Journal of applied biochemistry最新文献

Using an ultrafiltration cell, the continuous production of maltose and a mixture of other oligosaccharides was achieved by the combined action of alpha- or beta-amylase and phosphorylase. For the beta-amylase-phosphorylase system, a steady production of maltose was obtained until the beta-amylase was inactivated. For the alpha-amylase-phosphorylase system, it was difficult to maintain a stable production of oligosaccharides, probably due to the destruction of substrate glycogen by random cleavage by alpha-amylase.

Adjuvant arthritis was induced in rats by the injection of Mycobacterium tuberculosis, and its severity was scored according to the macroscopic findings of the legs, tails, and ears. The average score so obtained was lower when the rats also received indomethacin (1.5 mg/kg/day). The depression of the albumin/globulin ratio was inhibited significantly by the administration of indomethacin. The levels of acid phosphatase and beta-glucuronidase were elevated after the injection of an adjuvant, but they decreased to some extent in rats administered indomethacin. The levels of thiobarbituric acid (TBA)-reactive substances in the sera and synovia were elevated at 2 weeks after the injection of adjuvant and decreased thereafter. In rats administered 1.5 mg/kg of indomethacin, the increase in both serum and synovial levels of TBA reactants was inhibited significantly. These observations suggest that the aggravation of adjuvant arthritis may be associated with lipid peroxidation and that indomethacin may, in part, exert its anti-inflammatory effect by preventing lipid peroxide-induced damage of the synovial membrane.

Antibiotic-cyclopeptide bacitracin covalently bound to Sepharose proved to be an efficient general ligand for affinity chromatography of aspartyl, serine, and metalloproteinases from various sources. The yields of purified enzymes varied from 50 to 180%. New experimental data extend the application of bacitracin-Sepharose for affinity chromatography of cysteine proteinases--papain, bromelain, and ficin. Hence, bacitracin acts as a ligand which more or less efficiently binds proteinases that belong to all the main classes of these enzymes. Bacitracin, being a weak proteinase inhibitor of broad specificity, interacts with the substrate-binding sites of proteinases, which explains its efficiency as a ligand.

Ligand-macromolecule interactions are studied by nonlinear regression analysis performed using a microcomputer (SYMAG-Micromachine 3000/Z). The basic phenomenon is described by the Clark equation B = (Formula: see text), where B and F represent bound and free ligand, respectively, Ni the total concentration of binding sites, and Ki their corresponding affinity constant. The programs of calculation have been extended also for Hill and for Adair equations using the Gauss algorithm described by E. E. Beaulieu and J. P. Raynaud [Eur. J. Biochem. 13, 293 (1970)]. A statistical test of F type is introduced to test the quality of the fit and compare the representations of the phenomenon using the different equations.

Homogeneous (as judged by data from gel filtration, ultracentrifugation, polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate (SDS), and isoelectric focusing) beta-xylosidase showing beta-D-xylosidase, beta-D-glucosidase, beta-D-galactosidase, and alpha-L-arabinosidase activities has been isolated from the hemicellulase preparation of the microscopic fungus Aspergillus niger 15 by ethanol fractionation and chromatography on Sephadex G-50, cellulose DE-52, and Sephadexes SP C-50 and G-200. The specific activity of the enzyme toward p-nitrophenyl-beta-D-xylopyranoside (p-NPX) increased 199-fold and was equal to 35.2 units/mg of protein; the activity yield was 43%. The sedimentation coefficient was equal to 10.6 S, and the molecular weight was 253,000 according to the gel filtration data and 122,000 according to the data from SDS electrophoresis. The isoelectric point was at pH 4.9. An amino acid analysis has shown that dicarboxylic and hydrophobic amino acids prevail in the enzyme. beta-Xylosidase had no carbohydrate component, and p-chloromercuribenzoate inhibited its activity. The temperature optimum of beta-xylosidase activity toward p-NPX was at 70 degrees C, and the pH optimum was 3.8-4.0. The enzyme was stable at pH 3 to 8 and did not lose its activity for 1 h at temperatures up to 50 degrees C. D-Xylose was found to be a competitive inhibitor of the beta-D-xylosidase activity of the enzyme with Ki = 2.9 mM. beta-Xylosidase showed transglycosylase activity.

A new separation method using high-voltage capillary electrophoresis was applied to the analysis of several nucleotides. This system consisted of a micro-bore glass capillary column of 80-micron i.d., a high-voltage power supply, and a uv detector set at 254 nm. Seven nucleotides (cyclic AMP, AMP, ADP, ATP, GMP, GDP, and GTP) were separated completely from each other in 0.02 M phosphate buffer (pH 7) containing 0.5% ethylene glycol by applying about 150 V/cm. The theoretical plate number for AMP was nearly 45,000. A good separation of nucleotides in biological samples of rat blood, liver, and kidney was attained under the same conditions as above. Concentrations of nucleotides in these biological samples were measured by using pyridoxamine as an internal standard.

Mitomycin C (MMC) was conjugated to horse anti-rat alpha-fetoprotein (AFP) antibody at the N-1a position through a glutaric acid-derived spacer arm. In vitro studies showed that MMC was spontaneously and slowly released from the conjugate. The conjugate caused a greater inhibition of both the in vitro and in vivo tumor growth of the AFP-producing rat hepatoma AH66 than did a mixture of anti-rat AFP antibody and MMC or a similar conjugate of MMC with normal horse immunoglobulin.

Most of the chromogen formed when peroxidized material is heated with thiobarbituric acid (TBA) can be ascribed to a colored complex formed between malondialdehyde (MDA) and TBA. Even when little MDA is present, large amounts of MDA-TBA adduct can be formed. This is because lipid peroxides break down to release MDA during the test conditions. Iron is not essential for the breakdown of the peroxides but is essential for the formation of TBA reactivity. This can be related to the ability of iron to decompose lipid peroxides with the release of peroxy radicals, which are precursors of MDA. These peroxy radicals, when released, can initiate further peroxidation during the heating stage of the TBA test. Fatty acids in the absence of lipid peroxides do not undergo significant peroxidation during the acid-heating stage of the TBA test.