RESUMO
Lager beers are traditionally made at lower temperatures (6-14 degrees C) than ales (15-25 degrees C). At low temperatures, lager strains (Saccharomyces pastorianus) ferment faster than ale strains (Saccharomyces cerevisiae). Two lager and two ale strains had similar maltose transport activities at 20 degrees C, but at 0 degrees C the lager strains had fivefold greater activity. AGT1, MTT1 and MALx1 are major maltose transporter genes. In nine tested lager strains, the AGT1 genes contained premature stop codons. None of five tested ale strains had this defect. All tested lager strains, but no ale strain, contained MTT1 genes. When functional AGT1 from an ale strain was expressed in a lager strain, the resultant maltose transport activity had the high temperature dependence characteristic of ale yeasts. Lager yeast MTT1 and MALx1 genes were expressed in a maltose-negative laboratory strain of S. cerevisiae. The resultant Mtt1 transport activity had low temperature dependence and the Malx1 activity had high temperature dependence. Faster fermentation at low temperature by lager strains than ale strains may result from their different maltose transporters. The loss of Agt1 transporters during the evolution of lager strains may have provided plasma membrane space for the Mtt1 transporters that perform better at a low temperature.
Assuntos
Bebidas Alcoólicas/microbiologia , Maltose/metabolismo , Saccharomyces/metabolismo , Saccharomyces/efeitos da radiação , Temperatura , Transporte Biológico/efeitos da radiação , Fermentação/efeitos da radiação , Proteínas Fúngicas/genética , Proteínas Fúngicas/efeitos da radiação , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/efeitos da radiação , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/efeitos da radiação , Simportadores/genética , Simportadores/efeitos da radiaçãoRESUMO
Glucose is the crucial metabolic fluid for the brain, and the transport of this nutrient from blood to brain is limited by the blood-brain barrier (BBB) GLUT1 glucose transporter. The activity of this transporter is altered in different pathophysiological conditions including Alzheimer's disease. The expression of the BBB-GLUT1 gene is directed by brain trophic factors, and the brain-derived peptide preparation Cerebrolysin (Cl, EBEWE, Austria), used in the treatment of Alzheimer's disease, increases the BBB-GLUT1 mRNA stability and the expression of the BBB-GLUT1 gene. In the present investigation, Cl markedly increased (p < 0.001) the expression of a BBB-GLUT1 reporter gene, named clone 753, that contains an important regulatory cis-acting element involved in the stabilization of this transcript in brain endothelial cultured cells (ECL). In experiments with a reporter gene lacking this regulatory element, Cl produced only a minimal fraction of the effect observed with clone 753. UV-cross linking/PAGE experiments showed that the GLUT1 transcript reacts with ECL cytosolic proteins to form a RNA/protein complex of approximately 80 kDa. The abundance of this cis/trans acting complex was found to be increased in Cl-treated cells. Overall, data presented here demonstrate that i) Cl increases the expression of a BBB-GLUT1-luciferase reporter gene containing a region of the 3'-untranslated region of BBB-GLUT1 mRNA with important regulatory cis-acting elements involved in the stabilization of this transcript, and ii) the increased expression of this BBB-GLUT1 reporter gene was associated with augmented abundance of a transacting factor that binds to the cis-acting element described in (i), suggesting that this association may be involved in the stabilization of GLUT1 mRNA induced by Cl.
Assuntos
Aminoácidos/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Química Encefálica/fisiologia , Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Nootrópicos/farmacologia , Regiões 3' não Traduzidas/biossíntese , Regiões 3' não Traduzidas/genética , Animais , Química Encefálica/efeitos dos fármacos , Química Encefálica/efeitos da radiação , Bovinos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Transportador de Glucose Tipo 1 , Proteínas de Transporte de Monossacarídeos/biossíntese , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/efeitos da radiação , Ratos , Transfecção , Raios UltravioletaRESUMO
Studies on human cell hybrids between a cervical carcinoma cell line, HeLa, and normal fibroblasts have indicated that their tumorigenicity is under the control of a putative tumor suppressor on chromosome 11. We have previously demonstrated that a tumorigenic cell hybrid CGL4 expresses a larger glucose transporter, GLUT1, due to altered glycosylation when compared to the nontumorigenic counterpart CGL1. In this study, we demonstrated this glycosylation change in GLUT1 in gamma-ray-induced tumorigenic mutants (GIMs) isolated from CGL1 cells as expressing a tumor-associated surface antigen, intestinal alkaline phosphatase. In contrast, GLUT1 in the gamma-irradiated nontumorigenic control cells (CONs) did not show this alteration. In accordance with this glycosylation change, affinity to 2-deoxyglucose in these GIM clones was increased by about twofold when compared to the nontumorigenic CONs. These results suggest a close correlation between the glycosylation change in GLUT1 with increased affinity to D-glucose and tumorigenicity of these human cell hybrids.
Assuntos
Transformação Celular Neoplásica/efeitos da radiação , Proteínas de Transporte de Monossacarídeos/metabolismo , Fosfatase Alcalina/metabolismo , Transporte Biológico , Desoxiglucose/metabolismo , Fibroblastos , Raios gama , Transportador de Glucose Tipo 1 , Glicosilação/efeitos da radiação , Células HeLa , Humanos , Células Híbridas , Cinética , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/efeitos da radiação , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificaçãoRESUMO
The radiation-inactivation size (RIS) of the rat renal brush-border membrane sodium/D-glucose cotransporter was estimated from the loss of transport activity in irradiated membrane vesicles. The RIS depended on the electrochemical conditions present when measuring transport activity. A RIS of 294 +/- 40 kDa was obtained when transport was measured in the presence of a sodium electrochemical gradient. Under sodium equilibrium conditions, the RIS was 84 +/- 25 kDa in the presence of a glucose gradient, and 92 +/- 20 kDa in its absence. In the absence of a sodium gradient, but in the presence of an electrical potential gradient, the RIS increased to 225 +/- 49 kDa. The 294 kDa result supports earlier suggestions that the Na+ gradient-dependent glucose transport activity is mediated by a tetramer. Individual monomers appear, however, to carry out glucose transport under equilibrium exchange conditions or when a glucose gradient serves as the only driving force. The electrical potential gradient-driven glucose transport RIS appears to involve three functional subunits.
Assuntos
Rim/química , Proteínas de Transporte de Monossacarídeos/química , Animais , Eletroquímica , Rim/efeitos da radiação , Masculino , Microvilosidades/química , Microvilosidades/efeitos da radiação , Proteínas de Transporte de Monossacarídeos/efeitos da radiação , Ratos , Ratos Sprague-DawleyRESUMO
The functional unit molecular size of the intestinal brush border membrane-bound Na+/glucose cotransporter was determined by radiation inactivation. Purified brush border membrane vesicles preserved in cryoprotectant buffer were irradiated (-135 degrees C) with high-energy electrons from a 13-MeV (1 eV = 1.602 x 10(-19) J) linear accelerator at doses from 0 to 70 Mrad (1 rad = 0.01 Gy). After each dose, the cotransporter was investigated with respect to (i) Na(+)-dependent transport activity and (ii) immunologic blot analysis with antibodies against the cloned rabbit intestinal cotransporter. Increasing radiation decreased the maximal Na(+)-dependent cotransporter activity Jmax without affecting apparent Km. The size of the transporting functional unit was 290 +/- 5 kDa. Immunologic blot analysis of brush border membranes gave a single band of Mr 70,000, which decreased in intensity with increased radiation dose and gave a target size of 66 +/- 11 kDa. We conclude that activity of the intestinal Na+/glucose cotransporter in situ in the brush border membrane requires the simultaneous presence of four intact, independent, identical subunits arranged as a homotetramer.
Assuntos
Intestino Delgado/metabolismo , Microvilosidades/metabolismo , Proteínas de Transporte de Monossacarídeos/isolamento & purificação , Animais , Western Blotting , Relação Dose-Resposta à Radiação , Substâncias Macromoleculares , Masculino , Modelos Estruturais , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas de Transporte de Monossacarídeos/efeitos da radiação , CoelhosRESUMO
Stereospecific glucose transport was assayed and characterized in bovine cardiac sarcolemmal vesicles. Sarcolemmal vesicles were incubated with D-[3H]glucose or L-[3H]glucose at 25 degrees C. The reaction was terminated by rapid addition of 4 mM HgCl2 and vesicles were immediately collected on glass fiber filters for quantification of accumulated [3H]glucose. Non-specific diffusion of L-[3H]glucose was never more than 11% of total D-[3H]glucose transport into the vesicles. Stereospecific uptake of D-[3H]glucose reached a maximum level by 20 s. Cytochalasin B (50 microM) inhibited specific transport of D-[3H]glucose to the level of that for non-specific diffusion. The vesicles exhibited saturable transport (Km = 9.3 mM; Vmax = 2.6 nmol/mg per s) and the transporter turnover number was 197 glucose molecules per transporter per s. The molecular sizes of the cytochalasin B binding protein and the D-glucose transport protein in sarcolemmal vesicles were estimated by radiation inactivation. These values were 77 and 101 kDa, respectively, and by the Wilcoxen Rank Sum Test were not significantly different from each other.
Assuntos
Proteínas de Transporte de Monossacarídeos/metabolismo , Miocárdio/ultraestrutura , Sarcolema/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/efeitos da radiação , Bovinos , Citocalasina B/farmacologia , Difusão , Glucose/metabolismo , Cinética , Peso Molecular , Proteínas de Transporte de Monossacarídeos/antagonistas & inibidores , Proteínas de Transporte de Monossacarídeos/efeitos da radiaçãoRESUMO
The in situ assembly states of the glucose transport carrier protein in the plasma membrane and in the intracellular (microsomal) storage pool of rat adipocytes were assessed by studying radiation-induced inactivation of the D-glucose-sensitive cytochalasin B binding activities. High energy radiation inactivated the glucose-sensitive cytochalasin B binding of each of these membrane preparations by reducing the total number of the binding sites without affecting the dissociation constant. The reduction in total number of binding sites was analyzed as a function of radiation dose based on target theory, from which a radiation-sensitive mass (target size) was calculated. When the plasma membranes of insulin-treated adipocytes were used, a target size of approximately 58,000 daltons was obtained. For adipocyte microsomal membranes, we obtained target sizes of approximately 112,000 and 109,000 daltons prior to and after insulin treatment, respectively. In the case of microsomal membranes, however, inactivation data showed anomalously low radiation sensitivities at low radiation doses, which may be interpreted as indicating the presence of a radiation-sensitive inhibitor. These results suggest that the adipocyte glucose transporter occurs as a monomer in the plasma membrane while existing in the intracellular reserve pool either as a homodimer or as a stoichiometric complex with a protein of an approximately equal size.
Assuntos
Tecido Adiposo/efeitos da radiação , Proteínas de Transporte de Monossacarídeos/efeitos da radiação , Tecido Adiposo/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Citocalasina B/metabolismo , Relação Dose-Resposta à Radiação , Cinética , Masculino , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Proteínas de Transporte de Monossacarídeos/antagonistas & inibidores , Ratos , Ratos EndogâmicosRESUMO
The human erythrocyte nucleoside and glucose transporters, identified previously as band 4.5 peptides (apparent Mr 66 000-45 000) on SDS-polyacrylamide gels, have been characterized in situ by radiation inactivation analysis. Target size analysis of lyophilized membranes indicates an apparent Mr of 110 000 +/- 12 000 and 124 000 +/- 11 000 for the nucleoside and glucose carriers, respectively. These data suggest that both transporters exist in the membrane as dimers.
Assuntos
Proteínas Sanguíneas/efeitos da radiação , Proteínas de Transporte/efeitos da radiação , Membrana Eritrocítica/efeitos da radiação , Proteínas de Membrana/efeitos da radiação , Proteínas de Transporte de Monossacarídeos/efeitos da radiação , Proteínas Sanguíneas/metabolismo , Relação Dose-Resposta à Radiação , Membrana Eritrocítica/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Monossacarídeos/sangue , Proteínas de Transporte de Nucleosídeos , Aceleradores de PartículasRESUMO
Low concentrations of glucose induce cultured kidney epithelial cells (LLC-PK1) to produce hexose transport proteins. We have investigated the effects of ionizing radiation on this induction process in plateau-phase cultures. The induced production of hexose transporters, requiring approximately 6 to 9 days for complete expression, can be inhibited by irradiation during the first 4 days. After the fourth postinduction day, radiation sensitivity decreases with almost no radiation effect on the induction of hexose transport apparent by the sixth day of the induction period. The D0 value associated with the induction block is approximately 25 Gy, a value which is considerably greater than that necessary to inhibit cell replication. Hexose transport, itself resistant to ionizing radiation at doses in excess of 100 Gy, is sensitive to cycloheximide throughout the induction period. The sensitivity to cycloheximide decreases during the last 2 days of the induction period, approximately 1 day after the reduction in radiosensitivity. Based on these properties hexose transport may be a convenient model for the study of radiation effects upon gene expression in this cell line.