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Flow injection determination of glucose, bile acid and ATP using immobilized enzyme reactor and chemiluminescent assay of NAD(P)H.

We have developed a chemiluminescent flow injection method for analysis of bile acid, glucose and ATP using the chemiluminescent assay of NADH using 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(IL)/microperoxidase (m-POD) system and immobilized enzyme reactors such as 3 alpha-hydroxysteroid dehydrogenase, glucose-dehydrogenase, hexokinase and glucose-6-phosphate dehydrogenase. The standard curves were obtained in the range of 5-100 pmol for bile acid, 0.5-5.0 nmol for glucose and 10(-7)-10(-5) mol/L for ATP. The coefficient of variation for each assay was not more than 4.1% for bile acid, 2.3% for glucose and 5.3% for ATP, respectively.

3-Hydroxysteroid Dehydrogenases↗

Alterations in some biochemical processes in the organism of rats being under the influence of chlorfenvinphos administered in diets with variable protein content.

The different doses of chlorfenvinphos given in diets with low-protein and optimal-protein level to young Wistar rats of both sexes were, after 10 or 30 days, without the significant influence on the activity of several serum enzymes used as diagnostic markers in determining the liver damage or disease, as for example:sorbitol dehydrogenase, glutamic dehydrogenase, glucosephosphate isomerase (PHI), aspartate and alanine aminotransferase. Not even important changes were found in the activity of aromatic amino acids aminotransferases in the brain and in protein content in the brain and liver of rats fed diets contaminated with chlorfenvinphos, irrespective of the protein concentration in the diet. Only in some cases at the highest concentration of chlorfenvinphos in the diets the decreased activity of aromatic amino acids aminotransferases appeared in the liver, more evident in low-protein rats. The decrease of the PHI activity in the brain and the inhibition of acetylcholinesterase activity in the serum and brain depended mainly on the amount of chlorfenvinphos in the diets and to a lower degree on the amount of protein. All changes caused by chlorfenvinphos normalized during two weeks after its elimination from the diets.

Acetylcholinesterase↗

Evidence against gene expression after meiosis in the male mouse.

Isozyme patterns in homogenates from various testicular cell types from mice were examined in an effort to ascertain whether the haploid genome is expressed during spermiogenesis. Male mice heterozygous for electrophoretic variants of several glycolytic enzymes were analyzed by starch gel electrophoresis. The enzymes examined were isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, and glucosephosphate isomerase. The isozyme patterns produced by these dimeric enzymes reflect the relative activity of genes in each cell type. These patterns reveal the presence or absence of the transcription of specific genes during spermiogenesis. We found that the genes encoding these enzymes continue to increase during spermiogenesis. Synthesis of these enzymes most likely continues in spermatids, but this synthesis must depend upon premeiotically produced mRNA. These data provide biochemical evidence for the hypothesis that the phenotype of the haploid mammalian gamete depends upon the preceding diploid genome and that a mechanism must exist for the long term post-transcriptional regulation of gene expression during spermiogenesis.

Animals↗

The activation of glucose dehydrogenase by p-chloromercuribenzoate.

p-Chloromercuribenzoate alters various reactions of rat liver glucose (hexose phosphate) dehydrogenase differently. The reagent has little effect on the glucose: NAD or the glucose: NADP oxidoreductases, doubles the rates of oxidations of galactose-6-phosphate and glucose-6-phosphate by NADP and greatly stimulates the oxidations of glucose-6-phosphate and galactose-6-phosphate by NAD. The reagent appears to react with a sulfhydryl group of the enzyme since activation is reversed and prevented by mercaptoethanol. The direct reaction of the reagent with the enzyme is indicated by its lower thermal stability in the presence of the p-chloromercuribenzoate. The size of the enzyme appears to be the same when determined by sucrose gradient centrifugation in the presence or absence of p-chloromercuribenzoate. In microsomes, the oxidation of NADH or NADPH hampers measurements of glucose dehydrogenase. Since p-chloromercuribenzoate inhibits microsomal oxidation of reduced nicontinamide nucleotides, it is possible to assay for glucose dehydrogenase accurately in the presence of the mercurial in microsomes and microsomal extracts and thus measure the effectiveness of a detergent in extracting the enzyme from microsomes.

Animals↗

Effect of temperature on diauxic growth with glucose and organic acids in Pseudomonas fluorescens.

Growth of Pseudomonas fluorescens in batch culture with glucose and organic acids resulted in typical diauxic responses at 30 degrees C but no detectable diauxic lag at 5 degrees C. At 30 degrees C, organic acids were preferentially utilized during the first growth phase. Glucose utilization was delayed until onset of the second growth phase. Systems involved in direct uptake and catabolism of glucose responded in a manner compatible with repression by malate and/or its metabolites and induction by glucose and/or its metabolites. The oxidative non-phosphorylated pathway, through gluconate and 2-ketogluconate (2-KG) as intermediates, was not induced during either growth phase. At 5 degrees C, growth with glucose and organic acids was biphasic but without diauxic lag. Organic acids were preferentially utilized during the first growth phase. Although carbon from glucose was not fully catabolized until onset of the second growth phase, glucose was oxidized to and accumulated extracellularly as gluconate and 2-KG during the first growth phase. No significant repression of glucose-catabolizing enzymes was observed during growth with organic acids in the presence of glucose. However, uptake activities for gluconate and 2-KG did not increase significantly until onset of the second growth phase. Thus, at low temperatures, psychotrophic P. fluorescens oxidized glucose to extracellular 2-KG, while growing on preferred carbon sources. The 2-KG was then catabolized after depletion of the organic acid.

Carboxylic Acids↗

Non-enzymatic synthesis of the coenzymes, uridine diphosphate glucose and cytidine diphosphate choline, and other phosphorylated metabolic intermediates.

The synthesis of uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P) has been accomplished under simulated prebiotic conditions using urea and cyanamide, two condensing agents considered to have been present on the primitive Earth. The synthesis of UDPG was carried out by reacting G1P and UTP at 70 degrees C for 24 hours in the presence of the condensing agents in an aqueous medium. CDP-choline was obtained under the same conditions by reacting choline phosphate and CTP X G1P and G6P were synthesized from glucose and inorganic phosphate at 70 degrees C for 16 hours. Separation and identification of the reaction products have been performed by paper chromatography, thin layer chromatography, enzymatic analysis and ion pair reverse phase high performance liquid chromatography. These results suggest that metabolic intermediates could have been synthesized on the primitive Earth from simple precursors by means of prebiotic condensing agents.

Choline↗

An enzymatic cycling procedure for beta-NADP+ generated by 3'-phosphodiesterase, 2':3'-cyclic nucleotide.

An enzymatic cycling procedure for beta-NADP+ generated by the enzyme 3'-phosphodiesterase, 2':3'-cyclic nucleotide (EC 3.1.4.37) from its substrate 2':3'-cyclic NADP+ is described. The enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and diaphorase (EC 1.8.1.4) are used to cycle the cofactor between its oxidized and reduced forms in the presence of glucose-6-phosphate and p-iodonitrotetrazolium violet (INT) with the concomitant production of colored INT-formazan, monitored at 492 nm. The amplification is about 400-fold per hour and is sensitive enough to detect 6 x 10(-13) mol of NADP(H). A simple procedure for the optimization of this cycling assay is also described. Conjugates to 3'-phosphodiesterase, 2':3'-cyclic nucleotide may be used in heterogeneous enzyme immunoassays for the detection of small quantities of haptens or proteins in biological fluids.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗