Labeling of the active subunit of cholera toxin from within the membrane bilayer.

Using the photoreactive glycolipid probe 12-(4-azido-2-nitrophenoxy)-stearoylglucosamine-[1-¹⁴C], we have effected the radiolabeling of the active A₁ subunit of cholera toxin from within the membrane bilayer. The membrane employed as a target was the envelope of Newcastle disease virus which contained the photoreactive probe. Radiolabeling of the A₁ subunit occurred after cholera toxin and virus were incubated together for 15 min at 37° and then irradiated at 366 nm for 1 min. Labeling of A₁ did not occur when cholera toxin was irradiated in a solution of probe without virus or when the 15 min incubation with virus was performed at 0° instead of at 37°.     

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells

Receptor sites for insulin on GH₃ cells were characterized. Uptake of ¹²⁵I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37°C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with ¹²⁵I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for ¹²⁵I-labeled insulin binding sites. ¹²⁵I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. ¹²⁵I-labeled insulin was degraded at both 4 and 37°C by GH₃ cells, but not by medium conditioned by these cells. After a 5 min incubation at 37°C, products of ¹²⁵I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of ¹²⁵I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of ¹²⁵I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of ¹²⁵I-labeled insulin, as well as intact hormone, were recovered from GH₃ cells. After 30 min incubation at 37°C, 80% of the cell-bound radioactivity was not extractable from GH₃ cells with acetic acid.     

Temperature-Mediated Interaction of Tetanus Toxin with Cerebral Neuron Cultures: Characterization of a Neuraminidase-Insensitive Toxin-Receptor Complex

Abstract: Energy-dependent internalization of ¹²⁵I-labeled tetanus toxin into cultured neural cells is shown to follow an energy-independent binding process. A three-step model, involving receptor-mediated binding followed by sequestration and internalization is proposed. In the first step, binding of toxin is enhanced in appearance under low ionic strength medium, at 0–4°C; it is suppressed, however, with increasing incubation temperature under physiological salt concentrations. Cell-bound toxin is displaced by approximately 35.5% when high-salt medium (physiological concentrations) is added to cells at 0–4°C; the effect is further amplified at 37°C. Addition of disialoganglioside G_D1b (1–5 μg/ml) also lowers the amount of cell-associated toxin. The fraction of ¹²⁵I-labeled toxin retained by the cells after exposure to high-salt medium at 0–4°C or after addition of G_D1b is operationally defined as sequestered toxin. This second step, characterized by a stable association of the toxin with the neural cells, is affected by both physiological salt and by 37°C conditions. Lastly, an energy-dependent phenomenon of firm association of tetanus toxin with neural cells, compatible with internalization, is described. The toxin residing in this fraction is bioactive and cannot be removed by salts, gangliosides, or by treatment with protease or neuraminidase. Binding, sequestration, and internalization are mutually dependent, as they are all blocked by pretreatment of cells with neuraminidase and by an enhanced energy-independent sequestration event, which results in enhanced tetanus toxin internalization by an energy-dependent process.     

Action of cholera toxin on dispersed acini from guinea pig pancreas

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of ⁴⁵Ca or cellular cyclic AMP. Binding of ¹²⁵I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of bindind of ¹²⁵I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in ⁴⁵Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretion or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Lectin and cholera toxin binding to guinea pig tumor (104C1) cell surfaces before and after glycosphingolipid incorporation

¹²⁵I-Labeled $\text{Dolichos biflorus}$ lectin and cholera toxin were used as probes for identification of Forssman- and GM1-type receptor sites on guinea pig tumor (104C1) cell surfaces. Increased binding of ¹²⁵I-labeled lectin and toxin to 104C1 cell surfaces was observed after the cells were treated with exogenous Forssman glycosphingolipid and GM1 ganglioside, respectively. Biosynthesis $\text{in vitro}$ of these two glycosphingolipids from their precursor molecules was established using a membrane preparation isolated from confluent cultures of guinea pig tumor 104C1 cells.     

Subcellular structures involved in internalization and degradation of epidermal growth factor

Epidermal Growth Factor (EGF), a small polypeptide which acts as a mitogen for many cell types, has previously been shown to bind to a specific plasma membrane receptor on 3T3 cells. If ¹²⁵I-EGF is bound to 3T3 cells for one hour at 4°C, it remains predominantly associated with the plasma membrane-containing fractions obtained by subjecting cell supernatants to equilibrium sedimentation on sucrose gradients. When binding is followed by a 10-minute incubation at 37°C, over 50% of the ¹²⁵I-EGF is associated with two internal membrane-containing peaks having higher densities than the plasma membrane. After one hour at 37°C, over 80% of the ¹²⁵I-EGF is degraded and removed from the cells. The most rapidly labeled internal peak corresponds in density to brain-coated vesicles (CVs). Antiserum prepared against coated vehicles from brain precipitates the ¹²⁵I-EGF in this peak. In addition, CVs containing ¹²⁵I-EGF can be co-purified from 3T3 cells exposed to ¹²⁵I-EGF, using brain as a carrier. Several lines of evidence suggest that the other ¹²⁵I-EGF-labeled intracellular peak is ¹²⁵I-EGF in lysosomes. These results provide kinetic and biochemical evidence for a unidirectional pathway for EGF catabolism by 3T3 cells. EGF first binds to the plasma membrane bound receptors, is then moved to the cytoplasm in CVs, and finally appears in lysosomes, where it is degraded and released from the cells. Ten-millimolar NH₄Cl blocks lysosomal hydrolysis of EGF almost completely. Subsequently, EGF internalization is inhibited. This finding suggests that the pathway for EGF internalization and degradation is tightly coupled.     

Action of cholera toxin on dispersed acini from guinea pig pancreas.

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of 45Ca or cellular cyclic AMP. Binding of 125I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of binding of 125I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in 45Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretin or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Interaction of rat liver ribosomal subunits with homologous Met-tRNAs

Rat liver 40 S ribosomal subunits, in the presence of magnesium ions, bind homologous, resolved Met-tRNAs in the absence of added exogenous proteins. The interaction of the aminoacyl-tRNAs with the particle is dependent on the concentration of magnesium ions in the incubation. At various Mg²⁺ concentrations examined, binding of the putative initiator Met-tRNA_i to 40 S subunits is greater than that observed with Met-tRNA_m. Also, binding of Met-tRNA_i to 40 S subunits is greater than that obtained with 40 S plus 60 S particles. The initial rate of formation of the 40 S·Met-tRNA_i complex is greater at 25 °C than at 37 or 4 °C; decay of the complex, which is observed after 15 min of incubation, is greater at 37 °C but it is slower if 60 S subunits are added after the complex has been formed. If 60 S subunits are added to the incubation with 40 S subunits at the start of the reaction, binding of Met-tRNA_i is inhibited; inhibition is also obtained if elongation (binding) factor EF-1 or stripped tRNAs (particularly tRNA^Met) are present in the incubation mixture containing 40 S subunits. Acetyl-Met-tRNA_i binds to 40 S·ApUpG complex to the same extent as unacetylated Met-tRNA_i and, after addition of 60 S subunits, reacts extensively with puromycin; the addition of elongation (translocation) factor EF-2 and GTP do not affect the extent of the puromycin reaction, suggesting that the acMet-tRNA_i is bound to a site on the 40 S subunits which becomes the P site on 80 S ribosomes.     

Testicular membranes with improved stability of the gonadotropin receptor

A plasma membrane fraction has been prepared from rat testis using an aqueous double-phase polymer system containing dextran, poly(ethylene glycol) 6000 and Zn²⁺. The membrane-associated gonadotropin receptor for lutropin and human choriogonadotropin can be markedly stabilized by a thawing-washing step of frozen membranes which prolongs the apparent half-life of the unoccupied membrane-associated receptors from less than 1 h at 37°C to greater than 5 h. Also, no degradation of ¹²⁵I-labeled human choriogonadotropin was detected following incubation with the membrane fraction. The equilibrium binding was characterized by an apparent association constant of 1.6 · 10¹⁰ M^?1 and a receptor content of 33 fmol/mg protein. Binding kinetic yielded an association rate constant of 1.0 · 10⁸ M^?1, while the dissociation rate constant for human choriogonadotropin was too low to be accurately determined under the conditions used. In contrast, ovine lutropin could be reversibly bound to the membranes leaving the previously occupied receptors available for binding by ¹²⁵I-labeled human choriogonadotropin.     

Subcellular distribution of iodothyronine 5′-deiodinase in cerebral cortex from hypothyroid rats

We have examined iodothyronine deiodination in subcellular fractions of cerebral cortex obtained from hypothyroid rats. Enzymatic activities were measured at 37°C in the presence of 20 mM dithiothreitol with ¹²⁵I-labeled T₄ and ¹²⁵I-labeled rT₃ as substrate for 5′-deiodination and ¹³¹I-labeled T₃ as the substrate for the 5-deiodinase. Reaction products were separated by descending paper and/or ion-exchange chromatography. Cerebral cortex subcellular fractions were also characterized by marker enzyme analysis and electron microscopy. Under optimal reaction conditions more than 80% of the 5′-deiodinase was recovered after fractionation. Both 5′-deiodinase and (Na⁺ +K⁺-ATPase showed similar subcellular distributions and were enriched approx. 3-fold in the easily sedimenting membrane fraction and nerve terminal plasma membranes. Crude microsomal membranes (6·10⁶g·min pellet) also showed 2-fold enrichment of these enzymes. Nuclei and isolated mitochondria were devoid of deiodinating activity. T₄ and T₃ 5-deiodinating activity was absent in the easily sedimenting membranes and present but not enriched in particulate fractions containing microsomal membranes. These data suggest that iodothyronine 5′-deiodinase is associated with plasma membrane fractions in the cerebral cortex.     

Ribosomal-membrane interaction: in vitro binding of ribosomes to microsomal membranes

Rat liver rough microsomal membranes were stripped of bound ribosomes by treatment with puromycin and high concentrations of monovalent ions. Ribosomal subunits labeled in the RNA were detached from rough microsomes by the same procedure, recombined into monomers, and then incubated with stripped membranes in a medium of low ionic strength (25 mm-KCl, 50 mm-Tris-HCl, 5 mm-MgCl₂). These ribosomes readily attached to the stripped membranes, as determined by isopycnic flotation of the reconstituted microsomes. The binding reaction was complete after incubation for five minutes at 37 °C, but also proceeded at 0 °C, at a lower rate. Scatchard plots showed a binding constant of ~8 × 10⁷m^?1 and ~5 × 10^?8 mol binding sites per gram of membrane protein. Native rough microsomes showed a much lower binding capacity at 0 °C than stripped rough microsomes, but showed considerable uptake of ribosomes at 37 °C. Smooth microsomes, treated for stripping and incubated at 0 °C, accepted less than half as many ribosomes as stripped rough microsomes. Erythrocyte ghosts were incapable of binding ribosomes. Microsomal binding sites were heat sensitive, were destroyed by a brief incubation with a mixture of trypsin and chymotrypsin in the cold, and were unaffected by incubation with phospholipase C.Ribosome binding was decreased by increasing the concentration of monovalent ions and was strongly inhibited by 10^?4m-aurintricarboxylic acid. Experiments with purified ribosomal subunits revealed that at concentrations of monovalent ions close to physiological concentrations (100 to 150 mm-KCl), microsomal binding sites had a greater affinity for 60 S than for 40 S subunits.Stripped rough microsomes were also capable of accepting polysomes obtained from rough microsomes by detergent treatment. Although this binding presumably involves the correct membrane binding sites, polypeptides discharged from re-bound polymers were not transferred to the vesicular cavities, as in native microsomes. The released polypeptides remained firmly associated with the outer microsomal face, as shown by their accessibility to proteases.     

Binding and internalization of 125I-LDL in normal and mutant human fibroblasts. A quantitative autoradiographic study.

Using a quantitative EM autoradiographic technique, we have visualized the membrane binding and receptor-mediated uptake of low density lipoprotein (LDL) in human fibroblasts. The initial binding was restricted to the plasma membrane (2 h of incubation at 4 °C) and approx. 62% of the grains could be localized to coated pits in the plasma membrane. When the incubations were carried out at 37 °C, ¹²⁵I radioactivity was found both on the membrane and within the cell and predominantly localized on or within lysosomes. In cells from the patient J. D., a familial hypercholesterolemic homozygote with an internalization defect, initial binding of ¹²⁵I-LDL was restricted to the plasma membrane but not preferentially localized to coated segments of the plasma membrane. After incubation for 30 min at 37 °C, the membrane bound ¹²⁵I-LDL in J. D. cells was not internalized. These data confirm results obtained with ferritin-labeled LDL and illustrate the complementary application of two different morphologic probes, each of which offers special advantages for special problems.     

Influence of lectins on the binding of 125I-labeled EGF to human fibroblasts.

Lectins that interact with mannose (concanavalin A), galactose (ricin, abrin), or N-acetylglucosamine (wheat germ agglutinin) block ¹²⁵I-labeled EGF binding to the surface of cultured human fibroblasts at 37° or 5°. Lectins specific for fucose or N-acetylgalactosamine, soybean agglutinin or gorse lectin, respectively, do not interfere with growth factor binding. The inhibition of ¹²⁵I-labeled EGF binding by concanavalin A at 37° or 5° could be reversed rapidly by the addition of α-methyl mannoside. The results suggest that the fibroblast membrane receptor for EGF is, or is closely associated with, a glycoprotein or glycolipid that contains mannose, galactose and N-acetylglucosamine residues.     

Binding, internalization and degradation of colony-stimulating factor by peritoneal exudate macrophages

Iodinated colony-stimulating factor produced by L-cells (¹²⁵I-CSF-1) binds specifically to murine peritoneal exudate macrophages. At 37°C, the cell-bound ¹²⁵I-CSF-1 was internalized and degraded very rapidly, with the appearance of radioactive iodotyrosine in the medium. At 0°C, the cell-bound ¹²⁵I-CSF-1 was not internalized and degraded, nor did it dissociate from the membrane. The internalization and degradation at 37°C could be blocked or reduced by the presence of phenylglyoxal, methylamine and NH₄Cl. The chemical nature of the CSF-1 binding site is polypeptide as judged by its sensitivity to trypsin treatment. After the binding and degradation of unlabeled CSF-1, the exudate cells were no longer able to rebind freshly added ¹²⁵I-CSF-1, indicating the removal of CSF-1 binding site. The binding capacity of these cells, however, could be restored by prolonged incubation at 37°C but not at 0°C in culture medium containing fetal calf serum.     

Actions of cholera toxin on dispersed Chief cells from guinea pig stomach

Although much is known about the actions of cholera toxin on intestinal and extra-gastrointestinal tissues, almost nothing is known about the interaction of this toxin with cells in the stomach. In the present study, we prepared 125I-labeled cholera toxin (1900 Ci/mmol) and examined the binding of this radioligand to dispersed Chief cells from guinea pig stomach. Moreover, we examined the actions of cholera toxin on cellular cAMP and pepsinogen secretion from Chief cells. Binding of 125I-labeled cholera toxin could be detected within 5 min, was maximal by 60 min, and was increased by increasing the radioligand or cell concentrations. Inhibition of binding by unlabeled toxin indicated a dissociation constant of 3 nM and 8.7 X 10(5) cholera toxin receptors per Chief cell. In contrast to the rapidity of binding, a cholera toxin-induced increase in cAMP and pepsinogen secretion was not detected until 30-45 min of incubation. A 3 to 6-fold increase in cAMP and pepsinogen secretion was observed with maximal concentrations of cholera toxin. Binding of 125I-labeled cholera toxin and the toxin's actions on cAMP and pepsinogen secretion were inhibited by the B subunit of the toxin. Binding was not altered by other agents that have been shown to stimulate pepsinogen secretion (carbachol, CCK-8, secretin, vasoactive intestinal peptide, prostaglandin E1, or forskolin). These data indicate that Chief cells from guinea pig stomach possess a specific class of cholera toxin receptors. Binding of cholera toxin to these receptors causes an increase in cellular cAMP that stimulates pepsinogen secretion.     

Nicotinic acetylcholine receptor of mammalian brain: naja toxin binding to subcellular fractions of rat brain

Abstract— Subcellular fractions of rat brain cortex bound 6.8 ± 0.4 fmol of ¹²⁵I-labeled α-naja toxin per milligram of protein. Toxin binding was saturable and specific e.g. nicotinic agonist and antagonist blockable. Regions in rat brain varied in saturable, specific toxin binding with hippocampus, olfactory area and corpus striatum displaying greatest toxin binding. We conclude that ¹²⁵I-α-naja toxin is a suitable probe for nicotinic cholinergic receptor in brain.     

Saturable binding to cell membranes of the presynanptic neurotoxin, β-Bungarotoxin

Brief exposure to the protein neurotoxin, β-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using ¹²⁵I-labeled toxin. At low membrane protein concentration ¹²⁵I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration ¹²⁵I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes.In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of ¹²⁵I-labeled toxin binding sites on brain mitochondrial prepartions ($\text{3.3 ± 0.3}\text{pmol/mg}$) exceeded by a factor of ten the density on liver mitochondrial preparations ($\text{0.3 ± 0.05}\text{pmol/mg}$). It is also shown that the labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude the β-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.     

Synthesis of a photoreactive, radiolabelled derivative of the oligosaccharide of GM1 ganglioside.

A photoreactive, radioiodinatable derivative of the oligosaccharide (GM1OS) of ganglioside GM1 was synthesized as follows: GM1OS was generated from GM1 by ozonolysis and alkaline fragmentation, and reductively aminated to GM1OSNH2 (1-amino-1-deoxymonosialogangliotetraitol). The latter compound was then reacted with N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA) to form GM1OSNH-ASA [1-(4-azidosalicoylamido)-1-deoxymonosialogangliotetraitol], which was radioiodinated and further purified. To test the [125I]GM1OSNH-IASA [1-(4-iodoazidosalicoylamido)-1-deoxymonosialogangliotetraitol+ ++] as a probe for ganglioside-binding proteins, the derivative was incubated with cholera toxin, which specifically binds GM1, followed by photolysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The probe only labelled the B or binding subunit of cholera toxin, but not the A or adenylyl cyclase activating subunit. Labelling was inhibited by excess GM1OS, but not by the oligosaccharides from gangliosides GD1a and GD1b. [125I]GM1OSNH-IASA and analogous oligosaccharide derivatives may be valuable probes for detecting ganglioside-binding proteins.     

Temporal behavior of abrin in the intoxication of chinese hamster cells (line CHO)

Abrin, a potent cytotoxin, was utilized as a probe to elucidate the mechanism by which external proteins are delivered to the cytoplasm of mammalian cells. Abrin bound rapidly to the surface receptors of the Chinese hamster cells (line CHO) and appeared to be internalized immediately without any significant lag. The maximum level of abrin internalization was achieved within eight minutes, based on both biochemical and electron microscopic autoradiographic studies with [¹²⁵|] abrin. About 10% of the silver grains of internalized [¹²⁵|] abrin were associated with vesicular structures, irrespective of the incubation time. Inhibition of protein synthesis began 30 minutes postincubation, and this latent period was not dependent on extracellular toxin concentration. SDS-polyacrylamide gel electrophoresis of the internalized [¹²⁵|] abrin indicated that internalized abrin molecules remained intact even after two hours of incubation.