Measuring hexose transport in suspended cells.
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3-O-Methylglucose↗
Biomedical subjects
J Gliemann
Publications and source records attributed to J Gliemann.
Effect of chemotactic factors on hexose transport in polymorphonuclear leucocytes.
Transport of the nonmetabolizable glucose analogue, 3-O-methylglucose, was assessed in human polymorphonuclear leucocytes with or without the chemotactic peptide N-formylmethionylleucylphenylalanine (fMet-Leu-Phe). The peptide increased entry of labelled 3-O-methylglucose about 5-fold and the intracellular distribution space about 70%. The half-time of equilibration was 3 s in the treated cells. Similar effects were observed with zymosan-treated serum (containing the chemotactic factor C5a), with arachidonic acid, calcium ionophore A23187 and phorbol myristate acetate. However, the chemotactic protein, thrombin, had no effect, even though binding to high-affinity receptors was demonstrated. Km for zero-trans entry of 3-O-methylglucose was about 1 mM and fMet-Leu-Phe increased Vmax from 5 to about 25 amol.s-1.cell-1. Similar values were obtained from incubations for a few seconds with glucose and 2-deoxyglucose. The rate of 2-deoxyglucose uptake (8 min incubations) was limited by the transport step at substrate concentrations lower than approx. 0.1 mM, whereas the phosphorylation step became rate-limiting at higher concentrations. Thus, 2-deoxyglucose uptake can only be taken as a measure of transport at a tracer concentration. It is concluded that chemotactic factors can, but do not necessarily, increase the maximal transport velocity of hexoses entering the polymorphonuclear leucocyte via the glucose transporter.
3-O-Methylglucose↗
Three different conformational states of pregnancy zone protein identified by monoclonal antibodies.
Human pregnancy zone protein (PZP), related to human alpha 2-macroglobulin, forms dimeric/tetrameric (360/720 kDa) species. PZP binds proteinases which cause the cleavage of internal thiol esters in the molecule. Both the binding of proteinases, i.e. chymotrypsin, (CT) to PZP, forming PZP.CT complexes, or reaction with methylamine (MA) forming PZP.MA complexes, cause transition to a new similar conformational state. Reactivity of selected monoclonal antibodies against PZP towards the three PZP derivatives demonstrated differences in the reactivity pattern. PZP and PZP.MA share one determinant, which is missing on the PZP.CT complex. PZP after transition to PZP.CT, but not to PZP.MA, presents a neodeterminant detected by one of six monoclonal antibodies. The findings demonstrate that at least three different conformational states exist for PZP and its derivatives. Access to discriminating immunochemical tools makes possible an evaluation of the relative abundance of the different complexes in vivo.
Antibodies, Monoclonal↗
Human hepatocytes exhibit receptors for alpha 2-macroglobulin and pregnancy zone protein-proteinase complexes.
Hepatocytes were isolated by application of the two-step collagenase technique to pieces of human liver. 125I-labelled alpha 2-macroglobulin-trypsin complex bound to hepatocytes at 4 degrees C with a half time of approximately 4.5 h. At near equilibrium half of the receptors were saturated at an alpha 2-macroglobulin-trypsin complex concentration of about 60 pmol 1(-1) and the Scatchard plot was linear. Dissociation of the labelled complex was slow (T1/2 = 24 h) at low receptor occupancies. At high receptor occupancies dissociation was biphasic with a rate constant (K-1) for the initial rapid phase of about 2.4 x 10(-2) min-1. Labelled alpha 2-macroglobulin-trypsin complex bound at 4 degrees C was rapidly internalized at 37 degrees C (T1/2 = 1.9 min), and in 3.5 h approximately 10% of the label was released into the medium in a trichloroacetic acid-soluble form. At 37 degrees C, 125I alpha 2-macroglobulin-trypsin was taken up by hepatocytes and trichloroacetic acid soluble radioactivity appeared in the medium following a sigmoidal curve. Similar results were obtained with 125I-pregnancy zone protein-chymotrypsin complex. At 4 degrees C, hepatocytes bound nearly equal amounts of labelled alpha 2-macroglobulin-trypsin and pregnancy zone protein-chymotrypsin complex, and a large excess (100 nmol 1(-1) of one of the macroglobulins could almost completely abolish binding of trace amounts (5-20 pmol 1(-1] of the other. The present findings strongly suggest that the hepatocyte is of major importance for removal of alpha 2-macroglobulin- and pregnancy zone protein-proteinase complex in humans, in agreement with previous results in rats and mice.
Biological Transport, Active↗
Cell association and degradation of pregnancy zone protein-chymotrypsin complex in cultured human monocytes.
125I-labelled pregnancy zone protein complexed with chymotrypsin (PZP-ct) bound to freshly isolated and to cultured human monocytes. Binding of uncomplexed 125I-pregnancy zone protein (PZP) was less than 20% of complex binding. At 4 degrees C, labelled PZP-ct complex bound to cultured monocytes with a half-time of about 4 h. Dissociation at 4 degrees C was slow at low receptor occupancies (t 1/2 greater than 24 h). At high receptor occupancies, dissociation was biphasic (initial k-1 = 8 X 10(-3) min-1) and 85% of the cell-associated label had dissociated within 24 h. At near equilibrium, binding of 20 pmol/l 125I-PZP-ct was half maximally inhibited at a ligand concentration of about 450 pmol/l. The Scatchard plot was linear giving an estimated concentration of 2 X 10(4) receptors/cell. Ligand bound at 4 degrees C was rapidly internalized at 37 degrees C (half-time = 0.5 min) and after a 5-10 min lag time, radioactivity comprising monoiodotyrosine was released into the medium following a sigmoidal curve. At 37 degrees C the uptake of 125I-PZP-ct complex was initially linear and within 15 min internalized label accumulated, reaching a steady state at about 85% of the cell-associated radioactivity. Following a lag time of 15 min, radioactivity soluble in trichloroacetic acid appeared in the medium. Similar results were obtained with 125I-alpha 2-macroglobulin-trypsin complex (alpha 2M-T). It is concluded that high affinity receptors mediate binding, uptake and degradation of pregnancy zone protein--and alpha 2-macroglobulin-proteinase complex in human monocyte-macrophages.
Cells, Cultured↗
Binding and receptor-mediated endocytosis of pregnancy zone protein-proteinase complex in rat macrophages.
125I-labelled pregnancy zone protein complex was injected intravenously in rats and after 6 min uptake into cells of the liver and spleen was determined by electron microscopic autoradiography. The liver took up 68% of the injected radioactivity; 61% was in the hepatocytes and 7% was in the liver macrophages (Kupffer cells). The spleen took up 3-4% and nearly all the radioactivity was in the macrophages of the red pulp. The uptake per cell volume was several times higher in the macrophage than in the hepatocyte. The radioactivity associated with macrophages was largely in endocytotic vacuoles and lysosomes. Binding of labelled pregnancy zone protein complex to peritoneal macrophages at 4 degrees C was 2-3 times higher than binding of the homologous alpha 2-macroglobulin complex. The two ligands competed for binding to the same receptors and the difference was due to a higher affinity of the pregnancy zone protein complex (Kd approx. 60 pM). After binding to the receptor, this ligand was internalised within 2-3 min at 37 degrees C and radioactivity inside the cells largely represented intact pregnancy zone protein complex. Radioactivity was released from the cell as iodotyrosine after a lag time of about 10 min. It is concluded that pregnancy zone protein complex is bound with a high affinity to the alpha 2-macroglobulin receptors in rat macrophages followed by receptor-mediated endocytosis and degradation of the ligand in the lysosomes.
Animals↗
Rat plasma alpha 1-inhibitor3 binds to receptors for alpha 2-macroglobulin.
The cellular binding and uptake was studied for alpha 1-inhibitor3, a monomeric 200 kDa proteinase inhibitor present in rat plasma. After intravenous injection in the rat the inhibitor disappeared from the circulation with a half-time of 2.5 min when complexed with chymotrypsin, whereas the half-time for uncomplexed inhibitor was more than 60 min. 6 min after the injection of labelled complex, 83% was in the liver and 2.5% in the spleen. In vitro experiments at 4 degrees C with isolated hepatocytes and peritoneal macrophages showed binding to the previously described receptors which bind and internalize the tetrameric rat and human alpha 2-macroglobulin-proteinase complexes. The binding affinities were similar for the two types of complexes and binding was followed by uptake and degradation of the labelled complex when the cells were warmed to 37 degrees C. The binding of uncomplexed alpha 1-inhibitor3 was low and did not increase following treatment with methylamine in spite of cleavage of the internal thiol ester. alpha 1-Inhibitor3-methylamine was changed to the receptor binding form when treated with chymotrypsin which caused the cleavage of at least one peptide bond in the bait region.
Acute-Phase Proteins↗
Re-examination of hexose-transporter inhibition and labelling by hexose isothiocyanates.
We have re-examined hexose-transport inhibition by hexose isothiocyanates and find that the inhibition is incomplete, probably because of decomposition of the reagent. The inhibition type is 'mixed', because hexose-transporter ligands such as maltose and cytochalasin B only give partial protection from inhibition. This suggests that a liganded-transporter-hexose isothiocyanate ternary complex is formed. We have compared the labelling of red-blood-cell membranes by [14C]MITC (D-maltose isothiocyanate) with the labelling obtained using a photoaffinity probe (ASA-BMPA [2-N-(4-azidosalicyloyl)-1,3-bis-(D-mannos-4'-yloxy)-2 -propylamine]) which gives specific labelling of the hexose transporter in band 4.5. [14C]MITC gives a partially D-glucose-displaceable labelling of a band 3 component in the same cell preparations which show ASA-BMPA labelling of band 4.5. This eliminates the possibility that band 4.5 labelling can only occur when the HITC (hexose isothiocyanate) binding protein in band 3 is proteolysed. HITC pretreatment does not decrease ASA-BMPA labelling of the exofacial site of band 4.5. This is also consistent with the formation of ternary complex. However, HITC pretreatment inhibits both reversible and photoactivated covalent [3H]cytochalasin B binding to band 4.5. These results suggest that, in the intact cell, interactions between a band 3 HITC-binding component and the inside cytochalasin B-binding site on the hexose transporter in band 4.5 may occur.
Affinity Labels↗
Enhancement of glucose transport by insulin at 37 degrees C in rat adipocytes is accounted for by increased Vmax.
The kinetics of 3-O-methyl-D-glucose and D-glucose transport was reinvestigated at 37 degrees C in rat adipocytes exhibiting very slow transport rates in the absence of insulin and approximately 30-fold insulin stimulation. The apparent maximal velocity for 3-O-methylglucose was increased at least 15-fold from 0.04 +/- 0.01 to 0.73 +/- 0.17 mmol.l-1.s-1 (SD, n = 7). The apparent half saturation constant was decreased by insulin from 8.1 +/- 1.6 mmol/l to 3.3 +/- 0.8 mmol/l. However, approximately 10% of the 3-O-methylglucose at a very low concentration was transferred by routes other than the glucose transporter, e.g. via nonmediated diffusion or via the fructose transporter, and the estimated value for the half saturation constant was therefore too high in the nonstimulated cells. After appropriate correction this value was estimated as 5.0 +/- 1.8 mmol/l. The inhibition constant of 3-O-methylglucose on the conversion of 12 mumol/l. 14C labelled glucose to cell-associated products was about 4 mmol/l and was not changed significantly by insulin. Transport experiments with labelled glucose gave results similar to those obtained for transport of 3-O-methylglucose. It is concluded that insulin can cause a 15- to 30-fold increase in the maximal velocity for transport of 3-O-methylglucose and glucose, and that a concomitant decrease in the half saturation constant, if present, is insignificant.
3-O-Methylglucose↗
Binding of alpha-2-macroglobulin trypsin complex to human monocytes in culture.
125I-labelled alpha 2-macroglobulin-trypsin (alpha 2MT) complex bound specifically to freshly isolated human blood monocytes at 4 degrees C but not to B or T lymphocytes, polymorphonuclear leucocytes, erythrocytes or thrombocytes. Binding of 12 pmol/l labelled alpha 2MT to freshly isolated monocytes was low. However, when monocytes were cultured in vitro, binding increased, reaching 10- to 20-fold higher specific binding by 2-4 weeks. Non-specific binding was absent. Considerable variations were observed in binding to monocytes from different cultures (individuals). The half-time of 125I-labelled alpha 2MT complex association to 14-days-old monocyte cultures was about 6 h at 4 degrees C. Dissociation of labelled complex after the addition of a saturing concentration of unlabelled complex was biphasic. About half of the labelled complex dissociated with a half-time of about 1 h, whereas the other half dissociated extremely slowly. At near steady state, half of the receptors were occupied at a complex concentration of about 200 pmol/l and Scatchard analysis showed that the data were adequately described by assuming one class of receptors. It is concluded that human monocytes express a marked increase in binding of alpha 2M complex when developing to macrophage-like cells in tissue cultures.
Cells, Cultured↗
Transport of glucose and fructose in rat hepatocytes at 37 degrees C.
The kinetic parameters for transport of the nonmetabolizable glucose analogue 3-O-methyl-D-glucose and the relationship between transport and metabolism of D-glucose and D-fructose were determined in isolated rat hepatocytes at 37 degrees C and pH 7.4. 3-O-Methylglucose at a very low concentration (0.1 mM) equilibrated with the intracellular water with a rate constant of 0.41 s-1. Km for equilibrium exchange entry was 5.5 mM and Vmax was 2.2 mM X s-1 and similar results were obtained when using the zero-trans entry protocol. The rate constant for entry of tracer D-glucose was 0.15 s-1 and Km for glucose was about 20 mM. The phosphorylation rate for D-glucose was much slower than the transport rate. The rate constant for D-fructose entry was about 0.04 s-1, the apparent Km was about 100 mM and Vmax about 5 mM X s-1. The concentration dependence of 3-O-methylglucose inhibition of labelled fructose transport revealed biphasic kinetics indicating that fructose was transferred by both the glucose transporter and a fructose transporter. At concentrations lower than 1 mM, fructose metabolism appeared to be limited by the transport step.
3-O-Methylglucose↗
Evidence for binding of human pregnancy zone protein-proteinase complex to alpha 2-macroglobulin receptors.
125I-labelled human pregnancy zone protein complexed with chymotrypsin was removed from the circulation with a half-time of 2.3 min after intravenous injection in rats. After 6 min about 67% of the label was present in the liver and about 3% was in the spleen, both in male and in female pregnant rats. The half-time of removal was more than 30 min for native pregnancy zone protein. Uptake into other organs, including placentae and feti, was negligible. 30 pM labelled pregnancy zone protein X chymotrypsin was specifically bound to rat hepatocytes and adipocytes and to human fibroblasts and monocyte-derived macrophages at 4 degrees C. Binding was almost completely abolished by a saturating concentration of unlabelled alpha 2-macroglobulin X trypsin. Binding of 15 pM labelled macroglobulin complex was completely abolished by a saturating concentration of pregnancy zone protein X chymotrypsin. In rat hepatocytes, binding of pregnancy zone protein complex was lower than that of alpha 2-macroglobulin complex at low ligand concentrations. Half-maximal receptor occupancy was obtained with about 300 pM pregnancy zone protein complex. Unlabelled alpha 2-macroglobulin or pregnancy zone protein complex failed to accelerate dissociation of the labelled pregnancy zone protein complex under conditions where dissociation of alpha 2-macroglobulin was markedly enhanced. It is concluded that pregnancy zone protein and alpha 2-macroglobulin complexes bind to the same receptors. The quantitative differences may be related to the fact that alpha 2-macroglobulin is a tetramer whereas the functional unit of pregnancy zone protein is probably a dimer.
Animals↗
Domain structure of human alpha 2-macroglobulin. Characterization of a receptor-binding domain obtained by digestion with papain.
Digestion of methylamine-treated alpha 2-macroglobulin (alpha 2M X MA) with catalytic amounts of papain at pH 4.5 has been investigated. Cleavage of Lys(1313)-Glu resulted in two major products, which could be separated by gel chromatography: a large disulfide bridged fragment set nearly the size of intact alpha 2M X MA, and an 18 kDa fragment, constituting the carboxy-terminal domain of alpha 2M. This domain contained the receptor recognition site, exposed as a result of cleavage of the internal beta-cysteinyl-gamma-glutamyl thiol esters in alpha 2M. Compared with alpha 2M-trypsin complex the apparent affinity for binding to rat hepatocyte receptors was 0.1 and 2% at 4 and 37 degrees C, respectively. The receptor-binding domain presumably forms a compact globular beta-barrel-type structure, stable at pH 2.5-9.0. Chemical modification experiments suggest that receptor binding is contributed by a determinant formed by the precise folding of the polypeptide chain.
Animals↗
Coypu insulin. Primary structure, conformation and biological properties of a hystricomorph rodent insulin.
Insulin from a hystricomorph rodent, coypu (Myocaster coypus), was isolated and purified to near homogeneity. Like the other insulins that have been characterized in this Suborder of Rodentia, coypu insulin also exhibits a very low (3%) biological potency, relative to pig insulin, on lipogenesis in isolated rat fat-cells. The receptor-binding affinity is significantly higher (5-8%) in rat fat-cells, in rat liver plasma membranes and in pig liver cells, indicating that the efficacy of coypu insulin on receptors is about 2-fold lower than that of pig insulin. The primary structures of the oxidized A- and B-chains were determined, and our sequence analysis confirms a previous report [Smith (1972) Diabetes 21, Suppl. 2, 457-460] that the C-terminus of the A-chain is extended by a single residue (i.e. aspartate-A22), in contrast with most other insulin sequences, which terminate at residue A21. In spite of a large number of amino acid substitutions (relative to mammalian insulins), computer-graphics model-building studies suggest a similar spatial arrangement for coypu insulin to that for pig insulin. The substitution of the zinc-co-ordinating site (B10-His----Gln) along with various substitutions on the intermolecular surfaces involved in the formation of higher aggregates are consistent with the observation that this insulin is predominantly 'monomeric' in nature. The c.d. spectrum of coypu insulin is relatively similar to those of casiragua insulin and of bovine insulin at low concentration.
Amino Acid Sequence↗
Characterization of receptors for alpha 2-macroglobulin-trypsin complex in rat hepatocytes.
High-affinity receptors for alpha 2-macroglobulin-trypsin complex were demonstrated in rat hepatocytes at 4 degrees C. The dissociation rate constant for the labelled complex was very small at low receptor occupancies, approx. 4 X 10(-4) min-1. Dissociation was biphasic at high receptor occupancies with a rate constant for the rapid phase of about 2 X 10(-2) min-1. At near-equilibrium, half of the receptors were saturated at a complex concentration of 150 pM, and the Scatchard plot was concave upwards. Thus, the binding shows complex kinetics with the probable involvement of negative cooperativity. Binding of the labelled complex was not influenced by galactose, mannose, mannose phosphate or fucoidin, whereas it was abolished in the absence of extracellular Ca2+ and inhibited by bacitracin. Approx. 70% of the labelled complex bound at 4 degrees C was rapidly internalized (kint about 3 X 10(-1) min-1) after being warmed to 37 degrees C. Radioactivity released from the cells at 37 degrees C comprised intact labelled complex and iodide. The complex was initially released at a rapid rate (k-1 about 1 X 10(-1) min-1) from about 25% of the cell-bound pool. This probably represents dissociation from the receptors. A slow phase of release followed, so that half of the bound pool was finally released as intact complex. Iodide release followed a sigmoidal curve after a 20 min lag period. Thus, specific high-affinity receptors mediate the internalization and eventual degradation of alpha 2-macroglobulin-proteinase complex into hepatocytes.
Animals↗
Facilitated transport of 3-O-methyl-D-glucose in human polymorphonuclear leukocytes.
Transport of the nonmetabolizable hexose analogue 3-O-methyl-D-glucose (30MG) was measured in human polymorphonuclear leukocytes at 37 degrees C, pH 7.4. 3OMG at very low concentration (0.05 mM) equilibrated with the intracellular water with a rate constant of about 0.08 s-1. Transport of 3OMG in the presence of 20 microM cytochalasin B and transport of L-glucose were insignificant. Countertransport of 14C-labelled 3OMG was demonstrated. Exchange of 3OMG between the extracellular and intracellular water showed saturation with a Km of about 4 mM. Thus, the transport of 3OMG is mediated almost exclusively by facilitated diffusion.
Biological Transport↗
Uptake of rat and human alpha 2-macroglobulin-trypsin complexes into rat and human cells.
Uptake of rat and human alpha 2-macroglobulin-trypsin complexes was measured in rat hepatocytes, rat and human adipocytes and human fibroblasts. Uptake and degradation of 125I-labelled rat complex were about one-third of that of the human complex in the various isolated cell types. In rat hepatocytes, the apparent Km for cell association of the rat complex was about 16 nM as compared to about 6 nM for the human complex. The Vmax values were similar, about 1 X 10(4) molecules X cell-1 X min-1. Thus, rat alpha 2-macroglobulin (an acute-phase protein) complexed with trypsin follows the same pathways of uptake as the human homologue, although with a somewhat lower affinity for the uptake system.
Adipose Tissue↗
The plasma clearance of human alpha 2-macroglobulin-trypsin complex in the rat is mainly accounted for by uptake into hepatocytes.
Rats were given intravenous injections of 125I-labelled human alpha 2-macroglobulin X trypsin. The half-time of disappearance of radioactivity from arterial blood was 2 min. External counting showed that radioactivity in the liver was maximal by 10 min and then decreased slowly. 87% of the injected dose was recovered in the liver by 10 min. Light- and electron microscopic autoradiography carried out on samples of liver fixed with glutaraldehyde 3 min or 30 min after the injection showed that 85-90% of the grains were over the hepatocytes and 4-9% were over the Kupffer cells. Thus, uptake into hepatocytes, and not into Kupffer cells as believed previously, appears to account for the major part of the uptake of alpha 2-macroglobulin X trypsin by the liver and thereby for its rapid removal from the blood.
Animals↗