Journal of applied biochemistry最新文献

Cysteamine determination is performed by using a coupled enzymatic system. The reaction product between cysteamine and bromopyruvate inhibits competitively and specifically D-amino acid oxidase activity. A reaction system allowing the quantitative assay of cysteamine at the nanomole level has been worked out. The standard curve is linear and the assay is useful in determining the cysteamine concentration in tissues and biological samples. The method is fast and of simple execution. The cysteamine concentration in several mammalian organs is also reported.

Antibody such as anti-alpha-fetoprotein was immobilized on cellulose fibril sheets by radiation polymerization of hydrophilic monomers. The nature of the immobilized anti-alpha-fetoprotein sheets was examined by measuring the enzyme activity in an antibody-antigen complex reaction. The relationship between the activity and the polymerization condition was studied. The degree of hydration of the polymer matrix giving a maximum activity appeared to be about 0.2. The activity varied with monomer concentration and with monomer composition in copolymerization of acrylate and diacrylate monomer. The activity was increased by the addition of polyethylene glycol diacrylate monomers, in which the activity increased with increasing length of the oxyethylene unit chain.

beta-Galactosidase has been isolated from Lactobacillus helveticus of a strain isolated from natural starters for the manufacture of Argentine hard cheeses and its properties have been studied. The enzyme was purified 14-fold (by chromatography on DEAE-cellulose and Sepharose 6B-DEAE-cellulose columns and by affinity chromatography in agarose-p-aminophenyl-beta-D-thiogalactoside). The purified extract exhibited a single band following polyacrylamide gel electrophoresis. Maximum enzymatic activity was observed at 42 degrees C and pH 6.5 in 50 mM phosphate buffer. At pH values substantially different from the optimum, a positive cooperativity between substrate molecules was observed. The Km's for o-nitrophenylgalactoside (ONPG) and ONPG + 10 mM of lactose were 4.46 X 10(-5) and 8.9 X 10(-5) M, respectively. Glucose, galactose, galactose 6-phosphate, and lactate acted as noncompetitive inhibitors; MgCl2 protected the enzyme from thermal denaturation. The activation energy of enzymatic hydrolysis of ONPG was 11,400 cal/mol. The Mr was estimated to be 250,000. It is an oligomeric enzyme made of 4 subunits of Mr 65,000.

Albumin has been purified by chromatography using standardized procedures with different starting materials depending on previous fractionation. Plasma can be used directly after cryoprecipitation and Factor IX adsorption or after isolation of IgG. The plasma was centrifuged and then desalted on Sephadex G-25 Coarse, the pH was adjusted, and the euglobulins were precipitated before ion-exchange chromatography on DEAE- and CM-Sepharose CL-6B. This was followed by concentration by ultrafiltration, gel filtration on Sephacryl S-200, and final concentration and formulation. The albumin obtained was 99% pure and contained less than 1% of aggregated protein.

Glucoamylase (alpha-1,4-glucan glucohydrolase, EC 3.2.1.3) from fungal sources is one of the microbial glycoproteins that has received considerable attention particularly because it is used in the commercial production of dextrose. Several investigators have isolated glucoamylase from various fungal sources. In many instances the presence of more than one form of enzyme is common. The enzymes from most sources have pH optima between 4 and 5 and exhibit maximum activity between 40 and 60 degrees C. The enzyme does not require any cofactors for activity or for stability. The enzyme has an Mr between 48,000 and 80,000 and usually has no subunit structure. The amino acid composition of multiple forms of glucoamylases differ in general, but all of them are glycoproteins. The carbohydrate content of the enzyme ranges from 3 to 30% containing mainly mannose, but glucose, galactose, and in some instances glucosamine and xylose are also present. In the enzyme from Aspergillus the carbohydrate structures are present as mono-, di-, tri-, and tetrasaccharide units linked O-glycosidically through mannose to the hydroxyl groups of serine and threonine. In the enzyme from Rhizopus part of the carbohydrate is present as disaccharide (Man-Man-) units linked O-glycosidically and the remainder is present as large heterosaccharide structures attached by N-glycosidic linkages involving aspargine and glucosamine. Carbohydrate moieties seem to have no influence on the enzyme activity or antigenicity but appear to stabilize the enzyme by preserving the three-dimensional structure.

The covalent attachment of polyethylene glycol (PEG) to beta-glucosidase from sweet almonds and alpha-galactosidase from green coffee beans results in alterations of their catalytic properties and masking of specific determinant sites on the enzymes. Both enzymes have increased Km and decreased Vmax values against their respective p-nitrophenyl substrate analogs after PEG attachment. When PEG is attached to 30% of alpha-galactosidase epsilon-amino groups, 12% activity remains against ceramide trihexoside, while its ability to convert type B erythrocytes to type H specificity is lost. However, it still is able to cleave terminal galactose residues from human saliva blood group substance B. PEG-beta-glucosidase (38%) did not elicit the production of complement-fixing antibodies, nor did it react with antibodies produced against the native enzyme. Antibody and lectin-specific binding were lost from both modified enzymes (PEG-beta-glucosidase and PEG-alpha-galactosidase). After conjugation with PEG, beta-glucosidase lost its ability to bind to concanavalin A-Sepharose. Antibodies directed against native alpha-galactosidase blocked its enzyme activity, but lost their ability to inhibit activity in progressively higher modified preparations of the enzyme. Antisera against PEG-alpha-galactosidase (53%) did not inhibit enzyme activity in any alpha-galactosidase or PEG-alpha-galactosidase preparation. These results indicate that PEG tends to cover lectin-specific carbohydrate moieties and antigenic determinants and that these sites probably remain cryptic during in vivo processing of PEG-enzymes.

Glutathione synthetase was purified about 60-fold with 8.5% of activity yield from the cell extracts of Escherichia coli C600 cells transformed with a recombinant plasmid for the glutathione synthetase gene of E. coli B. The purified enzyme had a Mr of 152,000 and was composed of four identical subunits each with a Mr of 38,000. The Km values of the enzyme for gamma-glutamylcysteine, glycine, and ATP were 2.6, 2.0, and 1.8 mM, respectively. The enzyme was most active at pH 8.5 and at 45 degrees C and required divalent cations such as Mg2+, Mn2+, and Co2+ for activity. The activity was inhibited by oxidized glutathione (Ki = 4.4 mM). Reduced glutathione showed no effect on glutathione synthetase activity.

The basic chemistry of the reactions leading to light emission in the firefly and in bacteria are briefly reviewed. With excess firefly reagents, the light intensity is proportional to the ATP concentration. For this reason, the reagents have been used for ATP determination in a number of important biological systems. A number of such applications are reviewed. With excess bacterial reagents, the light intensity is directly proportional to the reduced pyridine nucleotide concentration (NADH or NADPH). The applications of this system for studying reactions involving dehydrogenases using NAD or NADP as electron acceptors are presented. Many assays have now been developed using enzymes immobilized on Sepharose. The advantages of using the immobilized enzymes are greater stability of the immobilized enzymes over the soluble forms; increased sensitivity of detection relative to the soluble forms, and reusability of the immobilized enzymes. A comparison of the immobilized bioluminescent assay for 7 alpha-hydroxysteroid with gas-liquid chromatography and radioimmunoassay is presented. Coimmobilized enzymes can be packed in a flow cell and used in an automated instrument with good reproducibility. It is likely that future developments of bioluminescent assays for ATP or NAD(P)H will be with immobilized enzymes using an automated instrument.

Erythrocytes can be made to entrap enzymes, lipids, drugs, pesticides, and large macromolecules by a variety of encapsulation procedures. Methods of encapsulation include hypotonic dilution and dialysis, lipid fusion, electrical hemolysis, and chemical perturbation. The survival of carrier erythrocytes is dependent on the method of encapsulation as is the encapsulation efficiency. Research has led to utilization of carrier cells for drug and enzyme-replacement therapies, for incorporation of DNA and antibodies into living cells, and for enhancement of the oxygen-carrying capacity of hemoglobin. Many of the possible uses of erythrocytes as carriers are explored.