Michaelis-Menten kinetics of galactose elimination by the isolated perfused pig liver

1976 ◽  
Vol 230 (5) ◽  
pp. 1302-1313 ◽  
Author(s):  
S Keiding ◽  
S Johansen ◽  
K Winkler ◽  
K Tonnesen ◽  
N Tygstrup
Keyword(s):  
Irreversible Process ◽  
Experimental Uncertainty ◽  
Maximal Rate ◽  
Michaelis Constant ◽  
Pig Liver ◽  
Blood Concentrations ◽  
Saturation Kinetics ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Galactose Elimination

The relation between galactose elimination rates and blood concentrations in the isolated perfused pig liver was analyzed by a mathematical kinetic model. It assumes that the substrate, under steady-state conditions, is removed from the blood that flows through the sinusoids by an irreversible process which follows Michaelis-Menten (i.e., saturation) kinetics. The experiments consisted of successive periods with constant infusions of galactose. The model fitted the data to within the experimental uncertainty. The estimated maximal rate (Vmax) ranged from 0.34 to 0.57 mmol-min(-1)-kg(-1) liver, and the Michaelis constant, Km, ranged from 0.12 to 0.30 mmol-liter(-1) plasma water in nine experiments. The ratio between the galactose concentration in hepatocyte water and plasma water was not significantly different from 1.0, indicating that membrane transport is not rate limiting for the elimination of galactose. In experiments with increasing concentrations of galactose in hepatocyte water and approximately saturated elimination rates, the concentrations of galactose 1-phosphate, UDPgalactose, and UDPglucose remained essentially constant. This indicates that the phosphorylation of galactose to galactose 1-phosphate is the rate-determining process.

Effect of insulin on kinetics of sugar transport in heart muscle.

1978 ◽  
Vol 234 (1) ◽  
pp. E70 ◽  
Author(s):  
J Y Cheung ◽  
C Conover ◽  
D M Regen ◽  
C F Whitfield ◽  
H E Morgan
Keyword(s):  
Heart Muscle ◽  
Sugar Transport ◽  
Exchange Constant ◽  
Maximal Rate ◽  
Transport Parameters ◽  
Michaelis Constant ◽  
Perfused Rat Heart ◽  
Equilibrium Exchange ◽  
Kinetics Of ◽  
Intracellular Glucose

Insulin increased the maximal rate of sugar transport in the perfused rat heart, but had essentially no effect on the Michaelis constant of sugar entry or half-maximal constant for equilibrium exchange. In control hearts, the following kinetic parameters of 3-O-methylglucose transport were measured: Michaelis constant for entry, 7-10 mM; equilibrium exchange constant, 7 mM; and activity constant (Vmax/Km) from 0.02 to 0.1 ml/g.min. In insulin-treated hearts, these values were 6 mM, 3 MM, and 2.2 ml/g.min, respectively. These changes in transport constants were consistent with a model in which 1) sequestered carrier was released by the hormone or 2) carrier movement, in all forms and directions, was accelerated. Measurements of glucose transport in control hearts indicated that the Michaelis constant for entry was 4 mM and the activity constant, 0.5 ml/g.min. In insulin-treated hearts, quantitation of transport parameters was prevented by accumulation of intracellular glucose.

Kinetics of the sarcolemmal lactate carrier in single heart cells using BCECF to measure pHi

1994 ◽  
Vol 267 (5) ◽  
pp. H1759-H1769 ◽  
Author(s):  
X. Wang ◽  
A. J. Levi ◽  
A. P. Halestrap
Keyword(s):  
Guinea Pig ◽  
Specific Inhibitor ◽  
Limiting Factor ◽  
Heart Cells ◽  
Michaelis Constant ◽  
Hypoxic Conditions ◽  
Rapid Fall ◽  
Rat Myocytes ◽  
Rate Limiting ◽  
Kinetics Of

The pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was used to measure lactate transport in single cardiac myocytes. Addition of lactate externally caused a rapid fall of intracellular pH (pHi), which was largely inhibited by 5 mM alpha-cyano-4-hydroxycinnamate (CHC), a specific inhibitor of the lactate carrier. Stilbene disulfonates such as 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) only partially inhibited the response, with inhibition being greater in guinea pig than rat myocytes. The data are consistent with two isoforms of the lactate carrier, one sensitive and one insensitive to DBDS, coexisting within a single myocyte and both having a stoichiometry of 1 lactate:1 proton. The initial rate of pHi fall was used to determine carrier kinetics. Rat myocytes had a Michaelis constant (Km) for external L-lactate of 2.74 mM and a Km for external pyruvate of 0.2 mM. Guinea pig cells had a Km for external L-lactate of 2.2 mM. Kinetics of lactate efflux were also evaluated using the rate of pHi recovery on removing external lactate. The Km and maximal rate values for efflux were both threefold higher than for influx and were related to each other and the transmembrane pH gradient as predicted by the Haldane relationship. It is suggested that under hypoxic conditions, the carrier may be the rate-limiting factor for lactate extrusion.

scholarly journals Effect of thrombomodulin on the kinetics of the interaction of thrombin with substrates and inhibitors

1986 ◽  
Vol 237 (1) ◽  
pp. 243-251 ◽  
Author(s):  
J Hofsteenge ◽  
H Taguchi ◽  
S R Stone
Keyword(s):  
Dissociation Constant ◽  
Antithrombin Iii ◽  
Dissociation Constants ◽  
Competitive Inhibitor ◽  
Mean Value ◽  
Similar Amount ◽  
Michaelis Constant ◽  
Saturation Kinetics ◽  
Slow Binding Inhibitor ◽  
Kinetics Of

Thrombomodulin decreased by 20-30% the Michaelis constant of two tripeptidyl p-nitroanilide substrates of thrombin. Thrombomodulin increased the rate of inactivation of thrombin by two peptidyl chloromethane inhibitors by a similar amount. This effect appeared to be due to a decrease in the dissociation constants of the inhibitors. An improved method for the separation of fibrinopeptides A and B by h.p.l.c. was developed, and this method was used to study the effect of thrombomodulin on the thrombin-catalysed cleavage of fibrinogen. In this reaction, thrombomodulin was a competitive inhibitor with respect to the A alpha-chain of fibrinogen. The release of fibrinopeptide B was also inhibited by thrombomodulin. Analysis of the inhibition caused by thrombomodulin with respect to fibrinopeptides A and B yielded the same dissociation constant for the thrombin-thrombomodulin complex. In the presence of thrombomodulin, the rate of inactivation of thrombin by antithrombin III was stimulated 4-fold. This stimulation showed saturation kinetics with respect to thrombomodulin. Thrombomodulin was found to compete with hirudin for a binding site on thrombin. As a result of this competition, hirudin became a slow-binding inhibitor of thrombin at high thrombomodulin concentrations. Estimates of the dissociation constant for thrombomodulin were obtained in several of the above experiments, and the weighted mean value was 0.7 nM.

Functional Fractionation of Platelets: Aggregation Kinetics and Glycoprotein Labeling of Differing Platelet Populations

1982 ◽  
Vol 48 (02) ◽  
pp. 211-216 ◽  
Author(s):  
V M Haver ◽  
A R L Gear
Keyword(s):  
Galactose Oxidase ◽  
Aggregation Kinetics ◽  
Maximal Rate ◽  
Membrane Glycoproteins ◽  
Whole Cells ◽  
Reversible Aggregation ◽  
Significant Difference ◽  
Small Doses ◽  
Major Loss ◽  
Kinetics Of

SummaryPlatelet heterogeneity has been studied with a technique called functional fractionation which employs gentle centrifugation to yield subpopulations (“reactive” and “less-reactive” platelets) after exposure to small doses of aggregating agent. Aggregation kinetics of the different platelet populations were investigated by quenched-flow aggregometry. The large, “reactive” platelets were more sensitive to ADP (Ka = 1.74 μM) than the smaller “less-reactive” platelets (Ka = 4.08 μM). However, their maximal rate of aggregation (Vmax, % of platelets aggregating per sec) of 23.3 was significantly lower than the “less-reactive” platelets (Vmax = 34.7). The “reactive” platelets had a 2.2 fold higher level of cyclic AMP.Platelet glycoproteins were labeled using the neuraminidase-galactose oxidase – [H3]-NaBH4 technique. When platelets were labeled after reversible aggregation, the “reactive” platelets showed a two-fold decrease in labeling efficiency (versus control platelets). However, examination of whole cells or membrane preparations from reversibly aggregated platelets revealed no significant difference in Coomassie or PAS (Schiff) staining.These results suggest that the large, “reactive” platelets are more sensitive to ADP but are not hyperaggregable in a kinetic sense. Reversible aggregation may cause a re-orientation of membrane glycoproteins that is apparently not characterized by a major loss of glycoprotein material.

Kinetics of cyclization of S-ethoxycarbonylmethylisothiouronium chloride to 2-imino-4-thiazolidone

1979 ◽  
Vol 44 (3) ◽  
pp. 912-917 ◽  
Author(s):  
Vladimír Macháček ◽  
Said A. El-bahai ◽  
Vojeslav Štěrba
Keyword(s):  
Hydrochloric Acid ◽  
Dilute Hydrochloric Acid ◽  
Base Catalysis ◽  
General Base ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Kinetics Of Formation ◽  
Acid Catalyzed ◽  
Rate Limiting ◽  
Kinetics Of

Kinetics of formation of 2-imino-4-thiazolidone from S-ethoxycarbonylmethylisothiouronium chloride has been studied in aqueous buffers and dilute hydrochloric acid. The reaction is subject to general base catalysis, the β value being 0.65. Its rate limiting step consists in acid-catalyzed splitting off of ethoxide ion from dipolar tetrahedral intermediate. At pH < 2 formation of this intermediate becomes rate-limiting; rate constant of its formation is 2 . 104 s-1.

Polarographic studies on renaturation of urea-denatured human serum albumin

1989 ◽  
Vol 54 (2) ◽  
pp. 536-543 ◽  
Author(s):  
Josef Chmelík ◽  
Pavel Anzenbacher ◽  
Vítěz Kalous
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Irreversible Process ◽  
State Model ◽  
Native Protein ◽  
Main Components ◽  
Two State Model ◽  
Kinetics Of

The renaturation of the two main components of human serum albumin, i.e. of mercaptalbumin and nonmercaptalbumin, was studied polarographically. It has been demonstrated that renaturation of both proteins after 1-min denaturation in 8M urea is reversible. By contrast, renaturation after 200 min denaturation in 8M urea is an irreversible process; the characteristics of renatured mercaptalbumin differ more from the properties of the native protein than the characteristics of nonmercaptalbumin. The studies of the kinetics of renaturation of both proteins have shown that the renaturation can be represented by a two-state model. This means that the existence of stable intermediary products during the renaturation process was not determined polarographically.

Kinetics and mechanism of cyclization of N-(2-methoxycarbonylphenyl)-N’-methylsulphonamide to 3-methyl-(1H)-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide

1991 ◽  
Vol 56 (8) ◽  
pp. 1701-1710 ◽  
Author(s):  
Jaromír Kaválek ◽  
Vladimír Macháček ◽  
Miloš Sedlák ◽  
Vojeslav Štěrba
Keyword(s):  
Potassium Hydroxide ◽  
Acid Catalysis ◽  
Potassium Hydroxide Solution ◽  
Hydroxide Solution ◽  
General Base ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Cyclization Kinetics ◽  
Rate Limiting ◽  
Kinetics Of

The cyclization kinetics of N-(2-methylcarbonylphenyl)-N’-methylsulfonamide (IIb) into 3-methyl-(1H)-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (Ib) has been studied in ethanolamine, morpholine, and butylamine buffers and in potassium hydroxide solution. The cyclization is subject to general base and general acid catalysis. The value of the Bronsted coefficient β is about 0.1, which indicates that splitting off of the proton from negatively charged tetrahedral intermediate represents the rate-limiting and thermodynamically favourable step. In the solutions of potassium hydroxide the cyclization of dianion of the starting ester IIb probably becomes the rate-limiting step.

Kinetic analysis of mechanism of intestinal Na+-dependent sugar transport

1985 ◽  
Vol 248 (5) ◽  
pp. C498-C509 ◽  
Author(s):  
D. Restrepo ◽  
G. A. Kimmich
Keyword(s):  
Experimental Data ◽  
Steady State ◽  
Sugar Transport ◽  
Enzyme Kinetic ◽  
Steady State Distribution ◽  
State Distribution ◽  
Least Squares Fit ◽  
Coupling Stoichiometry ◽  
Rate Limiting ◽  
Kinetics Of

Zero-trans kinetics of Na+-sugar cotransport were investigated. Sugar influx was measured at various sodium and sugar concentrations in K+-loaded cells treated with rotenone and valinomycin. Sugar influx follows Michaelis-Menten kinetics as a function of sugar concentration but not as a function of Na+ concentration. Nine models with 1:1 or 2:1 sodium:sugar stoichiometry were considered. The flux equations for these models were solved assuming steady-state distribution of carrier forms and that translocation across the membrane is rate limiting. Classical enzyme kinetic methods and a least-squares fit of flux equations to the experimental data were used to assess the fit of the different models. Four models can be discarded on this basis. Of the remaining models, we discard two on the basis of the trans sodium dependence and the coupling stoichiometry [G. A. Kimmich and J. Randles, Am. J. Physiol. 247 (Cell Physiol. 16): C74-C82, 1984]. The remaining models are terter ordered mechanisms with sodium debinding first at the trans side. If transfer across the membrane is rate limiting, the binding order can be determined to be sodium:sugar:sodium.

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