractions of the acylase showing activity towards N-chloroacetyl-gamma-L-glutamyl-beta-naphthylamide were found in human kidney, liver and intestine. The two fractions isolated from liver differ in substrate specificity, heat resistance, response to metal ions, inhibition by deaminated dipeptides, and in molecular weight. They differ also from other N-acylamino acid amidohydrolases: aminoacylase (EC 3.5.1.14) and aspartoacylase (EC 3.5.1.15).

Two molecular forms of penicillin amidase synthesized by Escherichia coli.

The Swatek's method was further simplified for the assay of penicillin amidase activity. The absorbance of colour obtained during determination of 6-aminopenicillanic acid was dependent on concentration of 4-dimethylaminobenzaldehyde and on temperature. Antiodies induced in rabbits with one molecular form of penicillin amidase from E. coli PCM 271 (PA-1 or PA-2) did not cross-react with the other amidase form. No differences in substrate specificity on inactivation with SDS and in alkaline medium between the two amidase forms were observed. Concentrated urea inactivated PA-2 irreversibly and PA-1 reversibly. N-Bromosuccinimide inactivated almost completely only PA-1. Two E. coli PCM 271 strain variants were separated by microbial selection. Each of them produced only one amidase form. Also two amidase forms were found in cells of E. coli ATCC 11105, whereas E. coli ATCC 9636 and ATCC 9637 synthesize only PA-1.

Aminoacylase from Micrococcus agilis.

Intracellular aminoacylase from Micrococcus agilis CCM 2131 was purified 430-fold with a 23% yield. The purified enzyme was homogeneous on polyacrylamide-gel electrophoresis and it smolecular weight was 58000. The enzyme hydrolysed stereospecifically a number of acylated L-amino acids. Its activity towards N-acetyl-L-phenylglycine was strongly inhibited by 1,10-phenanthroline, N-bromosuccinimide and mercaptoethanol, and was inhibited competitively by glycylglycine.

Activity and polymorphism of the cobalt-activated acylase in tissues of rodents during development.

The activity of cobalt-activated acylase, measured towards N-chloroacetyl- and N-butyryl-gamma-L-glutamyl-beta-naphthylamide, was found in all tissues of the adult animals. In the kidney, liver and small intestine of adult guinea-pig and rat two fractions differing in electrophoretic mobility (fractions 1 and 2) were present. The early foetus contained fraction 2, sometimes accompanied by fraction 3 which later disappeared; on further development of the foetus, fraction 1 appeared. Fraction 1 was distinctly activated by cobalt ions; fractions 2 and 3 were strongly inhibited by deaminated leucylphenylalanine. In the guinea-pig, the molecular weight of the three fractions ranged from 43000 to 59000.

Colorimetric method for assay of serum gamma-glutamyltransferase activity with some L-gamma-glutamyl-carboxyanilides.

A simple method for the assay of serum gamma-glutamyltransferase activity with soluble L-gamma-glutamyl-carboxyanilides is presented. It is based on transformation of aminobenzoic acid liberated by the enzyme into colour complex with 4-dimethyl-aminobenzaldehyde or with 4-dimethylaminocinnamaldehyde in acid medium. Among three carboxyanilides studied the highest enzyme activity was noted with L-gamma-glutamyl-3-carboxyanilide and no activity with L-gamma-glutamyl-2-carboxyanilide. A good correlation was demonstrated for serum gamma-glutamyltransferase activity determined by the new colorimetric method and by the standard one with L-gamma-glutamyl-3-carboxy-4-nitroanilide.

A new fluorimetric method for the determination of gamma-glutamyltransferase activity in blood serum.

Using 7-(gamma-L-glutamyl)-4-methyl-coumarylamide as the fluorogenic substrate a simple and sensitive method for the assay of transferase activity of gamma-glutamyl-transferase in human blood serum and some other biological fluids is described. The substrate was synthesized with a good yield by the King and Kidd method. Close correlation and good agreement was noted between activities measured by the fluorimetric method and by the old colorimetric one in which gamma-L-glutamyl-p-nitroanilide was used.

The use of gamma-glutamyl-p-aminobenzoic acid as the substrate for determination of gamma-glutamyltranspeptidase activity in blood serum.

By the phthaloyl method less toxic and readily soluble gamma-L-glutamyl-p-aminobenzoic acid was synthesized. This substance was used as a substrate for gamma-glutamyltranspeptidase activity assay in blood serum and urine. Close correlation was shown between the results obtained with the new method and with the old one which used gamma-L-glutamyl-p-nitroanilide.

Molecular forms of cobalt-activated acylase in human tissues and serum of patients with viral hepatitis.

Two molecular forms of cobalt-activated acylase present in human tissues and one or three in sera of patients with viral hepatitis were noted. They have different substrate specificity. Only form 2 is strongly inhibited by alpha-hydroxyisocaproyl-tyrosine and -phenylalanine. Electrophoretic migrations of all enzyme forms are different from those of aminoacylase. Immunoglobulin antiform 2 of the acylase does not precipitate other forms of cobalt-activated acylase or aminoacylase.

Acyl derivatives of p-aminobenzoic acid as new substrates for the assay of serum acylase activity.

A simple colorimetric method for the assay of cobalt-activated acylase activity in human serum using new and less toxic N-chloroacetyl-gamma-L-glutamyl-p-aminobenzoic acid as substrate has been described. The values obtained with this method are almost the same as with the previously described method using naphthylamide substrate.

Detection and assay of some human proteins by immunoconcentration technique.

For detection and semi-quantitative assays of some human proteins by immunoconcentration techniques, specific antibodies covalently bound to nylon microparticles and then captured in the form of small dots on glass microfibre disc, were used. The discs were placed on a water absorbant material in a simple plastic device. For the technique, antibodies labeled with peroxidase were also applied. The use of several chromogenic substrates for peroxidase was checked in the technique.

The use of normal filter paper for "dot-immunobinding assay" of some antibodies.

Normal filter papers were used for specific detection of tiny amounts of antibodies against two penicillin amidase molecular forms from Escherichia coli, monoclonal antibodies of IgG and IgM class against lipopolysaccharide from Citrobacter O36 and IgM antibody against glycoprotein N from human red blood cells. For colour detection of antibodies bound to proper antigens adsorbed on the paper, second antibody-horse radish peroxidase conjugates and hydrogen peroxide with 4-chloronaphthol were applied. The use of filter paper for dot-immunobinding assay of antibodies gave similar results to those obtained with expensive nitrocellulose sheets used by other authors.

Separation of two fractions from rabbit anti-BPTI antibody by affinity chromatography.

Non-competitive enzyme-linked immunosorbent assay (ELISA) for rabbit anti-BPTI antibody is described. The antibody was purified by affinity chromatography on Sepharose-BPTI column and then separated into two antibody fractions on Sepharose-trypsin (BPTI) column. The fraction I antibody, not retarded on the second biosorbent column, efficiently inactivated BPTI, but the fraction II antibody, eluted from the column with glycine buffer, pH 2.2 was firmly bound to active BPTI. This proves that BPTI has at least two epitopes.

Cobalt-activated acylase in serum of patients with viral hepatitis.

Newly synthesized and non-toxic acyl derivatives of p-aminobenzoic acid were used as substrates indiagnostic kit for assay of cobalt-activated acylase activity. The enzyme activity, in serum of patients with viral hepatitis, depends on time, type and treatment of the disease and also on age and sex of patients. The presence of HBs antigen has no influence on it. In the patient sera 1-3 molecular forms of the enzyme were found but in the liver of healthy or sick individuals two forms were noted. Using alpha-hydroxy-isocaproyl-tyrosine covalently coupled to Sepharose 4B as a bioadsorbent; the form 2 of acylase from human liver was isolated and separated from the form 1, aminoacylase and aspartyl acylase. Specific immunoglobulins anti-form 2 does not react with other forms of the enzyme either in serum or in the liver.

Isolation of human lutropin from woman urine and comparison of its properties with pituitary hormone.

Lutropin was isolated from woman preovulatory urine by immunoaffinity chromatography using a column with monoclonal antibody (mAb) anti-beta hLH(37) coupled to Sepharose CL 4B. As it was demonstrated the isolated hormone, as well as pituitary lutropin, were separated in SDS-PAGE into several well visible fractions with 30-94 kDa molecular mass and scarcely visible fractions with 14-20 kDa. All fractions reacted only with mAb anti-alpha hCG(99)-HRP but not with mAb anti-beta hLH-HRP. Pretreatment of pituitary lutropin with PN-Gase F did not affect its electrophoretic pattern. After boiling the hormone with SDS and beta ME no fractions in SDS-PAGE were observed. No substantial differences in affinity chromatography on Con A-Sepharose between pituitary and urinary lutropin were noted. Some differences between these two hormone preparations were observed in assays performed with several ELISA variants. Using two pairs of mAbs anti-beta hLH for ELISA technique no hormone was assayed in urine samples from women, collected between 12 and 16 days of menstrual cycles.

Purification of gamma-glutamyltransferases by immunoaffinity chromatography and some properties of the enzymes.

Immunoadsorbents were obtained by coupling antibodies to Sepharose 4 B activated with cyanogen bromide. Thus immobilized antibody directed against bovine kidney gamma-glutamyltransferase was used for immunoaffinity chromatography of the enzyme from bovine kidney and liver, from cow milk and from sheep kidney and liver. Immobilized anti-rat kidney gamma-glutamyltransferase antibody was used for purification of the enzyme from rat kidney, mouse kidney, hamster kidney and rat Morris hepatoma 5123D. Yields of the protease-solubilized gamma-glutamyltransferases isolated on immunoadsorbents columns were usually over 60%. The purified enzymes were almost homogenous in polyacrylamide gel electrophoresis. The enzymes showed different molecular weights and electrophoretic mobilities. The effect of antibodies on affinity of the enzymes to substrate and inhibition by synthetic anthglutin and its isomers were studied.

The use of antibodies labelled with dyes for fast and simple assay of some human proteins by immunofiltration technique.

Immunofiltration technique with polyclonal and monoclonal antibodies for semi-quantitative assays of human albumin, chorionic gonadotropin, immunoglobulin G and transferrin was elaborated. An amount of antibody was immobolized in the form of 6 radially located small bars on a dry test filter made of glass microfibre sheet. The other amount of antibody, used in solution, was labelled with some dyes like commercial disperse dyes, colloidal elements, formazans and polypyrrole. Number of colour bars appearing on the test filter showed ranged of analyte concentration. Good results were obtained using antibodies labelled with colloidal gold, Disperse Red 11 and formazan from MTT. Assays with monoclonal antibodies were more sensitive than with polyclonal antibodies.

Enzyme-linked immunosorbent assay (ELISA) of penicillin amidases.

Non-competitive, sandwich enzyme immunoassay for both penicillin amidases from Escherichia coli is described. The assay involves the use of monospecific antibodies and their conjugates. The amidases inactivated by heating and by acid- or alkali-treatment cannot be assayed. Reproducible results for each amidase were achieved within 5-6 hours in the range of 3-500 ng/ml (0.25-40 mU/ml) and coefficient of variation was 13%.