Direct Enzymatic Analysis Research Articles

A highly selective amperometric biosensor array for the simultaneous determination of glutamate, glucose, choline, acetylcholine, lactate and pyruvate

The work was aimed at the development of a biosensor array for the simultaneous determination of six solutes (glutamate, glucose, choline, acetylcholine, lactate, and pyruvate) in aqueous solutions. Enzymes selective for these substrates were immobilized on the surface of amperometric platinum disc electrodes and served as bioselective elements of a biosensor array. Direct enzymatic analysis by the developed biosensors provided high sensitivity to the tested substrates (limits of detection were 1–5 μM). The linear ranges of the biosensors were from 0.001–0.01 mM to 0.2–2.5 mM. The influence of solution pH, ionic strength and buffer capacity on the biosensor responses was investigated; the conditions for simultaneous operation of all the bioselective elements were optimized. The absence of any cross-influence of the substrates of enzymatic systems used was shown as well as a high selectivity of the biosensors and the absence of any impact of interfering substances (ascorbic acid, dopamine, cysteine, paracetamol). The developed biosensor array had good response reproducibility and storage stability. The array is suitable for rapid (0.5–1 min) and simple simultaneous determination of glutamate, glucose, choline, acetylcholine, lactate, and pyruvate in aqueous (biological) samples; furthermore, the creation of a single chip with six sensitive elements is possible as well as the addition of other biosensors.

Mutational analysis of substrate specificity in a Citrus paradisi flavonol 3-O-glucosyltransferase

Citrus paradisi 3-O-glucosyltransferase (Cp3GT, Genbank Protein ID: ACS15351) and Citrus sinensis 3-O-glucosyltransferase (Cs3GT, Genbank Protein ID: AAS00612.2) share 95% amino acid sequence identity. Cp3GT was previously established as a flavonol-specific 3-O-glucosyltransferase by direct enzymatic analysis. Cs3GT is annotated as a flavonoid-3-O-glucosyltransferase and predicted to use anthocyanidins as substrates based on gene expression analysis correlated with the accumulation of anthocyanins in C. sinensis cv. Tarocco, a blood orange variety. Mutant enzymes in which amino acids found in Cs3GT were substituted for position equivalent residues in Cp3GT were generated, heterologously expressed in yeast, and characterized for substrate specificity. Structure–function relationships were investigated for wild type and mutant glucosyltransferases by homology modelling using a crystallized Vitis vinifera anthocyanidin/flavonol 3-O-GT (PDB: 2C9Z) as template and subsequent substrate docking. All enzymes showed similar patterns for optimal temperature, pH, and UDP/metal ion inhibition with differences observed in kinetic parameters. Although changes in the activity of the mutant proteins as compared to wild type were observed, cyanidin was never efficiently accepted as a substrate.

Enzymatic Determination of Citric Acid in Honey by Using Polyvinylpolypyrrolidone Clarification.

To characterize honey types, a citric acid determination may be useful. A citric acid determination on honey was carried out with previous polyvinylpolypyrrolidone (PVPP) clarification followed by the Boehringer-Mannheim GmbH enzymatic test. The sample solution was prepared from 2 g of honey in 100 mL of Milli-Q water. A volume of 10 mL of this sample was clarified with PVPP stirring for 1 min and filtered. The enzymatic determination was measured spectrophotometrically at 340 nm, using citrate lyase, L-malate dehydrogenase, and L-lactate dehydrogenase. With these conditions, there were no observed interference effects. The proposed method improves precision [coefficient of variation (CV) between 0.26% and 1.60%] and recovery (between 98.0% and 100.9%) on the direct enzymatic analysis (% CV between 1.02 and 2.66 and recovery between 84.0% and 115.6%). Furthermore, the cost was reduced 70% using a microtest. The method was applied to 20 honeys of Galicia (northwestern Spain), and the results ranged between 44.2 and 827.0 mg of citric acid/kg of honey (mean = 192.9 mg/kg).

Deficiency of methylthioadenosine phosphorylase in human leukemic cells in vivo

Cells from 20 patients with leukemia and 9 with solid tumors were assayed for the enzyme methylthioadenosine phosphorylase, which function in both purine and polyamine metabolism in rapidly dividing cells. As determined by autoradiography of viable cells, and by direct enzymatic analysis, samples from one individual with pre-T-cell acute lymphoblastic leukemia and one with common acute lymphoblastic leukemia were methylthioadenosine phosphorylase deficient. In contrast, other leukemias of similar antigenic phenotype, as well as normal peripheral blood lymphocytes, thymic lymphocytes, and normal bone marrow cells, had substantial methylthioadenosine phosphorylase activity. This evidence suggests that the complete absence of methylthioadenosine phosphorylase distinguishes some leukemic cells in vivo from their nonmalignant counterparts.

Deficiency of methylthioadenosine phosphorylase in human leukemic cells in vivo

AbstractCells from 20 patients with leukemia and 9 with solid tumors were assayed for the enzyme methylthioadenosine phosphorylase, which function in both purine and polyamine metabolism in rapidly dividing cells. As determined by autoradiography of viable cells, and by direct enzymatic analysis, samples from one individual with pre-T-cell acute lymphoblastic leukemia and one with common acute lymphoblastic leukemia were methylthioadenosine phosphorylase deficient. In contrast, other leukemias of similar antigenic phenotype, as well as normal peripheral blood lymphocytes, thymic lymphocytes, and normal bone marrow cells, had substantial methylthioadenosine phosphorylase activity. This evidence suggests that the complete absence of methylthioadenosine phosphorylase distinguishes some leukemic cells in vivo from their nonmalignant counterparts.

Queen bee venom contains much less phospholipase than worker bee venom

Phospholipase A 2 in queen bee venom was measured by direct enzymatic analysis, by the wheal/flare reaction of an allergic individual, and by translation of the venom gland mRNA in a wheat germ system. These analyses showed that on a weight for weight basis, venom from queen bees contains no more than one fortieth of the phospholipase activity present in the venom from mature worker bees. The formation of phospholipase A 2 is thus subject to different control mechanisms in these castes of indentical genotype.

Oxidative phosphorylation and ultrastructural transformation in mitochondria in the intact ascites tumor cell.

We have examined the ultrastructure of mitochondria as it relates to energy metabolism in the intact cell. Oxidative phosphorylation was induced in ultrastructurally intact Ehrlich ascites tumor cells by rapidly generating intracellular adenosine diphosphate from endogenous adenosine triphosphate by the addition of 2-deoxyglucose. The occurrence of oxidative phosphorylation was ascertained indirectly by continuous and synchronous monitoring of respiratory rate, fluorescence of pyridine nucleotide, and 90 degrees light-scattering. Oxidative phosphorylation was confirmed by direct enzymatic analysis of intracellular adenine nucleotides and by determination of intracellular inorganic orthophosphate. Microsamples of cells rapidly fixed for electron microscopy revealed that, in addition to oxidative phosphorylation, an orthodox --> condensed ultrastructural transformation occurred in the mitochondria of all cells in less than 6 sec after the generation of adenosine diphosphate by 2-deoxyglucose. A 90 degrees light-scattering increase, which also occurs at this time, showed a t (1/2) of only 25 sec which agreed temporally with a slower orthodox --> maximally condensed mitochondrial transformation. Neither oxidative phosphorylation nor ultrastructural transformation could be initiated in mitochondria in intact cells by the intracellular generation of adenosine diphosphate in the presence of uncouplers of oxidative phosphorylation. Partial and complete inhibition of oxidative phosphorylation by oligomycin resulted in a positive relationship to partial and complete inhibition of 2-deoxyglucose-induced ultrastructural transformation in the mitochondria in these cells. The data presented reveal that an orthodox --> condensed ultrastructural transformation is linked to induced oxidative phosphorylation in mitochondria in the intact ascites tumor cell.

Identification of reduced nicotinamide adenine dinucleotide phosphate-dependent aldehyde reductase in a Rhodotorula strain.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-aldehyde reductase was isolated in 24% yield and 66-fold purification from a dl-glyceraldehyde-grown Rhodotorula species. This enzyme was specific for NADPH, and d-, l-, or dl-glyceraldehyde were equally good substrates. Other substrates had activities as follows: methylglyoxal, 50%; fructose, 33%; d- and l-arabinose, 12%; d-xylose, 8%; d-glucose, 5%; d-erythrose and d-threose, 0 to 5%. The product from the reduction of dl-glyceraldehyde was glycerol, as shown by high voltage electrophoresis, paper chromatography, and direct enzymatic analysis. Kinetic studies gave K(m) values of 0.89 mm and 0.013 mm for dl-glyceraldehyde and NADPH, respectively. An optimal pH range of 6.3 to 6.7 was found for maximal activity. Reduction of NADP(+) by glycerol was not demonstrable. This Rhodotorula NADPH-aldehyde reductase activity was compared to similar enzymes from other sources.