Preparation and characterization of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and related stilbene disulfonates

4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and other 4,4'-stilbene-2,2'-disulfonate derivatives used as reagents in histochemistry and physiology have been prepared in their E isomeric form, and rearranged to the Z isomers by irradiation with visible light. Infrared, and 1H and 13C nuclear magnetic resonance spectra were recorded for these compounds, and used to establish the chemical structures. In particular, it was shown that the E-isomer of SITS decomposed in aqueous solution by hydrolysis of both the acetamido and isocyano groups yielding a diamine; disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) also decomposed in solution, while disodium 4,4'-dinitrostilbene-2,2'-sulfonate (DNDS) rearranged from the E-isomer to the Z-isomer when solutions were kept unprotected from light. These results indicate that benchworkers should not be surprised when commercial samples of such stilbenes contain large amounts of various types of impurities.     

Chloride permeability of membrane vesicles isolated from Torpedo californica electroplax.

The Cl- permeability of membrane vesicles prepared from the electric organ of the marine ray Torpedo californica was studied by means of radioactive tracer exchange and by measuring the changes in the scattered-light intensity caused by osmotically induced volume changes. Both types of experiments indicate that a substantial fraction of the vesicles is extremely permeable of Cl-. Furthermore, this permeability pathway is inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, a well-known inhibitory of anion transport in a variety of systems. The properties of this permeability pathway are consistent with its identification as the voltage-aged Cl- channel studied in planar bilayers.     

Inactivation of the Na,K-ATPase by modification of Lys-501 with 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS).

The sodium pump or Na,K-ATPase, maintains the Na+ and K+ gradients across eukaryotic cell membranes at the expense of ATP. Incubation of purified canine renal Na,K-ATPase with 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) inhibited the ATPase activity. Both the labeling of the protein and the loss of ATPase activity were prevented by co-incubation with ADP (acting as an ATP analog) or KCl. Only the alpha-subunit was labeled by SITS. The alpha-subunit from the inhibited enzyme was extensively digested with trypsin, and SITS-labeled peptides were purified by reverse-phase HPLC and sequenced. The amino acid sequence determined, His-Leu-Leu-Val-Met-X-Gly-Ala-Pro-Glu, indicated that SITS modifies Lys-501 (X) on the alpha-subunit of Na,K-ATPase.     

Inhibition of sulfate transport in ehrlich ascites tumor cells by 4-acetamido-4′-isothiocyano-stilbene-2,2′-disulfonic acid (SITS)

The effects of the nonpenetrating amino reactive reagent 4-acetamido-4′-isothiocyano-stilbene-2-2′-dilsulfonic acid (SITS) on anion transport (sulfate, chloride, and inorganic phosphate) were investigated in Ehrlich ascites tumor cells. Short time exposure to SITS produces a reversible inhibition (92%) of sulfate transport. The kinetics of interaction suggest that reversibly bound SITS competitively inhibits sulfate transport, Ki = 3 × 10^?6 M. Incubation of tumor cells with SITS (1 × 10^?4 M) for longer periods of time results in a time dependent irreversible inhibition of sulfate transport which obeys first order kinetics. The rate coefficient for the inactivation process is 0.040 min^?1. The kinetics of irreversible inhibition is best explained by the irreversible binding of SITS to the sulfate transport site, and therefore makes SITS a potentially useful probe for the quantitation of these sites in the tumor cell. The lack of effect of irreversibly bound SITS on either chloride or inorganic phosphate transport points to a specificity in the interaction of SITS with the tumor cell membrane, as well as indicating that an alternate pathway exists for the movement of these anions across the membrane.     

Acetylcholine receptor: characterization of the voltage-dependent regulatory (inhibitory) site for acetylcholine in membrane vesicles from Torpedo californica electroplax

Evidence for a voltage-dependent regulatory (inhibitory) site on the nicotinic acetylcholine receptor to which acetylcholine binds was obtained in membrane vesicles prepared from the Torpedo californica electric organ. Two rate coefficients, JA and alpha, which pertain to the receptor-controlled ion flux, were measured. A 1000-fold concentration range of acetylcholine was used in a transmembrane voltage (Vm) range from 0 to -48 mV under a voltage-clamped condition at pH 7.4, 1 degrees C. The following observations were made. (i) At low acetylcholine concentrations, the value of JA, the rate coefficient for ion translocation by the active (nondesensitized) state of the receptor, increased with increasing concentration. (ii) JA decreased at high acetylcholine concentrations. (iii) In contrast, alpha, the rate coefficient for receptor desensitization, did not show such a decrease. (iv) When the transmembrane potential of the vesicle membrane was changed to more negative values, the value of KR (the dissociation constant for binding of acetylcholine to the regulatory site) decreased by a factor of approximately 9 for a 25 mV change in Vm, while KI (the dissociation constant for binding of acetylcholine to the receptor site that controls channel opening) did not show such a change and has a value of 80 microM. When Vm is -48 mV, KR has a value of 8 microM. (v) The effect of a transmembrane voltage on the regulatory site was reversible and occurred within the time resolution (5 ms) of the quench-flow technique used in the measurements.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cholinergic ligands and local anesthetics on acetylcholine receptor enriched membrane preparations from Torpedo californica electroplax.

Pyrene was introduced in acetylcholine receptor (AcChR)-rich membrane preparations of Torpedo californica electroplax. The lifetime of the singlet excited state of pyrene was used to probe the properties of the hydrocarbon regions of the lipid bilayer as well as the possible perturbing effects of cholinomimetic agents on this region. After excitation with a single 15-ns pulse with a Q-switched ruby laser, the lifetime of the pyrene singlet excited state in the membranes was 200 ns. In desensitized membranes the pyrene fluorescence lifetimes remained unchanged when the cholinergic ligands carbamylcholine, d-tubocurarine, decamethonium, and hexamethonium, as well as α-bungarotoxin, were present. By contrast, the lifetime was shortened when local anesthetics were present. In sensitized membranes no changes in the pyrene lifetimes were detected when the membranes were converted from their resting state to a carbamylcholine-induced “desensitized state.” Water-soluble fluorescence quenchers affected the lifetime of pyrene in membranes. The second order rate constants for the pyrene-quencher interaction were used to detect changes in fluidity and/or membrane lipid accessibility to quenchers induced by ligands or anesthetics. No changes were detected in the quenching constants of nitromethane or Tl⁺ in the presence of cholinergic agents (with the exception of d-tubocurarine); on the other hand, a marked decrease in Tl⁺ accessibility was induced by the anesthetics procaine and tetracaine. Fluorescene dynamics measurements indicate that the hydrocarbon core of the bulk lipid in electroplax is not significantly affected by binding cholinergic ligands to membranebound AcChR. However, the hydrophobic region of the membrane is perturbed by both local anesthetics and one cholinergic ligand, d-tubocurarine. Pyrene was also incorporated into lipid vesicles prepared from T. californica electroplax lipids. The fluorescence lifetimes and quenching values of these lifetimes yielded results similar to those obtained with both sensitized and “desensitized” membrane preparations. The d-tubocurarine effect on the Tl⁺ quenching of the pyrene probe is ascribed to direct interaction of d-tubocurarine with the lipids. These findings favor a mechanism in which perturbation of the hydrophobic (lipid) environment of the AcChR in membranes by local anesthetics and even d-tubocurarine may influence the receptor conversion: sensitized state ? desensitized state.     

Primary structure of the human, membrane-associated Ca2+-binding protein p68 a novel member of a protein family.

cDNA clones encoding human 'p68', a membrane-associated Ca2+-binding protein, were isolated from a lambda gt11 expression library of the human T-leukaemia cell line J6, by using a rabbit antiserum against denatured purified lymphocyte p68, and from a liver cDNA library by using 32P-labelled p68 cDNA fragments. The amino acid sequence of p68, deduced from the sequences of overlapping cDNA clones, is described. The results show that p68 is closely related to a family of proteins which includes intracellular substrates of the EGF receptor and pp60src tyrosine kinases. The p68 amino acid sequence is internally repetitive, being constructed from eight repeats of varying lengths, each of which contains a highly conserved sequence. Multiple copies of the latter sequence are also present in the related proteins p36, lipocortin I and protein II. We discuss how the common structural features of these proteins may reflect common functions and, furthermore, how the eight repeat structure of p68 may have evolved. The sequences of independent cDNAs suggest that alternatively-spliced mRNAs could encode different p68 protein species. This suggestion is consistent with the observation that purified p68 migrates as a closely-spaced doublet when analysed by SDS-PAGE.     

Molecular and biochemical characterization of a novel oxysterol-binding protein (OSBP2) highly expressed in retina

We are interested in understanding the possible function(s) of the oxysterol-binding proteins in mediating oxysterol cytotoxicity in the retina. In this study we describe the cloning, localization, and biological activity of a novel oxysterol-binding protein (OSBP2), and complete the molecular characterization of the previously known OSBP1. Both OSBP genes contain 14 exons and have similar exon sizes and splice sites suggesting they may have arisen from a gene duplication event. OSBP1 is located in chromosome 11q12.1, and OSBP2 is located in 22q12. At the protein level they share 63% overall similarity and although they have unique N termini, both have similar pleckstrin homology domains within the N terminus region. Northern blot analyses indicate that OSBP1 is broadly expressed in human and monkey tissues. OSBP2 is detected mainly in retina, testis, and fetal liver. Western blot analysis using peptide antibodies specific to OSBP1 and OSBP2 detected the proteins in different subcellular fractions in the retinal monkey tissue. OSBP1 is detected mainly in the soluble or cytosolic fraction and nuclei whereas OSBP2 is detected exclusively in the detergent soluble fraction suggesting association with membranes. Immunohistochemical localization of OSBP1 and OSBP2 in the monkey retina placed these two proteins in similar but distinct areas of the inner retina. OSBP2 was found to bind 7-ketocholesterol but to have very little affinity for cholesterol or 25-hydroxycholesterol.     

The acetylcholine receptor as part of a protein complex in receptor-enriched membrane fragments from Torpedo californica electric tissue

The acetylcholine receptor from Torpedo californica electric tissue consisting of polypeptide chains of molecular weight 42000 (+/- 2000) is part of a protein complex. Cross-linking experiments with bifunctional reagents have shown that this complex has possibly a pentameric structure with a molecular weight of 270000 (+/- 30000). Besides the receptor subunit (alpha-chain), at least three further classes of polypeptide chains are part of the complex: beta (Mr 48000), gamma (Mr 62000) and delta (Mr 68000). This can be shown by cross-linking the proteins extracted from receptor-enriched membrane fractions with a cleavable reagent: From the 270000 molecular weight particle the four predominant polypeptide chains of the membrane, alpha, beta, gamma, and delta, can be obtained. The gamma-polypeptide chains appear to form a dimer connected by an inter-chain disulphide bridge.     

Synip: a novel insulin-regulated syntaxin 4-binding protein mediating GLUT4 translocation in adipocytes.

Insulin-stimulated glucose transport and GLUT4 translocation require regulated interactions between the v-SNARE, VAMP2, and the t-SNARE, syntaxin 4. We have isolated a novel syntaxin 4-binding protein, Synip, which specifically interacts with syntaxin 4. Insulin induces a dissociation of the Synip:syntaxin 4 complex due to an apparent decrease in the binding affinity of Synip for syntaxin 4. In contrast, the carboxyterminal domain of Synip does not dissociate from syntaxin 4 in response to insulin stimulation but inhibits glucose transport and GLUT4 translocation. These data implicate Synip as an insulin-regulated syntaxin 4-binding protein directly involved in the control of glucose transport and GLUT4 vesicle translocation.     

Purification of a novel UV-damaged-DNA binding protein highly specific for (6-4) photoproduct.

UV damage-specific binding proteins are considered to play important roles in early responses of cells irradiated with UV, including damage recognition in the DNA repair process. We have surveyed nuclear and cytoplasmic proteins which bind selectively to UV-irradiated DNA using an electrophoretic mobility shift assay. We detected four distinct binding activities with different mobilities in fractions separated from HeLa cells by heparin chromatography. Three of them were found in nuclear extracts and one in cytoplasmic extracts. We purified one of the binding factors from nuclear extracts to homogeneity, which was designated NF-10 (the 10th fraction of nuclear extract on heparin chromatography). It migrated as a 40 kDa polypeptide in SDS-PAGE, and bound to UV-irradiated double- stranded DNA but not to unirradiated DNA. The binding pattern of the NF-10 protein to DNA irradiated with UV corresponded to the induction kinetics of (6-4) photoproduct. Removal of (6-4) photoproducts from UV- irradiated DNA by (6-4) photoproduct-specific photolyase diminished the binding of NF-10 protein. These results suggest that the NF-10 protein binds to UV-damaged DNA through (6-4) photoproduct. Immunoblot analysis using a monoclonal antibody revealed that the NF-10 protein was expressed in cell lines from all complementation groups of xeroderma pigmentosum, indicating that the NF-10 protein is a novel UV-damaged-DNA binding protein.     

Identification of Glypican3 as a novel GLUT4-binding protein

Insulin stimulates glucose uptake in fat and muscle primarily by stimulating the translocation of vesicles containing facilitative glucose transporters, GLUT4, from intracellular compartments to the plasma membrane. Although cell surface externalization of GLUT4 is critical for glucose transport, the mechanism regulating cell surface GLUT4 remains unknown. Using a yeast two-hybrid screening system, we have screened GLUT4-binding proteins, and identified a novel glycosyl phosphatidyl inositol (GPI)-linked proteoglycan, Glypican3 (GPC3). We confirmed their interaction using immunoprecipitation and a GST pull-down assay. We also revealed that GPC3 and GLUT4 to co-localized at the plasma membrane, using immunofluorescent microscopy. Furthermore, we observed that glucose uptake in GPC3-overexpressing adipocytes was increased by 30% as compared to control cells. These findings suggest that GPC3 may play roles in glucose transport through GLUT4.     

A novel strategy for production of a highly expressed recombinant protein in an active form.

Under standard growth conditions, E. coli transformed with the high-level expression vector pMON5525 produces recombinant DMAPP/AMP transferase in inactive, insoluble complexes. We have produced large amounts of active, soluble protein by growing and inducing the cells under osmotic stress in the presence of sorbitol and glycyl betaine. This caused an increase of up to 427-fold in the active yield, and the disappearance of the protein from the pelletable fraction of cell extracts. This treatment may have wide applicability.     

Functional characterization of a novel serotonin receptor (5-HTap2) expressed in the CNS of Aplysia californica

Serotonin has been shown to be a neuromodulator in the Aplysia californica CNS. The diversity of serotonin actions is due to the existence of several different receptor subtypes. In this study we report the cloning of a full-length cDNA, coding for a novel serotonin receptor (5-HTap2). The receptor protein bears the characteristics of G protein-coupled receptors. It shares 68% and 34% of its amino acid sequence identity with the 5-HTlym receptor from Lymnaea stagnalis and the mammalian 5-HT1A receptor, respectively. When transfected in HEK 293 cells, 5-HTap2 was negatively coupled to adenylate cyclase. Ligand binding analysis indicated that the order of potencies of various drugs for the inhibition of [3H]LSD binding was: methiothepin > metergoline > 5-CT > PAPP > 5-HT > ketanserin > NAN-190 > 8-OH-DPAT > clozapine. RT-PCR amplification of RNA isolated from different tissues indicated that this receptor is expressed in the CNS and in bag cells. The expression of 5-HTap2 restricted to the CNS suggests an important role for this receptor in the modulation of neuronal functions in Aplysia. Moreover, the high expression of 5-HTap2 in the bag cells, associated with its pharmacological profile, suggests that this receptor may be implicated in modulating the afterdischarge during the egg-laying behavior.     

Effects of 4-acetamido-4'-isothiocyano-2,2-disulfonic stilbene on ion transport in turtle bladders.

The disulfonic stilbene (4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene) is found to be more potent than acetazolamide as an anion transport inhibitor in the turtle bladder, but less potent than acetazolamide as a carbonic anhydrase inhibitor. The anion-dependent (HCO-3,Cl-) moiety of the short-circuiting current is eliminated by 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene, but only after its addition to the serosal bathing fluid. Whereas 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene has no effect on Na+ transport across the bladder, it is more potent than ouabain as an inhibitor of microsomal (Na+ + K+)-ATPase of both turtle bladder and eel electric organ.     

Restin: a novel intermediate filament-associated protein highly expressed in the Reed-Sternberg cells of Hodgkin''s disease.

We have identified a cDNA coding for a protein of 160 kDa which is expressed in in vitro cultured human peripheral blood monocytes. The predicted amino acid sequence contains an alpha-helical rod domain possessing features characteristic of intermediate filament proteins. However, the immunocytochemical staining pattern, abundance and solubility in Triton X-100/high salt buffers suggest that this protein is probably only associated with the intermediate filament network and represents a new type of intermediate filament associated protein. In a survey of normal, inflammatory and human tumour tissue samples, this protein, which we have named restin, was found to be highly expressed in Reed-Sternberg cells, the tumoral cells diagnostic for Hodgkin's disease. We suggest that restin overexpression may be a contributing factor in the progression of Hodgkin's disease.