Component Of Glucose Transport Research Articles

Growth on glucose decreases cAMP-CRP activity while paradoxically increasing intracellular cAMP in the light-organ symbiont Vibrio fischeri.

Proteobacteria often co-ordinate responses to carbon sources using CRP and the second messenger cyclic 3', 5'-AMP (cAMP), which combine to control transcription of genes during growth on non-glucose substrates as part of the catabolite-repression response. Here we show that cAMP-CRP is active and important in Vibrio fischeri during colonization of its host squid Euprymna scolopes. Moreover, consistent with a classical role in catabolite repression, a cAMP-CRP-dependent reporter showed lower activity in cells grown in media amended with glucose rather than glycerol. Surprisingly though, intracellular cAMP levels were higher in glucose-grown cells. Mutant analyses were consistent with predictions that CyaA was responsible for cAMP generation, that the EIIA(Glc) component of glucose transport could enhance cAMP production and that the phophodiesterases CpdA and CpdP consumed intracellular and extracellular cAMP respectively. However, the observation of lower cAMP levels in glycerol-grown cells seemed best explained by changes in cAMP export, via an unknown mechanism. Our data also indicated that cAMP-CRP activity decreased during growth on glucose independently of crp's native transcriptional regulation or cAMP levels. We speculate that some unknown mechanism, perhaps carbon-source-dependent post-translational modulation of CRP, may help control cAMP-CRP activity in V.fischeri.

Basolateral glucose transport in distal segments of the dog nephron

The transport of glucose by canine thick ascending limbs (TAL) and inner medullary collecting ducts (IMCD) was studied using tubule suspensions and membrane vesicles. The uptake of D-[14C(U)]glucose by a suspension of intact TAL tubules was reduced largely by phloretin (Pt), moderately by phlorizin (Pz), and completely suppressed by a combination of both agents. A selective effect of Pz on the transport of [14C]alpha-methyl-D-glucoside, but not on 2-[3H]deoxyglucose, was also observed in TAL tubules. In contrast, glucose transport was unaffected by Pz but entirely suppressed by Pt alone in IMCD tubules. The metabolism of glucose was largely suppressed by Pt but unaffected by Pz in both types of tubules. Membrane vesicles were prepared from the red medulla and the white papilla or from TAL and IMCD tubules isolated from these tissues. Vesicle preparations from both tissues demonstrated a predominant carrier-mediated, sodium-independent, Pt- and cytochalasin B-sensitive glucose transport. Following purification of basolateral membrane on a Percoll gradient, the sodium-insensitive D-[14C(U)]glucose transport activity copurified with the activity of the basolateral marker Na(+)-K+ ATPase in both tissues. However, a small sodium-dependent and Pz-sensitive component of glucose transport was found in membrane vesicles prepared from the red medulla or from thick ascending limb tubules but not from the papilla nor collecting duct tubules. The kinetic analysis of the major sodium-independent processes showed that the affinity of the transporter for glucose was greater in collecting ducts (Km = 2.3 mM) than in thick ascending limbs (Km = 4.9 mM). We conclude that glucose gains access into the cells largely through a basolateral facilitated diffusion process in both segments. However a small sodium-glucose cotransport is also detected in membranes of TAL tubules. The transport of glucose presents an axial differentiation in the affinity of glucose transporters in the renal medulla, ensuring an adequate supply of glucose to the glycolytic inner medullary structures.

Beta-glucoside permease represses the bgl operon of Escherichia coli by phosphorylation of the antiterminator protein and also interacts with glucose-specific enzyme III, the key element in catabolite control.

The beta-glucoside (bgl) operon of Escherichia coli is subject to both positive control by transcriptional termination/antitermination and negative control by the beta-glucoside-specific transport protein, an integral membrane protein known as enzyme IIBgl. Previous results led us to speculate that enzyme IIBgl exerts its negative control by phosphorylating and thereby inactivating the antiterminator protein, BglG. Specifically, our model postulated that the transport protein enzyme IIBgl exhibits protein-phosphotransferase activity in the absence of beta-glucosides. We now present biochemical evidence that the phosphorylation of protein BglG does indeed occur in vivo and that it is accompanied by the loss of antitermination activity. BglG persists in the phosphorylated state in the absence of beta-glucosides but is rapidly dephosphorylated when beta-glucosides become available for transport. Our data also suggested specific interactions between the beta-glucoside transport protein and the glucose-specific enzyme III (enzyme IIIGlc), a component of glucose transport and a key element in regulation of catabolite repression. These observations indicate that enzyme IIIGlc may, in conjunction with enzyme IIBgl, modulate the transport of beta-glucosides and the phosphorylation of the antiterminator protein. In the absence of both sugars, when the catabolite-controlled promoter of the operon is derepressed, enzyme IIIGlc may mediate tight repression of antitermination.

Glucose transport in vesicles reconstituted from Saccharomyces cerevisiae membranes and liposomes.

Glucose transport activity was reconstituted into liposomes by the freeze-thaw-sonication procedure from unextracted Saccharomyces cerevisiae membranes and preformed phospholipid liposomes. Fluorescence-dequenching measurements with octadecylrhodamine B chloride (R18)-labeled membranes showed that the yeast membrane lipids are diluted by the liposome lipids after the freeze-thaw-sonication procedure. At lipid-to-protein ratios greater than 75:1, vesicles with single transporters were formed. Reconstituted specific activity was increased at least twofold if the liposomes contained 50 mol% cholesterol. A further increase in specific activity, from 3- to 10-fold, was achieved by fractionation of the membranes on a Renografin gradient before reconstitution. Examination of the fractions from the Renografin gradient by sodium dodecyl sulfate-gel electrophoresis showed a parallel enrichment of glucose transport activity and a number of proteins including one with an apparent Mr of ca. 60,000, which might be the glucose transporter. Finally, preliminary kinetic analysis of glucose transport activity in vesicles reconstituted at a high lipid-to-protein ratio gave a Vmax of ca. 2.8 mumol/mg of protein per min at 23 degrees C and a Km of ca. 8 mM. The latter value corresponds to the kinase-independent, low-affinity component of glucose transport observed in wild-type cells.

Studies on the transport of glucose from disaccharides by hamster small intestine in vitro. I. Evidence for a disaccharidase-related transport system

In the presence of saturating concentrations of free d-glucose, total glucose uptake was enhanced beyond the theoretical V for free glucose uptake when disaccharides were incubated with intestinal rings. This phenomenon was not seen when glucose 1-phosphate was the substrate. Analogs of d-glucose transport system, galactose and β-methyl glucoside, had an inhibitory effect on glucose uptake from sucrose and their uptake was in turn inhibited by the disaccharide. This inhibition was non-competitive. The effect of phlorizin on glucose uptake from sucrose was 2-fold, competitive at low concentrations and non-competitive at high concentrations. Sucrose did not induce counterflow of the preloaded β-methyl glucoside. These observations indicate that with a disaccharide as the substrate, there is a component of glucose transport which is in addition to the monoscaccharide-transport system and that this could arise as a consequence of the association of disaccharidases with the brush border membrane.