Isotopic non-stationary 13C gluconate tracer method for accurate determination of the pentose phosphate pathway split-ratio in Penicillium chrysogenum

2008 ◽  
Vol 10 (3-4) ◽  
pp. 178-186 ◽  
Author(s):  
Zheng Zhao ◽  
Karel Kuijvenhoven ◽  
Cor Ras ◽  
Walter M. van Gulik ◽  
Joseph J. Heijnen ◽  
...  
Keyword(s):  
Pentose Phosphate Pathway ◽  
Penicillium Chrysogenum ◽  
Accurate Determination ◽  
Pentose Phosphate ◽  
Tracer Method ◽  
Split Ratio

scholarly journals 13C-Labeled Gluconate Tracing as a Direct and Accurate Method for Determining the Pentose Phosphate Pathway Split Ratio in Penicillium chrysogenum

2006 ◽  
Vol 72 (7) ◽  
pp. 4743-4754 ◽  
Author(s):  
Roelco J. Kleijn ◽  
Wouter A. van Winden ◽  
Cor Ras ◽  
Walter M. van Gulik ◽  
Dick Schipper ◽  
...  
Keyword(s):  
Pentose Phosphate Pathway ◽  
Penicillium Chrysogenum ◽  
Accurate Determination ◽  
Pentose Phosphate ◽  
Penicillin G ◽  
Metabolic Parameter ◽  
Flux Analysis ◽  
Primary Metabolism ◽  
Tracer Method ◽  
Split Ratio

ABSTRACT In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-13C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a 13C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of 13C-labeled primary metabolites are reported for P. chrysogenum and used for a 13C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h−1 yielded comparable values for the gluconate tracer method and the 13C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the 13C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the 13C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.

Mass isotopomer study of the nonoxidative pathways of the pentose cycle with [1,2-13C2]glucose

1998 ◽  
Vol 274 (5) ◽  
pp. E843-E851 ◽  
Author(s):  
Wai-Nang Paul Lee ◽  
Laszlo G. Boros ◽  
Joaquim Puigjaner ◽  
Sara Bassilian ◽  
Shu Lim ◽  
...  
Keyword(s):  
Pentose Phosphate ◽  
Gas Chromatography Mass Spectrometry ◽  
Hep G2 ◽  
Tracer Method ◽  
Human Hepatoma Cells ◽  
Glucose Flux ◽  
Isotopomer Analysis ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Mass Isotopomer

We present a single-tracer method for the study of the pentose phosphate pathway (PPP) using [1,2-13C2]glucose and mass isotopomer analysis. The metabolism of [1,2-13C2]glucose by the glucose-6-phosphate dehydrogenase, transketolase (TK), and transaldolase (TA) reactions results in unique pentose and lactate isotopomers with either one or two13C substitutions. The distribution of these isotopomers was used to estimate parameters of the PPP using the model of Katz and Rognstad (J. Katz and R. Rognstad. Biochemistry 6: 2227–2247, 1967). Mass and position isotopomers of ribose, and lactate and palmitate (products from triose phosphate) from human hepatoma cells (Hep G2) incubated with 30% enriched [1,2-13C2]glucose were determined using gas chromatography-mass spectrometry. After 24–72 h incubation, 1.9% of lactate molecules in the medium contained one 13C substitution ( m 1) and 10% contained two 13C substitutions ( m 2). A similar m 1-to- m 2ratio was found in palmitate as expected. Pentose cycle (PC) activity determined from incubation with [1,2-13C2]glucose was 5.73 ± 0.52% of the glucose flux, which was identical to the value of PC (5.55 ± 0.73%) determined by separate incubations with [1-13C] and [6-13C]glucose.13C was found to be distributed in four ribose isotopomers ([1-13C]-, [5-13C]-, [1,2-13C2]-, and [4,5-13C2]ribose). The observed ribose isotopomer distribution was best matched with that provided from simulation by substituting 0.032 for TK and 0.85 for TA activity relative to glucose uptake into the model of Katz and Rognstad. The use of [1,2-13C2]glucose not only permits the determination of PC but also allows estimation of relative rates through the TK and TA reactions.

Assessment of DMSP turnover reveals a non-bioavailable pool of dissolved DMSP in coastal waters of the Gulf of Mexico

2016 ◽  
Vol 13 (2) ◽  
pp. 266 ◽  
Author(s):  
Chengxuan Li ◽  
Gui-Peng Yang ◽  
David J. Kieber ◽  
Jessie Motard-Côté ◽  
Ronald P. Kiene
Keyword(s):  
Coastal Waters ◽  
Loss Rate ◽  
Small Volume ◽  
Accurate Determination ◽  
Tracer Technique ◽  
Marine Microbes ◽  
Tracer Method ◽  
Consumption Rates ◽  
Inhibitor Method

Environmental context DMSP is one of the most important substrates for marine bacteria and its cycling contributes substantially to fluxes of carbon and sulfur in the ocean. Accurate determination of the concentration of DMSP available to bacteria is essential to quantifying DMSP consumption rates, and this work improves those determinations by identifying non-bioavailable pools of DMSP that have previously gone unrecognised. Improved estimates of DMSP consumption rates will lead to better understanding of its role in ocean food web and biogeochemical dynamics. Abstract Dissolved dimethylsulfoniopropionate (DMSPd) is an important substrate for marine microbes and a precursor of sulfur gases. We compared DMSPd turnover flux rates in coastal seawater measured with a 35S-DMSPd tracer to those obtained with the DMSP-uptake inhibitor glycine betaine (GBT). The 35S-DMSP tracer method yielded DMSPd turnover fluxes (35.7–215nM day–1) that were 1.7 to 152 times higher than those obtained in parallel samples with the GBT inhibitor method (0.34–21.6nM day–1). Tests confirmed that GBT functioned as planned by strongly inhibiting DMSPd degradation and that 35S-DMSPd gave accurate estimates of DMSPd loss rate constants. This left the initial DMSPd concentrations, determined by small volume drip filtration (SVDF) through Whatman GF/F filters (0.7-μm nominal retention) ([DMSPd]SVDF), as a potential cause of the discrepancy in rate estimates. Indeed, GF/F filtrate incubations showed that the initial [DMSPd]SVDF overestimated the bioavailable DMSPd concentrations for at least two reasons: (1) a significant fraction (10–37%) of DMSP passing through GF/F filters was in particles >0.2μm (likely bacteria) and therefore not dissolved, and (2) a significant pool (0.44–1.0nM) of operationally dissolved, non-particle DMSP ([DMSPd]<0.2μm), comprising 40–99% of [DMSPd]SVDF, was refractory to degradation on a time scale of days. The nature of this refractory DMSP is currently unknown. Accounting for DMSP-containing particles and the refractory DMSP pool in GF/F filtrates is necessary to obtain the true bioavailable DMSPd concentrations, which we estimate to be very low (0.006–1.0nM; mean of 0.41nM) in the coastal waters examined, and to avoid overestimation of DMSPd turnover fluxes when using the 35S-DMSP tracer technique.

scholarly journals The pentose phosphate pathway of glucose metabolism. Measurement of the non-oxidative reactions of the cycle

1968 ◽  
Vol 107 (6) ◽  
pp. 775-791 ◽  
Author(s):  
F. Novello ◽  
Patricia McLean
Keyword(s):  
Pentose Phosphate Pathway ◽  
Coupled System ◽  
Pentose Phosphate ◽  
Phosphogluconate Dehydrogenase ◽  
Tissue Extracts ◽  
Oxidative Reactions ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Rate Limiting ◽  
Made In

Methods for the quantitative determination of ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase, transketolase and transaldolase in tissue extracts are described. The determinations depend on the measurement of glyceraldehyde 3-phosphate by using the coupled system triose phosphate isomerase, α-glycero-phosphate dehydrogenase and NADH. By using additional purified enzymes transketolase, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase conditions could be arranged so that each enzyme in turn was made rate-limiting in the overall system. Transaldolase was measured with fructose 6-phosphate and erythrose 4-phosphate as substrates, and again glyceraldehyde 3-phosphate was measured by using the same coupled system. Measurements of the activities of the non-oxidative reactions of the pentose phosphate pathway were made in a variety of tissues and the values compared with those of the two oxidative steps catalysed by glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase.

A quantitative approach to inner shell losses

1978 ◽  
Vol 36 (1) ◽  
pp. 526-527 ◽  
Author(s):  
R.D. Leapman ◽  
P. Rez ◽  
D.F. Mayers
Keyword(s):  
Energy Transfer ◽  
Cross Section ◽  
Cross Sections ◽  
Accurate Determination ◽  
Background Intensity ◽  
Core Excitation ◽  
Quantitative Basis ◽  
Cross Section Differential ◽  
Bound Electrons

Microanalysis by EELS has been developing rapidly and though the general form of the spectrum is now understood there is a need to put the technique on a more quantitative basis (1,2). Certain aspects important for microanalysis include: (i) accurate determination of the partial cross sections, σx(α,ΔE) for core excitation when scattering lies inside collection angle a and energy range ΔE above the edge, (ii) behavior of the background intensity due to excitation of less strongly bound electrons, necessary for extrapolation beneath the signal of interest, (iii) departures from the simple hydrogenic K-edge seen in L and M losses, effecting σx and complicating microanalysis. Such problems might be approached empirically but here we describe how computation can elucidate the spectrum shape.The inelastic cross section differential with respect to energy transfer E and momentum transfer q for electrons of energy E0 and velocity v can be written as

Fast calibration of CBED patterns for quantitative analysis

1993 ◽  
Vol 51 ◽  
pp. 674-675
Author(s):  
M.A. Gribelyuk ◽  
M. Rühle
Keyword(s):  
Reciprocal Lattice ◽  
Accurate Determination ◽  
Incident Beam ◽  
Crystal Thickness ◽  
Structure Model ◽  
Beam Direction ◽  
Transmitted Beam ◽  
Reciprocal Vector ◽  
On Line

A new method is suggested for the accurate determination of the incident beam direction K, crystal thickness t and the coordinates of the basic reciprocal lattice vectors V1 and V2 (Fig. 1) of the ZOLZ plans in pixels of the digitized 2-D CBED pattern. For a given structure model and some estimated values Vest and Kest of some point O in the CBED pattern a set of line scans AkBk is chosen so that all the scans are located within CBED disks.The points on line scans AkBk are conjugate to those on A0B0 since they are shifted by the reciprocal vector gk with respect to each other. As many conjugate scans are considered as CBED disks fall into the energy filtered region of the experimental pattern. Electron intensities of the transmitted beam I0 and diffracted beams Igk for all points on conjugate scans are found as a function of crystal thickness t on the basis of the full dynamical calculation.

Computer-Assisted High-Re Solution TEM

1990 ◽  
Vol 48 (1) ◽  
pp. 162-163
Author(s):  
F.A. Ponce ◽  
H. Hikashi
Keyword(s):  
Signal To Noise Ratio ◽  
Accurate Determination ◽  
Computer Software ◽  
Video Camera ◽  
Image Contrast ◽  
Objective Lens ◽  
Computer Assisted ◽  
Optical Parameters ◽  
Astigmatism Correction

The determination of the atomic positions from HRTEM micrographs is only possible if the optical parameters are known to a certain accuracy, and reliable through-focus series are available to match the experimental images with calculated images of possible atomic models. The main limitation in interpreting images at the atomic level is the knowledge of the optical parameters such as beam alignment, astigmatism correction and defocus value. Under ordinary conditions, the uncertainty in these values is sufficiently large to prevent the accurate determination of the atomic positions. Therefore, in order to achieve the resolution power of the microscope (under 0.2nm) it is necessary to take extraordinary measures. The use of on line computers has been proposed [e.g.: 2-5] and used with certain amount of success.We have built a system that can perform operations in the range of one frame stored and analyzed per second. A schematic diagram of the system is shown in figure 1. A JEOL 4000EX microscope equipped with an external computer interface is directly linked to a SUN-3 computer. All electrical parameters in the microscope can be changed via this interface by the use of a set of commands. The image is received from a video camera. A commercial image processor improves the signal-to-noise ratio by recursively averaging with a time constant, usually set at 0.25 sec. The computer software is based on a multi-window system and is entirely mouse-driven. All operations can be performed by clicking the mouse on the appropiate windows and buttons. This capability leads to extreme friendliness, ease of operation, and high operator speeds. Image analysis can be done in various ways. Here, we have measured the image contrast and used it to optimize certain parameters. The system is designed to have instant access to: (a) x- and y- alignment coils, (b) x- and y- astigmatism correction coils, and (c) objective lens current. The algorithm is shown in figure 2. Figure 3 shows an example taken from a thin CdTe crystal. The image contrast is displayed for changing objective lens current (defocus value). The display is calibrated in angstroms. Images are stored on the disk and are accessible by clicking the data points in the graph. Some of the frame-store images are displayed in Fig. 4.

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