Chan Y. Jung

Target size of calcium pump protein from skeletal muscle sarcoplasmic reticulum.

The oligomeric size of calcium pump protein (CPP) in fast skeletal muscle sarcoplasmic reticulum membrane was determined using target theory analysis of radiation inactivation data. There was a parallel decrease of Ca2+-ATPase and calcium pumping activities with increasing radiation dose. The loss of staining intensity of the CPP band, observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, also correlated directly with the loss of activity. The target size molecular weight of the CPP in the normal sarcoplasmic reticulum membrane ranged between 210,000 and 250,000, which is consistent with a dimeric structure. Essentially the same size is obtained for the non-phosphorylated CPP or for the phosphoenzyme form generated from either ATP (E1 state) or inorganic phosphate (E2 state). Hence, the oligomeric state of the pump does not appear to change during the catalytic cycle. Similar results were obtained with reconstituted sarcoplasmic reticulum membrane vesicles with different lipid to protein ratios. We conclude that the CPP is a dimer in both native and reconstituted sarcoplasmic reticulum membranes. The target size of the calcium-binding protein (calsequestrin) was found to be 50,000 daltons, approximating a monomer.

Glucose transport carrier activities in extensively washed human red cell ghosts

Abstract Human red cell ghosts were prepared and extensively washed in the presence of divalent cations (Ca 2+ and Mg 2+ ) and their residual glucose transport carrier activities were studied. 99.18 % of the original cellular contents of hemoglobin were removed by the isolation procedures. The glucose carrier activities of such ghosts exhibited the following characteristics. (1) An extremely high permeability to d -glucose (2.0·10 −5 cm/sec at 24°) and to 2-deoxy- d -glucose (2.9·10 −5 cm/sec at 24°), compared with the permeability to d -mannitol (5.2·10 −8 cm/sec at 24°). (2) Stereospecificity towards various sugars; relative rates of permeation by 2-deoxy- d -glucose, d -glucose, d -mannose and d -ribose were 1.5, 1.0, 0.35 and 0.020, respectively. (3) Saturability (for d -glucose at 24°, a K m of 19.5 mM and a Φ max of 6.4 μmoles·cm −3 ·sec −2 ). (4) Sensitivity to stilbestrol (competitive inhibition with a K i of 4.69 μM at 24°) and to dinitrofluorobenzene (irreversible inhibition). (5) Transient uphill movements of d -glucose by the addition of a second sugar, 2-deoxy- d -glucose, a counter-transport phenomenon. (6) Relative insensitivity to change in pH, between 5.2 and 9.0. (7) High degree of temperature dependency with a negative Q 10 for K m at low temperatures. (8) A relative resistivity to heat inactivation up to 65°. In conclusion, this work provides (1) the first evidence that such purified ghosts maintain fully functional glucose carrier activity and (2) a new detailed description of the nature of the temperature dependence and heat denaturation of this carrier system in ghosts.

Depletion of mitochondrial DNA causes impaired glucose utilization and insulin resistance in L6 GLUT4myc myocytes.

Mitochondrial dysfunction contributes to a number of human diseases, such as hyperlipidemia, obesity, and diabetes. The mutation and reduction of mitochondrial DNA (mtDNA) have been suggested as factors in the pathogenesis of diabetes. To elucidate the association of cellular mtDNA content and insulin resistance, we produced L6 GLUT4myc myocytes depleted of mtDNA by long term treatment with ethidium bromide. L6 GLUT4myc cells cultured with 0.2 μg/ml ethidium bromide (termed depleted cells) revealed a marked decrease in cellular mtDNA and ATP content, concomitant with a lack of mRNAs encoded by mtDNA. Interestingly, the mtDNA-depleted cells showed a drastic decrease in basal and insulin-stimulated glucose uptake, indicating that L6 GLUT4myc cells develop impaired glucose utilization and insulin resistance. The repletion of mtDNA normalized basal and insulin-stimulated glucose uptake. The mRNA level and expression of insulin receptor substrate (IRS)-1 associated with insulin signaling were decreased by 76 and 90% in the depleted cells, respectively. The plasma membrane (PM) GLUT4 in the basal state was decreased, and the insulin-stimulated GLUT4 translocation to the PM was drastically reduced by mtDNA depletion. Moreover, insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B were drastically reduced in the depleted cells. Those changes returned to control levels after mtDNA repletion. Taken together, our data suggest that PM GLUT4 content and insulin signal pathway intermediates are modulated by the alteration of cellular mtDNA content, and the reductions in the expression of IRS-1 and insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B are associated with insulin resistance in the mtDNA-depleted L6 GLUT4myc myocytes.

Structural state of the Na+/D-glucose cotransporter in calf kidney brush-border membranes: target size analysis of Na+-dependent phlorizin binding and Na+-dependent D-glucose transport

Target sizes of the renal sodium-D-glucose cotransport system in brush-border membranes of calf kidney cortex were estimated by radiation inactivation. In brush-border vesicles irradiated at -50 degrees C with 1.5 MeV electron beams, sodium-dependent phlorizin binding, and Na+-dependent D-glucose tracer exchange decreased exponentially with increasing doses of radiation (0.4-4.4 Mrad). Inactivation of phlorizin binding was due to a reduction in the number of high-affinity phlorizin binding sites but not in their affinity. The molecular weight of the Na+-dependent phlorizin binding unit was estimated to be 230 000 +/- 38 000. From the tracer exchange experiments a molecular weight of 345 000 +/- 24 500 was calculated for the D-glucose transport unit. The validity of these target size measurements was established by concomitant measurements of two brush-border enzymes, alkaline phosphatase and gamma-glutamyltransferase, whose target sizes were found to be 68 570 +/- 2670 and 73 500 +/- 2270, respectively. These findings provide further evidence for the assumption that the sodium-D-glucose cotransport system is a multimeric structure, in which distinct complexes are responsible for phlorizin binding and D-glucose translocation.

Characteristics of the permeability barrier of human erythrocyte ghosts to non-electrolytes

Abstract The non-specific diffusion barrier of practically hemoglobin-free human erythrocyte ghosts prepared in hypotonic phosphate buffer was studied by measuring the permeabilities to water-soluble non-electrolytes. In contrast to pink ghost preparations, these white ghosts are completely permeable to mannitol or sucrose, indicating that their diffusion barrier is completely lost. Such leaky ghosts can be resealed effectively by incubation at 37°C in 0.1 isoosmolar balanced salt solution for 2 h. Temperature, Ca2+, osmolarity and ionic strength are all factors which appear to control the resealing process. Such resealed ghosts, however, never exhibit complete restoration of the diffusion barrier and the permeabilities to non-electrolytes are 10- to 50-fold higher than those of the native membranes. Unlike the native membranes, the permeabilities of the resealed ghosts are little affected by changes in temperature, the molecular size or lipid solubility of the permeants. It is concluded that the resealed white ghost membranes represent an altered form of the native membrane structure, having water-filled discontinuities through which the water soluble non-electrolytes diffuse freely. Possible structural aspects of such discontinuity and its genesis are discussed.

Adenosine and adenosine triphosphate modulate the substrate binding affinity of glucose transporter GLUT1 in vitro.

Evidence indicates that a large portion of the facilitative glucose transporter isoform GLUT1 in certain animal cells is kept inactive and activated in response to acute metabolic stresses. A reversible interaction of a certain inhibitor molecule with GLUT1 protein has been implicated in this process. In an effort to identify this putative GLUT1 inhibitor molecule, we studied here the effects of adenosine and adenosine triphosphate (ATP) on the binding of D-glucose to GLUT1 by assessing their abilities to displace cytochalasin B (CB), using purified GLUT1 in vesicles. At pH 7.4, adenosine competitively inhibited CB binding to GLUT1 and also reduced the substrate binding affinity by more than an order of magnitude, both with an apparent dissociation constant (K(D)) of 3.0 mM. ATP had no effect on CB and D-glucose binding to GLUT1, but reduced adenosine binding affinity to GLUT1 by 2-fold with a K(D) of 30 mM. At pH 3.6, however, ATP inhibited the CB binding nearly competitively, and increased the substrate binding affinity by 4--5-fold, both with an apparent K(D) of 1.22 mM. These findings clearly demonstrate that adenosine and ATP interact with GLUT1 in vitro and modulate its substrate binding affinity. They also suggest that adenosine and ATP may regulate GLUT1 intrinsic activity in certain cells where adenosine reduces the substrate-binding affinity while ATP increases the substrate-binding affinity by interfering with the adenosine effect and/or by enhancing the substrate-binding affinity at an acidic compartment.

Chromatographic characterization of nitrobenzylthioinosine binding proteins in band 4.5 of human erythrocytes: purification of a 40 kDa truncated nucleoside transporter

DEAE-column-purified band 4.5 polypeptides of human erythrocyte membranes are mostly glucose transporters with nucleoside transporters as a minor component. The purpose of the present work was to differentially identify and isolate the nucleoside transporters in band 4.5 free from glucose transporters. Equilibrium binding studies demonstrated that the band 4.5 preparation binds nibrobenzylthioinosine (NBTI), a potent nucleoside transport inhibitor, at two distinct sites, one with a high affinity (dissociation constant, KD of 1 nM) with a small capacity, BT (0.4 nmol/mg protein), and the other with a low affinity (KD of 15 microM) with a large BT (14-16 nmol/mg protein). The BT of the low-affinity site was equal to that of the cytochalasin B binding site in the preparation. A gel-filtration chromatography of band 4.5 photolabeled with [3H]NBTI and [3H]cytochalasin B identified three polypeptides of apparent Mr 55,000, 50,000 and 40,000. Of these, the 55 kDa polypeptide was specifically labeled by cytochalasin B (p55GT), indicating that it is a glucose transporter. Both the 50 and 40 kDa polypeptides were labeled with NBTI at low ligand concentrations (less than 0.1 microM), which was abolished by an excess (20 microM) of nitrobenzylthioguanosine, indicating that they are two forms (p50NT and p40NT, respectively) of the high affinity NBTI binding protein or nucleoside transporter. At higher (not less than 10 microM) NBTI concentrations, however, p55GT was also labeled with NBTI, indicating that the low-affinity NBTI binding is due to a glucose transporter. Treatment of band 4.5 with trypsin reduced the p50NT labeling with a concomitant and stoichiometric increase in the p40NT NBTI labeling without affecting the high-affinity NBTI binding of the preparation. These findings indicate that the nucleoside transporter is slightly smaller in mass than the glucose transporter and that trypsin digestion produces a truncated nucleoside transporter of apparent Mr 40,000 which retains the high-affinity NBTI binding activity of intact nucleoside transporter. Both p55GT and p50 NT were coeluted in a major protein fraction, P1 in the chromatography, while p40NT was eluted separately as a minor protein fraction, P1a. All three polypeptides formed mixed dimers, which were eluted in a fraction PO. We have purified and partially characterized the truncated nucleoside transporter, p40NT. The purified p40NT may be useful for biochemical characterization of the nucleoside transporter.

Hypertonic cryohemolysis and the cytoskeletal system.

Hypertonic cryohemolysis is defined as the lysis of erythrocytes in a hypertonic environment when the temperature is lowered from above 15-18 degrees C below that temperature. This has been found to be a general phenomenon (that is, whether the solute is charged or not), to exhibit interesting temperature characteristics and to be preventable by agents such as valinomycin which tend to dissipate the concentration gradient across the cell membrane. As yet, no clear information is available to translate this phenomenon to the molecular level and to relate it to current structure/function concepts in the erythrocyte membrane. In this study, data are presented which would indicate on the basis of two entirely separate methodologies that the spectrin-actin cytoskeletal framework is involved in this phenomenon. The first of these methodologies is based on radiation-induced ablation of cryohemolysis and indicates that an intact macromolecular complex of an order of 16000 000 daltons is required for cryohemolysis with hypertonic NaCl. The second methodology is based on selective cross-linking of spectrin and actin in the agent diamide, which resulted in concentration-dependent suppression of cryohemolysis. Polyacrylamide gel electrophoresis of the erythrocyte from diamide-treated cells showed intense protein aggregation with loss of spectrin-actin and bands 4.1, 4.2. We conclude that the spectrin-actin cytoskeletal system possibly including its interaction with phospholipids is the key to the phenomenon of hypertonic cryohemolysis.

Sodium-dependent glucose transport by cultured proximal tubule cells

The cotransport of sodium ion and alpha-methyl glucose, a non-metabolized hexose, was studied in rabbit proximal tubule cells cultured in defined medium. The rate of uptake of alpha-methyl glucose shows saturation kinetics, in which Km, but not Vmax, is dependent upon the Na+ concentration in the medium. The transport system was found to be of the high-affinity type, characteristic of the straight portion of the proximal tubule. Analysis of the rates of initial uptake within the context of a generalized cotransport model, suggests that two Na+ ions are bound in the activation of the hexose transport. The steady-state level of accumulation of alpha-methyl glucose also depends upon sodium concentration, consistent with the initial rate findings. The uptake of alpha-methyl glucose is inhibited by other sugars with the relative potencies of D-glucose greater than alpha-methyl glucose greater than D-galactose = 3-O methylglucose. L-Glucose, D-fructose, and D-mannose show no inhibition. Phlorizin inhibits the alpha-methyl glucose uptake with a Ki of 9 X 10(-6) M. Ouabain (10(-3) M) decreases the steady-state alpha-methyl glucose accumulation by 60%. In the absence of sodium, the accumulation of alpha-methyl glucose is 7-fold less than at 142 mM Na+, reaching a level comparable to the sodium-independent accumulation of 3-O-methyl-D-glucose. These findings are similar to those observed in the proximal tubule of the intact kidney.

Substrate-induced conformational change of human erythrocyte glucose transporter: inactivation by alkylating reagents.

The glucose transport carrier in human erythrocyte membranes, when transporting glucose, undergoes a conformation change. In an attempt to delineate the extent of this substrate-induced conformational change, transport inactivation by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, N-ethylmaleimide, iodoacetamide, and 2,4,6-trinitrobenzenesulfonic acid was examined in the presence and in the absence of D-glucose. All these alkylating agents inactivated the carrier. With each of these reagents, with the exception of trinitrobenzene-sulfonic acid, D-glucose modified the rate of inactivation as well as the activation enthalpy (delta H*) of the inactivation. The inactivation by trinitrobenzenesulfonic acid was not affected by the sugar. Based on these findings, it is suggested that the substrate-induced conformational change mostly occurs within the transmembrane hydrophobic domain while the hydrophilic extramembrane domains are largely outside of this change.