scholarly journals Use of a genetic variant to study the hexose transport properties of human skin fibroblasts

1990 ◽  
Vol 265 (3) ◽  
pp. 823-829 ◽  
Author(s):  
O T Mesmer ◽  
B A Gordon ◽  
C A Rupar ◽  
T C Y Lo
Keyword(s):  
Human Skin ◽  
Transport System ◽  
Cytochalasin B ◽  
Transport Systems ◽  
Hexose Transport ◽  
Glucose Starvation ◽  
Human Skin Fibroblasts ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Human skin fibroblasts from ‘normal’ subjects were found to possess at least two hexose transport systems. One system was responsible for the uptake of 2-deoxy-D-glucose (dGlc), D-glucose and D-galactose, whereas the other was responsible primarily for the uptake of 3-O-methyl-D-glucose (MeGlc). The transport of dGlc was the rate-limiting step in the uptake process; over 97% of the internalized dGlc was phosphorylated and the specific activity of hexokinase was several times higher than that for dGlc transport. The dGlc transport system was activated by glucose starvation, and was very sensitive to inhibition by cytochalasin B and energy uncouplers. Fibroblasts isolated from a patient with symptoms of hypoglycaemia were found to differ from their normal counterparts in the dGlc transport system. They exhibited a much higher transport affinity for dGlc, D-glucose and D-galactose, with no change in the respective transport capacity. Transport was not the rate-limiting step in dGlc uptake by these cells. Moreover, the patient's dGlc transport system was no longer sensitive to inhibition by cytochalasin B and energy uncouplers. This suggested that the intrinsic properties of the patient's dGlc transport system were altered. It should be noted that the patient's dGlc transport system could still be activated by glucose starvation. Despite the changes in the dGlc transport system, the MeGlc transport system in the patient's fibroblasts remained unaltered. The observed difference in the properties of the two hexose transport systems in the ‘normal’ and the patient's fibroblasts strongly suggests that the two transport systems may be coded or regulated by different genes. The present finding provides the first genetic evidence from naturally occurring fibroblasts indicating the presence of two different hexose transport systems.

Saturable and nonsaturable hexose uptake in cultured human skin fibroblasts

1978 ◽  
Vol 56 (2) ◽  
pp. 80-88 ◽  
Author(s):  
Ralph J. Germinario ◽  
Maureen Oliveira ◽  
Hyman Leung
Keyword(s):  
Human Skin ◽  
Cytochalasin B ◽  
Competitive Inhibition ◽  
Sugar Transport ◽  
Skin Fibroblasts ◽  
Sugar Uptake ◽  
Human Skin Fibroblasts ◽  
Rate Limiting Step ◽  
Hexose Uptake ◽  
Rate Limiting

The saturable transport of 2-deoxy-D-glucose across the cell membrane of cultured human skin fibroblasts was measured in sparse and confluent cultures. The contribution of nonsaturable sugar uptake to total sugar uptake was monitored by determining L-glucose uptake. The uptake of 2-deoxy-D-glucose was studied as a function of time and substrate concentration. Greater than 70% of transported 2-deoxy-D-glucose was phosphorylated after incubation for 2 min or less at all substrate concentrations employed (0.1 to 3.0 mM), and phosphorylation paralleled sugar uptake at these time intervals. Experiments with cytochalasin B demonstrated that an inhibition of transport was always paralleled by an equal inhibition of sugar phosphorylation.The kinetic constants for the uptake and phosphorylation of 2-deoxy-D-glucose and the inhibition of transport by competing sugars and cytochalasin B were calculated from Line-weaver-Burk plots. The Km and Vmax for saturable sugar uptake were calculated for sparse and confluent cultures after subtracting the contribution of nonsaturable sugar uptake. The resulting Km values for sugar uptake in the sparse and confluent cultures were 1.21 ± 0.04 and 0.88 ± 0.2 mM respectively. The corresponding Vmax values were 15.5 ± 1 nmol/mg protein∙min−1 for the sparse cultures and 10.1 ± 1 nmol/mg protein∙min−1 for the confluent cultures. In both sparse and confluent cultures, the Ki values for the competitive inhibition of sugar transport by D-glucose and 3-O-methyl-D-glucose were 0.8 and 2.7 mM respectively; the Ki value for the noncompetitive inhibition of sugar transport by cytochalasin B was 0.5 μM. The Km values for sugar phosphorylation by cell-free homogenates of sparse and confluent cultures were 0.57 ± 0.1 and 0.6 ± 0.1 mM respectively, while their respective Vmax values were 160 ± 53 and 139 ± 43 nmol/mg protein∙min−1.The data are in agreement with the concept that in cultured human skin fibroblasts sugar transport is the rate-limiting step in 2-deoxy-D-glucose metabolism and that phosphorylation is distinct from transport.

Rate-limiting steps for insulin-mediated glucose uptake into perfused rat hindlimb

1986 ◽  
Vol 250 (1) ◽  
pp. E100-E102 ◽  
Author(s):  
K. Kubo ◽  
J. E. Foley
Keyword(s):  
Glucose Uptake ◽  
Glucose Transport ◽  
Transport System ◽  
Glucose Concentration ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Glucose Transport System ◽  
Glucose Clearance ◽  
Rate Limiting ◽  
Uptake And Metabolism

To determine the glucose and insulin concentrations at which glucose transport is rate limiting for insulin-mediated glucose uptake and metabolism in muscle, glucose clearance was determined in the presence of glucose concentrations ranging from trace to 20 mM and in the absence or presence of insulin in the perfused rat hindlimb. In the absence of insulin and at submaximally stimulating insulin concentrations glucose clearance was constant up to 7 mM glucose and then decreased as the glucose concentration was raised. At maximally stimulating insulin concentrations glucose clearance was constant up to 2 mM glucose and then decreased. The decrease in glucose clearance between 2 and 7 mM glucose in the presence of maximally stimulating insulin concentrations could not be accounted for by competition among glucose molecules for the glucose transport system. The results suggest that at physiological glucose concentrations in the presence of maximally stimulating insulin concentrations the rate-limiting step for insulin-mediated glucose uptake and metabolism in muscle shifts from glucose transport to some step beyond transport.

Transport of fluorescein in trichomes of Lycopersicon esculentum

1982 ◽  
Vol 60 (4) ◽  
pp. 397-402 ◽  
Author(s):  
Gregor F. Barclay ◽  
Carol A. Peterson ◽  
Melvin T. Tyree
Keyword(s):  
Lycopersicon Esculentum ◽  
Cytoplasmic Streaming ◽  
Cytochalasin B ◽  
The Other ◽  
Square Root ◽  
Inhibiting Effect ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Other Hand ◽  
Rate Limiting

Translocation of the dye disodium fluorescein (uranin) in trichomes of Lycopersicon esculentum (tomato) was nonpolar and proportional to the square root of time. Inhibition of cytoplasmic streaming by cytochalasin B had no effect on the rate of dye movement. On the other hand, disruption of plasmodesmatal connections between adjacent cells by plasmolysis strongly diminished the rate of fluorescein translocation. Subsequent deplasmolysis of the cells did not remove the inhibiting effect of plasmolysis. The data are consistent with the interpretation that dye movement proceeds by diffusion, the rate-limiting step being transport through plasmodesmatal connections.

scholarly journals Cytochalasin B as a probe for the two hexose-transport systems in rat L6 myoblasts

1988 ◽  
Vol 251 (1) ◽  
pp. 63-72 ◽  
Author(s):  
S R Chen ◽  
T C Y Lo
Keyword(s):  
Plasma Membrane ◽  
Transport System ◽  
Binding Sites ◽  
Cytochalasin B ◽  
Transport Systems ◽  
Scatchard Analysis ◽  
Hexose Transport ◽  
Whole Cells ◽  
High Affinity ◽  
Binding Data

We have recently demonstrated that two hexose-transport systems are present in undifferentiated rat L6 myoblasts: D-glucose and 2-deoxy-D-glucose are preferentially transported by the high-affinity system, whereas 3-O-methyl-D-glucose is transported primarily by the low-affinity system. Mutant D23 is found to be defective only in the high-affinity hexose-transport system. The low-affinity transport system is much more sensitive to inhibition by cytochalasin B (CB). The present study examines the identity, properties and regulation of the CB-binding sites by measuring CB binding to both whole cells and plasma membrane. Scatchard analysis of the binding data revealed the presence of two CB-binding sites, namely CBH and CBL. These two sites differ not only in their affinity for CB, but their levels can also be differentially altered by various biochemical, physiological and genetic manipulations. CBL resembles the high-affinity hexose-transport system in that it is absent in mutant D23 and is present in larger quantities in glucose-starved cells. Moreover, CB binding to this site is inhibited by D-glucose and 2-deoxy-D-glucose, the preferred substrates of the high-affinity hexose-transport system. On the other hand, CBH is found to be unaltered in mutant D23, which also retains the normal low-affinity hexose-transport system. CBH also resembles the low-affinity transport system in that it is not elevated in glucose-starved cells. Furthermore, binding of CB to this site can be inhibited by 3-O-methyl-D-glucose, the preferred substrate of the low-affinity transport system. It should be noted that 2-deoxy-D-glucose does not have much effect on CBH, and vice versa. Studies with purified membrane preparations indicate that both CB-binding sites are present in similar ratios in the plasma membrane and the low-density microsomal fraction. Plasma-membrane studies also reveal that D-glucose 6-phosphate, but not 2-deoxy-D-glucose 6-phosphate, is very effective in activating CB binding. Data presented suggest that CB binding may be regulated by sugar analogues in an allosteric manner.

Basolateral 3-O-methylglucose transport by cultured kidney (LLC-PK1) epithelial cells

1992 ◽  
Vol 262 (3) ◽  
pp. F480-F487
Author(s):  
J. M. Mullin ◽  
L. M. Kofeldt ◽  
L. M. Russo ◽  
M. M. Hagee ◽  
A. H. Dantzig
Keyword(s):  
Cytochalasin B ◽  
Sugar Transport ◽  
Glucose Starvation ◽  
Rate Limiting Step ◽  
Incubation Periods ◽  
Transport Kinetic ◽  
Free Hydroxyl ◽  
Inhibition Studies ◽  
Rate Limiting ◽  
Stimulation Of

In previous work we demonstrated the similarity of basolateral sugar transport of LLC-PK1 renal epithelia to basolateral kidney sugar transport using 2-deoxy-D-glucose as a substrate. In this study we first examine a central limitation to use of 2-deoxyglucose for basolateral sugar transport study in LLC-PK1 epithelia, namely, a shift of the rate-limiting step in uptake from transport to phosphorylation. Use of 3-O-methylglucose avoids this complication because it is not phosphorylated. However, use of 3-O-methylglucose requires much shorter incubation periods to examine linear rates of uptake (steady state is reached by 60 s at 22 degrees C for 0.1 mM 3-O-methylglucose). As was true for 2-deoxyglucose, apical uptake of 3-O-methylglucose was only a fraction of total uptake. Basolateral uptake was characteristically more sensitive to phloretin and cytochalasin B inhibition, relative to phlorizin. Inhibition studies indicate a requirement for a free hydroxyl on C-1 carbon of the pyranose ring, as is characteristic for renal basolateral sugar transport. Kinetic analysis indicates a single transport system with a Km of 10.9 mM and Vmax of 17.2 pmol.micrograms DNA-1.15 s-1. Subconfluent, undifferentiated LLC-PK1 cells show a similar Km (12.7 mM) but a ninefold higher Vmax (166.2 pmol.micrograms DNA-1.15 s-1). Stimulation of 3-O-methylglucose transport rate in confluent cultures by phorbol ester is relatively small (less than 100%) compared with effects on other somatic cells. The uptake rate of 3-O-methylglucose is not affected by glucose starvation, but subsequent refeeding with glucose-containing medium does significantly stimulate uptake.

scholarly journals Monosaccharide transport into lactating-rat mammary acini

1982 ◽  
Vol 204 (2) ◽  
pp. 493-501 ◽  
Author(s):  
L C Threadgold ◽  
H G Coore ◽  
N J Kuhn
Keyword(s):  
Plasma Membrane ◽  
Cytochalasin B ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Monosaccharide Transport ◽  
Rat Mammary Gland ◽  
The Mean ◽  
Rate Limiting ◽  
Rate Of Consumption ◽  
Uptake And Release

The uptake and release of 3-O-methyl-D-[3H]glucose at 37 degrees C by acini, prepared from lactating-rat mammary gland with collagenase, was inhibited by glucose, phloretin, cytochalasin B, HgCl2 and low temperature. Uptake and phosphorylation of 2-deoxy-D-[3H]glucose, studied in greater detail, could be ascribed to a specific, saturable, inhibitable, process of apparent Km 16 mM and Vmax. approx. 56 nmol/min per mg of protein, plus a non-specific, non-inhibitable process that was monitored with [14C]fructose. The mean rate of uptake of 5 mM-2-deoxyglucose (16 nmol/min per mg of protein) was similar to the rate of consumption of 5 mM-glucose, suggesting that transport was a rate-limiting step in the overall metabolism of glucose. This accords with evidence for a glucose gradient across the plasma membrane.

Product Release is Not the Rate-Limiting Step during Cytochalasin B-Induced ATPase Activity of Monomeric Actin

1991 ◽  
Vol 46 (1-2) ◽  
pp. 139-144 ◽  
Author(s):  
Peter Dancker ◽  
Lore Hess ◽  
Karl Ritter
Keyword(s):  
Steady State ◽  
Atpase Activity ◽  
Rate Constant ◽  
Cytochalasin B ◽  
Single Turnover ◽  
Rate Limiting Step ◽  
Hydrolysis Products ◽  
Limiting Step ◽  
Rate Limiting ◽  
Hydrolysis Of

Abstract Under conditions where cytochalasin B induces ATPase activity of monomeric actin (0.3 mᴍ MgCl2, 1 mᴍ EGTA , 30 (μᴍ cytochalasin B, 1 mᴍ ATP) the rate constant of the ex­change of actin-bound ε-ATP for free ATP is about 4 -6 times faster than steady state ATPase activity. When a stoichiometric ATP -actin complex is extracted with PCA (single turnover ex­periment) the apparent rate constant of Pi generation is not faster than steady state ATPase activity. -The experiments suggest that the hydrolysis of actin-bound ATP and not the subse­quent release of hydrolysis products is rate-limiting during cytochalasin-induced ATPase activi­ty of actin.

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