Kinetics of p-aminohippurate secretion in the rabbit

1963 ◽  
Vol 205 (5) ◽  
pp. 1019-1024 ◽  
Author(s):  
E. C. Foulkes
Keyword(s):  
Steady State ◽  
Turnover Rate ◽  
Renal Cortex ◽  
Isotopic Equilibration ◽  
Kinetics Of ◽  
Carrier Mechanisms

A technique is proposed and tested which permits the calculation of the size and turnover rate of the p-aminohippurate (PAH) secretory pool in the renal cortex of the rabbit in vivo. The secretory pool is here defined as the cellular solute compartment with which secreted solute equilibrates during its transcellular movement from plasma into tubular urine. The calculations are based on the rate of isotopic equilibration of plasma and urine under steady-state conditions. From the influence of various drugs on the characteristics of the secretory pool, it is inferred that 1) similar PAH carrier mechanisms mediate influx into, and outflux from, the secretory pool, and 2) the PAH accumulated within the cells is coextensive with the secretory pool and is present in a free rather than bound form.

Measurement of leucine metabolism in man from a primed, continuous infusion of L-[1-3C]leucine

1980 ◽  
Vol 238 (5) ◽  
pp. E473-E479 ◽  
Author(s):  
D. E. Matthews ◽  
K. J. Motil ◽  
D. K. Rohrbaugh ◽  
J. F. Burke ◽  
V. R. Young ◽  
...  
Keyword(s):  
Steady State ◽  
Continuous Infusion ◽  
Co2 Production ◽  
Human Beings ◽  
Priming Dose ◽  
Leucine Metabolism ◽  
Kinetics Of ◽  
13Co2 Enrichment

Leucine metabolism in vivo can be determined from a primed, continuous infusion of L-[1-13C]leucine by measuring, at isotopic steady state, plasm [-13C]leucine enrichment, expired 13CO2 enrichment, and CO2 production rate. With an appropriate priming dose of L-[1-13C]leucine and NaH13CO3, isotopic steady state is reached in less than 2 h, and the infusion is completed in 4 h. The method can determine rates of leucine turnover, oxidation, and incorporation into protein with typical relative uncertainties of 2, 10, and 4%, respectively. The method requires no more than 1 ml of blood and uses stable isotope rather than radioisotope techniques. Thus, the method is applicable to studies of human beings of all ages. L-[1-13C]leucine may be infused with a second amino acid labeled with 15N for simultaneous determination of the kinetics of two amino acids.

scholarly journals Determination of steady-state protein breakdown rate in vivo by the disappearance of protein-bound tracer-labeled amino acids: a method applicable in humans

2013 ◽  
Vol 304 (8) ◽  
pp. E895-E907 ◽  
Author(s):  
Lars Holm ◽  
Bruce O'Rourke ◽  
David Ebenstein ◽  
Michael J. Toth ◽  
Rasmus Bechshoeft ◽  
...  
Keyword(s):  
Amino Acids ◽  
Steady State ◽  
Exponential Decay ◽  
Turnover Rate ◽  
Deuterium Oxide ◽  
Protein Breakdown ◽  
Breakdown Rate

A method to determine the rate of protein breakdown in individual proteins was developed and tested in rats and confirmed in humans, using administration of deuterium oxide and incorporation of the deuterium into alanine that was subsequently incorporated into body proteins. Measurement of the fractional breakdown rate of proteins was determined from the rate of disappearance of deuterated alanine from the proteins. The rate of disappearance of deuterated alanine from the proteins was calculated using an exponential decay, giving the fractional breakdown rate (FBR) of the proteins. The applicability of this protein-specific FBR approach is suitable for human in vivo experimentation. The labeling period of deuterium oxide administration is dependent on the turnover rate of the protein of interest.

Evaluation of the isotopic equilibration between lactate and pyruvate

1988 ◽  
Vol 254 (4) ◽  
pp. E532-E535 ◽  
Author(s):  
R. R. Wolfe ◽  
F. Jahoor ◽  
H. Miyoshi
Keyword(s):  
Steady State ◽  
Metabolic Flux ◽  
Isotopic Exchange ◽  
Whole Body ◽  
Isotopic Enrichment ◽  
Isotopic Tracer ◽  
Anesthetized Dogs ◽  
Isotopic Equilibration ◽  
Lactate And Pyruvate ◽  
Kinetics Of

When an isotopic tracer is infused for the purpose of determining the rate of turnover or oxidation of a substrate, it is assumed that the resulting isotopic enrichment by the tracer will reflect the kinetics of only the pool of interest. However, this may not be the case when carbon-labeled lactate is infused, since rapid isotopic exchange with the intracellular pyruvate and alanine pools could potentially occur. Therefore we have determined the extent of isotopic exchange occurring during the infusion of [3-13C]lactate into six anesthetized dogs. In the steady state, pyruvate enrichment was 91 +/- 2.2% (means +/- SE) of the lactate enrichment, and alanine enrichment was 81 +/- 3.3% of the pyruvate enrichment and 72 +/- 2.6% of the lactate enrichment. In contrast, when [3-13C]alanine was infused (n = 2), pyruvate (and lactate) enrichment was 9.9% of the alanine enrichment. We therefore conclude that there is rapid isotopic equilibration between lactate and pyruvate but that interaction with alanine reflects the true metabolic flux rates, rather than isotopic exchange. Consequently, lactate kinetics, as traditionally determined, more accurately reflect whole body pyruvate kinetics.

Glucose transport and equilibrium across alveolar-airway barrier of rat

1996 ◽  
Vol 270 (2) ◽  
pp. L183-L190 ◽  
Author(s):  
G. Saumon ◽  
G. Martet ◽  
P. Loiseau
Keyword(s):  
Steady State ◽  
Glucose Transport ◽  
Glucose Concentration ◽  
Epithelial Lining Fluid ◽  
Uptake Sites ◽  
Maximum Uptake Rate ◽  
Isolated Lungs ◽  
Kinetics Of ◽  
State Concentration

The glucose concentration in the epithelial lining fluid (ELF) results from a balance between cellular uptake and paracellular leakage. The present study examines whether the ELF glucose concentration can be predicted from the kinetics of glucose transport obtained in fluid-filled lungs. Isolated rat lungs were filled via the trachea with instillate containing 0-10 mM glucose; the perfusate glucose concentration was 10 mM. The rate of glucose removal from airspaces depended on luminal glucose concentration and was saturable [maximum uptake rate = 101 +/- 8.6 mumol.h-1.g dry lung wt-1; apparent Michaelis constant K(m) = 1.5 +/- 0.43 mM; R2 = 0.79]. Glucose removal was inhibited by phloridzin but not by phloretin or by inhibiting glycolysis. The steady-state concentration in fluid-filled lungs was estimated to be 0.15 +/- 0.034 mM. It agreed with that (< 1/20 plasma) calculated using glucose transport kinetics and paracellular permeability. The ELF glucose concentration obtained by bronchoalveolar lavage was 0.39 +/- 0.012 plasma in vivo and 0.39 +/- 0.021 perfusate in air-filled isolated lungs. The equilibrium ELF/perfusate distribution ratio of alpha-methyl-glucose was similar to that of glucose. Thus there is a major difference between the alveolar steady-state glucose concentration in air- and fluid-filled lungs despite similar mechanisms of airspace glucose removal. This suggests that glucose kinetics or access to uptake sites differ in air- and fluid-filled lungs.

scholarly journals Enhanced enzyme kinetics of reverse transcriptase variants cloned from animals infected with SIVmac239 lacking viral protein X

2020 ◽  
Vol 295 (50) ◽  
pp. 16975-16986
Author(s):  
Si'Ana A. Coggins ◽  
Dong-Hyun Kim ◽  
Raymond F. Schinazi ◽  
Ronald C. Desrosier ◽  
Baek Kim
Keyword(s):  
Steady State ◽  
Viral Protein ◽  
Catalytic Efficiency ◽  
Myeloid Cell ◽  
Immunodeficiency Virus ◽  
Protein X ◽  
Kinetics Of ◽  
Hiv 1

HIV Type 1 (HIV-1) and simian immunodeficiency virus (SIV) display differential replication kinetics in macrophages. This is because high expression levels of the active host deoxynucleotide triphosphohydrolase sterile α motif domain and histidine-aspartate domain–containing protein 1 (SAMHD1) deplete intracellular dNTPs, which restrict HIV-1 reverse transcription, and result in a restrictive infection in this myeloid cell type. Some SIVs overcome SAMHD1 restriction using viral protein X (Vpx), a viral accessory protein that induces proteasomal degradation of SAMHD1, increasing cellular dNTP concentrations and enabling efficient proviral DNA synthesis. We previously reported that SAMHD1-noncounteracting lentiviruses may have evolved to harbor RT proteins that efficiently polymerize DNA, even at low dNTP concentrations, to circumvent SAMHD1 restriction. Here we investigated whether RTs from SIVmac239 virus lacking a Vpx protein evolve during in vivo infection to more efficiently synthesize DNA at the low dNTP concentrations found in macrophages. Sequence analysis of RTs cloned from Vpx (+) and Vpx (−) SIVmac239–infected animals revealed that Vpx (−) RTs contained more extensive mutations than Vpx (+) RTs. Although the amino acid substitutions were dispersed indiscriminately across the protein, steady-state and pre-steady-state analysis demonstrated that selected SIVmac239 Vpx (−) RTs are characterized by higher catalytic efficiency and incorporation efficiency values than RTs cloned from SIVmac239 Vpx (+) infections. Overall, this study supports the possibility that the loss of Vpx may generate in vivo SIVmac239 RT variants that can counteract the limited availability of dNTP substrate in macrophages.

scholarly journals Evaluation of Cerebral Acetate Transport and Metabolic Rates in the Rat Brain in vivo Using 1H-[13C]-NMR

2010 ◽  
Vol 30 (6) ◽  
pp. 1200-1213 ◽  
Author(s):  
Anant B Patel ◽  
Robin A de Graaf ◽  
Douglas L Rothman ◽  
Kevin L Behar ◽  
Graeme F Mason
Keyword(s):  
Steady State ◽  
Tricarboxylic Acid Cycle ◽  
Exponential Fitting ◽  
Resonance Spectroscopy ◽  
Specific Substrate ◽  
Amino Acid Turnover ◽  
In Vivo Analysis ◽  
Kinetics Of

Acetate is a well-known astrocyte-specific substrate that has been used extensively to probe astrocytic function in vitro and in vivo. Analysis of amino acid turnover curves from 13C-acetate has been limited mainly for estimation of first-order rate constants from exponential fitting or calculation of relative rates from steady-state 13C enrichments. In this study, we used 1H-[13C]-Nuclear Magnetic Resonance spectroscopy with intravenous infusion of [2-13C]acetate-Na+ in vivo to measure the cerebral kinetics of acetate transport and utilization in anesthetized rats. Kinetics were assessed using a two-compartment (neuron/astrocyte) analysis of the 13C turnover curves of glutamate-C4 and glutamine-C4 from [2-13C]acetate-Na+, brain acetate levels, and the dependence of steady-state glutamine-C4 enrichment on blood acetate levels. The steady-state enrichment of glutamine-C4 increased with blood acetate concentration until 90% of plateau for plasma acetate of 4 to 5 mmol/L. Analysis assuming reversible, symmetric Michaelis–Menten kinetics for transport yielded 27±2mmol/L and 1.3±0.3 μmol/g/min for Kt and Tmax, respectively, and for utilization, 0.17±0.24 mmol/L and 0.14±0.02 μmol/g/min for KM_util and Vmax_util, respectively. The distribution space for acetate was only 0.32±0.12 mL/g, indicative of a large excluded volume. The astrocytic and neuronal tricarboxylic acid cycle fluxes were 0.37±0.03 μmol/g/min and 1.41±0.11 μmol/g/min, respectively; astrocytes thus comprised ∼21%±3% of total oxidative metabolism.

scholarly journals Microtubule dynamics in interphase cells.

1986 ◽  
Vol 102 (3) ◽  
pp. 1020-1031 ◽  
Author(s):  
E Schulze ◽  
M Kirschner
Keyword(s):  
Growth Rate ◽  
Steady State ◽  
Immunoelectron Microscopy ◽  
In Vitro Models ◽  
Interphase Cells ◽  
Microtubule Growth ◽  
Kinetics Of ◽  
Rapid Incorporation

The sites of microtubule growth and the kinetics of elongation have been studied in vivo by microinjection of biotin-labeled tubulin and subsequent visualization with immunocytochemical probes. Immunofluorescence and immunoelectron microscopy demonstrate that injected biotin-labeled subunits are incorporated into new segments of growth which are contiguous with unlabeled microtubules. Rapid incorporation occurs by elongation of existing microtubules and new nucleation off the centrosome. The growth rate is 3.6 micron/min and is independent of the concentration of injected labeled tubulin. This rate of incorporation together with turnover of existing microtubules leads to approximately 80% exchange in 15 min. The observed kinetics and pattern of microtubule turnover allow for an evaluation of the relevance of several in vitro models for steady-state dynamics to the in vivo situation. We have also observed a substantial population of quasi-stable microtubules that does not exchange subunits as rapidly as the majority of microtubules and may have specialized functions in the cell.

scholarly journals Transient kinetics of a cGMP-dependent cGMP-specific phosphodiesterase from Dictyostelium discoideum.

1984 ◽  
Vol 98 (2) ◽  
pp. 709-716 ◽  
Author(s):  
P J Van Haastert ◽  
M M Van Lookeren Campagne
Keyword(s):  
Steady State ◽  
Dictyostelium Discoideum ◽  
Binding Sites ◽  
Catalytic Site ◽  
Transient Kinetics ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Rate Of Activation ◽  
Stimulation Of

Chemotactic stimulation of Dictyostelium discoideum cells induces a fast transient increase of cGMP levels which reach a peak at 10 s. Prestimulation levels are recovered in approximately 30 s, which is achieved mainly by the action of a guanosine 3',5'-monophosphate cGMP-specific phosphodiesterase. This enzyme is activated about fourfold by low cGMP concentrations. The phosphodiesterase has two distinct cGMP-binding sites: a catalytic site and an activator site. cAMP does not bind to either site; inosine 3',5'-monophosphate (cIMP) binds only to the catalytic site, whereas 8-bromoguanosine 3',5'-monophosphate (c-b8-GMP) preferentially binds to the activator site. For detailed kinetical measurements we have used [3H]cIMP as the substrate and c-b8-GMP as the activator. c-b8-GMP activated the hydrolysis of [3H]cIMP by reducing the Km, whereas the Vmax was not altered. The hydrolysis of [3H]cIMP was measured at 5-s intervals by using a new method for the separation of 5'-nucleotides from cyclic nucleotides. The hydrolysis of [3H]cIMP by nonactivated enzyme or by preactivated enzyme was linear with time, which indicates that a steady state is reached at the catalytic site within 5 s after addition of the substrate. In contrast, the hydrolysis of [3H]cIMP immediately after activation by 0.1 microM c-b8-GMP was not linear with time, but increased in a quasi-exponential manner with a time constant of 21 s. This suggests that a steady state at the activator site is only reached in 30-45 s after addition of the activator. The on-rate of activation (k1) was 3 X 10(5) M-1s-1 for c-b8-GMP and 1.4 X 10(5) M-1s-1 for cGMP. The off-rate of activation (k-1) was 0.03 s-1 for both c-b8-GMP and cGMP. The significance of these kinetic constants for the chemoattractant-mediated cGMP response in vivo is discussed.

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