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R Horuk

Publications and source records attributed to R Horuk.

At least 91 records · Page 5Linked to original sources
PubMed1989

The interleukin-1 receptor in Raji human B-lymphoma cells. Molecular characterization and evidence for receptor-mediated activation of gene expression.

In a previous paper [Horuk, Huang, Covington & Newton (1987) J. Biol. Chem. 262, 16275-16278] we reported that there were fundamental differences in the biochemical properties of the interleukin-1 (IL-1) receptor between Raji and EL4 cell lines. In the present study we have investigated the basis for these differences. Kinetic studies measuring the on and off rates of IL-1 receptor binding revealed that the low-affinity IL-1-binding sites observed in Raji cells, compared with EL4 cells, result from a combination of a lower association rate and a higher dissociation rate in the Raji cells. The turnover of the Raji IL-1 receptor, measured by inhibiting protein synthesis with cycloheximide, was much faster than that of the EL4 IL-1 receptor, with a half-time of 2 h as against 5 h. Treatment of 125I-IL-1-labelled IL-1 receptors in Raji and EL4 cells with neuraminidase decreased their molecular mass by approx. 2-5 kDa as assessed by SDS/polyacrylamide-gel electrophoresis (PAGE). The covalently labelled IL-1 receptors in both cell types were sensitive to treatment with endoglycosidase F, which decreased their molecular mass on SDS/PAGE by 12-13 kDa. Incubation of Raji cells with maximally stimulating doses of IL-1 resulted in an increase in the nascent RNA levels of several genes, including the IL-2 receptor and the proto-oncogenes c-Ha-ras and c-myc.

B-Lymphocytes↗
PubMed1989

A rapid and direct method for the detection and quantification of interleukin-1 receptors using 96 well filtration plates.

A rapid method for the detection and quantification of interleukin-1 receptors in cultured cells has been developed. The receptor binding assay is carried out in sealed 96 well filtration plates. At the end of the incubation period the seal is removed and the cells are filtered under vacuum to separate free ligand from bound. After washing several times, individual wells are removed using a well punch and counted in a gamma counter. The method is rapid, accurate and capable of high sample throughput and should find wide application as a screen to evaluate IL-1-like drugs.

Cell Line↗
PubMed1989

Identification of IL-1 receptors on human monocytes.

The expression and functional analysis of IL-1 beta R on human monocytes were investigated. Binding of 125I-IL-1 to human monocytes was found to be specific and saturable. Scatchard plot analysis revealed a single class of receptors with a binding constant of 600 pM and a receptor density of approximately 100 binding sites per cell. At 37 degrees C 54% of the labeled ligand was internalized over 2 h of incubation. Addition of 0.2% sodium azide to the cells reduced ligand internalization to 9% of total bound. Cross-linking studies revealed that the IL-1R in human monocytes had a Mr of 80 kDa. The addition of IL-1 to monocytes caused changes in membrane Ag expression as assessed by flow cytometric analysis. The results of this study identify IL-1 receptors on monocytes and suggest that IL-1 may act as an effector molecule for monocytes by enhancing expression of Ag correlated with cell differentiation and immune function.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1988

Characterization of murine IL-1 beta. Isolation, expression, and purification.

One cDNA clone encoding a truncated murine IL-1 beta (M IL-1 beta) sequence was isolated from a murine macrophage cDNA library. We reconstituted the coding sequence of the 152-residue mature protein and expressed it in Escherichia coli. rM IL-1 beta was purified to homogeneity and characterized by oligonucleotide and NH2-terminal sequence analysis. Purified rM IL-1 beta exhibited biologic activity equivalent to 7.8 x 10(7) units/mg in the murine thymocyte proliferation assay and 9.9 x 10(3) units/mg in the human gingival fibroblast PGE2 production assay, indicative of species specificity. The isoelectric point of rM IL-1 was found to be 8.85. The circular dichroism spectrum revealed that the secondary structure of M IL-1 is indistinguishable from that of the human protein. Receptor binding studies indicated the rM IL-1 bound to murine EL-4.1 thymoma cells in a specific and dose-dependent fashion with an affinity of 32 pM. Competition binding data suggested that murine and human IL-1 compete for a single class of receptor. Antisera were generated in rabbits against both murine and human IL-1. Results of ELISA binding and antisera neutralization assays indicated that there are common antigenic sites between the two IL-1 beta molecules. These domains are of functional importance because they are capable of mediating the neutralization of biologic activity.

Amino Acid Sequence↗
PubMed1987

Biochemical characterization and subcellular distribution of the glucose transporter from rat brain microvessels.

This study describes the biochemical characterization and subcellular distribution of glucose transporters from isolated rat brain cortical microvessels. The D-glucose inhibitable [3H]cytochalasin B binding assay was used to quantitate glucose transporter binding sites in plasma membranes, high-density microsomes and low-density microsomes prepared from basal and insulin-stimulated cells. Incubation with insulin for 30 min increased the number of glucose transporters in the high-density microsomes by around 33% but had no effect on the number of glucose transporters in the plasma membrane or low-density microsomes. Prolonged incubation with insulin (2 h), however, resulted in a small but significant redistribution of glucose transporters to the low-density microsomes. Preincubation of cells with cycloheximide blocked this insulin-induced increase in glucose transporter number, suggesting that this effect of insulin was due to the synthesis of new glucose transport proteins. Specific labeling of glucose transporters was achieved by photoincorporation of [3H]cytochalasin B. Labeled membranes from all fractions contained a single D-glucose inhibitable peak, migrating with a molecular size of 55 kDa on SDS-polyacrylamide gel electrophoresis. Isoelectric focusing of the 55 kDa protein revealed one major peak of D-glucose inhibitable radioactivity focusing at pH 6.0 in all fractions.

Animals↗
PubMed1987

A biochemical and kinetic analysis of the interleukin-1 receptor. Evidence for differences in molecular properties of IL-1 receptors.

This study describes the biochemical characterization and kinetic analysis of the interleukin-1 (IL-1) receptor in Raji human B-lymphoma and EL4 murine T-lymphoma cells. The internalization of 125I-IL-1 was studied in both cell types by an acid extraction technique which removes surface bound ligand. At 37 degrees C, binding to Raji IL-1 receptors was almost entirely cell surface (91%). EL4 cells, in contrast, internalized 59% of ligand at this temperature and this was almost totally inhibited by sodium azide. Receptor binding studies showed that the B-cells had a lower binding affinity but much higher receptor density per cell (KD = 2.1 nM, Ro = 7709) than the T-cells (KD = 0.4 nM, Ro = 241). The receptor binding affinity of two IL-1 analogs, Glu-4 and clone 18, was determined in competitive binding studies. In the B-cells the analogs had binding affinities of 25 and 90%, respectively, whereas in the T-cells the affinities were 0.2 and 200%, respectively. Chemical cross-linking studies showed that the IL-1 receptor in B-cells had a lower molecular weight than that in T-cells (68 kDa compared to 80 kDa). In summary these studies demonstrate that structural differences exist between IL-1 receptors in Raji and EL4 cells.

Animals↗
PubMed1987

Muteins of human interleukin-1 that show enhanced bioactivities.

Using recombinant DNA techniques, we have made a series of amino-terminal muteins of human interleukin-1 (IL-1). Two of the muteins demonstrated 4-7-fold increase in bioactivity as compared to that of the native IL-1. The enhanced biological potency coincides with an increase in both receptor binding affinity and in vivo tumor inhibitory activity. By site specific mutagenesis, we have shown that the arginine at the fourth position of IL-1 is one of the key residues in the function of IL-1. Circular dichroism studies of the amino-terminus analogs showed little structural rearrangement. The change in bioactivity might be due to a change in the stability of the muteins, in the side chain interactions with receptors or in the minor change in folding near the receptor binding site.

Amino Acid Sequence↗
PubMed1987

Dissociation of insulin-stimulated glucose transport from the translocation of glucose carriers in rat adipose cells.

Cycloheximide, a potent inhibitor of protein synthesis, has been used to examine the relationship between recruitment of hexose carriers and the activation of glucose transport by insulin in rat adipocytes. Adipocytes were preincubated +/- cycloheximide for 90 min then +/- insulin for a further 30 min. We measured 3-O-methylglucose uptake in intact cells and in isolated plasma membrane vesicles. The concentration of glucose transporters in plasma membranes and low density microsomes was measured using a cytochalasin B binding assay. Cycloheximide had no affect on basal or insulin-stimulated 3-O-methylglucose uptake in intact cells or in plasma membrane vesicles. However, the number of glucose carriers in plasma membranes prepared from cells incubated with cycloheximide and insulin was markedly reduced compared to that from cells incubated with insulin alone (14 and 34 pmol/mg protein, respectively). Incubation of cells with cycloheximide alone did not change the concentration of glucose carriers in either plasma membranes or in low density microsomes compared to control cells. When isolated membranes were analyzed with an antiserum prepared against human erythrocyte glucose transporter, decreased cross-reactivity was observed in plasma membranes prepared from cycloheximide/insulin-treated cells compared to those from insulin cells. The present findings indicate that incubation of adipocytes with cycloheximide greatly reduces the number of hexose carriers in the plasma membrane of insulin-stimulated cells. Despite this reduction, insulin is still able to maximally stimulate glucose uptake. Thus, these data suggest an apparent dissociation between insulin stimulation of glucose transport activity and the recruitment of glucose carriers by the hormone.

3-O-Methylglucose↗
PubMed1987

Photolabeling of the adipocyte hexose carrier with an aryl azide derivative of maltose.

A nitrophenyl azide derivative of maltose, N-(4-azido-2-nitrophenyl)-maltosylamine (NAP-maltosylamine), was synthesized as a potential photoaffinity label for the hexose carrier of the rat adipocyte. This derivative inhibited 3-O-methylglucose uptake with a Ki of 1.3 mM in the dark, while that of maltose was 10.0 mM. Carbon-14-labeled maltose and NAP-maltosylamine entered adipocytes via the hexose carrier, the latter in a concentrative fashion. Photolysis of NAP-[14C]maltosylamine in the presence of an adipocyte low density microsomal membrane fraction labeled several electrophoretic bands. Among these are a 45 kDa band which showed features expected of the hexose carrier: its labeling was decreased 40% by D- but not L-glucose and pretreatment of intact adipocytes with insulin decreased labeling of the 45 kDa band by 10-40%, as predicted by the translocation theory of insulin-stimulated transport activation. These studies show the suitability of using carbon-1-modified sugar photoaffinity labels as probes for the hexose carrier and possibly of its regulation in rat adipocytes.

Adipose Tissue↗
PubMed1987

Human adipocyte glucose transport system. Biochemical and functional heterogeneity of hexose carriers.

We have investigated glucose transport proteins in isolated human adipocytes. Using the cytochalasin B binding assay to measure glucose transporters in subcellular membrane subfractions, we found that insulin induced translocation of intracellular glucose transporters to the cell surface. Isoelectric focusing of glucose transporters photolabeled with [3H]cytochalasin B revealed two distinct glucose transporter isoforms in low density microsomes focusing at pH 5.6 and pH 6.4, but only the pH 5.6 isoform was detectable in plasma membranes and only the pH 6.4 form was found in the high density microsomes. Insulin recruited only the pH 5.6 glucose transporter from the low density microsomes to the plasma membrane with no effect on the pH 6.4 transporter isoform. The results suggest that the pH 6.4 species is an immature form of the glucose transporter initially located in the high-density microsome fraction, which then migrates to the low-density microsomes where it matures (converted to pH 5.6 species) and becomes available for insulin-mediated recruitment to the plasma membrane.

Adipose Tissue↗
PubMed1986

Biochemical and functional characterization of the rat liver glucose-transport system. Comparisons with the adipocyte glucose-transport system.

The properties of the glucose-transport systems in rat adipocytes and hepatocytes were compared in cells prepared from the same animals. Hormones and other agents which cause a large stimulation of 3-O-methylglucose transport in adipocytes were without acute effect in hepatocytes. Hepatocytes displayed a lower affinity for 3-O-methylglucose (20 mM) and alternative substrates than adipocytes (6 mM), whereas inhibitor affinities were similar in both cell types. The concentration and distribution of glucose transporters were determined by Scatchard analysis of D-glucose-inhibitable [3H]cytochalasin B binding to subcellular fractions. In liver, most of the transporters were located in the plasma membrane (42 +/- 5 pmol/mg of protein) with a small amount (4 +/- 3 pmol/mg) in the low-density microsomal fraction ('microsomes'), the reverse of the situation in adipocytes. Glucose transporters were covalently labelled with [3H]cytochalasin B by using the photochemical cross-linking agent hydroxysuccinimidyl-4-azidobenzoate and analysed by SDS/polyacrylamide-gel electrophoresis. A single D-glucose-inhibitable peak with a molecular mass of 40-50 kDa was seen in both plasma membrane and low-density microsomes. This peak was further characterized by isoelectric focusing and revealed a single peak of specific [3H]cytochalasin B binding at pI 6.05 in both low-density microsomes and plasma membrane, compared with peaks at pI 6.4 and 5.6 in adipocyte membranes. In summary: the glucose-transport system in hepatocytes has a lower affinity and higher capacity than that in adipocytes, and is also not accurately modulated by insulin; the subcellular distribution of glucose transporters in the liver suggests that few intracellular transporters would be available for translocation; the liver transporter has a molecular mass similar to that of the adipocyte transporter; the liver glucose transporter exists as a single charged form (pI 6.05), compared with the multiple forms in adipocytes. This difference in charge could reflect a functionally important difference in molecular structure between the two cell types.

3-O-Methylglucose↗
PubMed1986

Identification and biochemical characterization of the plasma membrane glucose transporter of Leishmania donovani.

The plasma membrane glucose transporter of Leishmania donovani, an obligate intracellular protozoan parasite of humans, was specifically labeled, identified, and biochemically characterized. Cytochalasin B, a known inhibitor of D-glucose transport in mammalian cells, but not cytochalasin E inhibited the transport of 2-deoxy-D-glucose in the extracellular promastigote form of this organism. Hydroxysuccinimidyl-4-azido-benzoate was used to photochemically cross-link [3H] cytochalasin B to the glucose transporter in isolated surface membranes and plasma membrane vesicles of L. donovani promastigotes. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the covalently labeled glucose transporter migrated as a 20-30-kDa protein band. This band was eluted from SDS-PAGE gels and subsequently analyzed by isoelectric focusing. The latter revealed two major peaks focusing at pH 6.8 and 6.6 [3H]Cytochalasin B-labeled membrane activity was detergent-solubilized, bound to concanavalin A-agarose beads, and specifically eluted with alpha-methyl mannoside. Analysis of the eluted material by SDS-PAGE revealed a D-glucose-inhibitable cytochalasin B peak with an apparent Mr approximately 20,000. The cumulative results indicate that the glucose transporter of L. donovani promastigotes is a glycoprotein which contains mannose as its major carbohydrate constituent.

Animals↗
PubMed1986

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↗
PubMed1986

Rapid and effective transfer of integral membrane proteins from isoelectric focusing gels to nitrocellulose membranes.

A method describing the rapid and effective transfer of integral membrane protein from isoelectric focusing gels to nitrocellulose is described. Initial experiments were carried out with detergent-solubilized extracts of human erythrocyte membrane proteins. The effectiveness of the transfer was demonstrated by assaying for erythrocyte glucose transporter, an integral membrane protein, using specific antibodies followed by 125I-protein A and autoradiography. Several detergents including octyl glucoside, Triton X-100 and CHAPS were used in this study but only octyl glucoside effectively solubilized the glucose transporter and did not interfere with the electrotransfer of the protein. The glucose transporter separated on isoelectric focusing gels was effectively transferred after 2 h of electroblotting and was found to have an apparent pI of 6.4-6.5. These findings were substantiated by photolabeling red cell membranes with [3H]cytochalasin B in the presence or absence of D-glucose (which inhibits [3H]cytochalasin B binding to the glucose transporter) and separating the labeled proteins by two dimensional electrophoresis. With this procedure we identified a D-glucose sensitive 50-60 kDa protein focusing with an apparent pI of around pH 6.4-6.5.

Collodion↗
PubMed1986

Biochemical and functional heterogeneity of rat adipocyte glucose transporters.

We have studied the biochemical mechanism of insulin action on glucose transport in the rat adipocyte. Plasma membranes and low-density microsomes were prepared by differential ultracentrifugation of basal and insulin-stimulated cells. The photochemical cross-linking agent hydroxysuccinimidyl-4-azidobenzoate was used to covalently bind [3H]cytochalasin B to the glucose transporter which migrated as a 45-50-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing of the eluted 40-55-kDa proteins revealed two peaks of D-glucose-inhibitable [3H]cytochalasin B radioactivity focusing at pH 6.4 and 5.6 when low-density microsomes were used as the starting material. In contrast, only one D-glucose inhibitable peak, focusing at pH 5.6, was found in plasma membranes. Pretreatment of the cells with insulin led to a marked redistribution of the pH 5.6 form of the glucose transporter from low-density microsomes to plasma membranes with no effect on the pH 6.4 form of the glucose transporter. Following isolation from the isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels, both glucose transporter isoforms were shown to cross-react with an antiserum raised against the purified human erythrocyte glucose transporter. Following incubation of [3H]cytochalasin B-labeled low-density microsomal and plasma membranes with neuraminidase, the pH 5.6 transporter isoform was shifted on isoelectric focusing to a more basic pH, while the pH 6.4 isoform was not affected. These data demonstrate that: there is a heterogeneity of glucose transporter species in the intracellular pool while the plasma membrane transporters are more uniform in structure. The pH 5.6 glucose transporter isoform is translocated by insulin from the low-density microsomes to the plasma membrane but the pH 6.4 isoform is not sensitive to insulin. Differential sensitivity of the glucose transporter isoforms to neuraminidase suggests that the heterogeneity is at least partially due to differences in glycosylation state.

Adipose Tissue↗
PubMed1986

Blood-brain glucose transfer in diabetes mellitus. Decreased number of glucose transporters at blood-brain barrier.

This study describes the effects of streptozocin (STZ)-induced diabetes mellitus on the glucose-transporter system of the rat blood-brain barrier. Subcellular membrane fractions, i.e., plasma membranes and high- and low-density microsomes, were prepared from isolated brain microvessels derived from control and diabetic animals. The number of glucose transporters in each of the membrane fractions from both control and diabetic animals was determined by the D-glucose-inhibitable cytochalasin B-binding assay. The total number of glucose transporters was decreased by 43% in STZ-treated rats compared with controls (35 vs. 115 pmol/mg protein; P less than .05). The glucose-transporter number in plasma membranes was decreased by 50%, in high-density microsomes by 38%, and in low-density microsomes by 45%. Incubation of isolated microvessels from control animals with 7 microM insulin for 30 min at 37 degrees C led to a cycloheximide-sensitive 27% increase (P less than .05) in the number of transporters in high-density microsomes. This insulin effect was significantly diminished to 15% in the diabetic animals (P less than .05). In conclusion, STZ-induced diabetes decreases the glucose-transporter number in all subcellular membrane fractions derived from isolated rat brain microvessels, and the insulin-induced increase in de novo synthesis of glucose transporters in brain microvessels is diminished in these chronically diabetic animals.

Animals↗