Aluminium effects on pyridine nucleotide redox state in roots of Scots pine

After prolonged (3-9 weeks) hydroponic treatment of Scots pine seedlings with different concentrations (0.5-4.0 mM) of Al (AI(N0₃)₃), the levels of pyridine nucleotides were determined in root homogenates. After 3 weeks of Al stress, a significant decrease of the anabolic reduction charge (ARC: NADPH/(NADP⁺ + NADPH)) and an increase of the redox status (NAD(P)H/NAD(P)⁺), catabolic reduction charge (CRC: NADH/(NAD⁺ + NADH)) and phosphorylation capacity expressed as NADP⁺/NAD⁺ ratio was found in the 4.0 mM Al treatment. After 6 weeks, Al at concentrations of 0.5 and 1.0 mM induced an enhancement of the NADH level and a reduction of NADPH level, but the redox ratios were not changed significantly. After 9 weeks treatment with Al concentrations ranging from 0.5 to 4.0 mM, decreases of the relative level of NADP⁺, NADPH and NADH and increases of NAD⁺ were found. Consequently, the CRC, NAD(P)H/NAD(P)⁺ and NADP⁺/NAD⁺ ratios reached a minimum and ARC a maximum as compared to previous measurements.

Limitations in Xylose-Fermenting Saccharomyces cerevisiae, Made Evident through Comprehensive Metabolite Profiling and Thermodynamic Analysis

ABSTRACT Little is known about how the general lack of efficiency with which recombinant Saccharomyces cerevisiae strains utilize xylose affects the yeast metabolome. Quantitative metabolomics was therefore performed for two xylose-fermenting S. cerevisiae strains, BP000 and BP10001, both engineered to produce xylose reductase (XR), NAD+-dependent xylitol dehydrogenase and xylulose kinase, and the corresponding wild-type strain CEN.PK 113-7D, which is not able to metabolize xylose. Contrary to BP000 expressing an NADPH-preferring XR, BP10001 expresses an NADH-preferring XR. An updated protocol of liquid chromatography/tandem mass spectrometry that was validated by applying internal 13C-labeled metabolite standards was used to quantitatively determine intracellular pools of metabolites from the central carbon, energy, and redox metabolism and of eight amino acids. Metabolomic responses to different substrates, glucose (growth) or xylose (no growth), were analyzed for each strain. In BP000 and BP10001, flux through glycolysis was similarly reduced (∼27-fold) when xylose instead of glucose was metabolized. As a consequence, (i) most glycolytic metabolites were dramatically (≤120-fold) diluted and (ii) energy and anabolic reduction charges were affected due to decreased ATP/AMP ratios (3- to 4-fold) and reduced NADP+ levels (∼3-fold), respectively. Contrary to that in BP000, the catabolic reduction charge was not altered in BP10001. This was due mainly to different utilization of NADH by XRs in BP000 (44%) and BP10001 (97%). Thermodynamic analysis complemented by enzyme kinetic considerations suggested that activities of pentose phosphate pathway enzymes and the pool of fructose-6-phosphate are potential factors limiting xylose utilization. Coenzyme and ATP pools did not rate limit flux through xylose pathway enzymes.

Xylose metabolism in Pachysolen tannophilus: purification and properties of xylose reductase

Xylose reductase (xylitol: NADP oxidoreductase, EC 1.1.1.139) has been purified from D-xylose grown cells of the yeast Pachysolen tannophilus by application of DEAE-cellulose ion exchange chromatography, 2′,5′-ADP-Sepharose affinity chromatography, Biogel P200 gel filtration, and dextran blue Sepharose chromatography to approximately 95% homogeneity. It consists of a single polypeptide chain with a relative molecular weight of 35 000–40 000 and an isoelectric point of pH 4.9. The enzyme has a broad substrate specificity similar to that of aldose (or aldehyde) reductases from mammalian tissues. It exhibits Michaelis–Menten type kinetics (Km D-xylose, 162 mM; Km D-xylitol, 212 mM; Km NADPH, 0.059 mM; [Formula: see text], 0.071 mM). The enzyme is specific for NADPH; activity with NADH is below 0.5% of Vmax observed with NADPH. The reduction of xylose is inhibited by NADP, the anabolic reduction charge (NADPH/NADP + NADPH), and also in a complex manner by ATP. At physiological pH values the equilibrium is Keq = 10−10. The importance of these findings for the physiology of xylose fermentation by this yeast is discussed.

Pyridine nucleotide levels and ratios in Aspergillus niger

A reliable method for the extraction and assay of pyridine nucleotides (NAD, NADH, NADP, and NADPH) in filamentous fungi is presented. The method is applied to a study of the physiology of citric acid accumulation by Aspergillus niger. The measurement of "catabolic reduction charge" (NADH/(NAD + NADH)) and "anabolic reduction charge" (NADPH/(NADP + NADPH)) as a means of characterizing the metabolic state is discussed.

The Regulation of Glucose-6-phosphate Dehydrogenase in Chloroplasts

Glucose-6-phosphate Dehydrogenase, Reduction Charge, Pentosephosphate Cycle, Chloroplasts Glucose-6-phosphate dehydrogenase from intact pea chloroplasts is partially membrane bound and inactivated upon illumination. The inhibitory effect of light can be abolished by addition of methylviologen. Kinetic experiments with glucose-6-phosphate dehydrogenase reveal that, in the dark, the enzyme activity is strongly inhibited by the accumulation of NADPH. The inhibition of NADPH can be reversed by the addition of excess NADP+ . The non-Michaelis-Menten-type kinetics suggest that the enzyme is stringently regulated by the ratio of NADPH to NADP+ plus NADPH, i. e., the “reduction charge”. These observations seem to indicate that in the light the inhibition of glucose-6-phosphate dehydrogenase is due to a high reduction charge, whereas in the dark the enzyme is controlled by the metabolic demand for reducing equivalents.