scholarly journals The kinetics of consecutive enzyme reactions. The design of coupled assays and the temporal response of pathways

1984 ◽  
Vol 219 (3) ◽  
pp. 843-847 ◽  
Author(s):  
J S Easterby
Keyword(s):  
Steady State ◽  
Time Course ◽  
Enzyme Assays ◽  
Unexpected Finding ◽  
Temporal Response ◽  
Considerable Decrease ◽  
Transient Time ◽  
Time Required ◽  
Kinetics Of ◽  
State Concentration

A regime is proposed for the design of coupled enzyme assays in which auxiliary enzymes are added at concentrations proportional to their Km values. Under these conditions it is possible to calculate the complete time course of the assay including the time required for the system to approach its steady state. The consequence of increasing the number of coupling enzymes is shown to be a considerable decrease in time required to reach the steady state provided that the overall transient time remains the same. The method is extended to the general consideration of pathways and shows that pathways of the same length exhibit identical temporal responses provided that the units of concentration and time used are based on the steady-state concentration of intermediates and the transient time respectively. An unexpected finding is that increasing the number of intermediates in a pathway can decrease the time required to enter a steady state.

Anticonvulsants modify inactivation but not activation processes of sodium channels in Myxicola axons

1987 ◽  
Vol 65 (6) ◽  
pp. 1220-1225 ◽  
Author(s):  
C. L. Schauf
Keyword(s):  
Steady State ◽  
Time Course ◽  
Internal Surface ◽  
Slow Inactivation ◽  
Fast Inactivation ◽  
Inactivation Curve ◽  
Kinetics Of ◽  
Enzyme Application ◽  
Voltage Axis ◽  
Pronase Treatment

The effects of pronase and the anticonvulsant drugs diphenylhydantoin, bepridil, and sodium valproate on fast and slow Na+ inactivation were examined in cut-open Myxicola giant axons with loose patch-clamp electrodes applied to the internal surface. Pronase completely eliminated fast Na+ inactivation without affecting the kinetics of Na+ activation or the maximum Na+ conductance. The time and voltage dependences of slow inactivation following pronase treatment were identical to those measured before enzyme application in the same axons. All three anticonvulsants slowed the time course of recovery from fast Na+ inactivation in untreated axons, and shifted the steady-state fast inactivation curve in the hyperpolarizing direction along the voltage axis. Anticonvulsants enhanced steady-state slow inactivation and retarded recovery from slow inactivation in both untreated and pronase-treated axons. Although some quantitative differences were seen, the order of potency of the anticonvulsants on slow Na+ inactivation was the same as that for recovery from fast inactivation.

Theory and practical application of coupled enzyme reactions: one and two auxiliary enzymes

1984 ◽  
Vol 62 (10) ◽  
pp. 945-955 ◽  
Author(s):  
S. P. J. Brooks ◽  
T. Espinola ◽  
C. H. Suelter
Keyword(s):  
Steady State ◽  
Enzyme System ◽  
Enzyme Reaction ◽  
Mathematical Treatment ◽  
Steady State Concentration ◽  
Practical Application ◽  
Enzyme Reactions ◽  
Time Required ◽  
The Cost ◽  
State Concentration

An extended and practical set of equations which describe coupled enzyme reactions is presented. The mathematical treatment relies on two assumptions: (a) the rate of the primary enzyme reaction is constant and (b) the reverse reactions are negligible. The treatment leads to the development of new equations which relate the time required for the concentration of a reaction intermediate to reach a defined fraction of its steady-state concentration to the kinetic parameters of the enzymes when mutarotation of one of the intermediates does not occur. The new equations reduce to those previously derived when the steady-state concentration of the intermediate is small compared with its Km value. A method for minimizing the cost of the two auxiliary enzyme system is also provided.

Time course and kinetics of proximal tubular processing of insulin

1992 ◽  
Vol 262 (5) ◽  
pp. F813-F822 ◽  
Author(s):  
S. Nielsen
Keyword(s):  
Steady State ◽  
Time Course ◽  
Accumulation Rate ◽  
Compartmental Analysis ◽  
Proximal Tubules ◽  
Hydrolysis Rate ◽  
Time Courses ◽  
High Concentrations ◽  
Kinetics Of

The present study was undertaken to determine the time courses and kinetics of the subcellular processing of 125I-insulin in isolated and in vitro perfused proximal tubules. Morphometric analysis demonstrated well-preserved ultrastructure after 90 min of perfusion. After luminal perfusion for 90 min the absorption was constant with time and reached steady state within 5 min (177 +/- 7 fg.min-1.mm-1). Also the hydrolysis rate and tubular accumulation rate were constant and averaged 84 +/- 8 and 93 +/- 10 fg.min-1.mm-1, respectively. Free 125I appeared already within 5 min of perfusion and reached steady state within 10 min. From proximal tubules perfused with 125I-insulin for 30 min and chased for 60 min, a compartmental analysis revealed two compartments; half time (t1/2) for delivery of insulin to the lysosomes was determined to be 8.5 min, and t1/2 for lysosomal degradation was 72 min. The results demonstrated that internalization by endocytic invaginations, incorporation in endocytic vacuoles, fusion with lysosomes, and hydrolysis were rapid processes and reached maximum rates within few minutes. A significant transtubular transport of insulin to the peritubular compartment was determined to be a constant rate of 11.2 +/- 0.7 fg.min-1.mm-1. Perfusion of tubules with insulin at high concentrations in the perfusate revealed that the transport was dependent on the absorbed amount and not on the perfused load, compatible with transport through the cells and not via a paracellular mechanism. The intactness of the tight junctions was supported by the following: 1) [14C]inulin leak did not increase with time and 2) enzyme-free intercellular spaces were evident after perfusion for only 5 min with microperoxidase (mol wt of 1,700). The transported 125I-insulin was trichloroacetic acid precipitable and immunoprecipitable.

scholarly journals Simultaneous determination of the kinetics of cardiac output, systemic O2 delivery, and lung O2 uptake at exercise onset in men

2006 ◽  
Vol 290 (4) ◽  
pp. R1071-R1079 ◽  
Author(s):  
Frédéric Lador ◽  
Marcel Azabji Kenfack ◽  
Christian Moia ◽  
Michela Cautero ◽  
Denis R. Morel ◽  
...  
Keyword(s):  
Cardiac Output ◽  
Steady State ◽  
Phase I ◽  
Time Course ◽  
Hemoglobin Concentration ◽  
Two Phase ◽  
Rapid Phase ◽  
Steady State Method ◽  
Exercise Onset ◽  
Kinetics Of

We tested whether the kinetics of systemic O2 delivery (Q̇aO2) at exercise start was faster than that of lung O2 uptake (V̇o2), being dictated by that of cardiac output (Q̇), and whether changes in Q̇ would explain the postulated rapid phase of the V̇o2 increase. Simultaneous determinations of beat-by-beat (BBB) Q̇ and Q̇aO2, and breath-by-breath V̇o2 at the onset of constant load exercises at 50 and 100 W were obtained on six men (age 24.2 ± 3.2 years, maximal aerobic power 333 ± 61 W). V̇o2 was determined using Grønlund's algorithm. Q̇ was computed from BBB stroke volume (Qst, from arterial pulse pressure profiles) and heart rate ( fh, electrocardiograpy) and calibrated against a steady-state method. This, along with the time course of hemoglobin concentration and arterial O2 saturation (infrared oximetry) allowed computation of BBB Q̇aO2. The Q̇, Q̇aO2 and V̇o2 kinetics were analyzed with single and double exponential models. fh, Qst, Q̇, and V̇o2 increased upon exercise onset to reach a new steady state. The kinetics of Q̇aO2 had the same time constants as that of Q̇. The latter was twofold faster than that of V̇o2. The V̇o2 kinetics were faster than previously reported for muscle phosphocreatine decrease. Within a two-phase model, because of the Fick equation, the amplitude of phase I Q̇ changes fully explained the phase I of V̇o2 increase. We suggest that in unsteady states, lung V̇o2 is dissociated from muscle O2 consumption. The two components of Q̇ and Q̇aO2 kinetics may reflect vagal withdrawal and sympathetic activation.

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.

CONVECTIVE TRANSPORT OF AMMONIUM WITH NITRIFICATION IN SOIL

1971 ◽  
Vol 51 (3) ◽  
pp. 339-350 ◽  
Author(s):  
C. M. CHO
Keyword(s):  
Steady State ◽  
Soil Column ◽  
Reaction Rates ◽  
Convective Transport ◽  
Concentration Profiles ◽  
Nitrogen Transformations ◽  
First Order ◽  
Time Required ◽  
Solution Velocity ◽  
State Concentration

Dispersion equations with first-order reaction rates for nitrogen transformations were applied to describe the convective transport of NH4+ with nitrification and denitrification in soil. The equations describing the concentrations of NH4+, NO2− and NO3− as functions of position, time, rate constants, average solution velocity and the distribution coefficient of NH4+ between soil and soil solution were obtained. Several values of parameters were chosen to obtain the concentration profiles of NH4+, NO2− and NO3− along the soil column at several times. Steady-state concentration profiles and the time required to attain the steady state are discussed.

scholarly journals THE KINETICS OF PENETRATION

1939 ◽  
Vol 22 (4) ◽  
pp. 501-520 ◽  
Author(s):  
A. G. Jacques
Keyword(s):  
Steady State ◽  
Sea Water ◽  
Sodium Concentration ◽  
Permeability Constant ◽  
Ph Increase ◽  
Permeability Constants ◽  
Kinetics Of ◽  
State Concentration ◽  
Source Of Error ◽  
External Ph

The accumulation of ammonia takes place more rapidly in light than in darkness. The accumulation appears to go on until a steady state is attained. The steady state concentration of ammonia in the sap is about twice as great in light as in darkness. Both effects are possibly due to the fact that the external pH (and hence the concentration of undissociated ammonia) outside is raised by photosynthesis. Certain "permeability constants" have been calculated. These indicate that the rate is proportional to the concentration gradient across the protoplasm of NH4X which is formed by the interaction of NH3 or NH4OH and HX, an acid elaborated in the protoplasm. The results are interpreted to mean that HX is produced only at the sap-protoplasm interface and that on the average its concentration there is about 7 times as great as at the sea water-protoplasm interface. This ratio of HX at the two surfaces also explains why the concentration of undissociated ammonia in the steady state is about 7 times as great in the sea water as in the sap. The permeability constant P''' appears to be greater in the dark. This is possibly associated with an increase in the concentration of HX at both interfaces, the ratio at the two surfaces, however, remaining about the same. The pH of sap has been determined by a new method which avoids the loss of gas (CO2), an important source of error. The results indicate that the pH rises during accumulation but the extent of this rise is smaller than has hitherto been supposed. As in previous experiments, the entering ammonia displaced a practically equivalent amount of potassium from the sap and the sodium concentration remained fairly constant. It seems probable that the pH increase is due to the entrance of small amounts of NH3 or NH4OH in excess of the potassium lost as a base.

scholarly journals The effect of feedback on pathway transient response

1986 ◽  
Vol 233 (3) ◽  
pp. 871-875 ◽  
Author(s):  
J S Easterby
Keyword(s):  
Steady State ◽  
Metabolic Pathways ◽  
Transient Analysis ◽  
Steady States ◽  
Transient Time ◽  
Transient Behaviour ◽  
Steady State Flux ◽  
The Times ◽  
Time Required ◽  
State Functions

The effect of variation of the rate of input of material on the transient behaviour of metabolic pathways is examined. This reveals the existence of three transient times which make up the overall pathway transient. Two of these have been described previously and represent the times required for the accumulation of the free intermediate pool and the pool of enzyme-bound intermediate. They are state functions and as such are independent of the way in which the steady state was reached. The third is attributable to the variation in the rate of input of material to the pathway. It is dependent on three further factors. These are (a) the time required for the initial enzyme to reach its own steady state, (b) substrate depletion and (c) feedback. The description of the transient is: (Formula: see text) where V0 represents the rate of input and Vss represents the steady-state flux. The transient time associated with the transition between steady-states is shown to be a simple function of the transients for the establishment of each steady state from rest and may be expressed as: tau = tau b-Va/Vb . tau a where Va and Vb refer to the fluxes in the two steady states and tau a and tau b represent the transient times for the establishment of each of the steady-states from rest. The total pathway transient may now be completely defined as: (formula: see text) where summation over all intermediates, I, is implied. The significance of this to the analysis of pathway behaviour is discussed with more general examples of pathway transient analysis.

scholarly journals Determination of Michaelis parameters from differentials of progress curves

1983 ◽  
Vol 215 (3) ◽  
pp. 589-595 ◽  
Author(s):  
L C Petersen
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Kinetic Analysis ◽  
Cytochrome C Oxidase ◽  
Enzyme Assay ◽  
Serine Proteinases ◽  
Steady State Concentration ◽  
Kinetics Of ◽  
State Concentration

First differentials of progress curves are easily obtainable in many enzyme assay systems. Such curves may be more readily applicable to kinetic analysis than are the usual progress curves. The theory for this approach is developed, and simple graphical procedures for the determination of Michaelis parameters are indicated. By using an electronic differentiator device the application of the method is demonstrated on the kinetics of three different serine proteinases with various synthetic substrates. Whenever the steady-state concentration of an intermediate of the reaction is proportional to the rate, the transition of this intermediate in substrate-depletion experiments may be analysed in similar terms. This is demonstrated with cytochrome c oxidase kinetics. A number of other possible applications are discussed.

V˙o 2 kinetics of mild exercise are altered by RER

1999 ◽  
Vol 87 (6) ◽  
pp. 2097-2106 ◽  
Author(s):  
Paul A. Molé ◽  
James J. Hoffmann
Keyword(s):  
Steady State ◽  
Time Course ◽  
Respiratory Exchange Ratio ◽  
Short Term ◽  
Men And Women ◽  
Adult Men ◽  
Wide Range ◽  
Leg Cycling ◽  
Biexponential Model ◽  
Kinetics Of

We propose that variations in fat and carbohydrate (CHO) oxidation by working muscle alter O2 uptake (V˙o 2) kinetics. This hypothesis provides two predictions: 1) the kinetics should comprise two exponential components, one fast and the other slow, and 2) their contribution should change with variations in fat and CHO oxidation, as predicted by steady-state respiratory exchange ratio (RER). The purpose of this study was to test these predictions by evaluating theV˙o 2 kinetic model:V˙o 2( t) = αR + αF{1 − exp[( t − TD)/−τF]} + αC{1 − exp[( t − TD)/−τC]} for short-term, mild leg cycling in 38 women and 44 men, whereV˙o 2( t) describes the time course, αR is resting V˙o 2, t is time after onset of exercise, TD is time delay, αF and τF are asymptote and time constant, respectively, for the fast (fat) oxidative term, and αC and τC are the corresponding parameters for the slow (CHO) oxidative term. We found that 1) this biexponential model accurately described the V˙o 2kinetics over a wide range of RERs, 2) the contribution of the fast (αF, fat) component was inversely related to RER, whereas the slow (αC, CHO) component was positively related to RER, and 3) this assignment of the fast and slow terms accurately predicted steady-state respiratory quotient and CO2 output. Therefore, the kinetic model can quantify the dynamics of fat and CHO oxidation over the first 5–10 min of mild exercise in young adult men and women.

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