Effect of cytochalasin B on the adhesion of mouse peritoneal macrophages

The adhesion of normal mouse macrophages to glass surfaces was reduced by nontoxic levels (1-50 mug/ml) of cytochalasin B in combination with a centrifugal force (1,000-8,000 g). Macrophages nonspecifically activated by Corynebacterium acnes were also detached by this treatment, but less effectively. The effects of cytochalasin B treatment on these cells were shown to be reversible. After detachment, the cells reattached to glass, appeared morphologically normal, and behaved like untreated cells as judged by adhesion, acid phosphatase levels, and phagocytosis. The effect of cytochalasin B on several parameters of phagocytosis by normal macrophages was also examined. The results demonstrate that cytochalasin B can be used to detach macrophages from surfaces and suggest a functional relationship between phagocytosis and macrophage adhesion to surfaces. Furthermore, the effect of cytochalasin B on adhesion of phagocytic cells provides a probe for further investigation of the adhesion of cells to surfaces.     

The effect of cytochalasin B,colchicine and vinblastine on the adhesion of Trichomonas vaginalis to glass surfaces

The attachment of Trichomonas vaginalis to glass surfaces was studied in the presence of cytochalasin B, colchicine and vinblastine. Marked inhibition of adhesion was noted with high concentrations of cytochalasin B. Colchicine and vinblastine were without effect. These findings suggest that Trichomonas vaginalis adhesion is at least partially mediated by the extension of cellular probes, due to the action of cytochalasin-sensitive microfilaments.     

Role of divalent cations, pH, cytoskeleton components and surface charge on the adhesion of Trichomonas vaginalis to a polystyrene substrate

The process of adhesion of three different strains of Trichomonas vaginalis to a polystyrene substrate was analysed. The process of adhesion was dependent on the time of incubation and the pH of the phosphate-buffered solution (PBS) in which the parasites were suspended. The highest indices of adhesion were observed after an incubation time of 60 min at pH 6.6. The adhesion index increased when the parasites were incubated in the presence of culture media or when Ca++ or Mg++ was added to the PBS solution, whereas cytochalasin B, trypsin or neuraminidase reduced adhesion. Incubation of the parasites in the presence of poly-L-lysine facilitated the process of adhesion. Incubation of the parasites or polystyrene beads in the presence of poly-L-lysine led to important changes in their surface charge.     

Effects of temperature,metabolic and cytoskeletal inhibitors on the rate of BHK cell adhesion to polystyrene

Adhesion of baby hamster kidney fibroblasts (BHK cells) to a Falcon tissue culture flask was measured under various physiological conditions. While 75–80% of the fibroblasts adhere at temperatures from 19–50°, cellular adhesion decreased dramatically below 19°. Less than 10% of the cells adhere to the substratum even after prolonged incubations at temperatures of 8° or below. This lack of adhesion at low temperatures cannot be overcome by the application of increased gravitational force to the cells. No correlation exists between cellular ATP concentrations or respiration rates and the rate of cell adhesion to the substratum. One millimolar Na F and 1 mM 2,4 dinitrophenol together lower cellular ATP concentration by 95% but adhesion is reduced by only 50%. NaN₃ and KCN greatly lower cellular ATP concentrations without a corresponding inhibition of adhesion. Inhibition of cellular respiration by these compounds occurs at lower concentrations than does the inhibition of adhesion. Two micrograms/milliliters of cytochalasin B inhibits adhesion by 90%, 0.1 mM vinblastine sulphate or colchicine by less than 50% and 50 μg/ml colcemid by less than 30%. Fixing the cells with formaldehyde, hardening their membranes with ZnCl₂ or treating the cells with toluene, all cause an inhibition in adhesion. Again, application of increased gravitational force cannot overcome these latter inhibitions of BHK cell adhesion to the surface of the flasks.     

Facile platelet adhesion to collagen requires metabolic energy and actin polymerization and evokes intracellular free calcium mobilization

The attachment of platelets to collagen-coated microtiter plates at 20 degrees C was inhibited strongly by depletion of metabolic energy or by addition of cytochalasins and was slightly inhibited by the intracellular Ca2+ chelator BAPTA. In keeping with their respective potencies as inhibitors of actin polymerization, cytochalasins D and H were the most potent inhibitors of adhesion, while cytochalasin B was the least potent. Energy depletion, cytochalasin D or, to a much lesser extent, BAPTA also inhibited platelet adhesion to collagen in a suspension assay system at 37 degrees C. Collagen-induced platelet cytosolic Ca2+ mobilization was inhibited up to 70% by cytochalasin D and abolished by energy depletion or BAPTA. Elevation of intracellular platelet calcium by treatment with ionomycin had little effect on platelet adhesion to collagen. We propose that rapid platelet spreading along collagen fibers is both energy- and actin-dependent and necessary to produce maximal adhesion needed to elicit Ca2+ mobilization required for subsequent responses.     

Biochemical and functional heterogeneity of rat adipocyte glucose transporters

We have studied the biochemical mechanism of insulin action on glucose transport in the rat adipocyte. Plasma membranes and low-density microsomes were prepared by differential ultracentrifugation of basal and insulin-stimulated cells. The photochemical cross-linking agent hydroxysuccinimidyl-4-azidobenzoate was used to covalently bind [3H]cytochalasin B to the glucose transporter which migrated as a 45-50-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing of the eluted 40-55-kDa proteins revealed two peaks of D-glucose-inhibitable [3H]cytochalasin B radioactivity focusing at pH 6.4 and 5.6 when low-density microsomes were used as the starting material. In contrast, only one D-glucose inhibitable peak, focusing at pH 5.6, was found in plasma membranes. Pretreatment of the cells with insulin led to a marked redistribution of the pH 5.6 form of the glucose transporter from low-density microsomes to plasma membranes with no effect on the pH 6.4 form of the glucose transporter. Following isolation from the isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels, both glucose transporter isoforms were shown to cross-react with an antiserum raised against the purified human erythrocyte glucose transporter. Following incubation of [3H]cytochalasin B-labeled low-density microsomal and plasma membranes with neuraminidase, the pH 5.6 transporter isoform was shifted on isoelectric focusing to a more basic pH, while the pH 6.4 isoform was not affected. These data demonstrate that: there is a heterogeneity of glucose transporter species in the intracellular pool while the plasma membrane transporters are more uniform in structure. The pH 5.6 glucose transporter isoform is translocated by insulin from the low-density microsomes to the plasma membrane but the pH 6.4 isoform is not sensitive to insulin. Differential sensitivity of the glucose transporter isoforms to neuraminidase suggests that the heterogeneity is at least partially due to differences in glycosylation state.     

Inhibition by nucleosides of glucose-transport activity in human erythrocytes.

The interaction of nucleosides with the glucose carrier of human erythrocytes was examined by studying the effect of nucleosides on reversible cytochalasin B-binding activity and glucose transport. Adenosine, inosine and thymidine were more potent inhibitors of cytochalasin B binding to human erythrocyte membranes than was D-glucose [IC50 (concentration causing 50% inhibition) values of 10, 24, 28 and 38 mM respectively]. Moreover, low concentrations of thymidine and adenosine inhibited D-glucose-sensitive cytochalasin B binding in an apparently competitive manner. Thymidine, a nucleoside not metabolized by human erythrocytes, inhibited glucose influx by intact cells with an IC50 value of 9 mM when preincubated with the erythrocytes. In contrast, thymidine was an order of magnitude less potent as an inhibitor of glucose influx when added simultaneously with the radioactive glucose. Consistent with this finding was the demonstration that glucose influx by inside-out vesicles prepared from human erythrocytes was more susceptible to thymidine inhibition than glucose influx by right-side-out vesicles. These data, together with previous suggestions that cytochalasin B binds to the glucose carrier at the inner face of the membrane, indicate that nucleosides are capable of inhibiting glucose-transport activity by interacting at the cytoplasmic surface of the glucose transporter. Nucleosides may also exhibit a low-affinity interaction at the extracellular face of the glucose transporter.     

Effects of cytochalasin, phalloidin, and pH on the elongation of actin filaments

We used electron microscopy to measure the effects of cytochalasins, phalloidin, and pH on the rates of elongation at the barbed and pointed ends of actin filaments. In the case of the cytochalasins, we compared the effects on ATP- and ADP-actin monomers. Micromolar concentrations of either cytochalasin B (CB) or cytochalasin D (CD) inhibit elongation at both ends of the filament, about 95% at the barbed end and 50% at the pointed end, so that the two ends contribute about equally to the rate of growth. Half-maximal inhibition of elongation at the barbed end is at 0.1 microM CB and 0.02 microM CD for ATP-actin and at 0.1 microM CD for ADP-actin. At the pointed end, CD inhibits elongation by ATP-actin and ADP-actin about equally. At high (2 microM) concentrations, the cytochalasins reduce the association and dissociation rate constants in parallel for both ADP- and ATP-actin, so their effects on the critical concentrations are minimal. These observations confirm and extend those of Bonder and Mooseker [Bonder, E. M., & Mooseker, M. S. (1986) J. Cell Biol. 102, 282-288]. The dependence of the elongation rate on the concentration of both cytochalasin and actin can be explained quantitatively by a mechanism that includes the effects of cytochalasin binding to actin monomers [Godette, D. W., & Frieden, C. (1986) J. Biol. Chem. 261, 5974-5980] and a partial cap of the barbed end of the filament by the complex of ADP-actin and cytochalasin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of cytochalasin B on the testosterone synthesis by interstitial cells of rat testis

The present study examined the effects of cytochalasin B on various steps in the luteinizing hormone (LH)-stimulated increase in testosterone synthesis by collagenase-dispersed interstitial cells of adult rat testis. Cytochalasin B at a concentration range of 0.1–50 μM inhibited the LH-stimulated increase in testosterone synthesis in a dose-dependent manner. Both intracellular and medium (released) testosterone levels were reduced, thus indicating that the decrease was not due to the accumulation of testosterone inside the cell as a result of cytochalasin B treatment. Cytochalasin B also inhibited the 8-bromocyclic AMP and pregnenolone-stimulated testosterone synthesis in a similar dose-dependent manner. Cytochalasin B at the two higher doses (10 and 50 μM) also inhibited the LH-stimulated generation of cyclic AMP by interstitial cells. However, this drug had no effect on basal testosterone synthesis except at the highest concentration added.Previous studies on adrenocorticotropic hormone (ACTH)- and LH-stimulated increase in glucocorticoid and testosterone synthesis in adrenal and Leydig cells, respectively, demonstrated that cytochalasin B or anti-actin inhibited the transport of cholesterol into mitochondria. The present studies suggest that cytochalasin B inhibits at least two additional steps in the LH-stimulated increase in testosterone synthesis: (1) the generation of cyclic AMP at the level of the plasma membrane, and (2) the conversion of pregnenolone to the testosterone at the level of the smooth endoplasmic reticulum. It remains to be established whether these are direct effects of cytochalasin B, or whether they are mediated by disruption of microfilaments by cytochalasin B.     

Direct block of cloned hKv1.5 channel by cytochalasins, actin-disrupting agents

The action of cytochalasins, actin-disrupting agents on human Kv1.5 channel (hKv1.5) stably expressed in Ltk^– cells was investigated using the whole cell patch-clamp technique. Cytochalasin B inhibited hKv1.5 currents rapidly and reversibly at +60 mV in a concentration-dependent manner with an IC₅₀ of 4.2 µM. Cytochalasin A, which has a structure very similar to cytochalasin B, inhibited hKv1.5 (IC₅₀ of 1.4 µM at +60 mV). Pretreatment with other actin filament disruptors cytochalasin D and cytochalasin J, and an actin filament stabilizing agent phalloidin had no effect on the cytochalasin B-induced inhibition of hKv1.5 currents. Cytochalasin B accelerated the decay rate of inactivation for the hKv1.5 currents. Cytochalasin B-induced inhibition of the hKv1.5 channels was voltage dependent with a steep increase over the voltage range of the channel's opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. Cytochalasin B produced no significant effect on the steady-state activation or inactivation curves. The rate constants for association and dissociation of cytochalasin B were 3.7 µM/s and 7.5 s^–1, respectively. Cytochalasin B produced a use-dependent inhibition of hKv1.5 current that was consistent with the slow recovery from inactivation in the presence of the drug. Cytochalasin B (10 µM) also inhibited an ultrarapid delayed rectifier K⁺ current (I_K,ur) in human atrial myocytes. These results indicate that cytochalasin B primarily blocks activated hKv1.5 channels and endogenous I_K,ur in a cytoskeleton-independent manner as an open-channel blocker. voltage-gated K⁺ channel; heart; open channel block     

Activation of superoxide production and differential exocytosis in polymorphonuclear leukocytes by cytochalasins A, B, C, D and E. Effects of various ions

All of the common cytochalasins activate superoxide anion release and exocytosis of beta-N-acetylglucosaminidase and lysozyme from guinea-pig polymorphonuclear leukocytes (neutrophils) incubated in a buffered sucrose medium. Half-maximal activation of both processes is produced by approx. 0.2 microM cytochalasin A, C greater than 2 microM cytochalasin B greater than or equal to 4-5 microM cytochalasin D, E. While maximal rates of O2- release and extents of exocytosis require extracellular calcium (1-2 mM), replacing sucrose with monovalent cation chlorides is inhibitory to neutrophil activation by cytochalasins. Na+, K+ or choline inhibit either cytochalasin B- or E-stimulated O2- production with IC50 values of 5-10 mM and inhibition occurs whether Cl-, NO3- or SCN- is the anion added with Na+ or K+. Release of beta-N-acetylglucosaminidase in control or cytochalasin B-stimulated cells is inhibited by NaCl(IC50 approximately 10 mM), while cytochalasin E-stimulated exocytosis is reduced less and K+ or choline chloride are ineffective in inhibiting either cytochalasin B- or E-stimulated exocytosis. Release of beta-glucuronidase, myeloperoxidase or acid phosphatase from neutrophils incubated in buffered sucrose is not stimulated by cytochalasin B. Stimulation of either O2- or beta-N-acetylglucosaminidase release by low concentrations of cytochalasin A is followed by inhibition of each at higher concentrations. It appears that all cytochalasins can activate both NAD(P)H oxidase and selective degranulation of neutrophils incubated in salt-restricted media and that differential inhibition of these two processes by monovalent cations and/or anions is produced at some step(s) subsequent to cytochalasin interaction with the cell.     

Inhibition of intercellular adhesion by concanavalin A is associated with concanavalin A-mediated redistribution of surface receptors

The inhibition of adhesion between aggregates and layers of embryonic retinal cells by concanavalin A (Con A) and Con A-mediated rearrangements of Con A receptors on retinal cells were studied. A short incubation of aggregates and layers with 10 micrograms/ml Con A substantially reduced aggregate-to-layer adhesion in a subsequent assay without soluble lectin present. This effect of Con A was dose-dependent, temperature-sensitive, involved events subsequent to Con A binding, and was reduced by cytochalasin B. The inhibition produced by succinylated Con A was substantially increased by incubation with antibody to Con A. Visualization of ConA- receptor complexes by fluorescence microscopy revealed that binding of Con A induced clearing of Con A receptors from filopodia, flattened regions of growth cones, and the edges of axons. This clearing reaction was prevented by the same agents that reduced Con A's inhibition of cell adhesion: low temperature, succinylation of Con A, or cytochalasin B. Aggregate-layer adhesion was restored by releasing Con A at 37 degrees C. Inhibitors of protein and ATP synthesis did not prevent recovery of ability to make adhesions. However, release of Con A at lowered temperatures did not prevent recovery. The results suggest that intercellular adhesion is inhibited by events associated with redistribution of Con A-receptor complexes on retinal cells.     

The monosaccharide transport system of the human erythrocyte. Orientation upon reconstitution

Treatment of intact human erythrocytes with trypsin had no effect upon either the rate of hexose transport or the binding of cytochalasin B to the transport system. In contrast, proteolysis of inside-out vesicles prepared from human erythrocyte membranes inactivated both hexose transport and cytochalasin B binding. When purified hexose transporter, reconstituted into phospholipid vesicles of undetermined size, was treated with trypsin, approx. 50% of the cytochalasin B binding activity was lost. This loss correlated with a decrease in the amount of the transporter polypeptide, as assayed by gel electrophoresis. These results show that the orientation of the transporter can be established through trypsin treatment in conjunction with cytochalasin B binding. Small unilamellar vesicles containing transporter were prepared by sonication of larger species and by a cycle of cholate solubilization and removal of the detergent. In the former case, the transporter orients almost randomly, whereas in the latter approx. 75% of the transporters have the cytoplasmic domain extemal.     

INHIBITION OF PHAGOCYTOSIS AND PLASMA MEMBRANE MOBILITY OF THE CULTIVATED MACROPHAGE BY CYTOCHALASIN B : Role of Subplasmalemmal Microfilaments

Functional and morphologic effects of cytochalasin B on the cultivated macrophage were examined to determine the basis for plasma membrane movements of the type required for endocytosis and/or spreading on a substratum. Inhibition of phagocytosis and changes in cell shape by cytochalasin B exhibited nearly identical dose-response curves requiring 2–5 x 10^-6 M and 1–2 x 10^-5 M cytochalasin B to inhibit these functions by 50% and 100%, respectively. In contrast, hexose transport was ten times more sensitive to the drug requiring 2–3 x 10^-7 M cytochalasin B to achieve 50% inhibition of 2-deoxyglucose uptake. Inhibition of phagocytosis and changes in cell shape could not be explained solely by drug effects on hexose transport. Analysis of serial thin sections showed that cytochalasin B doses inhibitory for hexose transport had no effect on distribution or organization of either of the two subplasmalemmal microfilament types. However, cytochalasin B concentrations (2.0 x 10^-5 M) that inhibited phagocytosis and altered cell shape disorganized and/or disrupted oriented bundles of 40–50-Å subplasmalemmal microfilaments, but had no effect on the microfilamentous network. Comparative dose-response studies showing positive correlations among cytochalasin B effects on phagocytosis, changes in cell shape, and alterations in oriented subplasmalemmal microfilament bundles provide additional support for the hypothesis that microfilamentous structures play a role in translocation of plasma membrane required for endocytosis and cell motility.     

Treatment of neutrophils with cytochalasins converts rolling to stationary adhesion on P-selectin

We investigated how disruption of the actin cytoskeleton with cytochalasins modified adhesion of neutrophils rolling on a platelet monolayer in vitro at 37°C. When perfused at a wall shear stress of 0.1 Pa over rolling cells, cytochalasin B, cytochalasin D and dihydro-cytochalasin B each induced dose-dependent (∼1–10 μg/ml) conversion to stationary attachment over minutes. Stopping was associated with cell elongation to a teardrop shape. Increased deformability of cytochalasin-treated cells was independently evidenced by more rapid entry into a micropipette. Spherical shape and rolling were reestablished concurrently on washout of the cytochalasins, while increasing the shear stress in the range 0.2 to 1.0 Pa induced tear-drop-shaped cells to restart rolling even in the continued presence of cytochalasin. When cells were pretreated with cytochalasin B, they attached efficiently at 0.1 Pa, rolled initially and only stopped after ∼30 seconds when elongation had been established. Adhesion was selectin-mediated in the presence or absence of cytochalasin B, as judged by inhibition of attachment by antibody against P-selectin and failure of antibody against β2-integrin CD18 to influence adhesion. Cessation of rolling is unlikely to have arisen from an increase in adhesive contact area induced by deformation because stopped cells were found to be attached only at their pointed end. Failure of adhesive bonds to peel may have arisen because selectin ligands freed of cytoskeletal restraint were dragged into this tethered region and clustered there, and because force applied to bonds was influenced by the change in cell shape. These results suggest that cytoskeletal structure is an important modulator of dynamic adhesive responses of leukocytes, via effects on adhesion receptors and cellular mechanics. J. Cell. Physiol. 174:206–216, 1998. © 1998 Wiley-Liss, Inc.     

Adhesion of human skin fibroblasts : Effect of tetravalent, divalent and monovalent concanavalin A

Concanavalin A (ConA) pretreatment inhibited the adhesion of fibroblasts to plastic surface in a dose-dependent manner. The ConA effect was reversible and could be inhibited by α-methylmannoside. Pretreatment with cytochalasin B (CB) and colchicine increased the ConA effect. Divalent and monovalent ConA derivatives had no effect on the fibroblast adhesion. This indicates that ligand attachment to ConA receptors is not sufficient to prevent cell adhesion. The requirement of tetravalent ConA for inhibition of cell adhesion suggests that the decrease of lateral mobility of membrane components, which seems to be specific for tetravalent ConA, is responsible for the inhibition of cell adhesion. The enhancement of the ConA effect by colchicine and CB pretreatment suggests an involvement of microtubules and microfilaments in the mobility of ConA receptors of fibroblasts.     

Selective release of lysosomal hydrolases from phagocytic cells by cytochalasin B

1. Cytochalasin B (10mug/ml) enhances the release of rabbit polymorphonuclear leucocyte lysosomal acid hydrolases induced by retinol (vitamin A alcohol). 2. This effect is seen at doses of the vitamin that cause selective release of acid hydrolases and those causing more general enzyme release indicated by the loss of lactate dehydrogenase. 3. Cytochalasin B (2-50mug/ml) has no effect on the release of sedimentable acid hydrolases of intact granules obtained from disrupted polymorphonuclear leucocytes. 4. Cytochalasin B (2-10mug/ml) causes a time- and dose-dependent release of mouse peritoneal macrophage acid hydrolases. 5. This effect is selective at all doses of cytochalasin B used, since no release of lactate dehydrogenase, malate dehydrogenase and leucine 2-naphthylamidase was detected. 6. Treatment with cytochalasin B at doses of up to 10mug/ml for as long as 72h did not significantly change the total activities of any of the enzymes measured. 7. The lack of toxicity of cytochalasin B was shown by dye-exclusion tests and its failure to release radioactive colloidal gold stored in secondary lysosomes.     

Binding of [3H]ctyochalasin B and [3H]colchicine to isolated liver plasma membranes.

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([3H]cytochalasin B and [3H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [3H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB3H4. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insensitive to changes of pH or ionic strength. At 10(-6) M [3H]cytochalasin B, glucose of p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 A) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10(-5) M [3H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [3H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes. [3H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [3H]cytochalasin B binding and cytochalasin B stimulated 3H colchicine binding. [3H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Cytochalasin B-sensitive, sodium ion-dependent glucose transport in intestinal microvillous membrane

It was found that sodium ion-dependent glucose uptake by microvillous membrane (MVM) vesicles was partially inhibited by cytochalasin B with a half-maximum inhibition at ca. 10 microM. The MVM was photolabeled with [3]cytochalasin B. The Kd value and the maximum number of binding sites for cytochalasin B were ca. 8 microM and 70 pmol/mg protein, respectively. SDS-PAGE of the photolabeled MVM revealed 2 binding components. One was 86 K in Mr and the other 42 K. The binding of cytochalasin B to the 86 K component was affected neither by cytochalasin E nor by the presence of 0.5 M NaCl, but was depressed in the presence of 2-deoxy-D-glucose or phlorizin, which had no effect on the labeling of the 42 K component. These and other data suggested that the 86 K component might be responsible for a cytochalasin B-sensitive glucose transport in intestinal epithelial MVM.     

Photoaffinity labeling of the K562 cell membrane D-glucose transporter with cytochalasin B

D-glucose carrier protein in K562 cell membrane was studied by photoaffinity labeling with cytochalasin B. The saturable cytochalasin B binding in purified K562 cell membranes was 90 pmol/mg and 200 pmol/mg protein in the presence of D-glucose and D-sorbitol, respectively. More than half of the total cytochalasin B binding could be depressed by D-glucose. The results of SDS-PAGE analysis of K562 cell membranes after photoaffinity labeling at 0.1 microM cytochalasin B showed that the main peak of covalently bound [3H]-cytochalasin B was in the Mr range of 46-65 KDa. The label found in the peak was reduced by more than 50% in the presence of 0.5 M D-glucose, the inhibition similar being to that obtained in the binding experiment. This polypeptide has a slightly higher molecular weight than that of the human erythrocyte cell membrane.