Journal of applied biochemistry最新文献

The effect of bloodstream factors (flow rate and blood cellular elements) on the behavior of an experimental system simulating drug targeting to injured sites of vessel walls was studied. The system consisted of red blood cells carrying antibody to type I human collagen (drug carrier) and plastic tube with a collagen-coated inner surface (target). The tube was perfused with a 0.5% (v/v) suspension of 51Cr-labeled red blood cells at different linear flow rates and the red blood cell binding to the tube was determined by gamma-counting. It was demonstrated that an increase in the linear flow rate from 0 to 2 cm/s leads at first to increase of red blood cell binding from 2 X 10(5) to 7 X 10(5) cells/cm2 and then to the decrease of binding back to 2 X 10(5) cells/cm2. In the presence of 50% (v/v) of intact red blood cells the binding continuously increases from 2 X 10(5) to 2.5 X 10(6) cells/cm2 without a subsequent drop. On the basis of the obtained results it is concluded that the behavior of the systems for drug targeting in simple in vitro models can drastically differ from the conditions present in vivo.

The thermophilic enzyme 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP oxidoreductase, decarboxylating, EC 1.1.1.44) from Bacillus stearothermophilus was much more resistant to inactivation under different conditions of temperature, pH, guanidine-hydrochloride, and organic solvents (dioxane, dimethylformamide, acetone) than its mesophilic counterpart from yeast. In addition, the thermophilic enzyme largely withstands proteolysis with trypsin, chymotrypsin, and elastase when compared with the yeast enzyme. It is proposed that thermophilic enzymes are not only thermostable, but also generally more stable to most common protein denaturants than their mesophilic counterparts. Because of their remarkable stability, enzymes isolated from thermophilic microorganisms may be ideally suited for technological applications.

Heat-stable acetate kinase (AK) from Bacillus stearothermophilus was successfully immobilized covalently to Sepharose resin by several conventional methods including carbodiimide, hydroxysuccinimide, cyanogen bromide, and glutaraldehyde and also by a new method which utilizes a bifunctional ADP derivative as a spacer. The latter method gave a higher yield in terms of enzyme activity than the conventional methods. The properties and kinetics of the immobilized AK were studied batchwise and in a column. The Michaelis-Menten equation could be applied to the immobilized AK column. The apparent Km values of ADP and acetyl phosphate for immobilized AK were not significantly different from those for free AK. The pH-activity profile of immobilized AK was similar to that of free AK. The heat stability of immobilized AK was markedly improved as compared with free AK. The immobilized AK retained more than 80% of the initial activity after continuous operation at 30 degrees C for 1 month. It was shown that the immobilized AK from B. stearothermophilus could be utilized as an ATP regeneration system in the bioreactor.

Nine different maleimide compounds were evaluated for the conjugation of Fab' to horseradish peroxidase through thiol groups in the hinge. The compounds evaluated were succinimidyl maleimidoacetate (I), succinimidyl 4-maleimidobutyrate (II), succinimidyl 6-maleimidohexanoate (III), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (IV), succinimidyl m-maleimidobenzoate (V), succinimidyl 4-(p-maleimidophenyl)butyrate (VI), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (VII), sulfosuccinimidyl m-maleimidobenzoate (VIII), and sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (IX). Maleimide groups of I-IV and VII were fairly stable at pH 7.0 at 30 degrees C, while those of the other compounds were significantly decomposed. I-III and VII-IX were sufficiently soluble in the reaction mixture for the introduction of maleimide groups, while the others were more or less precipitated during the reaction. II and III were the most effective in the introduction of maleimide groups and gave the highest recovery of peroxidase in the conjugate, which reached 80%. From these results, II and III were judged to be the most useful, and IV and VII were judged to be fairly useful.

The effect of ethanol, 16,16-dimethyl prostaglandin E2 (DMPGE2), and ethanol after DMPGE2 pretreatment on the solubilization of protein, glycoprotein, and glycolipid constituents of gastric mucus was investigated. The Lucite chamber stomach-flap preparation was used in dogs whose basal H+ secretion was inhibited by intravenous cimetidine. Graded concentrations (5-80%) of ethanol produced a dose-dependent decrease in potential difference (PD) which was accompanied by an increase in the content of proteins, glycoproteins, and glycolipids of the instillates. This effect was most pronounced at 40% ethanol. Exposure of gastric mucosa to DMPGE2 at 0.01 microgram/ml had no effect on the transmucosal PD, and the content of investigated components in the instillates increased only slightly over the saline control levels. DMPGE2 at 0.1 microgram/ml, although it did not induce an evident fall of PD, evoked a moderate increase in glycoprotein and glycolipid liberation. Higher doses (above 1.0 microgram/ml) of DMPGE2 were associated with the fall of PD and the increased solubilization of proteins, glycoproteins, and glycolipids. Pretreatment of the mucosa with DMPGE2 in a dose of 1.0 microgram/ml diminished the liberation of the investigated components from the gastric mucosa by 5 and 10% ethanol, but did not prevent the changes evoked by higher concentrations (20-80%) of ethanol. These data indicate that DMPGE2 applied topically protects in part the gastric mucosa against the ethanol-induced solubilization of mucus constituents.

A series of substituted 1-thio-beta-D-maltopyranosides was synthesized and confirmed by elemental analysis, optical rotation, NMR, and liquid chromatography. These compounds were shown by several biochemical techniques to bind to the active site of alpha-amylase. Steady-state kinetic studies showed the compounds to be competitive inhibitors, with affinities lying within the range of the natural ligands, maltose and maltotriose. Affinity chromatography employing p-aminophenyl-1-thio-beta-D-maltopyranoside linked to Sepharose provides a relatively simple procedure for alpha-amylase purification. The binding of p-bromphenyl-1-thio-beta-D-maltoside was observed in crystals of alpha-amylase using X-ray crystallography, and through the use of difference Fourier analysis its interaction at 5.0-A resolution with the active site of the enzyme has been visualized. The inhibitor binds in a long, deep cleft that divides the two major domains of the enzyme. These studies are believed to provide a first step toward the rational design of ligands for the physiological regulation of starch breakdown and utilization through modulation of alpha-amylase activity.

Pseudomonas sp. ATCC 14676 produces glycocyaminase (EC 3.5.3.2) and guanidinobutyrase (EC 3.5.3.7). Taurocyamine (2-guanidinoethane sulfonate) is a gratuitous inducer of both of these amidinohydrolases. Mutants of this organism capable of utilizing taurocyamine as a nitrogen source were isolated directly from the wild-type cells after uv irradiation or treatment with N-methyl-N'-nitro-N-nitrosoguanidine; frequencies of mutations observed under appropriate conditions were above 10(-7). Strain U2-3-3, which was selected from the 11 isolated taurocyamine-utilizing strains, was proved to be derived from the wild-type strain. Both taurocyamine and 4-guanidinobutyrate were able to induce an enzyme of strain U2-3-3 that liberated urea from taurocyamine, whereas glycocyamine failed to induce the system. The activity of the enzyme toward taurocyamine was found to be about one-third of that toward guanidinobutyrate when both taurocyamine and guanidinobutyrate were used as inducer. These observations suggest that the enzyme of the mutant capable of hydrolyzing taurocyamine has emerged from guanidinobutyrase of the wild-type strain which hydrolyzes taurocyamine at a very low rate, probably as a result of a point mutation in the structural gene.

Five fragments from hexapeptide to decapeptide, corresponding to positions 16-25 of thymosin beta 9, were synthesized and their effects on low E-rosette-forming capacity with sheep erythrocytes of cells from lupus nephritis patients were compared with that of the undecapeptide (positions 16-26) of thymosin beta 9 by taking synthetic thymosin beta 9 as a standard. Two of the fragments (16-25 and 18-25) exhibited higher activity than that of the parent peptide (16-26). The other three sequences (17-25, 19-25, and 20-25) had no effect at concentrations as high as 10(-4) M.

The bioluminescent firefly luciferase assay for ATP was used to measure adenylate kinase activity in plasma. The formation of ATP from ADP was measured continuously in a coupled assay using a luminometer. Optimal analytical conditions were determined for the coupled reaction. The assay was used to follow accumulation of adenylate kinase in plasma of different preparations of stored red blood cells. Adenylate kinase was found to be released concomitantly with hemoglobin during aging. There was a high degree of correlation between the amount of accumulated hemoglobin and adenylate kinase. The assay was also used to measure lysis of stored platelets during aging.

A method for incorporating into proteins a nonradioactive Eu3+ label, which exhibits fluorescence of a long decay time in the presence of suitable ligands, is described. As an example of the use of this label the method has been developed to work as a sensitive protease assay. By hydrolyzing the Eu3+-labeled casein, bound to an insoluble matrix (Sepharose 4B or Affi-Gel 10), with proteases and measuring the Eu3+ released with a pulsed time-resolved fluorometer it was possible to detect as low as 2.5, 1.0, or 1.0 ng of alpha-chymotrypsin, trypsin, or subtilisin, respectively.