Measurement Of NAD Research Articles

Measurement of NAD(P)H and NADPH-Generating Enzymes.

Pyridine nucleotides (NAD(H) and NADP(H)) are key redox carriers in cells and may also have other functions related to stress. These two molecules are crucial in linking metabolism to electron transport chains in photosynthesis and respiration, but they are also critical for ensuring redox signaling and homeostasis during episodes of stress. This is especially the case for NADPH, which must be generated from its oxidized form, NADP+, by key dehydrogenases. Here, we describe methods that can be used to assay contents and redox states of NAD(H) and NADP(H), as well as simple assays to measure the capacity of two key NADPH-generating enzymes.

Abstract 2151: Monitoring AMP Concentration,the universal product of diverse enzymatic reactions

Abstract Many enzymatic reactions use Adenosine triphosphate (ATP) as a substrate and produce adenosine monophosphate (AMP) as the common product of these reactions. AMP also functions as a modulator of many enzymes and a key cellular metabolite regulating energy homeostasis and signal transduction. For example, by activating AMP-Dependent protein kinase (AMPK), it maintains the energy charge ratio at optimal level during metabolic activities; and its concentration is altered during disregulated disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. Here, we demonstrate the versatility of an enzyme coupled assay that quantifies the amount of AMP produced by any enzymatic reactions regardless of its substrates in a homogenous and high through-put formatted form. We successfully implemented it to enzyme reactions that use ATP as a substrate (aminoacyl tRNA synthetase and DNA ligase) by first depleting the remaining residual ATP substrate and converting AMP produced into ATP, the later can be quantified using luciferase/luciferin and generating light. We also tested this assay to measure direct activities of AMP generating enzymes that do not require ATP as substrate, including phosphodiesterases (cAMP) and E.coli DNA ligases (NAD+). In a further elaboration of the AMP-Glo platform, we coupled it to E.coli DNA ligase, enabling measurement of NAD+ and enzymes that use NAD+ like mono- and poly-ADP-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system we can measure the activities of sulfotransferases. In summary, we have developed a very strong platform to monitor AMP concentrations that is applicable to diverse enzymatic reactions. Citation Format: Said A. Goueli, Kevin Hsiao, Subhanjan Mondal. Monitoring AMP Concentration,the universal product of diverse enzymatic reactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2151.

Quantitative Survey of NAD+ Flux in Aged Mice

Decline of NAD+ is associated with aging and disease; hence this critical metabolite is proposed as a master regulator of age‐dependent pathology. NAD+ is found in all living cells and is an essential coenzyme that broadly impacts metabolism and protein covalent modification. NAD+ depletion contributes to mitochondrial dysfunction, which is a classic hallmark of aging. Redox state and the rates of synthesis and degradation of the NAD+ chemical backbone govern the cellular concentration of NAD+. NAD+ decline during aging reflects depletion of the total NAD(H) pool, rather a shift in the NADH/NAD+ ratio, suggesting defective NAD+ synthesis and over‐consumption of NAD+ by cleaving enzymes. Concentration measurements are ill‐suited to distinguish these possibilities. Thus, there is a critical need to examine NAD+ metabolic flux, i.e. the rates of production and consumption of the NAD+ chemical backbone. To this end, we used isotope‐labeled NAD+ precursors, mass spectrometry, and quantitative modeling to determine NAD+ fluxes in young and aged mice. Tissue‐specific measurements of NAD+ labeling enabled organ‐specific assessment of NAD+ fluxes. These experiments identified accelerated NAD+ production and consumption in aging, and thus rational targets for intervention. Understanding of NAD+ fluxes will lay the foundation for understanding how NAD+ homeostasis is achieved: What are the primary control mechanisms that determine proper NAD+ concentration and fluxes? How do these go wrong in aging and disease?Support or Funding InformationHHMI Hanna H. Gray Fellows Program Burroughs Wellcome Fund Postdoctoral Enrichment ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Quantifying the cellular NAD+ metabolome using a tandem liquid chromatography mass spectrometry approach.

Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that serves as a key hydride transfer coenzyme for several oxidoreductases. It is also the substrate for intracellular secondary messenger signalling by CD38 glycohydrolases, DNA repair by poly(adenosine diphosphate ribose) polymerase, and epigenetic regulation of gene expression by a class of histone deacetylase enzymes known as sirtuins. The measurement of NAD+ and its related metabolites (hereafter, the NAD+ metabolome) represents an important indicator of cellular function. A study was performed to develop a sensitive, selective, robust, reproducible, and rapid method for the concurrent quantitative determination of intracellular levels of the NAD+ metabolome in glial and oocyte cell extracts using liquid chromatography coupled to mass spectrometry (LC/MS/MS). The metabolites were separated on a versatile amino column using a dual HILIC-RP gradient with heated electrospray (HESI) tandem mass spectrometry detection in mixed polarity multiple reaction monitoring mode. Quantification of 17 metabolites in the NAD+ metabolome in U251 human astroglioma cells could be achieved. Changes in NAD+ metabolism in U251 cell line, and murine oocytes under different culture conditions were also investigated. This method can be used as a sensitive profiling tool, tailoring chromatography for metabolites that express significant pathophysiological changes in several disease conditions and is indispensable for targeted analysis.

Extraction and measurement of NAD(P)(+) and NAD(P)H.

Nicotinamide adenine dinucleotides are critical redox-active substrates for countless catabolic and anabolic reactions. Ratios of NAD(+) to NADH and NADP(+) to NADPH are therefore considered key indicators of the overall intracellular redox potential and metabolic state. These ratios can be measured in bulk conditions using a highly sensitive enzyme cycling-based colorimetric assay (detection limit at or below 0.05 μM or 1 pmol) following a simple extraction procedure involving solutions of acid and base. Special considerations are necessary to avoid measurement artifacts caused by the presence of endogenous redox-active metabolites, such as phenazines made by diverse Pseudomonas species (see Chapter 25).

Imaging the Glucagon-Secreting Alpha Cells

Pancreatic islets secrete insulin and glucagon, two critical hormones for the blood glucose homeostasis. Islets are composed mainly of insulin-secreting beta-cells (∼80%) and glucagon-secreting alpha-cells (10-15%). Typically, insulin is secreted after a meal to reduce blood glucose levels, whereas glucagon is released in times of starvation. In order for insulin and glucagon to exert their opposite effects, glucose must also have an inverse effect on the secretory function of the two cell types. While the mechanisms of glucose-stimulated insulin secretion by beta-cells are fairly well understood, the study of the mechanisms underlying glucagon secretion has been hindered by the lack of reliable methods to distinguish between alpha- and beta-cells. Thus, it is not clear how glucose suppresses glucagon secretion. There is still little agreement as to whether this inhibition is a direct effect on alpha-cells or mediated by a paracrine inhibition from beta-cells via secretion of molecules such as insulin, Zn2+, GABA.Recently, a fragment of a rat glucagon promoter has been successfully used to specifically drive the expression of fluorescent proteins in mouse alpha-cells. Thus, it is now possible to easily identify glucagon-secreting cells within isolated islets or to flow sort a purified population of alpha-cells. Here, we report the use of dynamic fluorescence imaging techniques on these transgenic islets and cells. In particular, we investigated how glucose affects 1) alpha-cell metabolism via measurement of NAD(P)H autofluorescence by two-photon excitation microscopy; 2) alpha-cell intracellular calcium concentrations, via the loading of alpha-cells expressing red fluorescent proteins (tdRFP) with the calcium indicator dye Fluo4-AM; and 3) alpha-cell membrane potential, via application of the fluorescent probe DiSBAC2(3). The results of these biophysical measurements will also be compared to parallel glucagon secretion in response to glucose from both intact islets and sorted alpha-cells.

SUGAR-DEPENDENT6 Encodes a Mitochondrial Flavin Adenine Dinucleotide-Dependent Glycerol-3-P Dehydrogenase, Which Is Required for Glycerol Catabolism and Postgerminative Seedling Growth in Arabidopsis

The aim of this study was to clone and characterize the SUGAR-DEPENDENT6 (SDP6) gene, which is essential for postgerminative growth in Arabidopsis (Arabidopsis thaliana). Mutant alleles of sdp6 were able to break down triacylglycerol following seed germination but failed to accumulate soluble sugars, suggesting that they had a defect in gluconeogenesis. Map-based cloning of SDP6 revealed that it encodes a mitochondrial flavin adenine dinucleotide (FAD)-dependent glycerol-3-P (G3P) dehydrogenase:ubiquinone oxidoreductase called FAD-GPDH. This gene has previously been proposed to play a role both in the break down of glycerol (derived from triacylglycerol) and in NAD(+)/NADH homeostasis. Germinated seeds of sdp6 were severely impaired in the metabolism of [U-(14)C]glycerol to CO(2) and accumulated high levels of G3P. These data suggest that SDP6 is essential for glycerol catabolism. The activity of the glycolytic enzyme phosphoglucose isomerase is competitively inhibited by G3P in vitro. We show that phosphoglucose isomerase is likely to be inhibited in vivo because there is a 6-fold reduction in the transfer of (14)C-label into the opposing hexosyl moiety of sucrose when [U-(14)C]glucose or [U-(14)C]fructose is fed to sdp6 seedlings. A block in gluconeogenesis, at the level of hexose phosphate isomerization, would account for the arrested seedling growth phenotype of sdp6 and explain its rescue by sucrose and glucose but not by fructose. Measurements of NAD(+) and NADH levels in sdp6 seedlings also suggest that NAD(+)/NADH homeostasis is altered, and this observation is consistent with the hypothesis that SDP6 participates in a mitochondrial G3P shuttle by cooperating with the cytosolic NAD-dependent GPDH protein GPDHC1.

Investigation of Transport Mechanisms and Regulation of Intracellular Zn2+ in Pancreatic α-Cells

During insulin secretion, pancreatic alpha-cells are exposed to Zn(2+) released from insulin-containing secretory granules. Although maintenance of Zn(2+) homeostasis is critical for cell survival and glucagon secretion, very little is known about Zn(2+)-transporting pathways and the regulation of Zn(2+) in alpha-cells. To examine the effect of Zn(2+) on glucagon secretion and possible mechanisms controlling the intracellular Zn(2+) level ([Zn(2+)](i)), we employed a glucagon-producing cell line (alpha-TC6) and mouse islets where non-beta-cells were identified using islets expressing green fluorescent protein exclusively in beta-cells. In this study, we first confirmed that Zn(2+) treatment resulted in the inhibition of glucagon secretion in alpha-TC6 cells and mouse islets in vitro. The inhibition of secretion was not likely via activation of K(ATP) channels by Zn(2+). We then determined that Zn(2+) was transported into alpha-cells and was able to accumulate under both low and high glucose conditions, as well as upon depolarization of cells with KCl. The nonselective Ca(2+) channel blocker Gd(3+) partially inhibited Zn(2+) influx in alpha-TC cells, whereas the L-type voltage-gated Ca(2+) channel inhibitor nitrendipine failed to block Zn(2+) accumulation. To investigate Zn(2+) transport further, we profiled alpha-cells for Zn(2+) transporter transcripts from the two families that work in opposite directions, SLC39 (ZIP, Zrt/Irt-like protein) and SLC30 (ZnT, Zn(2+) transporter). We observed that Zip1, Zip10, and Zip14 were the most abundantly expressed Zips and ZnT4, ZnT5, and ZnT8 the dominant ZnTs. Because the redox state of cells is also a major regulator of [Zn(2+)](i), we examined the effects of oxidizing agents on Zn(2+) mobilization within alpha-cells. 2,2'-Dithiodipyridine (-SH group oxidant), menadione (superoxide generator), and SIN-1 (3-morpholinosydnonimine) (peroxynitrite generator) all increased [Zn(2+)](i) in alpha-cells. Together these results demonstrate that Zn(2+) inhibits glucagon secretion, and it is transported into alpha-cells in part through Ca(2+) channels. Zn(2+) transporters and the redox state also modulate [Zn(2+)](i).

Enhancement of Streptolysin O Activity and Intrinsic Cytotoxic Effects of the Group A Streptococcal Toxin, NAD-Glycohydrolase

Streptolysin O (SLO) is a cholesterol-dependent cytolysin produced by the important human pathogen, group A Streptococcus (Streptococcus pyogenes or GAS). In addition to its cytolytic activity, SLO mediates the translocation of GAS NAD-glycohydrolase (NADase) into human epithelial cells in vitro. Production of both NADase and SLO is associated with augmented host cell injury beyond that produced by SLO alone, but the mechanism of enhanced cytotoxicity is not known. We have now shown that expression of NADase together with SLO dramatically enhanced the lytic activity of GAS culture supernatants for erythrocytes but had no effect on SLO-mediated poration of synthetic cholesterol-rich liposomes. This result revealed a previously unknown contribution of NADase to the cytolytic activity associated with GAS production of SLO. Purified recombinant SLO bound NADase in vitro, supporting a specific, physical interaction of the two proteins. Exposure of human keratinocytes to wild-type GAS, but not to a NADase-deficient mutant strain, resulted in profound depletion of cellular NAD+ and ATP. Furthermore, expression of recombinant GAS NADase in yeast, in the absence of SLO, induced growth arrest, depletion of NAD+ and ATP, and cell death. These findings have provided evidence that the augmentation of SLO-mediated cytotoxicity by NADase is a consequence of depletion of host cell energy stores through the enzymatic action of NADase. Together, the results have provided mechanistic insight into the cytotoxic effects of a unique bipartite bacterial toxin.

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Regulation of blood glucose is a fundamental homeostasis in the body. Insulin is released from pancreatic beta-cells in response to changes in blood glucose, the defect of which leads to impaired insulin secretion and diabetes mellitus. Pancreatic beta-cells that release insulin occupy approximately 70% of the islet cells, while alpha, delta, PP-cells are also present in islets. Therefore, analysis of beta-cells is a direct approach for studying mechanisms of insulin secretion. Insulin secretion is regulated by cytosolic Ca(2+) in beta-cells, and its concentration and localization can be measured in real time by fluorescence imaging using indicators. Glucose metabolism is assessed by measurements of NAD(P)H by its autofluorescence. Furthermore, beta-cells are equipped with ion channels, which transduce glucose-evoked metabolic signals to electric signals. Electrophysiological analysis by patch clamp techniques detects the activity of various ion channels and membrane potential. Several beta-cell lines including HIT, MIN6, INS1, RIN, and beta TC are used; however, they do not necessarily retain normal responsiveness to glucose. Therefore, analysis of physiologic functions of beta-cells requires the use of acutely isolated or primary cultured beta-cells from normal animals. The methods for preparation of islets and single beta-cells using collagenase can be applied to a variety of animals of small to large sizes, which can produce islets and beta-cells with physiological responsiveness to glucose. These primary cultured beta-cells can be used for elucidating signal transduction mechanisms, and evaluating effects of drugs, providing excellent tools for physiological, pharmacological, and disease-oriented studies.

Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012

In the present study we sought to determine the ability of the chemiluminescence dye 8-amino-5-chloro-7-phenylpyridol[3,4- d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012) to detect superoxide in different biological systems. In human whole blood or isolated leukocytes, the sensitivity of the luminol analogue L-012 to detect superoxide was higher as compared with luminol, lucigenin, coelenterazine, and the fluorescence dye dihydroethidine. In isolated leukocytes as well as aortic rings from control (New Zealand White) and hyperlipidemic (Watanabe heritable hyperlipidemic) rabbits, L-012-enhanced chemiluminescence was successful in detecting differences in superoxide formation under basal conditions and on stimulation with the direct activator of protein kinase C, phorbol 12,13-dibutyrate (PDBu). The effects of PDBu were abrogated by gliotoxin and inhibitors of protein kinase C such as chelerythrine, identifying NAD(P)H oxidase as the significant superoxide source. Experiments using electron paramagnetic resonance and the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline- N-oxide revealed that in contrast to lucigenin, L-012 is not subject to redox cycling. These findings indicate that L-012-enhanced chemiluminescence represents a sensitive and reliable probe to detect superoxide in whole blood, inflammatory cells, and vascular tissue.

Reduction of oxidative stress and AT1 receptor expression by the selective oestrogen receptor modulator idoxifene.

1. The beneficial vasoprotective effects of oestrogens are hampered by their side effects on secondary sexual organs. Selective oestrogen receptor modulators (SERM) such as idoxifene may exert beneficial vascular effects without influencing cancerogenesis in breast or uterus. 2. In order to investigate vascular effects of selective oestrogen receptor modulators, we examined the impact of idoxifene on production of reactive oxygen species as well as AT1 receptor expression in vascular smooth muscle cells (VSMC). 3. Idoxifene caused a concentration- and time-dependent down-regulation of AT1 receptor mRNA expression, as assessed by Northern analysis. The maximal effect was reached with 10 micromol l(-1) idoxifene after a 4 h incubation period (33+/-7% of control levels). Western blots showed a similar down-regulation of AT1 receptor protein to 36+/-11% of control levels. 4. Confocal laserscanning microscopy using the redox sensitive marker 2',7'-dichlorofluorescein (DCF) and measurement of NAD(P)H oxidase activity in cell homogenates revealed that idoxifene effectively blunted the angiotensin II-induced production of reactive oxygen species. 5. In order to investigate the signal transduction involved in SERM-induced modulation of AT1 receptor expression, VSMC were preincubation with PD98059, genistein, wortmannin, or N(omega)-Nitro-L-arginine. The results suggested that idoxifene caused AT1 receptor down-regulation through nitric oxide-dependent pathways. 6. In conclusion, idoxifene reduces angiotensin II-evoked oxidative stress in VSMC. This could in part be explained by idoxifene-induced down-regulation of AT1 receptor expression. These results demonstrate that the selective oestrogen receptor modulator idoxifene may exert beneficial vascular effects which could be useful for therapeutic regimen in postmenopausal women at risk for cardiovascular diseases.

Monitoring NAD(P)H autofluorescence to assess mitochondrial metabolic functions in rat hippocampal–entorhinal cortex slices

Changes in neuronal energy metabolism, mitochondrial functions and homeostasis of reactive oxygen species are often supposed to induce alterations in neuronal activity in hippocampal slice models. In order to investigate the NAD(P)H autofluorescence signal in brain slice models, methods to monitor NAD(P)H signal in isolated mitochondria as described by Chance et al. [J. Biol. Chem. 254 (1979) 4764] and dissociated neurons as described by Duchen [Biochem. J. 283 (1992) 41] were adapted to recording conditions required for brain slices. Considering different experimental questions, we established an approach to monitor NAD(P)H autofluorescence signals from hippocampal slices of 400 μm thickness under either submerged or interface conditions. Therefore the procedure described here allows the measurement of NAD(P)H autofluorescence under conditions typically required in electrophysiological experiments. Depolarization of plasma membrane caused by electrical stimulation or application of glutamate (100 μM) resulted in a characteristic initial decrease followed by a long-lasting increase in the NAD(P)H autofluorescence signal. H 2O 2 (100 μM) evoked a strong NAD(P)H signal decrease indicating direct oxidation to the nonfluorescencend NAD(P) +. In contrast, the increase in NAD(P)H signal that followed a brief inhibition of mitochondrial respiratory chain complex I using rotenone (1 μM) indicated an accumulation of NAD(P)H. However, in presence of rotenone (1 μM) electrically evoked long-lasting NAD(P)H signal overshoot decreased progressively, due to a negative feedback of accumulated NAD(P)H to the citrate cycle. A comparable reduction in NAD(P)H signal increase were observed during low-Mg 2+ induced epileptiform activity, indicating a relative energy failure. In conclusion, the method presented here allows to monitor NAD(P)H autofluorescence signals to gain insight into the coupling of neuronal activity, energy metabolism and mitochondrial function in brain slice models.

Pancreatic islets cultured on extracellular matrix: An excellent preparation for microfluorometry

We have examined the use of extracellular matrix (ECM) as a substratum for mouse pancreatic islet cultures with special reference to microfluorometry work. We find the techniques for obtaining and maintaining ECM-producing cell cultures and coating of glass straightforward. The islets attached readily to ECM-coated glass and with time spread out. Cultures were probed for glucose responses after one day, one week and two weeks. Upon stimulation with 10 mM glucose, the well-established initial changes in [Ca2+]i, (monitored with Fura-2), as well as oscillations upon prolonged exposure, were observed. At 20 mM glucose only an elevated level without oscillations was seen. Similar responses were observed during the two weeks in culture. After one week in culture or more, single cell measurements of qualitative changes in [Ca2+]i and cell membranepotential (monitored with the dye bis-(1,3-dibutyl-barbituric acid) trimethine oxonol (DiBAC4(3)) could be performed concurrently on single cells at the islet periphery with ordinary fluorescence microscopy equipment. Simultaneous measurements of NAD(P)H and flavine adenine dinucleotide (FAD) fluorescence as well as measurements of rhodamine 123 fluorescence for mitochondrial membrane potential showed stable metabolic responses to glucose over the entire culture period. The islets had clearly elevated insulin secretion at 10 and 20 mM glucose compared to the secretion at 3 mM. In conclusion, mouse pancreatic islets cultured for up to two weeks on ECM-coated glass coverslips provide a functionally well- preserved and reproducible preparation for microfluorometry. The largest benefit of the method is that whole islet responses as well as single cell responses at the islet periphery can be monitored, using the same preparation.

Ubiquinone-1 Induces External Deamino-NADH Oxidation in Potato Tuber Mitochondria.

The addition of ubiquinone-1 (UQ-1) induced Ca2+-independent oxidation of deamino-NADH and NADH by intact potato (Solanum tuberosum L. cv Bintje) tuber mitochondria. The induced oxidation was coupled to the generation of a membrane potential. Measurements of NAD+-malate dehydrogenase activity indicated that the permeability of the inner mitochondrial membrane to NADH and deamino-NADH was not altered by the addition of UQ-1. We conclude that UQ-1-induced external deamino-NADH oxidation is due to a change in specificity of the external rotenone-insensitive NADH dehydrogenase. The addition of UQ-1 also induced rotenone-insensitive oxidation of deamino-NADH by inside-out submitochondrial particles, but whether this was due to a change in the specificity of the internal rotenone-insensitive NAD(P)H dehydrogenase or to a bypass in complex I could not be determined.

On-line measurement of culture fluorescence during cultivation of Methylomonas mucosa

This paper reports on the measurement of NAD(P)H dependent culture fluorescence during the batch culture of Methylomonas mucosa, a potential polysaccharide producer. The results obtained reveal that there is a high correlation between the fluorescence level and the cell concentration. The logistic equation was also found to be applicable for representing the biomass concentration data during the course of fermentation. A model was then presented for predicting both the biomass concentration and the culture fluorescence with high fidelity.

Effect of fatty acids and ketones on the activity of pyruvate dehydrogenase in skeletal-muscle mitochondria.

The presence of palmitoyl-L-carnitine and acetoacetate (separately) decreased flux through pyruvate dehydrogenase in isolated mitochondria from rat hind-limb muscle. The effect of acetoacetate was dependent on the presence of 2-oxoglutarate and Ca2+. Palmitoylcarnitine, but not acetoacetate, also decreased the mitochondrial content of active dephospho-pyruvate dehydrogenase (PDHA). This effect was large only in the presence of EGTA. Addition of Ca2+-EGTA buffers stabilizing pCa values of 6.48 or lower gave near-maximal values of PDHA content, irrespective of the presence of fatty acids or ketones when mitochondria were incubated under the same conditions used for the flux studies, i.e. at low concentrations of pyruvate. There was, however, a minor decrement in PDHA content in response to palmitoylcarnitine oxidation when the substrate was L-glutamate plus L-malate. Measurement of NAD+, NADH, CoA and acetyl-CoA in mitochondrial extracts in general showed decreases in [NAD+]/[NADH] and [CoA]/[acetyl-CoA] ratios in response to the oxidation of palmitoylcarnitine and acetoacetate, providing a mechanism for both decreased PDHA content and feedback inhibition of the enzyme in the PDHA form. However, only changes in [CoA]/[acetyl-CoA] ratio appear to underlie the decreased PDHA content on addition of palmitoylcarnitine when mitochondria are incubated with L-glutamate plus L-malate (and no pyruvate) as substrate. The effect of palmitoylcarnitine oxidation on flux through pyruvate dehydrogenase and on PDHA content is less marked in skeletal-muscle mitochondria than in cardiac-muscle mitochondria. This may reflect the less active oxidation of palmitoylcarnitine by skeletal-muscle mitochondria, as judged by State-3 rates of O2 uptake. In addition, Ca2+ concentration is of even greater significance in pyruvate dehydrogenase interconversion in skeletal-muscle mitochondria than in cardiac-muscle mitochondria.

A colorimetric method for assay of glutamate synthase in leaf tissue of Vigna mungo

Glutamine-2-oxo-glutarate aminotransferase (GOGAT) or glutamate synthase is generally assayed in terms of the disappearance of NAD(P)H from the assay medium. We describe a new technique which allows colorimetric measurement of the enzyme activity in cell-free extracts and whole tissue of Vigna mungo leaves in terms of disappearance of the substrate (α-ketoglutarate). The technique is offered as an alternative, more convenient method for assay of GOGAT. This also eliminates the use of more sophisticated ultraviolet range instrumentation required for measurement of NAD(P)H.

On-line measurements of culture fluorescence: Method and application

In this paper a new probe allowing the measurement of NAD(P)H-dependent culture fluorescence in a bioreactor is presented. This sterilizable probe can be inserted in every bioreactor using a standard fitting of 25 mm. Under well defined conditions high specificity and sensitivity as well as high stability are further advantages of this probe. Application examples are given to demonstrate the operation possibilities of this fluorescence probe. In batch growth the culture fluorescence can be used for on-line estimation of biomass concentration. Metabolic alterations due to substrate of oxygen deficiency can easily be detected by fluorometric measurements. In kinetic studies the fluorescence probe is of great use because of a very small time delay.

An enzymatic cycling method for nicotinamide-adenine dinucleotide with malic and alcohol dehydrogenases

A method for measuring nicotinamide-adenine dinucleotide by enzymatic cycling is described which uses malic and alcohol dehydrogenases (EC 1.1.1.37, and EC 1.1.1.1) for the enzyme couple. After cycling, malate is measured with either malic dehydrogenase or malic enzyme (EC 1.1.1.40). The method has a number of advantages compared to those previously described. The cycling rate is high (greater than 30 000/hr); blank values are low; the reaction is linear over a wide range of NAD concentrations; and the terminal indicator reaction requires only one step. In addition the system is well suited for double cycling. This was shown by measurements of NAD in nuclei and cytoplasm from single dorsal root ganglion cells (rabbit). The overall amplification in this case was about 1 000 000.