Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding domain of forskolin in the adipocyte/muscle-type glucose transporter (GLUT-4) was localized with the aid of the photoreactive derivative, [125I]IAPS-forskolin (3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin). Plasma membranes from insulin-treated rat adipocytes containing predominantly the GLUT-4 isoform were irradiated with UV light in the presence of [125I]IAPS-forskolin. The covalently labeled glucose transporters were isolated by immunoprecipitation with specific antiserum and partially digested with trypsin and elastase. The fragments were separated by gel electrophoresis, transferred on to nitrocellulose membranes, and identified by direct autoradiography and by immunoassay with antiserum against a peptide sequence corresponding to the C-terminus of GLUT-4. Digestion with a high-purity grade trypsin generated two photolabeled fragments with apparent molecular weights of 21 and 16 kDa. Since the antiserum detected two fragments with identical electrophoretic mobility, both labeled fragments appeared to contain the intact C-terminus of GLUT-4. In contrast, digestion with elastase generated only one photolabeled fragment with intact C-terminus at 21 kDa, and a smaller unlabeled fragment with intact C-terminus at 15 kDa. A less pure trypsin preparation generated two labeled (21 and 17 kDa) and one unlabeled (15 kDa) fragment with intact C-terminus. These data suggest that the site of covalent binding of IAPS-forskolin in the GLUT-4 is located within a region of 1-6 kDa defined by the difference between the unlabeled C-terminal fragment (15 kDa) and the labeled fragments (21, 17 and 16 kDa). Based on a tentative allocation of the fragments to the sequence of the GLUT-4, it is suggested that the covalent binding site of IAPS-forskolin is located between the membrane spanning helices 7-9, possibly in the proximity of helix 9.
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PMID:Localization of the binding domain of the inhibitory ligand forskolin in the glucose transporter GLUT-4 by photolabeling, proteolytic cleavage and a site-specific antiserum. 142 Feb 53

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.
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PMID:Chromatographic characterization of nitrobenzylthioinosine binding proteins in band 4.5 of human erythrocytes: purification of a 40 kDa truncated nucleoside transporter. 222 99

The synthesis of 2-N-[4-(1'-azitrifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-++ +yloxy)-2- propylamine (ATB-BMPA) is described. This compound was used as an exofacial probe for the human erythrocyte glucose-transport system. A new method is described for directly estimating the affinity for exofacial ligands which bind to the erythrocyte glucose transporter. By using this equilibrium-binding method, the Ki for ATB-BMPA was found to be 338 +/- 37 microM at 0 degrees C and 368 +/- 59 microM at 20 degrees C. This was similar to the concentration of ATB-BMPA required to half-maximally inhibit D-galactose uptake (Ki = 297 +/- 53 microM). The new photoaffinity reagent labelled the glucose transporter in intact cells but, because of its improved selectivity, was also used to label the glucose transporter in isolated erythrocyte membranes. The ATB-BMPA-labelled glucose transporter was 80% immunoprecipitated by anti-(GLUT1-C-terminal peptide) antibody, which shows that the GLUT1 glucose transporter is the major isoform present in erythrocytes. The labelling of the glucose transporter at its exofacial site, and the adoption of an outward-facing conformation, renders the transport system resistant to thermolysin and trypsin treatment. Trypsin treatment of the unlabelled glucose transporter in erythrocyte membranes produced an 18 kDa fragment which was subsequently labelled by ATB-BMPA, but had low affinity for this exofacial ligand. This suggests that the trypsin-treated transporter adopts an inward-facing conformation. The ability of D-glucose to displace ATB-BMPA from the native transporter and from the 18 kDa trypsin fragment have been compared. The D-glucose concentration which was required to obtain half-maximal inhibition of ATB-BMPA labelling was 6-fold lower for the 18 kDa tryptic fragment.
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PMID:Exofacial photolabelling of the human erythrocyte glucose transporter with an azitrifluoroethylbenzoyl-substituted bismannose. 239 55

Phosphorylation of the insulin-regulatable glucose transporter (IRGT) is increased by incubating rat adipocytes with isoproterenol or by incubating microsomal membranes with cAMP-dependent protein kinase. To attempt to locate the sites of phosphorylation, the IRGT (apparent Mr = 46,000) was immunoprecipitated from 32P-labeled adipocytes and cleaved with CNBr or trypsin. Essentially all of the 32P could be recovered in a single CNBr fragment, denoted CB-T (Mr = 8,000), which bound a polyclonal antibody (R820) against a peptide having the sequence of the last 12 amino acids in the COOH terminus of the IRGT. 32P-Labeling of the IRGT was also confined to CB-T when membranes were incubated with [gamma-32P]ATP and cAMP-dependent protein kinase. Isoproterenol increased phosphorylation of CB-T, but insulin was without effect. To resolve phosphorylation sites further, IRGT from 32P-labeled cells was subjected to exhaustive proteolysis with trypsin. Samples were applied to a C-18 column, and 32P-labeled fragments were resolved into three peak fractions by elution with an increasing gradient of acetonitrile. [32P]Phosphoserine was the only phosphoamino acid detected in any of the peaks. Peak III contained approximately 80% of the 32P and was increased by isoproterenol. Almost all of the 32P introduced by cAMP-dependent protein kinase in vitro eluted in Peak III. In all cases, the 32P-labeled species in Peak III were quantitatively immunoprecipitated by R820. Digesting the peptide(s) in Peak III with V8 protease generated a single peak of 32P which eluted at lower acetonitrile than Peak III and contained 32P-labeled species that did not interact with R820. Automated Edman degradation indicated that the serine residue in Peak III phosphorylated by cAMP-dependent protein kinase was the 3rd or 4th residue from the NH2 terminus of the peptide. These findings indicate that phosphorylation of the IRGT is restricted to the presumed intracellular domain at the COOH terminus and that Ser488 is a major site phosphorylated both by cAMP-dependent protein kinase in vitro and in response to isoproterenol in vivo.
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PMID:Phosphorylation of the glucose transporter in rat adipocytes. Identification of the intracellular domain at the carboxyl terminus as a target for phosphorylation in intact-cells and in vitro. 240 83

ATP has been reported to affect glucose transport in human erythrocytes and resealed erythrocyte ghosts [Jacquez, J. A. (1983) Biochim. Biophys. Acta 727, 367-378; Jensen, M. R., & Brahm, J. (1987) Biochim. Biophys. Acta 900, 282-290]. In more detailed studies, effects of micromolar levels of ATP on transport in ghosts and inside-out vesicles, and on the fluorescence of ghosts and the purified glucose transporter [Carruthers, A. (1986) Biochemistry 25, 3592-3602; Hebert, D. N., & Carruthers, A. (1986) J. Biol. Chem. 261, 10093-10099; Carruthers, A. (1986) J. Biol. Chem. 261, 11028-11037], have been interpreted as supporting a model in which ATP regulates the catalytic properties of the transporter. Both allosteric and covalent effects of ATP were proposed; among the allosteric effects was a 60% reduction in the Km for zero-trans uptake. In order to test whether allosteric ATP regulation of the transporter occurs, we reconstituted glucose transport activity into liposomes using erythrocyte membranes without detergent treatment. The effects of ATP, present either outside, inside, or both inside and outside the liposomes, on the transport activity were examined. Effects of ATP on trypsin-treated liposomes, which have only a single orientation of active transporters, were also tested. While the model predicts activation by ATP, only inhibition was observed. This was significant only at millimolar concentrations of ATP, in contrast to the previously reported effects at micromolar levels, and was primarily on the extracellular surface of the transporter. In addition, the ATP effects on reconstituted transport were nonspecific, with similar effects produced by tripolyphosphate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:ATP does not regulate the reconstituted glucose transporter. 274 44

RNA transcripts encoding the complete human glucose transporter (GT) or fragments of GT corresponding to the NH2-terminal 340 amino acids (GT-N) or the COOH-terminal 148 amino acids (GT-C) were synthesized in vitro from SP6 plasmids. The corresponding polypeptides were synthesized in reticulocyte or wheat germ cell-free systems and their insertion into pancreatic microsomes was assessed by endoglycosidase H and trypsin digestion or alkaline extraction of membranes. The following observations were made: both GT and GT-N can insert posttranslationally into microsomes; GT contains at least two distinct signal sequences; both co- and posttranslational insertion of GT-N are mediated by a signal recognition particle (SRP)-SRP receptor-dependent mechanism; and SRP is required both for targeting the polypeptide to the membrane and for initiating the actual insertion process. We propose a model for the biosynthesis of GT and proteins of similar membrane topology which does not require a tight ribosome-membrane interaction.
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PMID:The human glucose transporter can insert posttranslationally into microsomes. 300 42

The glucose transporter has been identified in a variety of mammalian cell membranes using a photoactivatable carrier-free radioiodinated derivative of forskolin, 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n ([125I]IAPS-forskolin) at 1-3 nM. The membranes that were photolabelled with [125I]IAPS-forskolin were human placental membranes, rat cortical and cerebellar synaptic membranes, rat cardiac sarcolemmal membranes, rat adipocyte plasma membranes, smooth-muscle membranes, and S49 wild-type (WT) lymphoma-cell membranes. The glucose transporter in plasma membranes prepared from the insulin-responsive rat cardiac sarcolemmal cells, rat adipocytes and smooth-muscle cells were determined to be approx. 45 kDa by SDS/polyacrylamide-gel electrophoresis (PAGE). Photolysis of human placental membranes, rat cortical and cerebellar synaptic membranes, and WT lymphoma membranes with [125I]-IAPS-forskolin, followed by SDS/PAGE, indicated specific derivatization of a broad band (43-55 kDa) in placental membranes and a narrower band (approx. 45 kDa) in synaptic membranes and WT lymphoma membranes. Digestion of the [125I]IAPS-forskolin-labelled placental and WT lymphoma membranes with endo-beta-galactosidase showed a reduction in the apparent molecular mass of the radiolabelled band to approx. 40 kDa. The membranes that were photolabelled with [125I]IAPS-forskolin and trypsin-treated produced a radiolabelled proteolytic fragment with an apparent molecular mass of 18 kDa. [125I]IAPS-forskolin is a highly effective probe for identifying low levels of glucose transporters in mammalian tissues.
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PMID:Identification of the glucose transporter in mammalian cell membranes with a 125I-forskolin photoaffinity label. 306 59

Tryptic digestion has been used to investigate the conformational changes associated with substrate translocation by the human erythrocyte glucose transporter. The effects of substrates and inhibitors of transport on the rates of tryptic cleavage at the cytoplasmic surface of the membrane have confirmed previous observations that this protein can adopt at least two conformations. In the presence of phloretin or 4,6-O-ethylidene-D-glucose, the rate of cleavage is slowed. Because these inhibitors bind preferentially at the extracellular surface of the transporter, their effects must result from a conformational change rather than from steric hindrance. A conformational change must also be responsible for the effect of the physiological substrate D-glucose, which is to increase the rate of cleavage. The regions of the protein involved in the conformational changes include both of the large cytoplasmic regions that are cleaved by trypsin: these are the central hydrophilic region of the sequence (residues 213-269) and the hydrophilic C-terminal region (residues 457-492).
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PMID:Proteolytic dissection as a probe of conformational changes in the human erythrocyte glucose transport protein. 322 21

The transport function and orientation of the reconstituted human erythrocyte glucose transporter was studied with liposomes made with bovine brain lipid or Escherichia coli lipid. Reconstitution was achieved by a simple octyl glucoside dilution method. The reconstituted transporters with either lipid showed identical counterflow transport activity, the same response to various inhibitors, and characteristic cytochalasin B (CB) labeling. Functional location and purification of the glucose transporter was performed by using gel-permeation high-performance liquid chromatography with octyl glucoside-containing buffer. The reconstituted transport activity was associated only with band 4.5 protein (preactin) and not with band 3 protein. Both CB binding and transport function of the reconstituted transporters were resistant to trypsin but susceptible to chymotrypsin digestion. However, both trypsin and chymotrypsin treatment of unsealed ghosts completely eliminated the CB labeling and transport function of the glucose transporter. In our reconstitution system the glucose transporters maintained a normal asymmetrical (right-side-out) orientation and good transport function. A specific monoclonal antibody against the glucose transporter inhibited CB labeling of the transporters on unsealed ghosts. This was not found with the reconstituted system; however, after freeze-thawing there was a significant inhibition of CB binding by the antibody. These findings suggest that the CB-binding site of the reconstituted transporter is on the inner side of the proteoliposomes.
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PMID:Human erythrocyte glucose transporter: normal asymmetric orientation and function in liposomes. 351 73

Phloridzin-insensitive, Na+-independent D-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed a Kt of 48 mM and a Vmax of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lower Kt of 23 mM and a Vmax of 0.20 nmol glucose/mg protein/sec. D-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. The Ki for cytochalasin B as an inhibitor of glucose transport was 0.11 microM and the KD for binding to the carrier was 0.08 microM. D-glucose-sensitive sensitive binding of cytochalasin B to the membrane preparation was maximized with L- and D-glucose concentrations of 1.25 M. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na+-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and the Kt for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.
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PMID:The Na+-independent D-glucose transporter in the enterocyte basolateral membrane: orientation and cytochalasin B binding characteristics. 362 59


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