Journal of applied biochemistry最新文献

The effect of oxygenated sterols on platelet aggregation induced by thrombin and ADP has been studied. All oxygenated sterols tested with a hydroxyl group on the side chain enhanced thrombin-induced aggregation at 25 microM. In the case of ADP-induced aggregation, however, only 22S-hydroxycholesterol[(22S)-5-cholestene-3 beta,22-diol] enhanced the aggregation, and 22R-hydroxycholesterol[(22R)-5-cholestene-3 beta,22-diol], 24S-hydroxycholesterol[(24S)-5-cholestene-3 beta,24-diol], and 25-hydroxycholesterol[5-cholestene-3 beta,25-diol] inhibited it. These effects were observed within 10 min after the addition of oxygenated sterols to platelet suspensions in plasma.

An engineering model was successfully developed for an ATP regeneration system by using two enzymes, acetate kinase (AK) and adenylate kinase (AdK), both obtained from the thermophile Bacillus stearothermophilus. This model is composed of five units: a substrate unit consisting of substrate solutions--AMP, ATP, and acetyl phosphate (AcOP)--an enzymatic reactor unit consisting of AK and AdK immobilized to Sepharose 4B, an auto sampler unit, an analytical unit made up of high-performance liquid chromatography, and a control unit made up of a microcomputer. Operation of the four units could be systematically controlled by the microcomputer. Fundamental, operational conditions were examined using this engineering model. The conversion of AMP to ATP concentration and space velocity (SV). The minimum amount of ATP, which is required to obtain the 100% conversion of AMP to ATP, was determined to be about 4% of AMP concentration. The conversion of AMP to ATP was controlled effectively by changing the SV value. Based on the above experimental data, the continuous operation of an ATP regeneration system was tested at pH 7.5 and 30 degrees C under the conditions of 1.59 mM AMP, 0.084 mM ATP, and 5.0 mM AcOP. It was found that the conversion of AMP to ATP was more than 99% over a period of 6 days without changing SV.

The effect of local gamma-ray irradiation on the enzymatic activity of superoxide dismutase in salivary glands was examined. Mice received a single dose of 60Co gamma-ray irradiation in the range of 3 to 24 Gy to their neck regions. The enzymatic activity in salivary glands was decreased significantly at the 7th day and almost recovered at the 14th day after irradiation with over 6 Gy. The elevation of the lipid peroxide levels in the submandibular gland followed by the leakage of superoxide dismutase from the glands into the blood plasma was observed after irradiation, indicating radiation-induced membrane damage of salivary glands.

beta-Galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) purified from Aspergillus oryzae was modified with 2,4,6-trichloro-s-triazine derivatives of polyethylene glycol (activated BPEG) having molecular weights of 600, 1500, 2000, and 4000. Polyethylene glycol derivatives were attached to 6 of the 12 amino groups exposed on the surface of the enzyme. Upon modification, the enzymatic activity for a water-soluble substrate, o-nitrophenyl beta-D-galactopyranoside, was reduced with increasing molecular weight of the activated BPEG. On the contrary, the enzymatic activity for another substrate, 4-methylumbelliferyl beta-D-galactopyranoside, was increased upon modification. The Michaelis constants of native and modified enzymes for these two substrates were virtually the same. The effect of the modification was more marked in the enzymatic hydrolysis of the beta-galactosidic bond of amphipathic substrates. A fluorescent analog of naturally occurring galactocerebroside, 1-O-galactosyl-2-N-(1-dimethylaminonaphthalene-5-sulfonyl)-sphingosine, was hydrolyzed more rapidly by the modified enzyme than by the native one. The enzyme modified with activated BPEG of 1500 Da had the highest activity for this substrate. The beta-galactosidic bond of the terminal galactose of GM1-ganglioside (II3NeuAcGgOse4Cer, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminosyl)-galactosyl -glucosylceramide) was cleaved by the modified but not by the native enzyme.

Horse liver alcohol dehydrogenase (EC 1.1.1.1) accepts a wide structural range of substrates but exhibits a well-defined and predictable stereospecificity. The enzyme was immobilized on CNBr-activated Sepharose 4B. The immobilized preparation was used to oxidize the enantiomeric pair of cis-1,2-bis(hydroxymethyl)-3-cyclopentene enantioselectively to a mixture of two diastereoisomeric chiral lactones. The two diastereoisomeric products are readily separated and each was isolated with an optical yield of greater than 99%.

The filamentous fungus, Aspergillus niger, efficiently converted cassava polysaccharides to mycelial mass, simple sugars, and acids during the course of its growth. A typical 70-ml culture broth containing 2% cassava polysaccharides yielded 0.38 g dry mycelial mass, 1.14 mmol reducing sugars, and 1.17 meq acids at the end of 42 h. About 70% of the initial total carbohydrate in the medium was degraded during the same period. The sugars and acids in the culture broths were analyzed by HPLC on a single Aminex HPX-87 column at 55 degrees C, using 0.013 N H2SO4 as the eluting solvent. Cassava polysaccharides were degraded to oligosaccharides, maltotriose, maltose, and glucose beyond the 20-h growth periods, with maltotriose emerging as the major simple sugar. The appearance of citric, malic, gluconic, succinic, and fumaric acids accounted mostly for the decreasing pH in the growth media. Formation of carbohydrate species in the culture broths was closely related to the biosynthesis and secretion of several carbohydrases by A. niger. The extracellular carbohydrases were separated and identified by chromatofocusing and polyacrylamide gel electrophoresis to be amyloglucosidase (EC 3.1.2.3), alpha-amylase (EC 3.2.1.1), and alpha-glucosidase (EC 3.2.1.20), respectively.

A facile hybridoma procedure has been used to generate monoclonal antibodies to alpha- and beta-subunits of human chorionic gonadotropin (HCG). The procedure is based on the method of Davis et al. (1982, J. Immunol. Methods 50, 161) and involves the use of a semisolid medium containing methylcellulose for the initial cloning of hybrid cells following immunization and cell fusion. Seven to ten days after cell fusion, viable hybrid clones were removed for subculture in a liquid medium containing RPMI 1640 and 15% fetal calf serum. Initial screening of hybrid cell lines that secrete antibodies to HCG was performed on microplate enzyme-linked immunoassay (ELISA) using HCG-coated microtiter plates. The specificity of these antibodies to either alpha- or beta-subunits was determined by the sodium dodecyl sulfate-gel/protein blot radioimmunobinding method which separates alpha- and beta-subunits of HCG on nitrocellulose strips for radioimmunobinding assay. As a result of this study, it has been possible to generate about 272 hybrid cell lines that secrete antibodies reacting with either the alpha- or beta-subunit of HCG in about 5 weeks. The association constants and cross-reactivities to luteinizing hormone for some of the HCG monoclonal antibodies were determined. The high affinity and specificity of these monoclonal antibodies permit their clinical application in a sensitive sandwich solid-phase enzyme-linked and radioimmunoassay of HCG.

The structural integrity of RNA as antigen in immunological reactions with serum as the source of antibody is endangered because of RNase activity. Aurine tricarboxylic acid (ATA) in its polymeric form has been used to overcome this problem by inhibiting serum RNase. At low concentrations, the polymer completely arrests the RNA degrading activity. An appreciable increase in antibody activity was observed with an anti-RNA serum pretreated with ATA polymer.