Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.8.1.4 (diaphorase)
2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method is described for increasing the response of enzyme immunoassays employing alkaline phosphatase as the label initiating 2 sequential catalytic reactions. First, NADP is dephosphorylated to produce NAD, which catalytically activates a specific redox-cycle involving the enzymes alcohol dehydrogenase and diaphorase. During each turn of the cycle 1 molecule of a tetrazolium salt is reduced to an intensely coloured formazan. The method is capable of detecting as little as 0.01 amol alkaline phosphatase, and when applied to an immunoassay for TSH a sensitivity (zero + 2.5 standard deviations) of 0.0013 mIU/l was obtained.
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PMID:Enzyme amplification for immunoassays. Detection limit of one hundredth of an attomole. 351 23

Human liver BCKADH complex was purified. On SDS-polyacrylamide gel electrophoresis, the purified enzyme complex gave three major bands having molecular weights of 51,000, 46,000, and 36,000, and one minor band with a molecular weight of 55,000. The minor band corresponded in molecular weight to lipoamide oxidoreductase which was purified separately. The purified BCKADH represented only approximately 20% of the maximum activity when assayed without addition of exogenous lipoamide oxidoreductase, indicating that lipoamide oxidoreductase component was readily dissociable from the complex. The BCKADH effectively oxidized all of KIV, KIC, and KMV, yielding apparent Km values in the range of 14-17 microM for those alpha-keto acids. Vmax values obtained were 0.86, 0.61, and 0.51 mumole NADH produced/min/mg of protein for KIV, KIC, and KMV, respectively, in the presence of excess amount of lipoamide oxidoreductase. This ratio of Vmax values was practically identical to those of specific activities obtained with respective branched-chain alpha-keto acids at each purification step. The enzyme complex also oxidized pyruvate and alpha-ketoglutarate to a lesser extent. Kinetic experiments gave Km values of 0.98 and 2.9 mM for pyruvate and alpha-ketoglutarate, respectively, with Vmax of 0.43 and 0.08 mumole NADH produced/min/mg of protein. NAD and CoASH were absolutely required for the reaction. Km values for NAD and CoASH were estimated to be 47 and 25 microM, respectively.
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PMID:Purification and characterization of human liver branched-chain alpha-keto acid dehydrogenase complex. 359 87

The results of quantitative histoenzymologic investigations of succinate dehydrogenase, lactate dehydrogenase and NAD-diaphorase in cardiomyocytes of dogs with acute experimental arterial occlusion in ischemic and postischemic periods are reviewed. An increased activity of dehydrogenases in the early periods (3,6 h) of ischemia and during recirculation was established, with its noticeable reduction at later terms (9,12 h). Medical correction of postischemic disorders was shown to improve cardiomyocyte metabolism.
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PMID:[Changes in the dehydrogenase activity of cardiomyocytes during acute experimental arterial occlusion of extremities]. 373 May 53

The different flavoproteins contributing to flavin fluorescence of isolated rat liver mitochondria have distinct excitation and emission spectra. The NAD-linked flavin component was identified as alpha-lipoamide dehydrogenase, while the non-NAD-linked component was found to be electron transfer flavoprotein. The differences in excitation and emission properties of the mitochondrial flavoproteins permit selective recording of their redox state changes in isolated mitochondria.
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PMID:Spectral properties of fluorescent flavoproteins of isolated rat liver mitochondria. 375 88

The fluorometric assay for formate in serum was modified by pretreating samples with acetonitrile (1:1) precipitation; substituting p-iodonitrotetrazolium violet (INT) for resazurin; and by combining the cofactor (NAD), coupled enzyme (diaphorase), and secondary substrate (INT) into one reagent. Formate is oxidized by formate dehydrogenase producing NADH which reduces INT via diaphorase to a visible red-colored endpoint that can be measured on a spectrophotometer at 500 nm. Previous problems with fluorometric endpoint methods are eliminated when using this modified procedure: calibration is linear rather than nonlinear; blanking is rarely needed due to the acetonitrile sample preparation; dynamic range is expanded up to 10-fold; a simple spectrometer rather than a fluorometer is used; and the number of steps is reduced. The method is demonstrated to be linear, specific, sensitive, precise, and accurate.
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PMID:Improved enzymic assay for serum formate with colorimetric endpoint. 375 27

Study of the activity of some myocardial enzymes in sudden death cases with alcoholic cardiomyopathy (ACMP) was made by quantitative histochemical methods. The decrease of dehydrogenase activity of succinate, lactate, beta-hydroxybutyrate, alpha-glycerophosphate and NAD-diaphorase was found in line with the increase of the activity of glucose-6-phosphate dehydrogenase, alcohol dehydrogenase and catalase versus control (myocardium of those who died of trauma). Disorders of major metabolic processes in the myocardium may occasionally lead to electrical instability resulting in ventricular fibrillation and sudden cardiac death. In almost 80% of sudden cardiac deaths in ACMP foci of acute myocardial ischemia are found, that can lead to ventricular fibrillation with lethal outcome.
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PMID:[Histoenzymologic characteristics of the myocardium in sudden death in patients with alcoholic cardiomyopathy]. 380 Jun 78

Intrinsic NADPH diaphorase activity is a component of the membrane-bound NAD(P)H:O2 oxidoreductase of human neutrophils. NADH-specific diaphorase activity is also present in membrane fractions rich in oxidoreductase activity. Studies were undertaken to determine whether the NADH diaphorase might also be intrinsic to the oxidoreductase. The latter diaphorase was freed from the membrane by detergent extraction and partially purified approximately 80-fold. Its apparent molecular weight following solubilization in deoxycholate and Tween-20 was 204 000 +/- 10 000. The specific activity of the partially purified diaphorase with ferricyanide as electron acceptor was 7.6 X 10(3) mU/mg protein, its pH optimum was 7.0, and its Km for NADH was 13 microM. It is completely devoid of NADPH diaphorase activity, lacks the capacity to reduce molecular oxygen, yet readily reduces ferricyanide, 2,6-dichlorophenolindophenol and ferricytochrome c. Whereas the NADH diaphorase was freed from the particulate fraction of cell lysates by extraction in 10 mM Tris-HCl buffer (pH 8.6) made up in 15% glycerol and 0.5% Tween-20, NADPH-dependent diaphorase and superoxide-generating activities also present in the membrane were not. These observations make it unlikely that the principal membrane-bound NADH diaphorase found in human neutrophils is a component of the NAD(P)H:O2 oxidoreductase, despite its common association in the same particulate fraction of cell lysates.
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PMID:Purification and resolution of NADH diaphorase activity from NADPH diaphorase-linked: O2 oxidoreductase activity of human neutrophils. 384 37

The clonal study of L cell culture has shown that the clone-forming cells are heterogeneous both in form and in the activities of enzymes (succinate dehydrogenase, lactate dehydrogenase, NAD- and NADP-diaphorase) which were determined by histochemical methods. The morphological heterogeneity is characteristic for clones with not less than 10 cells manifesting itself earlier and heterogeneity as to the activity of the studied enzymes--later, in clones with more than 15-20 cells.
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PMID:[Heterogeneity of L-line cells in the early stages of clone development]. 384 12

Up to now, more than 40.000 determinations of urinary estrogens (E1 + E2) have been carried out in routine clinical analysis by the enzymatic method using estradiol dehydrogenase. This method makes use of the transhydrogenating activity of the placental enzyme: this enzyme transfers hydrogen from NADP to NAD with recycling of the specific substrate (E1 + E2). For several years the necessary reagents have been commercially available in the form of a kit. Nonetheless, various improvements have been made to the measurement of reduced NAD, which accumulates in the reaction medium and is directly proportional to the concentration of the two estrogens. Three protocols are available at present: Spectrophotometric measurement at 340 nm (initial technique); Colorimetric measurement at 492 nm. The pink colour measured arises from the reduction of a tetrazolium salt (INT) by reduced NAD in a coupled system using diaphorase; Measurement by bioluminescence of the light energy liberated on the reduction of flavin derivatives by NADH. The reaction is mediated by various enzymes isolated from marine bacteria (FMN oxidoreductase and luciferase) in the presence of an aliphatic aldehyde (decanal). The procedure for each of these protocols is described as well as the means for controlling the linearity of the reaction. The choice of protocol is determined by the biological fluid available, the speed of response desired and the cost of the analysis.
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PMID:[Various protocols for determining estrogens by the enzymatic method using estradiol dehydrogenase. Respective procedures and advantages]. 386 35

The fluorescence signal of flavoproteins of rat liver mitochondria was investigated to determine the respective contributions of the various flavoenzymes. About 50% of the overall signal were found to be NAD-linked and caused by alpha-lipoamide dehydrogenase flavin (Em7.4 = -283 mV). Roughly 25% were due to a flavoprotein reducible in a non-NAD-linked reaction. This fluorescent flavoenzyme (Em7.4 = -52 mV) has been tentatively identified as a flavoprotein of the fatty-acid-oxidizing system, most probably the electron transfer flavoprotein. The remaining 25% of the signal are accounted for by flavoenzymes which are reducible by dithionite only. These flavoenzymes were not involved in the flavoprotein fluorescence alterations accompanying changes in electron flow through the respiratory chain. Contributions of other mitochondrial flavoproteins such as succinate dehydrogenase, NADH dehydrogenase, alpha-glycerophosphate dehydrogenase, proline dehydrogenase, and choline oxidase, to the overall flavin fluorescence signal of isolated rat liver mitochondria can be neglected.
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PMID:Contribution of different enzymes to flavoprotein fluorescence of isolated rat liver mitochondria. 402 66


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