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PubMed1999

The correlation and prediction of the solubility of compounds in water using an amended solvation energy relationship.

The aqueous solubility of liquids and solids, as log S(W), has been correlated with an amended solvation equation that incorporates a term in Sigma alpha(2)(H) x Sigma beta(2)(H), where the latter are the hydrogen bond acidity and basicity of the solutes, respectively. Application to a training set of 594 compounds led to a correlation equation with a standard deviation, SD, of 0.56 log units. For a test set of 65 compounds, the SD was 0.50 log units, and for a combined correlation equation for 659 compounds, the SD was 0.56 log units. The correlation equations enable the factors that influence aqueous solubility to be revealed. The hydrogen-bond propensity of a compound always leads to an increase in solubility, even though the Sigma alpha(2)(H) x Sigma beta(2)(H) term opposes solubility due to interactions in the liquid or solid. Increase in solute dipolarity/polarizability increases solubility, whereas an increase in solute excess molar refraction, and especially, volume decrease solubility. The solubility of Bronsted acids and bases is discussed, and corrections for the fraction of neutral species in the saturated solution are graphically presented.

Algorithms↗

PubMed1995

Estimation of recombinant bovine somatotropin solubility by excluded-volume interaction with polyethylene glycols.

PURPOSE: The potential to estimate protein solubilities, with limited protein, by excluded-volume interactions was evaluated using polyethylene glycols (PEG) and recombinant bovine Somatotropin (rbSt). METHODS: Solutions of rbSt were prepared at concentrations significantly below saturation solubility. Subsequently, varying amounts of PEG were added to force protein precipitation. Following centrifugation, the protein concentration in the supernatant was assayed by reversed-phase HPLC, where a logarithmic relationship between solubility and % PEG was observed. RESULTS: An apparent protein solubility in the absence of PEG was determined by extrapolation and compared well with values measured by conventional approaches. Slopes of log solubility versus % PEG curves were consistent with excluded-volume principles and depended on the molecular weight of the PEG used. Furthermore, the precipitation process proved to be reversible, allowing for recovery of intact protein. Solubility-pH profiles obtained in the presence of PEG greatly reduced the quantities of protein needed and compared favorably with profiles in the absence of PEG. CONCLUSIONS: Thus, it appears feasible and practical, with certain limitations, to obtain solubility estimates of proteins by volume-exclusion methods with limited supplies of protein.

Animals↗

PubMed1999

Vitamin E-TPGS increases absorption flux of an HIV protease inhibitor by enhancing its solubility and permeability.

PURPOSE: To investigate the effect of vitamin E-TPGS, d-alpha-tocopheryl polyethylene glycol 1000 succinate, on the solubility and permeability of amprenavir, a potent HIV protease inhibitor. METHODS: The aqueous solubility of amprenavir was measured as a function of vitamin E-TPGS concentration. Directional transport through Caco-2 cell monolayers was determined in the presence and absence of vitamin E-TPGS and P-glycoprotein inhibitors. Absorption flux was estimated from Caco-2 cell permeability and aqueous solubility. RESULTS: The solubility of amprenavir in a pH 7 buffer at 37 degrees C was 0.036+/-0.007 mg/mL. The solubility linearly increased with increasing vitamin E-TPGS concentration (above 0.2 mg/mL). Polarized transport was demonstrated in the basolateral to apical direction, exceeding apical to basolateral transport by a factor of 6. The active efflux system was inhibited by vitamin E-TPGS and known P-glycoprotein inhibitors verapamil and GF120918. CONCLUSIONS: The solubility of amprenavir was improved in the presence of vitamin E-TPGS through micelle solubilization. Vitamin E-TPGS inhibits the efflux system and enhances the permeability of amprenavir. Overall, vitamin E-TPGS enhanced the absorption flux of amprenavir by increasing its solubility and permeability. The enhancement is essential to the development of the novel soft gelatin capsule formulation of amprenavir for use in the clinic.

Algorithms↗

PubMed2003

In vitro permeability of poorly aqueous soluble compounds using different solubilizers in the PAMPA assay with liquid chromatography/mass spectrometry detection.

PURPOSE: This study compares the use of UV-VIS detection with liquid chromatography/mass spectrometry (LC/MS) detection for the PAMPA (Parallel Artificial Membrane Permeability Assay) permeability determination of compounds in the drug discovery stage. LC/MS detection offers a selective and sensitive method for the determination of the PAMPA permeability for compounds that do not contain a UV chromophore or possess a low UV extinction coefficient. To enhance the reliability of our permeability measurements for compounds with low aqueous solubility, we demonstrated the use of LC/MS detection as a means for facilitating the study of solubilizing agents to enhance aqueous solubility that normally would interfere with UV-VIS detection. In doing so, the PAMPA assay can be expanded to study the in vitro permeability of poorly water soluble compounds and evaluate the effects of solubilizers' on the membrane permeability of different compounds. This might be useful in selecting solubilizers for poorly water soluble compounds to be used for further in vivo studies. METHODS: A diverse set of 20 drugs using UV-VIS detection were compared with data using LC/MS detection. A PAMPA screening method was designed which used solubilizers (Brij 35, Cremophor EL, ethanol, and Tween 80) for compounds with low aqueous solubility. The stability of the artificial membrane was determined using various solubilizer concentrations (0.1-5% w/v) to ensure the phospholipid membrane was not disrupted. Two compounds, amiodarone and miconazole, with low aqueous solubility yielding an undetected response in the PAMPA assay using UV-VIS detection were subjected to the different solubilizing agents and their PAMPA permeability was measured using LC/MS detection. RESULTS: Most of the compounds showed similar PAMPA permeability using the two detection systems. However, for compounds lacking a UV chromophore or with a low UV extinction coefficient, LC/MS was the detection method of choice for determination of PAMPA permeability values. LC/MS also gave reliable quantification data for compounds containing impurities, as well as compounds that were not stable during the assay. Although many solubilizers were found to interfere with UV-VIS detection, the LC/MS approach was applicable to determine the permeability values of compounds with normally low aqueous solubility. CONCLUSIONS: LC/MS detection offered greater sensitivity and selectivity as compared with UV-VIS detection for the PAMPA assay. With this added versatility in detection, PAMPA can be used in both discovery and pre-formulation applications, which has not been described before.

Gas Chromatography-Mass Spectrometry↗

PubMed2004

A quantitative structure-property relationship for predicting drug solubility in PEG 400/water cosolvent systems.

PURPOSE: A quantitative structure-property relationship (QSPR) was developed to predict drug solubility in binary mixtures of polyethylene glycol (PEG) 400 and water. The ability of the QSPR model to predict solubility was assessed and compared to the classic log-linear cosolvency model. METHODS: The solubility of 122 drugs, ranging in log P from -2.4 to 7.5, was determined in 0%, 25%, 50%, and 75% PEG (v/v in water) by the shake-flask method. Solubility data from 84 drugs were fit by linear regression using the following molecular descriptors: molecular weight, volume, radius of gyration, density, number of rotatable bonds, hydrogen-bond donors, and hydrogen-bond acceptors. The multiple linear regression model was optimized by a genetic algorithm guided selection method. The remaining 38 compounds were used to test the predictability of the model. RESULTS: QSPR-based models developed at each volume fraction with the training set compounds showed a reasonable correlation coefficient (r) of approximately 0.9 and a root mean square (rms) error of <0.5 log unit. The model predicted solubility values of approximately 78% of the testing set compounds within 1 log unit. The log-linear model was as effective as the QSPR-based model in predicting the testing set solubilities; however, many drugs, as expected, showed significant deviation from log-linearity. CONCLUSIONS: The QSPR model requires only the chemical structure of the drug and has utility for guiding vehicle identification for early preclinical in vivo studies, especially when compound availability is limited and experimental data such as aqueous solubility and melting point are unknown. When experimental data are available, the log-linear model was verified to be a useful predictive tool.

Models, Chemical↗

PubMed2004

Biodegradable self-assembling PEG-copolymer as vehicle for poorly water-soluble drugs.

PURPOSE: To develop self-assembling systems increasing the solubility of poorly water-soluble drugs. METHODS: Low molecular weight liquid biodegradable copolymers were synthesized by ring-opening polymerization using caprolactone (CAP) and trimethylenecarbonate (TMC) as monomers. Various initiators were evaluated. The emulsifying and self-assembling properties were investigated by a water titration method. The self-assembling systems were characterized for size, shape, isotropic behavior, cloud point, surface charge, and critical micellar concentration in order to optimize the polymer synthesis. Finally, the improvement of solubility of model drugs was assessed. RESULTS: Only diblock monomethyl ether PEG-CAP/TMC copolymers synthesized with monomethyl ether polyethyleneglycol 550 to 2000 as initiator have shown self-assembling properties: upon dilution, these copolymers formed an isotropically clear solution with droplet sizes in the range of 20 to 100 nm. The hypothesis that these diblock polymers form micelles was confirmed by their low critical micellar concentration (10(-5) g/ml). The copolymers initated with mmePEG750 had a higher cloud point and better colloidal stability than those initiated with mmePEG 550. The solubility of the poorly water-soluble drugs was increased by 1 to 2 orders of magnitude. Good reproducibility was observed from batch to batch. CONCLUSIONS: The polyester diblock copolymer mmePEG750-CAP/TMC forms spontaneously stable micelles in aqueous medium and increases the solubility of lipophilic drugs. They are very promising vehicles for the oral delivery of poorly water-soluble drugs.

Biocompatible Materials↗

PubMed1981

Cytotoxic and clastogenic effects of soluble and insoluble compounds containing hexavalent and trivalent chromium.

Cr(III) and Cr(VI) compounds of varying solubilities have been tested in vitro for their ability to inhibit cell growth and nucleic acid and protein syntheses in BHK cells, to induce alterations in the mitotic cycle in HEp cells, and to increase the frequency of chromosomal aberrations and sister chromatid exchanges (SCE) in CHO cells. All Cr(VI) compounds, and particularly those containing soluble Cr(VI), such as potassium dichromate and zinc yellow, differentially inhibit macromolecular syntheses in BKH cells, that of DNA being always the most affected. Among Cr(III) compounds, which generally have very low cytotoxicity, chromite is particularly active, and inhibits cell growth and DNA synthesis even more than the poorly soluble Cr(VI) compounds. Preincubation in growth medium, with or without metabolizing cell cultures, solubilizes considerable amounts of Cr(VI) from zinc yellow and chromite, but significant amounts are also obtained from the most insoluble Cr(VI) pigments. When BHK cells are treated with such preincubated solutions, reduction of soluble Cr(VI) to Cr(III) by cell metabolites is seen with all Cr(VI) compounds, accompanied by decreased cytotoxicity. The same differences between Cr(VI) and Cr(III) compounds apply to the cytotoxic effects on mitosis of HEp cells and the clastogenic effects on CHO cells. The activity of chromite, the only Cr(III) pigment capable of significantly increasing the frequency of SCE, is due to contamination with soluble Cr(VI). In contrast to the very low cytotoxicity of Cr(III), much higher chromium levels are detected in the cells incubated with soluble Cr(III) than with the same concentrations of soluble Cr(VI). 50% and 75% of chromium accumulated in the cells during treatments with Cr(VI) and Cr(III) respectively remains firmly bound to the cells, even when they are incubated for up to 48 h in normal growth medium. Chromium accumulated in the cells after treatment with Cr(III) is most probably bound to the cell membrane, whereas some of the Cr(VI) is transported through the cell membrane and reduced in the cell nucleus. The results of the present investigation are in agreement with those obtained with the same Cr(VI) and Cr(III) compounds in mutagenicity assays in bacteria and carcinogenicity tests in rodents. A re-evaluation of the mechanisms of chromium carcinogenisis is proposed.

Animals↗

PubMed1968

The cell wall of Bacillus licheniformis N.C.T.C. 6346. Isolation of low-molecular-weight fragments from the soluble mucopeptide.

1. Soluble mucopeptide was prepared by lysozyme treatment of acid-extracted walls of Bacillus licheniformis N.C.T.C. 6346 and separated into fractions differing in molecular size by chromatography on Sephadex G-25 and G-50. 2. About 16% of the weight of soluble mucopeptide has a weight-average molecular weight in excess of 20000. About one half has a weight-average molecular weight of less than 2000 and the balance of soluble mucopeptide is of intermediate size. 3. In the mucopeptide fractions isolated from Sephadex there is a correlation between the weight-average molecular weight, the number of non-reducing muramic acid residues and the proportion of diaminopimelic acid residues recovered after treatment with 1-fluoro-2,4-dinitrobenzene. 4. The extent of cross-linking between peptide side chains is relatively low, even in mucopeptide material of the large molecular size. 5. The small amount of residual phosphorus present in preparations of B. licheniformis soluble mucopeptide remains associated mainly with mucopeptide material of large molecular size. 6. The mucopeptide components of lowest molecular weight are not produced as artifacts during the preparation of soluble mucopeptide, but are apparently incorporated in the insoluble mucopeptide present in walls of exponentially growing cells. 7. Soluble mucopeptide isolated in a complex with acidic polymers after lysozyme treatment of walls of B. licheniformis N.C.T.C. 6346 and Bacillus subtilis W23 retains a high molecular weight when the covalent bonds between mucopeptide and the acidic polymers are broken. 8. Pure fragments were isolated from B. licheniformis soluble mucopeptide. A major component, C1, of the material of smallest size is made up of one residue each of N-acetylglucosamine, N-acetylmuramic acid, l-alanine, glutamic acid and diaminopimelic acid. The N-acetylglucosamine is in beta-glycosidic linkage with a reducing N-acetylmuramic acid residue. The peptide unit is probably amidated. A quantitatively minor component, C2, has amino acid and amino sugar composition identical with that of component C1, but probably lacks an amide group. Another fragment, B1, is made up of two molecules of component C1 or C2 that are joined together through a molecule of d-alanine.

Amino Acids↗

PubMed1991

The soluble 'low-Km' 5'-nucleotidase of rat kidney represents solubilized ecto-5'-nucleotidase.

A soluble 'low-Km' 5'-nucleotidase has been described previously in several organs. It has been presumed to be of cytosolic origin and thus to play a role in the intracellular production of adenosine. Its catalytic properties are similar to those of the ecto-5'-nucleotidase of cell membranes. In the present study we compared molecular properties of the two enzymes in the kidney of the rat. The Mr of the main peak of soluble 'low-Km' 5'-nucleotidase in gel-filtration chromatography was similar to that of the ecto-5'-nucleotidase solubilized by a phosphatidylinositol-specific phospholipase C from renal brush-border membranes. In phase-partition experiments using Triton X-114, the soluble enzyme appeared to be hydrophobic. Its hydrophobicity was decreased on treatment with a phosphatidylinositol-specific phospholipase C, suggesting that the soluble 'low-Km' 5'-nucleotidase contains the phosphatidylinositol anchor which is characteristic for the ecto-enzyme. An anti-ecto-5'-nucleotidase antiserum provoked an almost complete inhibition of the soluble enzyme. Immunoblotting using anti-ecto-5'-nucleotidase antiserum revealed in the high-speed supernatants a polypeptide with a similar Mr to the subunit of the ecto-5'-nucleotidase. The soluble 'low-Km' 5'-nucleotidase, like the ecto-5'-nucleotidase, bound specifically to concanavalin A. We conclude that the soluble 'low-Km' 5'-nucleotidase is not a cytosolic enzyme, but that it most probably originates from the solubilization of the ecto-5'-nucleotidase, and that it therefore cannot participate in the intracellular production of adenosine.

5'-Nucleotidase↗

PubMed1998

Suppression of immunoglobulin secretion by soluble annexin II.

Annexin II (AII) belongs to a family of glycoproteins that bind negatively charged phospholipids in the presence of calcium. The annexins exert various biological functions. We have previously shown that soluble AII suppresses mitogen-induced lymphoproliferation in vitro. In this study we address the question of whether soluble AII may also affect immunoglobulin secretion. Mononuclear cells from peripheral blood were stimulated with pokeweed mitogen in vitro and immunoglobulin-secreting cells were quantified using an ELISPOT assay. Retroplacental serum and soluble AII significantly inhibited secretion of IgG and IgM when added at concentrations that did not affect lymphoproliferation or cell viability. The inhibitory effect was dose- and time dependent. Significant suppression was observed when soluble AII was added at concentrations of 0.1 and 0.01 microg/ml. The strongest inhibition was observed when soluble AII or retroplacental serum was added initially. The data demonstrate that soluble AII can suppress immunoglobulin secretion in vitro. AII seems to be a potent immunosuppressive substance. The presence of high levels of soluble AII in retroplacental serum may indicate a possible immunomodulatory role in normal pregnancy.

Animals↗

PubMed2002

Extra terminal residues have a profound effect on the folding and solubility of a Plasmodium falciparum sexual stage-specific protein over-expressed in Escherichia coli.

The presence of extra N- and C- terminal residues can play a major role in the stability, solubility and yield of recombinant proteins. Pfg27 is a 27K soluble protein that is essential for sexual development in Plasmodium falciparum. It was over-expressed using the pMAL-p2 vector as a fusion protein with the maltose binding protein. Six different constructs were made and each of the fusion proteins were expressed and purified. Our results show that the fusion proteins were labile and only partially soluble in five of the constructs resulting in very poor yields. Intriguingly, in the sixth construct, the yield of soluble fusion protein with an extended carboxyl terminus of 17 residues was several fold higher. Various constructs with either N-terminal or smaller C-terminal extensions failed to produce any soluble fusion protein. Furthermore, all five constructs produced Pfg27 that precipitated after protease cleavage from its fusion partner. The sixth construct, which produced soluble protein in high yields, also gave highly stable and soluble Pfg27 after cleavage of the fusion. These results indicate that extra amino acid residues at the termini of over-expressed proteins can have a significant effect on the folding of proteins expressed in E. coli. Our data suggest the potential for development of a novel methodology, which will entail construction of fusion proteins with maltose binding protein as a chaperone on the N-terminus and a C-terminal 'solubilization tag'. This system may allow large-scale production of those proteins that have a tendency to misfold during expression.

Amino Acid Sequence↗

PubMed1997

Increased solubility of high-molecular-mass neurofilament subunit by suppression of dephosphorylation: its relation to axonal transport.

To investigate the role of phosphorylation in the turnover and transport of neurofilament (NF) proteins in vivo, we studied their solubility properties and axonal transport in the rat sciatic nerve using phosphatase inhibitors to minimize dephosphorylation during preparation. About 20% of the 200-kDa subunit (NF-H) in the axon was soluble in the 1% Triton-containing buffer under the present conditions, whereas this amount was less and more variable in the absence of phosphatase inhibitors. The 68-kDa subunit (NF-L) was exclusively insoluble and not affected by the inhibitors. Such selective solubilization of NF-H by phosphorylation differed significantly from the in vitro phosphorylation with cyclic AMP-dependent protein kinase, which resulted in NF disassembly. The carboxy-terminal phosphorylation state of NF-H probed with the phosphorylation-sensitive antibodies was also not directly related to solubility. The solubility of NF-H did not differ along the nerve. In contrast, the solubility of L-[35S]methionine-labeled, transported NF-H was lowest at the peak of radioactivity. Higher solubility at the leading edge, regardless of its location along the nerve, indicates that NF-H solubility is positively correlated with the rate of NF transport.

Animals↗

PubMed2003

Soluble interleukin-2 receptors increase during the active periods in cluster headache.

OBJECTIVE: To investigate whether cytokines are altered during the active period of cluster headache. BACKGROUND: Patients with cluster headache show activation of the hypothalamus in PET studies and via endocrinologic parameters. Data also suggest an inflammatory process occurs in cluster headache. A connection between the presumed inflammatory cause, an immunological activation, and the hypothalamus could be generated by certain cytokines. DESIGN AND METHODS: ELISA was used to determine the serum levels of soluble interleukin-2 receptors, interleukin-1, interleukin-6, and 2 soluble interleukin-6 receptors (sIL-6R and soluble gp130) in 18 patients with cluster headache (6 women and 12 men) during the cluster period and in 17 healthy controls who were headache-free (3 women and 14 men). RESULTS: Patients with cluster headache had significantly increased soluble interleukin-2 receptors (413.6+/-223 U/mL vs. 290.0+/-112 U/mL; P <.05) compared with controls. Serum levels of interleukin-1 (0.29+/-0.30 pg/mL vs. 0.13+/-0.13 pg/mL, n.s.), interleukin-6 (0.87+/-0.6 pg/mL vs. 0.91+/-0.7 pg/ml; n.s.), soluble interleukin-6 receptors (33,131+/-8,349 pg/mL vs. 35,063+/-7,606 pg/mL; n.s.), or soluble gp130 (289+/-59 pg/mL vs. 283+/-20 pg/mL; n.s.) did not differ between the 2 groups, although patients with cluster tended to have higher interleukin-1 values. CONCLUSIONS: Because elevated soluble interleukin-2 receptors indicate T cell activation, our findings suggest immune activation during cluster headache. Because interleukin-2 can activate the hypothalamus and stimulate the release of Corticotropin-releasing Factor (CRF), interleukin-2 could link a putative immunological cause of cluster headache with the observed hypothalamic activation. Systemic changes of interleukin-1 or the interleukin-6 system do not seem to play a role in cluster headache, as no alterations of serum levels were observed. Even so, unchanged serum levels do not exclude limited local production.

Adult↗

PubMed2002

High serum concentrations of soluble E-selectin correlate with obesity but not fat distribution in patients with type 2 diabetes mellitus.

Serum concentrations of soluble adhesion molecules, eg, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin are elevated in patients with type 2 diabetes. However, little is known about the role of obesity or abnormal fat distribution in inducing upregulation of adhesion molecules. To investigate this issue, soluble ICAM-1, VCAM-1, and E-selectin levels were evaluated in 40 obese and 30 nonobese patients with type 2 diabetes. Both groups were matched for age, sex, and glycosylated hemoglobin (HbA(1c)) levels. Computed tomography (CT) was used to measure the abdominal subcutaneous and visceral fat areas. Soluble ICAM-1 and VCAM-1 levels did not differ significantly between obese and nonobese patients. However, serum concentrations of soluble E-selectin were significantly higher in obese than in nonobese patients (90 +/- 7 v 56 +/- 4 ng/mL, P <.01). Soluble E-selectin levels significantly correlated with body mass index, subcutaneous fat area, and visceral fat area (Rho = 0.48, 0.37, and 0.30, respectively). Stepwise multiple regression analysis showed that body mass index (F = 16.7), but not subcutaneous and visceral fat areas (F = 0.29 and 0.01, respectively), significantly and independently correlated with soluble E-selectin levels. Our results suggest that obesity may induce endothelial activation or increased shedding of cell surface E-selectin that leads to subsequent increase in soluble E-selectin levels. The high serum concentrations of E-selectin closely correlated with increased total fat volume, but not with regional fat distribution.

Abdomen↗

PubMed1985

Identification of a distinct soluble subunit of an intermediate filament protein: tetrameric vimentin from living cells.

Intermediate-sized filaments (IF) are among the most insoluble intracellular protein polymer structures. We have analyzed the small amounts of soluble vimentin, an IF protein, present in cytosol fractions obtained from lysis of cultured cells [rat RVF-SM cells, simian virus 40-transformed human fibroblasts, and human rhabdomyosarcoma (RD line) cells]. The molecular form of this soluble vimentin was determined by sucrose density gradient centrifugation, using vimentin-specific antibodies for subsequent ELISA and immunoblotting analyses. The majority of the soluble vimentin appeared in a distinct form indistinguishable in its sedimentation behavior from reconstituted tetrameric subunits of purified vimentin arrested at low ionic strength. The tetrameric coiled-coil nature of the soluble form of vimentin was indicated by the digestion pattern with chymotrypsin and by chemical crosslinking with copper-1,10-phenanthroline and dimethylsuberimidate. The competence of this soluble vimentin to assemble into IF at higher salt concentrations was demonstrated by electron microscopy. Pulse-chase experiments showed that the soluble form was not an exclusively posttranslational intermediate. We propose that in the living cell a small pool of a distinct soluble tetrameric form of vimentin exists which may exchange with polymeric IF vimentin.

Animals↗

PubMed1988

Soluble interleukin 2 receptors are released from the cell surface of normal murine B lymphocytes stimulated with interleukin 5.

Murine T and B lymphocytes can be induced to release soluble interleukin 2 receptors (IL2Rs). This receptor is believed to be a truncated form of the 55-kDa chain of the cell-membrane-associated receptor. It has been speculated that this receptor may play an important immunoregulatory role by binding to interleukin 2 (IL-2). We report here that interleukin 5 can induce normal murine B cells to release soluble IL2Rs. This extends our finding that interleukin 5 similarly can induce murine B cells to express functional cell-surface-associated IL2Rs. Two possible mechanisms of release of soluble IL2Rs have been suggested. Soluble IL2R could be synthesized as a secretory form of the receptor lacking the transmembrane domain or by cleavage of the extracellular domain of the cell-surface-associated IL2R at the cell surface. To investigate which mechanism was operative, we radioiodinated the cell surface of normal murine splenocytes that had been cultured for 1 day with Con A to stimulate the expression of cell-surface-associated IL2Rs and the release of soluble IL2Rs. Under the conditions used, radiolabeling of internal proteins was not apparent. Labeled cells were then recultured with Con A, conditioned medium was taken from replicate cultures at various times after radioiodination, and the specific radioactivity of released soluble IL2Rs was determined by ELISA and RIA. We demonstrate that the specific radioactivity and the kinetics of change of the specific radioactivity are consistent with the hypothesis that the soluble IL2Rs are derived from the cell-surface-associated IL2Rs rather than being released in a secretory form.

Animals↗

PubMed1991

Identification and molecular cloning of a soluble human granulocyte-macrophage colony-stimulating factor receptor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in hematopoiesis and host defense via interaction with specific cell-surface receptors in target tissues. We identified a truncated, soluble form of the low-affinity GM-CSF receptor (GMR) in chorio-carcinoma cells. Low-affinity GMR cDNAs encoding both the membrane-bound and soluble receptors were obtained by PCR using primers corresponding to the published sequence. Clones encoding the soluble receptor were identical in sequence to the membrane-bound form but contained a 97-nucleotide internal deletion. The amino acid sequence of this deleted cDNA predicts a protein that lacks the 84 C-terminal amino acids of the membrane-bound receptor, including the transmembrane and cytoplasmic domains, and contains 16 different amino acids at its C terminus. Expression of the soluble GMR cDNA in murine psi-AM cells as well as GM-CSF-dependent myeloid 32Dc13 cells produced a secreted protein that retained its capacity to bind GM-CSF in solution. RNase protection analysis indicates that this variant cDNA is derived from a naturally occurring mRNA. Soluble receptors have been identified for several other hematopoietin receptors and may be a general feature of this class. The striking similarity between the soluble form of the GMR and other hematopoietin receptors suggests that soluble binding proteins may play an important role in regulating the broad spectrum of biological responses mediated by these cytokines.

Amino Acid Sequence↗

PubMed1998

Platelets interact with soluble and insoluble collagens through characteristically different reactions.

Platelet interaction with soluble and insoluble collagens was characterized through binding studies. In contrast to resting platelets, cells reacted with activators, TS2/16 (integrin alpha2 beta1-activating antibody), thrombin, collagen-related peptide, or ADP, exhibited specific soluble collagen binding that is Mg2+-dependent, but inhibited by prostaglandin I2, Ca2+, and Gi9 (anti-integrin alpha2 beta1 antibody). Each platelet has 1500-3500 soluble collagen binding sites, with a dissociation constant of 3. 5-9 x 10(-8) M. This is the first study to show the specific binding of soluble collagen to platelets; our data strongly suggest that the receptor is integrin alpha2 beta1 after it becomes activated upon platelet activation. These results suggest that activation of platelets transforms integrin alpha2 beta1 to a state with higher affinity binding sites for soluble collagen. The soluble collagen-platelet interaction was compared with the platelet interaction with fibrillar collagen, which has until now not been demonstrated to bind specifically to platelets. Here, we demonstrated specific, biphasic fibrillar collagen binding. One phase is rapid and metal ion-independent, and accounts for most of the binding. The other phase is slow and Mg2+-dependent. The characteristic differences in the specific bindings of soluble and fibrous collagens demonstrate the different contributions of two different collagen receptors.

Animals↗