Km For Efflux Research Articles

Influence of Apolipoprotein (Apo) A-I Structure on Nascent High Density Lipoprotein (HDL) Particle Size Distribution

The principal protein of high density lipoprotein (HDL), apolipoprotein (apo) A-I, in the lipid-free state contains two tertiary structure domains comprising an N-terminal helix bundle and a less organized C-terminal domain. It is not known how the properties of these domains modulate the formation and size distribution of apoA-I-containing nascent HDL particles created by ATP-binding cassette transporter A1 (ABCA1)-mediated efflux of cellular phospholipid and cholesterol. To address this issue, proteins corresponding to the two domains of human apoA-I (residues 1-189 and 190-243) and mouse apoA-I (residues 1-186 and 187-240) together with some human/mouse domain hybrids were examined for their abilities to form HDL particles when incubated with either ABCA1-expressing cells or phospholipid multilamellar vesicles. Incubation of human apoA-I with cells gave rise to two sizes of HDL particles (hydrodynamic diameter, 8 and 10 nm), and removal or disruption of the C-terminal domain eliminated the formation of the smaller particle. Variations in apoA-I domain structure and physical properties exerted similar effects on the rates of formation and sizes of HDL particles created by either spontaneous solubilization of phospholipid multilamellar vesicles or the ABCA1-mediated efflux of cellular lipids. It follows that the sizes of nascent HDL particles are determined at the point at which cellular phospholipid and cholesterol are solubilized by apoA-I; apparently, this is the rate-determining step in the overall ABCA1-mediated cellular lipid efflux process. The stability of the apoA-I N-terminal helix bundle domain and the hydrophobicity of the C-terminal domain are important determinants of both nascent HDL particle size and their rate of formation.

Carrier-mediated uptake and excretion of bromosulfophthalein-glutathione in perfused rat liver: A multiple indicator dilution study

The hepatic removal of the glutathione conjugate of bromosulfophthalein (BSPGSH) was studied in the single-pass perfused rat liver with the multiple indicator dilution (MID) technique against various background concentrations of BSPGSH (20 to 214 mumol/L) over which nonlinear binding to both plasma (albumin) and tissue proteins with two classes of binding sites was found. A bolus containing 51Cr-labeled red blood cell (a vascular reference), [125I]albumin and [14C]sucrose (large and small molecular weight interstitial references, respectively), D2O (a cellular space reference), and [3H]BSPGSH was injected into the portal vein during steady-state. The eliminated fraction of dose, obtained by subtracting the survival fraction of [3H]BSPGSH in plasma from one, corresponded to the steady state extraction ratio (E) with bulk data, which declined from 0.74 +/- 0.04 to 0.27 +/- 0.01 with concentration. The major portion of the tracer outflow profile was a throughput component, which is the proportion of tracer that did not enter liver cells during its transit through the liver. The influx, efflux, and sequestration coefficients, evaluated with previously developed barrier-limited models, provided the corresponding influx (k1), efflux (k-1) and excretion (kseq) rate constants. Concentration-dependent influx (Vmax = 83 nmol min-1 g-1 and Km = 3.7 mumol/L), efflux (Vmax = 15 nmol min-1 g-1 and Km = 1.8 mumol/L), and excretion (Vmax = 94 nmol min-1 g-1 and Km = 1.8 mumol/L) were obtained for BSPGSH, when Km values are expressed in terms of the unbound concentrations. In these calculations, the observed unbound tissue concentration was not used for estimation of the Vmax and Km for efflux and excretion because of overestimation, because of the presence of highly concentrated BSPGSH in ductular elements present in liver homogenates; rather, the unbound tissue concentration was calculated from the influx, efflux, and removal rate coefficients. Because of carrier-mediated entry, the unbound tissue concentration does not equal the unbound plasma concentration, and kinetic parameters for BSPGSH excretion could be alternately estimated when the rate of excretion or net rate of loss of BSPGSH from plasma was regressed against the estimated tissue unbound concentration. This yielded a Vmax of 97 nmol min-1 g-1 and a Km of 3.6 mumol/L, values similar to those obtained from MID.(ABSTRACT TRUNCATED AT 400 WORDS)

Copper transport and kinetics in cultured C6 rat glioma cells.

C6 rat glioma cells accumulate and efflux 67Cu. Both processes showed saturation kinetics with increasing 67Cu concentration. The Michaelis constant (Km) for uptake was 0.63 +/- 0.14 microM; maximum velocity (Vmax) was 3.29 +/- 0.57 pmol Cu.mg protein-1.min-1. The Km for efflux was 0.15 +/- 0.06 microM; Vmax was 1.08 +/- 0.71 pmol Cu.mg protein-1.min-1. p-Chloromercuribenzoate (p-CMB) totally blocked 67Cu efflux but had no effect on Km or Vmax of uptake. Total 67Cu in the cell after 50 min was partitioned equally between particulate and soluble fractions. p-CMB-treated cells accumulated more 67Cu, but < 10% was bound to the particulate (membrane) fraction. Pb also increased 67Cu accumulation without affecting Km and Vmax of 67Cu uptake. These data suggest that carriers for import and export of Cu in C6 cells are distinct or operate in two different cellular environments. Efflux is a sulfhydryl-dependent process subject to inhibition by Pb. The data are consistent with a P-type ATPase in the efflux of Cu from cells and the potential for Pb to inhibit the efflux mechanism.

Hepatobiliary transport of glutathione and glutathione conjugate in rats with hereditary hyperbilirubinemia.

TR- mutant rats have an autosomal recessive mutation that is expressed as a severely impaired hepatobiliary secretion of organic anions like bilirubin-(di)glucuronide and dibromosulphthalein (DBSP). In this paper, the hepatobiliary transport of glutathione and a glutathione conjugate was studied in normal Wistar rats and TR- rats. It was shown that glutathione is virtually absent from the bile of TR- rats. In the isolated, perfused liver the secretion of glutathione and the glutathione conjugate, dinitrophenyl-glutathione (GS-DNP), from hepatocyte to bile is severely impaired, whereas the sinusoidal secretion from liver to blood is not affected. The secretion of GS-DNP was also studied in isolated hepatocytes. The secretion of GS-DNP from cells isolated from TR- rat liver was significantly slower than from normal hepatocytes. Efflux of GS-DNP was a saturable process with respect to intracellular GS-DNP concentration: Vmax and Km for efflux from TR- cells was 498 nmol/min.g dry wt and 3.3 mM, respectively, as compared with 1514 nmol/min.g dry wt and 0.92 mM in normal hepatocytes. These results suggest that the canalicular transport system for glutathione and glutathione conjugates is severely impaired in TR- rats, whereas sinusoidal efflux is unaffected. Because the defect also comes to expression in isolated hepatocytes, efflux of GS-DNP from normal hepatocytes must predominantly be mediated by the canalicular transport mechanism, which is deficient in TR- rats.

Direct evidence for ATP modulation of sugar transport in human erythrocyte ghosts.

Sugar transport in human erythrocyte ghosts is modulated by low molecular weight factors present in red cell cytosol that induce an asymmetry in Michaelis and velocity constants for sugar entry and exit (Carruthers, A., and Melchior, D. L. (1983) Biochim. Biophys. Acta 728, 254-266). This study examines the possibility that ATP is the transport-modulating factor. The intracellular factor must satisfy at least three criteria. It must reduce Km and Vmax for sugar efflux from inside-out red cell membrane vesicles. It should increase Km for efflux from red cell ghosts. It should have a molecular weight of less than 10 kDa. These criteria are satisfied by ATP. AMP, ADP, GTP, UTP, and ITP are without effect on sugar transport. The following results support the view that the cytosolic factor is ATP. Red cell lysate (obtained by hypotonic lysis of red cells) is unable to modify transport following dialysis against ATP-free medium. The ability of lysate to modify transport is retained following acid extraction. ATP depletion of acid-extracted lysate by treatment with apyrase results in the loss of transport-modulating potency. Myokinase partly restores both the ATP content and the ability of ATP-depleted (apyrase-treated) lysate to modify transport. Addition of ATP to ATP-depleted lysate mimics the ability to myokinase to restore the transport-modulating potency of lysate. ATP is without effect on the number and molecular size of D-glucose-sensitive cytochalasin B-binding proteins in the red cell membrane. These findings demonstrate that the transport-modulating potency of red cell cytosol is quantitatively accounted for by intracellular ATP which acts to modify the catalytic activity of plasmalemmal transporters.

The mechanism of pantothenate transport by rat liver parenchymal cells in primary culture.

The mechanism of pantothenate transport across the plasma membrane was investigated with initial velocity studies of [14C]pantothenate uptake and efflux in rat liver parenchymal cells maintained in primary culture. At 116 mM sodium, double-reciprocal plots of the initial velocity of uptake versus [pantothenate] were linear from 0.3 to 36.5 microM pantothenate and gave an apparent Km,pant of 11 +/- 2 microM. The rate of pantothenate uptake at 0 [sodium] was about 14% of the rate at 116 mM sodium, and the reciprocal of the apparent Km,pant was a linear function of [sodium]. Vmax obtained by extrapolation to infinite [pantothenate] was independent of [sodium]. Ouabain, gramicidin D, cyanide, azide, and 2,4-dinitrophenol inhibited uptake, but preloading cells with pantothenate did not. Pantothenate derivatives or carboxylic acids were only weak inhibitors of uptake. Efflux was measured in cells preloaded with [14C]pantothenate. The apparent Km for efflux was 85 +/- 29 microM, and the rate of efflux was unaffected by addition of pantothenate, sodium, ouabain, gramicidin D, or 2,4-dinitrophenol to the external medium. These features are consistent with a mechanism for pantothenate transport in which sodium and pantothenate are cotransported in a 1:1 ratio on a carrier highly specific for pantothenate; sodium decreases the apparent Km for pantothenate, and a sodium-carrier complex forms only on the intracellular side of the membrane.

Kinetic properties of the reconstituted glucose transporter from human erythrocytes.

The kinetic parameters of D-glucose transport in liposomes reconstituted with the purified glucose transporter were determined. Net uptake and efflux both had Km values of 0.7 to 1.2 mM and Vmax values of 1.6 mumol/mg of protein/min. Equilibrium exchange had a Km of 35 mM and a Vmax of 50 mumol/mg of protein/min. By separating the liposomes from unreconstituted protein using density centrifugation, the Vmax of exchange was increased to 86 mumol/mg of protein/min, about 3 times that of the erythrocyte membrane. Trypsin, which inhibits erythrocyte glucose transport only from the cytoplasmic side, inhibited reconstituted transport activity about 40% when added externally. With internal treatment as well, the inhibition was about 80%. This suggests that the reconstituted transporter is oriented about equally in both directions. Antibody prepared against the purified transporter inhibits transport to a maximum of about 50%, also indicating a scrambled orientation. External trypsin treatment decreased the Km for uptake and increased the Km for efflux, consistent with asymmetric kinetic parameters for the two faces of the transporter. However, the calculated Km values are lower than those reported for erythrocytes. Phloretin and diethylstilbestrol inhibit the reconstituted transporter. However, they bind to liposomes, producing anomalous results under some experimental conditions. When this binding is taken into account, phloretin inhibits completely and symmetrically. The binding accounts for the apparent asymmetric effects of phloretin reported by others. The inhibitory effects of mercuric ions are consistent with action at two classes of binding sites. Treatment with trypsin increases the sensitivity to Hg2+, indicating that the more sensitive site is on the external face of the transporter.

Kinetics of lactose transport into Escherichia coli in the presence and absence of a protonmotive force

The kinetics of galactoside movements into and out of whole cells of Escherichia coli under metabolizing and metabolically-inhibited conditions were described by Winkler and Wilson [ 11. They found that the inhibition of metabolism caused the very high app. Km for efflux to fall to the lower values characteristic of the app. Km for infhrx, whereas the app. Km for influx was not detectably changed. These results were substantially confirmed in [2], but in a neglected paper [3], the presence of uncoupler was reported to cause the app. Km for influx of thiomethylgalactoside to increase to a much higher value. In the presence of a protonmotive force membrane vesicles accumulate lactose with an app. Km similar to that for influx into whole cells [5,6]. In the absence of added respirable substrate there is essentially no protonmotive force and galactosides are not accumulated, though they may still bind to the carrier [6]. Under the latter conditions, the binding constant for lactose was 200-fold greater than the app. Km for active uptake [6]. Similar results have been reported in [7]. This paper describes experiments designed to examine the kinetics of the lactose carrier in the absence of a protonmotive force but under conditions of net transport. The results rule out the possibility that the difference between the app. Km and Kd observed in [6] could be due to measurements being made under different conditions (i.e., that the app.

Influence of exogenous sugars and polyols on C1-influx and efflux by the ascomycete Neocosmospora vasinfecta.

Glucose and other transportable sugars and polyols inhibited Cl- influx very soon after addition to mycelium in the process of Cl- accumulation. Under the usual experimental conditions (0.1 mM KCl, glucose greater than or equal to 2 mM) the mean percentage of inhibition of Cl- influx by glucose was 54.1 +/- 8.0 (+/- standard error; N = 26). Transport of the exogenous carbohydrate was necessary for inhibition of Cl- influx. Thus, the estimated Ki for glucose inhibition of Cl- influx (28 muM) was close to the Km for glucose transport; glycerol did not inhibit Cl- influx unless it was itself transported, and the degree of inhibition exerted by various carbohydrates correlated with their uptake rates. Inhibition was not caused by the accumulated sugar itself, as high levels (ca. 60 mM) of intramycelial 3-O-methylglucose gave rise to a stimulation of Cl- influx when the exogenous sugar was removed. It is suggested that interaction of Cl- and carbohydrate transport arises from competition for a common energy-coupling mechanism in the cell membrane. Both glucose and 3-O-methylglucose elicited Cl- efflux, but the maximal Cl- efflux rates were observed only after 40 min of incubation and only in the presence of the readily metabolizable glucose. Removal of the exogenous glucose, even after maximal Cl- efflux had been established, resulted in the rapid cessation of efflux. Studies under anaerobic conditions gave further evidence that glucose uptake was necessary and that efflux was not due to temporary depletion of energy reserves. It is proposed that glucose-induced leakage of Cl- is due to reversal of the Cl- uptake system, even though the Km for efflux is much greater than that for influx.

Transport of succinate in Escherichia coli. III. Biochemical and genetic studies of the mechanism of transport in membrane vesicles.

The D-lactate oxidation dependent transport of succinate in membrane vesicles of an Escherichia coli strain lacking succinate dehydrogenase and fumarate reductase is inhibited by several categories of compounds. One category consists of compounds that are electron transport inhibitors (Amytal, Dicumarol, and mercurials), the second of compounds that act as competitive inhibitors of D-lactate dehydrogenase (oxamate and β-chlorolactate), the third of reagents that inhibit the Ca2+–Mg2+-activated ATPase (dicyclohexylcarbodiimide and pyrophosphate), and the fourth of compounds that tap off electrons from the respiratory chain (2,6-dichlorophenolindophenol). None of the succinate transport inhibitors, including mercurials like p-chloromercuribenzoate, interfere with the binding of succinate to the presumed membrane carriers.Membrane preparations from mutants of E. coli lacking D-lactate dehydrogenase are unable to transport succinate in the presence of D-lactate. Whole cells of these mutants, however, take up succinate normally. This observation suggests that D-lactate oxidation is not obligatorily linked in vivo to the uptake of succinate although the possibility is not excluded that transport in such mutants may be linked to some other dehydrogenase. Mutants having altered levels of ATPase, or membrane preparations made from such cells also have greatly reduced capacity to transport succinate. This observation coupled with the finding that ATPase inhibitors block dicarboxylate transport suggests involvement of ATPase in an unknown way in the concentrative uptake of succinate.With the exception of oxamate, β-chlorolactate (competitive inhibitors of D-lactate oxidation), and dicyclohexylcarbodiimide, all of the inhibitors of succinate uptake (including p-chloromercuribenzoate) cause an immediate efflux of preloaded succinate from membrane vesicles. Efflux is also caused by proton conducting reagents. The Km for efflux is 1.9 mM. This value is to be compared with the Km for influx, which is only about 0.02 mM.The weight of evidence favors the view that the active transport of succinate in vesicles occurs as a result of an energization of the membranes by the passage of electrons, although alternate oxidation and reduction of the succinate carrier as a mechanism for transport has not been definitely ruled out.