Robert T. Sorbi

Expression of a lipocalin in Pichia pastoris: secretion, purification and binding activity of a recombinant mouse major urinary protein

The proteins of the mouse major urinary protein complex (MUP), members of the lipocalin family, bind volatile pheromones and interact with the vomeronasal neuroepithelium of the olfactory system. We report the expression of a MUP protein using its native signal sequence for secretion in the methylotrophic yeast, Pichia pastoris. Mature recombinant MUP (rMUP) is secreted at a concentration of 270 mg/l in minimal medium and it is isolated from the culture supernatant by one step ion‐exchange chromatography in a nearly pure form. Binding activity, tested with an odorant molecule which displays high affinity for native MUP, indicates that rMUP has a behavior similar to the native one. This finding suggests that the protein, and in particular its hydrophobic binding pocket, is properly folded.

Effect of strong illumination on the ion efflux from the isolated discs of frog photoreceptors.

(1) Low levels of illumination do not modify the efflux of the radioisotopes 22Na, 86Rb, 36Cl, and 45Ca from the isolated discs of the photoreceptors of the frog (Rana Catesbeiana). (2) The effluxes of 22Na+, 86Rb+ and 36Cl- increase when the discs are illuminated with more than 10(4) erg/cm2 per s for a few minutes. There is no effect on the efflux of 45Ca2+ or of [14C]urea. (3) The effect is greater for monochromatic lights of wavelengths in the shorter region of the spectrum. (4) The effect is also present in bleached visual membranes.

Reversible unfolding of bovine odorant binding protein induced by guanidinium hydrochloride at neutral pH.

An analysis of the unfolding and refolding curves at equilibrium of dimeric bovine odorant binding protein (bOBP) has been performed. Unfolding induced by guanidinium chloride (GdnHCl) is completely reversible as far as structure and ligand binding capacity are concerned. The transition curves, as obtained by fluorescence and ellipticity measurements, are very similar and have the same protein concentration-independent midpoint (C1/2 approximately 2.6 M). This result implies a sequential, rather than a concerted, unfolding mechanism, with the involvement of an intermediate. However, since it has not been detected, this intermediate must be present in small amounts or have the same optical properties of either native or denatured protein. The thermodynamic best fit parameters, obtained according to a simple two-state model, are: deltaG degrees un,w = 5.0 +/- 0.6 kcal mol(-1), m = 1.9 +/- 0.2 kcal mol(-1) M(-1) and C1/2 = 2.6 +/- 0.1 M. The presence of the ligand dihydromyrcenol has a stabilising effect against unfolding by GdnHCl, with an extrapolated deltaG degrees un,w of 22.2 +/- 0.9 kcal mol(-1), a cooperative index of 3.2 +/- 0.3 and a midpoint of 4.6 +/- 0.4 M. The refolding curves, recorded after 24 h from dilution of denaturant are not yet at equilibrium: they show an apparently lower midpoint (C1/2 = 2.2 M), but tend to overlap the unfolding curve after several days. In contrast to chromatographic unfolding data, which fail to reveal the presence of folded intermediates, chromatographic refolding data as a function of time clearly show a rapid formation of folded monomers, followed by a slower step leading to folded dimers. Therefore, according to this result, we believe that the preferential unfolding/refolding mechanism is one in which dimer dissociation occurs before unfolding rather than the reverse.

Diltiazem at high concentration increases the ionic permeability of biological membranes

SummaryThe effects of diltiazem, a drug which inhibits the calcium channels in cardiac muscle as well as the light-sensitive channels in photoreceptor cells, were studied on ionic fluxes in both membrane and intact cell preparations. Diltiazem nonselectively increased the ionic permeability to both anions and cations in photoreceptor rod outer segment and synaptic membrane vesicles as well as in intact erythrocytes. Under our conditions, the estimated threshold for the diltiazem effect varied between 12.5 and 200 μm. In each case the concentration dependence exhibited the sigmoidal shape characteristic of positive cooperativity. The effect of diltiazem on ionic fluxes from phospholipid vesicles were strongly influenced by phospholipid composition and membrane charge. By contrast, diltiazem inhibited the efflux of86Rb from photoreceptor cells of intact aspartate-isolated retina, an effect opposite to that of diltiazem on ionic permeabilities in photoreceptor membrane vesicle preparations.These data raise serious doubts on the specificity of diltiazem as a calcium channel blocker or as a cGMP channel blocker when used at concentrations higher than 10 μm.

The interaction of 4', 6-diamidino-2-phenylindole (DAPI) with pepsin.

We recently found that the fluorescent dye DAPI, well-known for its use with nucleic acids, is also able to interact with proteins as well as ordered phospholipids assemblies. The interaction of DAPI with pepsin under different conditions of pH and ionic strength was studied with fluorescence and circular dichroism techniques. From a comparison of the results obtained, the interaction appears to be rather tight and specific, dependent on both electrostatic and hydrophobic forces, and able to probe the tridimensional conformation of the protein.

Characterization of a Deswapped Triple Mutant Bovine Odorant Binding Protein

The stability and functionality of GCC-bOBP, a monomeric triple mutant of bovine odorant binding protein, was investigated, in the presence of denaturant and in acidic pH conditions, by both protein and 1-aminoanthracene ligand fluorescence measurements, and compared to that of both bovine and porcine wild type homologues. Complete reversibility of unfolding was observed, though refolding was characterized by hysteresis. Molecular dynamics simulations, performed to detect possible structural changes of the monomeric scaffold related to the presence of the ligand, pointed out the stability of the β-barrel lipocalin scaffold.

Chapter 18 Modulation of Sodium Conductance in Photoreceptor Membranes by Calcium Ions and cGMP

Publisher Summary This chapter discusses the modulation of sodium conductance in photoreceptor membranes by calcium ions and cyclic guanosine 3´,5´-monophosphate (cGMP). Ca 2+ is the soluble transmitter that is needed to link the disk membranes with the plasma membrane of the rod outer segment (ROS) in the process of photoreceptor excitation. The data obtained by either increasing or lowering the cytoplasmic Ca 2+ activity strongly support the idea that Ca 2+ can modulate plasma membrane permeability to Na + . A light-induced increase of the calcium activity has been recorded in the extracellular fluid surrounding the outer segments in isolated retinas. The hypothesis involving cCMP in visual excitation suggests that in the dark plasma membrane, permeability is high because of the cGMP-dependent phosphorylation of some protein(s) related to membrane permeability to Na + . A direct relationship between plasma membrane permeability to Na + and the endogenous cGMP content of isolated ROS is discussed in the chapter. cCMP in the micromolar range can modulate the Na + permeability of the disk membrane. cGMP may affect the plasma membrane in the same way as the disk membranes; that is, it increases the Na + conductance.

Search for a physiological role of cyclic GMP metabolism in the photoreceptors

Data on the time-course of the light-activation of the cyclic GMP phosphodiesterase and of the GTPase, and results on the influence of cyclic GMP on the disc membrane permeability are presented. On the basis of the kinetic data, it is not possible to separate the light-activation of these two enzymes from the early steps of photoreceptor transduction. In addition, the cyclic GMP increases the permeability of the disc membranes, indicating that a decrease of the endogenous cyclic GMP concentration, consequent to the light-activation of the phosphodiesterase, can decrease the membrane permeability shortly after illumination.

Effect of fluoride on the phosphodiesterase of bovine photoreceptors

In the absence of the specific hormone, fluoride is able to activate the adenylate cyclase because it interacts with the GTP-binding protein. It has been reported that fluoride activates also the phosphodiesterase of the light-sensitive enzymatic cascade in dark-adapted retinal rod outer segments, but there is no indication that the GTP-binding protein is involved in this process or not. We show here that also in the photoreceptor system fluoride does interact with the GTP-binding protein in order to activate the phosphodiesterase in the dark. Further, we show evidences that fluoride solubilizes the GTP-binding protein in the dark and that the resulting complex activates the phosphodiesterase in dark-adapted rod outer segment membranes.

More about the cyclic GMP effects on the disc membranes of vertebrate photoreceptors

Protein tyrosine phosphorylation (PTP) has recently been implicated in normal and abnormal cell growth processes. We now report high PTP activity in both normal human retina and in cultured human Y-79 cells. The highest sp. activity was found in the 27,000 g particulate fraction. Treatment of this fraction with detergent dispersed about 60~ of the activity into the supernatant; in both fractions, half maximal activity was seen at 10#M ATP. In the detergent supernatant, GTP was as good a substrate as ATP. However, in the particulate fraction, the activity with GTP was 5-fold lower than that with ATP. In both fractions, the activity with 5 mM MnCI 2 was 3to 5-times higher than that with 10 mM MgC12. With Y-79 cells, addition of a partially purified retinoblastoma-derived growth factor (RDGF) increased PTP in endogenous proteins of the particulate fraction but decreased phosphorylation of exogenous substrate (casein). The endogenous activity of the detergent supernatant was unaffected by RDGF, although the activity toward casein was reduced. Thus, these cells contain a well-defined PTP activity with phosphorylation of endogenous particulate protein(s) controlled by a growth factor elaborated by the cells themselves indicating a possible role for PTP in tumor cell growth. However, since the PTP activity in normal retina is comparable to that seen in Y-79 cells, other, as yet unknown, roles for this activity must be considered.