Quenching of intrinsic tryptophan fluorescence in membranes of rat pituitary cells.

The GH4C1 strain of hormone-producing rat pituitary cells has specific receptors for the tripeptide thyrotropin-releasing hormone (TRH). Membranes prepared from GH4C1 cells show intrinsic tryptophan fluorescence which was quenched by low concentrations (10--100 nM) of TRH and Ntau-methyl TRH but not by biologically inactive analogs of TRH. Membranes from GH4C1 cells were subjected to thermal denaturation. A conformational transition was noted above 40 degrees C and an irreversible denaturation was observed at 52 degrees C. TRH-induced quenching of intrinsic fluorescence was lost completely in membranes previously incubated for 10 min at 30 degrees C while loss of [3H]-TRH binding was only about 20% at this temperature. Collisional quenching by iodide revealed that about 38% of the tryptophanyl residues in GH4C1 membranes were exposed to solvent. Quenching by TRH occurred with a shift in wavelength maximum from 336 to 342 nm suggesting that few of the tryptophanyl residues quenched by the tripeptide are totally exposed. Membranes prepared from cells preincubated with 20 nM TRH for 48 h, in which TRH receptors were decreased to 30% of control values, showed no quenching of tryptophan fluorescence in response to freshly added TRH. We conclude that the TRH-receptor interaction in GH4C1 cells is associated with a change in membrane conformation that can be measured by differential spectrofluorometry of intrinsic tryptophan fluorescence.     

Intrinsic tryptophan fluorescence of membranes of GH3 pituitary cells: quenching by thyrotropin-releasing hormone.

The intrinsic tryptophan fluorescence of membranes prepared from the GH₃ strain of hormone-producing pituitary cells was monitored by spectrofluorometry. Membranes of GH₃ cells have specific receptors which bind thyrotropin-releasing hormone (TRH). When TRH binds to GH₃ membranes there is quenching of tryptophan fluorescence. The kinetics of the change in fluorescence of GH₃ membranes and of TRH binding are similar. In addition, the concentration of TRH required to produce a half-maximum change in fluorescence is 10 nM, and that required for half-maximum binding of TRH to receptors is 11 nM. Inactive TRH analogs which do not bind to TRH receptors likewise do not alter GH₃ membrane fluorescence, and a pituitary cell strain which lacks TRH receptors does not change membrane fluorescence on incubation with TRH. We conclude that the TRH-receptor interaction in GH₃ membranes is associated with a change in membrane conformation that is readily measured by differential spectrofluorometry.     

Hydrocortisone increases the number of receptors for thyrotropin-releasing hormone on pituitary cells in culture.

Hydrocortisone (cortisol) increased the binding of thyrotropin-releasing hormone (TRH) to specific membrane receptors in 4 clonal strains of rat pituitary cells. At the highest effective cortisol concentration (3–5 × 10^?6 M), the increase was observed within 6–8 hr and became maximal (140 to 160% of control binding) by 18–24 hr. Half-maximum stimulation occurred in serum-containing medium at 9 × 10^?8 M cortisol, and a significant increase in TRH binding was seen at 3 × 10^?8 M. Equilibrium binding studies showed that enhanced TRH binding was explained by an increase in receptor number with no change in affinity. Similar effects were seen with Dexamethasone, but no increase in TRH binding was noted when testosterone, methyltestosterone, progesterone, estradiol or the antiestrogen Lilly 88571 were added to the culture medium. Cortisol treatment did not cause the appearance of specific TRH binding sites in cell strains previously shown to lack receptors for the tripeptide (F₄C₁, GH₁2C₁ and R₅ cells). When added cortisol was removed from medium, receptor number decayed to control values with a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of about 30 hr. Previous studies have shown that TRH receptors in GH-cells can be down-modulated by TRH and thyroid hormones; the present findings demonstrate that glucocorticoid hormones can increase the number of TRH receptors in GH-cells.     

Structural determinants of the intrinsic fluorescence emission in notexin and phospholipase A2 enzymes

Fluorescence measurements of the homologous proteins, notexin and PLA₂ enzymes fromNaja naja atra, Naja nigricollis, and Hemachatus haemachatus venoms, showed that the wavelength of maximum emission and the quantum yield of their intrinsic fluorescence emission spectra were different. To verify the factors which affected their fluorescence characteristics, the dynamics of tryptophan residues in those homologous proteins were studied by quenching with acrylamide, iodide, and cesium. The degrees of exposure of tryptophanyl groups in notexin and PLA₂ enzymes assessed by acrylamide quenching were found to be the major factor that determined their fluorescence characteristics. However, the positively charged groups surrounding tryptophan residues of PLA₂ enzymes fromN. naja atra andN. nigricollis venoms might affect the quantum yield of their fluorophores. Tryptophan residues of notexin were in an environment with less fluctuation, which did not allow free diffusion of ionic quencher. This might render its typtophan residues to fluoresce at a shorter wavelength. These results suggested that the structural determinants affecting the intrinsic fluorescence emission of homologous proteins can be easily assessed by quenching studies.     

Interaction of bovine serum albumin and long-chain imidazolium ionic liquid measured by fluorescence spectra and surface tension

The binding of long-chain imidazolium ionic liquid (IL), 1-tetradecyl-3-methylimidazolium bromide (C₁₄mimBr) to bovine serum albumin (BSA) was investigated by fluorescence spectra and surface tension. Fluorescence spectra show that tryptophan (Trp) residues, one of the intrinsic fluorophores in BSA, are buried in a hydrophobic microenvironment with the addition of C₁₄mimBr, which induces the denaturation of BSA. Moreover, the fluorescence quenching mechanism was determined to be static quenching. The equilibrium constant (K) and the number of binding sites (n) were calculated based on the results of fluorescence measurement. The critical aggregation concentration (CAC) and critical micelle concentration (CMC) under different BSA concentrations at various temperatures were investigated based on the surface tension plots. Surface tension indicates that C₁₄mimBr binds to BSA through electrostatic attraction at low C₁₄mimBr concentrations (below CMC) and through hydrophobic interaction at high C₁₄mimBr concentrations (above CMC). Additionally, the thermodynamic parameters of micelle formation were determined. This study provides an understanding of the binding of C₁₄mimBr to BSA.     

Quenching of the fluorescence of proteins by silver nitrate

The mechanisms by which Ag⁺ may quench protein tryptophanyl fluorescence have been studied. A 1:1 Ag⁺-tryptophan complex was detected spectrophotometrically and shown to have a k_a = 6.5 × 10³ M^?1. The complex was nonfluorescent. Ag⁺ and NO₃^? each caused collisional quenching which proceeded at nearly diffusion-controlled rates in a series of indole-containing compounds. Analysis of the rates by means of Stern-Volmer plots and lifetime measurements showed also that charge and the presence of salt influence the quenching rate constants.The fluorescence of nonsulfhydryl proteins was quenched by AgNO₃ only in concentrations needed for Stern-Volmer quenching of simple indole model compounds. However, the plots for protein quenching were generally nonlinear, a reflection of the heterogeneity of tryptophanyl residues. AgNO₃ quenching increased the polarization of protein fluorescence and decreased the lifetime. Rotational relaxation times were determined from Perrin plots of reciprocal polarization vs fluorescence intensity in the presence of various amounts of AgNO₃.The fluorescence of the sulfhydryl proteins ovalbumin, yeast, and equine liver alcohol dehydrogenases was strongly quenched by AgNO₃ in parallel with the formation of Ag⁺-mercaptide bonds. The quenching of fluorescence of sulfhydryl proteins was exhibited even in 8 m urea, thus ruling out conformational change as a major basis for the quenching. It was found that Ag⁺ mercaptide bond formation was accompanied by development of an ultraviolet absorption band. The reaction of Ag⁺ with cysteine, for example, could be followed spectrophotometrically. The uv absorption of different silver mercaptides varied with the compound and pH.Since the uv absorption of Ag⁺-mercaptides extended up to 340 nm, and was also found in Ag⁺-treated sulfhydryl proteins, energy transfer from excited tryptophans seemed a reasonable basis for the observed fluorescence quenching. This possibility was confirmed by calculation of Förster critical transfer distances for a variety of donor-acceptor (Ag⁺-mercaptide) pairs.The lifetime of sulfhydryl protein fluorescence was decreased by AgNO₃, but the emission spectrum was relatively little affected, in contrast to previously reported quenching by Hg²⁺. Additional mechanisms of fluorescence alteration by Ag⁺ in proteins (e.g., “heavy atom” effect, conformational changes, enhancement of sulfhydryl quenching) are also considered.The spectral effects of Ag⁺ interaction with proteins have the following practical applications:determination of —SH groups; probe of accessibility of binding sites and tryptophan-sulfhydryl distances; determination of rotational relaxation times by Perrin plots of reciprocal polarization vs lifetime; kinetic studies of Ag⁺ interaction with proteins.     

Stereological and biochemical analysis of prolactin and growth hormone secreting rat pituitary cells in culture

Summary The secretion of prolactin is increased by treatment of prolactin producing rat pituitary cells with the hypothalamic tripeptide thyroliberin. To investigate the underlying mechanisms we used three closely related rat pituitary tumor cell strains (GH₁2C₁, GH₃ and GH₄C₁), which synthesize and spontaneously secrete prolactin and/or growth hormone. Growth hormone and prolactin released into the culture medium over a period of 24 h were measured by radioimmunoassay. Initial rates of synthesis were measured by immunoprecipitation of intracellular growth hormone and prolactin after incubation of cell cultures with ³H-leucine. The observed increase in prolactin synthesis and release was correlated with morphological effects of thyroliberin treatment. The volume density of Golgi complexes and the volume and surface densities of rough endoplasmic reticulum were compared in untreated cells and thyroliberin treated cells. As normal distribution could not be assumed, the non-parametric rank test of Wilcoxon was used whereby the densities calculated for each cell section were ranked. Alle three morphological parameters increased after thyroliberin treatment in cells secreting prolactin only (GH₄C₁), implying that the increase of prolactin secretion, at lest in part, is due to increased prolactin synthesis.     

Fluorescence quenching of tryptophan by trifluoroacetamide

Trifluoroacetamide was found to be a good quencher of tryptophan fluorescence, and the quenching was shown to proceed via both a dynamic and a static process. The respective quenching constants were determined by the measurement of the decrease of the fluorescence lifetime in the presence of the quencher. The static and the bimolecular rate quenching constants of N-acetyltryptophanamide are equal to 0.34 1·mol^?1 and 1.9·10⁹ 1·mol^?1·s^?1, respectively. These values indicate that trifluoroacetamide is an efficient quencher of tryptophan fluorescence. This conclusion is also supported by a complete quenching of bovine serum albumin and wheat germ agglutinin fluorescence. In the case of lysozyme, trifluoroacetamide quenches the fluorescence of tryptophan residues which fluoresce with a maximum at 348 nm but not the buried tryptophan residues which fluoresce with a maximum at 333 nm. Trifluoroacetamide quenching of wheat germ agglutinin emission confirms the homogeneity and the high accessibility of emitting tryptophan residues, in agreement with a previous report (Privat, J.P. and Monsigny, M. (1975) Eur. J. Biochem. 60, 555–567). The tryptophan fluorescence decay of wheat germ agglutinin is biexponential even in the presence of the quencher; the static and bimolecular rate quenching constants are equal to 0.22 1·mol^?1 and 092·10⁹ 1·mol^?1·^?1, respectively. In the presence of a specific lectin ligand, the methyldi-N,N′-trifluoroacetyl-β- chitobioside, the quenching of wheat germ agglutinin fluorescence involves a direct contact between tryptophan residues and trifluoroacetamido groups of the ligand and in contrast with the quenching induced by free trifluoroacetamide shows that the tryptophan fluorescence is not fully quenched.     

Defective thyroliberin-induced prolactin synthesis and release in a hybrid GH strain

Summary The hybrid GH cell strain, 928-9b, isolated from PRL+ (prolactin [PRL] producing) GH₄Cl and PRL (PRL non-producing) FIBGH12CI cells, has specific TRH (thyroliberin) receptors, yet does not respond to this peptide hormone. Unlike the parent strain, GH₄Cl, TRH does not stimulate synthesis or release of PRL in the hybrid strain. In contrast, treatment of 928-9b cells with another peptide, EGF (epidermal growth factor), stimulates both release and synthesis of PRL. The number of EGF receptors in the hybrid strain (2.5 × 10³/cell) and the affinity of these receptors for ligand (2.2 nM) are comparable to that of the parent strain, GH₄C₁. The EGF dose response curve is also essentially the same for parent and hybrid cells for the enhancement of PRL production. A 3-8-fold enhancement of PRL production is observed and 1/2 maximal enhancement occurs at approximately 5 × 10¹¹ M EGF for both strains. TRH does not have any potentiating effect on EGF-induced stimulation of PRL release or PRL synthesis in the hybrid strain. Although EGF and TRH have similar biological effects in responsive GH cells, binding of one hormone to its receptors does not modulate the binding of the heterologous hormone. These findings demonstrate that more than one effect of TRH is defective in 928-9b cells even though EGF responses are intact. This suggests that 1) TRH-stimulated PRL release and TRH-stimulated PRL production have a common intermediate step, and 2) TRH and EGF have a different mechanism of action in GH cells.     

Steroid-protein interactions. Fluorescence quenching of progesterone-binding globulin and alpha1-acid glycoprotein upon binding of steroids.

The influence of progesterone and four other steroids on the intrinsic fluorescence of progesterone-binding globulin was investigated. The corresponding effect of progesterone on α₁-acid glycoprotein was also studied. The intrinsic fluorescence of the progesterone-binding globulin and of α₁-acid glycoprotein was quenched by about 60 and 17%, respectively, upon forming stoichiometric complexes with progesterone. Graphical analysis of fluorescence quenching titrations with progesterone gave affinity constants at 23 °C of 2 × 10⁹m^?1 for progesterone-binding globulin and 1 × 10⁶m^?1 for α₁-acid glycoprotein. With progesterone-binding globulin, affinity constants of 1 × 10⁹m^?1 were determined for desoxycorticosterone, 1 × 10⁸m^?1 for testosterone, and 2 × 10⁶m^?1 for cortisol. The fluorescence quenching of PBG by 5-pregnen-3β-ol-20-one, 5α-pregnanedione, and 5β-pregnanedione, steroids lacking the Δ⁴-3-keto grouping, was too small to be evaluated; however, binding of the pregnanediones to progesterone-binding globulins was demonstrated when the progesterone-progesterone-binding globulin complex was “unquenched” as a result of competitive displacement of progesterone by addition of the pregnanediones. The quenching phenomenon is assumed to be mainly due to radiationless transfer from protein to the near uv (n → π1) absorption band of steroids containing the Δ⁴-3-keto chromophore.     

Probing the interactions of hemoglobin with antioxidant flavonoids via fluorescence spectroscopy and molecular modeling studies

Steady state and time resolved fluorescence spectroscopy, combined with molecular modeling computations, have been used to explore the interactions of two therapeutically important flavonoids, fisetin (3,7,3′,4′-OH-flavone) and 3-hydroxyflavone (3-HF), with normal human hemoglobin (HbA). Distinctive ‘two color’ fluorescence signatures and fairly high fluorescence anisotropy (r = 0.12-0.28) of fisetin and 3-HF reveal their specific interactions with HbA. Binding constants estimated from the fluorescence studies were ≈ 4.00 × 10⁴ M^− 1 and 9.83 × 10³ M^− 1 for fisetin and 3-HF respectively. Specific interactions with HbA were further confirmed from flavonoid-induced static quenching of the protein tryptophan fluorescence as indicated by: (a) bimolecular quenching constant K_q ? diffusion controlled limit (b) closely matched values of Stern-Volmer quenching constant and binding constant (c) τ_o/τ ≈ 1 (where τ_o and τ are the unquenched and quenched tryptophan fluorescence lifetimes respectively). Molecular docking and electrostatic surface potential calculations reveal contrasting binding modes of fisetin and 3-HF with HbA.     

Fluorescence quenching due to mercuric ion interaction with aromatic amino acids and proteins

Mercuric ion interacts with indoles, including tryptophan, to produce complexes whose absorption spectra are broader, less structured, and red-shifted as compared with those of the parent compound. Fluorescence and phosphorescence are totally quenched. In a survey of the effect of transition metal ions on tryptophan fluorescence, the strong quenching by Hg²⁺ was unique among the uncolored ions. Mercuric nitrate quenched the fluorescence of practically every protein tested, but the sensitivity to quenching varied with the protein. Ovalbumin was the most sensitive to quenching by Hg²⁺, over 70% of the intrinsic fluorescence being quenched by 2 moles of mercuric ion. Difference absorption spectra show that sulfhydryl groups are attacked by these reagents and Hg²⁺ is, in addition, perturbing the environment near some tryptophans. In contrast to Hg²⁺, Zn²⁺ had negligible effect on protein fluorescence. The emission spectra of proteins which were partly quenched by mercuric ion showed shifts in their maxima to higher or lower wavelengths. This suggests that mercuric ion quenched certain tryptophans more than others, and supports the idea that protein fluorescence is heterogeneous and arises from tryptophans in different microenvironments.     

Role of calcium in the thyrotropin-releasing hormone-stimulated release of prolactin from pituitary cells in culture.

Thyrotropin-releasing hormone (TRH) or 50 mM K⁺ stimulated the acute release of prolactin from the GH₄C₁ strain of rat pituitary cells in culture. The enhanced release of prolactin was inhibited in a dose-related manner by the Ca⁺² antagonist Co⁺² (2.0 to 0.5 mM) as well as by the Ca⁺² chelator EGTA (1.0 mM). Co⁺² also reduced spontaneous basal prolactin release. There was partial reversal of the inhibitory effect of Co⁺² (2.0 mM) by Ca⁺² (2.0 mM) and complete reversal of the inhibitory effect of EGTA (1.0 mM) by Ca⁺² (2.0 mM). The enhanced release of prolactin stimulated by 50 mM K⁺ was maximal by 10–20 minutes in medium containing 0.67 to 0.74 mM Ca⁺². Na⁺ (50 mM) did not mimic the effect of high K⁺. We conclude that Ca⁺² is an essential cation in mediating the actions of high external K⁺ and TRH on the release of prolactin by GH₄C₁ cells.     

Structural determinants of the intrinsic fluorescence emission in notexin and phospholipase A2 enzymes

Fluorescence measurements of the homologous proteins, notexin and PLA₂ enzymes fromNaja naja atra, Naja nigricollis, and Hemachatus haemachatus venoms, showed that the wavelength of maximum emission and the quantum yield of their intrinsic fluorescence emission spectra were different. To verify the factors which affected their fluorescence characteristics, the dynamics of tryptophan residues in those homologous proteins were studied by quenching with acrylamide, iodide, and cesium. The degrees of exposure of tryptophanyl groups in notexin and PLA₂ enzymes assessed by acrylamide quenching were found to be the major factor that determined their fluorescence characteristics. However, the positively charged groups surrounding tryptophan residues of PLA₂ enzymes fromN. naja atra andN. nigricollis venoms might affect the quantum yield of their fluorophores. Tryptophan residues of notexin were in an environment with less fluctuation, which did not allow free diffusion of ionic quencher. This might render its typtophan residues to fluoresce at a shorter wavelength. These results suggested that the structural determinants affecting the intrinsic fluorescence emission of homologous proteins can be easily assessed by quenching studies.     

Time resolved spectroscgpy of tryptopkyl fluorescence of yeast 3-phosphoglycerate kinase

The tryptophyl fluorescence emission of yeast 3-phosphoglycerate kinase decreases from pH 3.9 to pH 7.2 following a normal titration curve with an apparent pK of 4.7. The fluorescence decays have been determined at both extreme pH by photocounting pulse fluorimetry and have been found to vary with the emission wavelength. A quantitative analysis of these results according to a previously described method allows to determine the emission characteristics of the two tryptophan residues present in the protein molecule. At pH 3.9, one of the tryptophan residues is responsible for only 13% of the total fluorescence emission. This first residue has a lifetime τ₁= 0.6 ns and a maximum fluorescence wavelength λ_2max = 332 nm. The second tryptophan residue exhibits two lifetimes τ₂₁= 3.1 ns and τ₂₂= 7.0 ns (λ_2max= 338 nm). In agreement with the attribution of τ₂₁and τ₃₂ to the same tryptophan residue, the ratio β = C₂₁/C₂₂ of the normalized amplitudes is constant along the fluorescence emission spectrum. At pH 7.2, the two tryptophan residues contribute almost equally tc the protein fluorescence. The decay time of tryptophan 1 is 0.4 ns. The other emission parameters are the same as those determined at pH 3.9. We conclude that the fluorescence quenching in the range pH 3.9 to pH 8.0 comes essentially from the formation of a non emitting internal ground state complex between the tryptophan having the longest decay times and a neighbouring protein chemical group. The intrinsic pK of this group and the equilibrium constant of the irternal complex can be estimated. The quenching group is thought to be a carboxylate anion. Excitation transfers between the two tryptophyl residues of the protein molecule appear to have a small efficiency.     

Some aspects of studies of thermal transitions in proteins by means of their intrinsic fluorescence

The changes in intrinsic fluorescence parameters induced by thermal transitions in proteins are developed on the background of the common thermal fluorescence quenching due to an activation of collisions between the excited chromophores and neighbouring quenching groups. Two methods of separation of the thermai quenching and conformational change contributions to the temperature dependence of the fluorescence parameters are presented. One is based on the use of the linearity of the plots of the reciprocal fluorescence quantum yield, l/q, vs. the t/η ratio (T. temperature; η, solvent viscosity) for native proteins containing a single fluorescing chromophore (T.L. Bushueva, E.P. Busel and E.A. Burstein, Biochim. Biophys. Acta 534 (1978) 141). The other method is based on a consideration of the phase plots for the tryptophan fluorescence of proteins (fluorescence intensity at a fixed wavelength vs. intensity at any other fixed wavelength). The methods have been used for a study of the thermal transitions in Mg²⁺-loaded whiting parvalbumin (tryptophan fluorescence), Mg²⁺-loaded pike parvalbumins pI 4.2 (tyrosine fluorescence) and pI 5.0 (phenylalanine fluorescence), and Ca²⁺-loaded bovine α-lactalbumin (tryptophan fluorescence). The thermal denaturation curves for the parvalbumins show two-stepped character. The main change of the protein conformation occurs at the higher temperature step. Comparison of the fluorescence data with the microcalorimetry results shows that the maxima of the asymmetric heat sorption peaks for pike parvalbumins correlate with the mid-points of the higher temperature steps of the fluorimetric curves.     

Interaction of calcium and cyclic nucleotides in thyroliberin-stimulated prolactin release

The object of the present study was to determine the relative importance of Ca++ and cyclic nucleotides as “second messengers” in thyroliberin (TRH)-mediated prolactin (PRL) release in the GH₃ and GH₄ rat pituitary tumor cell lines. PRL, cyclic adenosine 3': 5'-monophosphate (cAMP), and cyclic guanosine 3': 5'-monophosphate (cGMP) were measured by radioimmunoassay (RIA) following TRH stimulation. TRH increased PRL release and cAMP levels in GH₃ and GH₄ cells, but cGMP increases were variable. Treatment with 1 mM theophylline increased PRL release and raised cAMP and cGMP. Addition of TRH to theophylline-pretreated cells produced further significant increases in PRL release without any additional increases in cAMP and cGMP. Co++, a Ca++ antagonist, abolished TRH-induced PRL release in a dose-dependent manner. The Co++ inhibition was partially reversed by Ca++ in GH₃ or GH₄ cells. Furthermore, the Ca++ ionophore A23187 stimulated PRL release. We conclude that Ca++ is the primary “second messenger” for TRH-mediated PRL release from GH₃ or GH₄ cells.     

Similarity Between Rat Brain Nicotinic α-Bungarotoxin Receptors and Stably Expressed α-Bungarotoxin Binding Sites

Abstract: The present results demonstrate stable expression of α-bungarotoxin (α-BGT) binding sites by cells of the GH₄C₁ rat pituitary clonal line. Wild-type GH₄C₁ cells do not express α-BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor α2, α3, α4, α5, α7, β2, or β3 subunits. However, GH₄C₁ cells stably transfected with rat nicotinic receptor α7 cDNA (α7/GH₄C₁ cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of the predicted size. The α7/GH₄C₁ cells also express saturable, high-affinity binding sites for ¹²⁵I-labeled α-BGT, with a K_D of 0.4 nM and B_max of 3.2 fmol/10⁶ intact cells. ¹²⁵I-α-BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or α7/GH₄C₁ cells. Furthermore, K_D and K_i values for ¹²⁵I-α-BGT binding sites on intact α7/GH₄C₁ cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the α-BGT binding sites expressed in α7/GH₄C₁ cells was similar to that of the native brain α-BGT receptor. Chronic exposure of α7/GH₄C₁ cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of α-BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of α-BGT binding sites in transfected α7/GH₄C₁ cells resemble those for brain nicotinic α-BGT receptors. If the heterologously expressed α-BGT binding sites in the present study are composed solely of α7 subunits, the results could suggest that the rat brain α-BGT receptor has a similar homooligomeric structure. Alternatively, if α-BGT binding sites exist as heterooligomers of α7 plus some other previously identified or novel subunit(s), the data would indicate that the α7 subunits play a major role in determining properties of the α-BGT receptor.     

Structural characteristics of thermostable immunogenic outer membrane protein from Salmonella enterica serovar Typhi

In this work, we explored the acid-induced unfolding pathway of non-porin outer membrane protein (OMP), an immunogenic protein from Salmonella Typhi, by monitoring the conformational changes over a pH range of 1.0–7.0 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, and dynamic light scattering. The spectroscopic measurements showed that OMP in its native state at pH 7.0 exists in more stable and compact conformation. In contrast, at pH 2.0, OMP retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii, and nearly four-fold increase in ANS fluorescence with respect to the native state, indicating that MG state exists at pH 2.0. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of a partially unfolded state between native and unfolded state. The effect of pH on the conformation and thermostability of OMP points towards its heat resistance at neutral pH (T _m?~?69 °C at pH 7.0, monitored by change in MRE_{222 nm}). Acid unfolded state was also characterized by the lack of a cooperative thermal transition. All these results suggested that acid-induced unfolded state of OMP at pH 2.0 represented the molten globule state. The chemical denaturation studies with GuHCl and urea as denaturants showed dissimilar results. The chemical unfolding experiments showed that in both far-UV CD and fluorescence measurements, GuHCl is more efficient than urea. GuHCl is characterized by low C _m (~1 M), while urea is characterized by high C _m (~3 M). The fully unfolded states were reached at 2 M GuHCl and 4 M urea concentration, respectively. This study adds to several key considerations of importance in the development of therapeutic agents against typhoid fever for clinical purposes.     

Saccharin induces morphological changes and enhances prolactin production in GH4C1 cells

Summary The artificial sweetener saccharin inhibits binding of epidermal growth factor (EGF) to cultured rat pituitary tumor cells (GH₄C₁ cells). Saccharin also causes morphological alterations in these cells, resulting in pronounced elongation, stretching, and firmer attachment of cells to the culture dishes. These alterations in cell shape are similar to those observed after treatment of GH₄C₁ cells with EGF and with thyrotropin-releasing hormone (TRH), both of which enhance prolactin (PRL) production in these cells. After assaying for PRL in saccharin-treated cultures, it was observed that this sweetener is also capable of stimulating PRL production two-to sixfold in a dose-dependent manner. Enhancement of PRL production can be observed at 0.5 mM saccharin, yet this is 10 times less than the saccharin concentration required to alter cell shape. These effects of saccharin on cell morphology and on PRL production are reversible in GH₄C₁ cell cultures. When added to cultures along with maximal concentrations of EGF or TRH, the effects of saccharin on PRL production are additive, suggesting that the actions of saccharin are mediated by a somewhat different pathway from that of the peptide hormones. Pulse labeling studies indicate that the enhancement of PRL production is highly specific inasmuch as saccharin was found to decrease the overall rate of protein synthesis in these cells. Saccharin also causes a decrease in the rate of DNA synthesis under these treatment conditions. Mitomycin C, which similarly inhibited DNA synthesis, had no effect on cell morphology or PRL production. This investigation was supported by a Faculty Research Grant from Wheaton College