Journal of applied biochemistry最新文献

Pronase, proteinase K, carboxypeptidases A and B, aminopeptidase M, intestinal mucosa exopeptidases and prolidase, immobilized to derivatized controlled-pore glass beads, were used in a study of total enzymic hydrolysis of proteins. The combined use of immobilized enzymatic and acid hydrolysis, for assessment of protein quality, will give a more accurate chemical score than that afforded by acid hydrolysis alone. Amino acid analysis of enzymic hydrolysates of native protein substrates (beta-lactoglobulin and insulin) yielded 92% of the theoretical values and 103% of the values observed for standard acid hydrolysates. These results suggest that using a combination of immobilized proteases in concert gives essentially total hydrolysis of protein substrates in a time period (18-24 h) comparable to conventional acid hydrolysis methods.

A method for the flow injection analysis of glucose and uric acid in serum using immobilized enzymes in column form and chemiluminescence detection is described. The method is based on the determination of chemiluminescence formed by the reaction of a luminol-ferricyanide mixture with hydrogen peroxide which is produced by the action of the respective oxidases on glucose and uric acid. Glucose or uric acid in serum were determined with 1 microliter of the sample at a speed of 120 samples/h without carryover and at an assay time of approximately 10 s. The immobilized glucose oxidase column measured only 1.0 X 5 mm, and the immobilized uricase column 1.0 X 20 mm. The present method gave perfect linearity of the data up to 4.0 g glucose per liter or 0.10 g uric acid per liter with satisfactory precision, reproducibility, and accurate reaction recoveries. Furthermore, the present method was hardly affected by ascorbic acid, while the peroxidase-linked colorimetric method is usually influenced significantly by ascorbic acid. Both column reactors showed good operational stability for a 2-month period, during which time they were repeatedly used for analyses over 2000 times. The results on glucose and uric acid correlated satisfactorily with those obtained by other well-established methods.

Both atrophy and hemorrhage were observed in the testis exposed to two acute subcutaneous injections of Cd (0.012 mmol/kg). The hemorrhage was accompanied by the enhancement of lipoperoxide concentration in the testis. These changes were prevented by the simultaneous injection of 0.024 mmol/kg of Se as SeO2, and testicular Zn, Fe, and glutathione levels recovered to the control level. Se stimulated the uptake of Cd to the testis. Se itself did not show any influence on the level of Zn, Fe, and glutathione. Our observations suggest that the preventive effect of Se is an inactivation of Cd in the testis and that testicular Cd concentration alone cannot be used to assess lipid peroxidation. The compounds linked to the glutathione system or related to the membrane integrity may contribute to peroxidation.

The present study was undertaken to compare the enzymatic oscillations induced in three major organs, spleen, kidney, and liver, of mice by the active and inactive forms of an aminopeptidase inhibitor, bestatin. Although bestatin caused sine curve-type oscillations of enzymatic activities in spleen and kidney, its isomer, possessing no enzyme-inhibiting actions and no biological actions, did not show any typical enzymatic changes. Such typical oscillations were not elicited by either one of those two agents in liver, in spite of the apparent difference in the type of oscillations induced by them in this organ. These observations were taken to indicate that the enzymatic oscillations in vivo caused by bestatin are closely related to its aminopeptidase-inhibiting actions seen in vitro and that the immunomodifying actions of this agent are based on its effects on enzymes sensitive to it, possibly involving immunoresponsive cells.

The reaction of succinamidopropyl-glass with nonaqueous thionyl chloride followed by washing with water produces an activated surface which reacts with amino and thiol groups under nondenaturing conditions. Subsequent treatment of the covalently immobilized species with dilute hydroxylamine at pH 7 and room temperature releases about 80% of those molecules attached through amino groups and about 50% of those attached through thiol groups. The succinamidopropyl group appears to be a minimum requirement for such surface reactivity. The derivatized glass beads are stable during storage, and immobilization is achieved by simply contacting the surface with a solution of the protein or biochemical species to be attached. Moreover, the activated succinamidopropyl sites can be dispersed within an inert surface of glycerolpropyl sites. Such dispersion improves the subsequent release of immobilized protein and should provide a useful technique for preparation of a wide variety of specific matrices for affinity chromatography. It was possible to obtain, in solution, molecules which had been refolded from a completely denatured state in an immobilized form; however, extensive unfolding of proteins in strong denaturants reduced by roughly 50% the amount of protein which could be subsequently released.

A simple method for enzyme immobilization using a monoclonal antibody raised against the enzyme was studied, taking guinea pig liver transglutaminase as an example. This method consists of two steps; a conjugation of monoclonal antibodies, which bind with the enzyme without inhibitory effect on the activity, with agarose gel bead supports, and immobilization of enzyme proteins onto the conjugated monoclonal antibodies through the antigen-antibody immunoreaction. The following are the advantages of this method. (i) Activity recovery after the immobilization was very high, (ii) enzyme proteins in crude enzyme preparation could be immobilized specifically, and (iii) immobilized enzymes could be replaced easily by fresh enzyme proteins through a urea-buffer treatment.

A highly sensitive sandwich enzyme immunoassay for a Ca2+-dependent cysteine proteinase (calpain I) and its specific endogenous inhibitor protein (calpastatin) was developed. The calpain I and calpastatin used as immunogens were purified from human erythrocytes. Anti-calpastatin antisera having sufficiently high titer were obtained only when the immunogen was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay method was principally based on the report by M. Imagawa et al. (1982, J. Appl. Biochem. 4, 41-57), using a specific antibody-coated polystyrene ball and horseradish peroxidase-conjugated Fab' fragment of the antibody. The sensitivity was 0.1 ng of calpain I or calpastatin per assay tube. Starting with 50 microliter of the hemolysate from human erythrocytes, the method permitted direct and simultaneous determination of calpain I and calpastatin, without prior separation of these two enzymatically counteracting components by chromatography. The present method as applied to the erythrocytes from 14 healthy adults gave 120-170 micrograms for calpain I and 164-211 micrograms for calpastatin per gram of hemoglobin, respectively.

The effect of linoleic acid hydroperoxide on cultured endothelial cells from human umbilical vein was examined morphologically. Incubation of the cells with a low concentration (0.5 nmol/ml) of linoleic acid hydroperoxide for 3 h caused a slight decrease in electron density of the mitochondrial matrix. Upon an increase of the concentration to 1.0 nmol/ml, the damage became more pronounced, and enlargement and dilatation of the rough-surfaced endoplasmic reticulum were observed. At higher concentrations (5 and 7.5 nmol/ml), dilatation of the rough-surfaced endoplasmic reticulum became further pronounced, and many vacuoles were observed in the cells.

Anti-adenylate antibodies were elicited in rabbits using a conjugate of adenylic acid and bovine serum albumin as immunogen. The specificity of the antibodies was determined by monitoring the inhibition of the binding of [3H]AMP to the antibodies by various nonradioactive nucleic acid components, using a nitrocellulose filter assay. The antibodies were found to be directed against the whole molecule of AMP. They were reactive to polyadenylic acid, RNA, and denatured DNA. The hapten-specific antibodies were isolated by employing an AMP-AH-Sepharose affinity column.