Determining the binding capability of the mouse major urinary proteins using 2-naphthol as a fluorescent probe.

The mouse major urinary proteins (MUPs) are an ensemble of isoforms secreted by adult male mice and involved in sexual olfactory communication. MUPs belong to the lipocalin superfamily, whose conserved structure is a beta-barrel made of eight antiparallel beta-strands forming a hydrophobic pocket that accommodates small organic molecules. A detailed knowledge of the molecular mechanism associated to the binding of those molecules can guide protein engineering to devise mutated proteins where the ligand specificity, binding affinity, and release rate can be modulated. Proteins with such peculiar properties may have interesting biotechnological applications for pest control, as well as in food and cosmetic industries. In this work, we demonstrate that the fluorescent molecule 2-naphthol binds to the natural ligand's binding site of MUPs with high affinity. In addition, we show that 2-naphthol binds to MUPs in its protonated form, that its fluorescence is blue-shifted, and the quantum yield is increased, thus confirming the high hydrophobicity of the protein pocket and the absence of proton acceptors inside the binding site. At large the results presented, besides demonstrating that the use of 2-naphthol provides a convenient and quick method for testing MUPs binding activity and to ascertain the quality of the protein preparation, suggest that MUPs can represent an interesting system for studying the photophysical characteristics of fluorescent molecules in a highly hydrophobic environment.

Solution structure of a recombinant mouse major urinary protein.

Major urinary proteins (MUPs) form an ensemble of protein isoforms which are expressed and secreted by sexually mature male mice only. They belong to the lipocalin superfamily and share with other members of this family the capacity to bind hydrophobic molecules, some of which are odorants. MUPs, either associated with or free of their natural ligands, play an important role in the reproductive cycle of these rodents by acting as pheromones. In fact, they are able to interact with receptors in the vomeronasal organ of the female mice, inducing hormonal and physiological responses by an as yet unknown mechanism. In order to investigate the structural and dynamical features of these proteins in solution, one of the various wild-type isoforms (rMUP: 162 residues) was cloned and subsequently isotopically labeled. The complete 1H, 13C and 15N resonance assignment of that isoform, achieved by using a variety of multidimensional heteronuclear NMR experiments, has been reported recently. Here, we describe the refined high-resolution three-dimensional solution structure of rMUP in the native state, obtained by a combination of distance geometry and energy minimization calculations based on 2362 NOE-derived distance restraints. A comparison with the crystal structure of the wild-type MUPs reveals, aside from minor differences, a close resemblance in both secondary structure and overall topology. The secondary structure of the protein consists of eight antiparallel beta-strands forming a single beta-sheet and an alpha-helix in the C-terminal region. In addition, there are several helical and hairpin turns distributed throughout the protein sequence, mostly connecting the beta-strands. The tertiary fold of the beta-sheet creates a beta-barrel, common to all members of the lipocalin superfamily. The shape of the beta-barrel resembles a calyx, lined inside by mostly hydrophobic residues that are instrumental for the binding and transport of small nonpolar ligand molecules.

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.

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.

Diltiazem at high concentration increases the ionic permeability of biological membranes.

The 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 microM. 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 of 86Rb 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 microM.

Regulation of cyclic GMP binding to retinal rod membranes by calcium.

The apparently cooperative binding of 8-(5-thioacetamidofluorescein)-cGMP (SAF-cGMP) to cGMP-binding sites of the rod outer segments is regulated by Ca2+ in the 0.1-1 microM activity range. High Ca2+ reduces, and low Ca2+ increases the affinity of SAF-cGMP binding. This regulation involves only intrinsic membrane components. It is proposed that an allosteric regulation of cGMP binding by Ca2+ can contribute to photoreceptor potential adaptation.

Homology between the pyrazine-binding protein from nasal mucosa and major urinary proteins.

Sequence analysis of the pyrazine-binding protein from bovine olfactory mucosa reveals marked homology with a family of proteins of unknown function found in the urine of the adult male mouse and rat. In view of the dramatic biological responses to odorants transmitted in male rodent urines, it is proposed that these proteins play important roles in some aspects of odor transmission and reception.

Immunocytochemical localization of pyrazine-binding protein in bovine nasal mucosa.

Polyclonal antibodies have been raised against purified bovine pyrazine-binding protein, a protein that binds the odorant 2-isobutyl-3-methoxypyrazine. These antibodies have been utilized in immunocytochemical experiments to localize the pyrazine-binding protein in bovine nasal mucosa. Tissue fragments, macroscopically identified as olfactory and respiratory mucosa, were fixed in Bouin's fluid and embedded in paraffin. Consecutive serial sections were processed for immunofluorescence studies and restained either with haematoxylin-eosin or with periodic acid Schiff-Alcian Blue. In both olfactory and respiratory mucosa, only seromucous tubulo-acinar glands were specifically labelled. These glands are located in the lamina propria underlying typical respiratory epithelium, even in those tissues that are macroscopically defined as olfactory mucosa.

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.

Binding stoichiometry of a fluorescent cGMP analogue to membranes of retinal rod outer segments.

The high-affinity binding of the cGMP analogue 8-(5-thioacetamidofluorescein)-cGMP to rod outer segment membranes depleted of peripherally bound proteins has been defined by equilibrium dialysis (mean +/- SD): membranes contain about one cGMP binding site per 130 rhodopsin molecules; the concentration of free ligand for half saturation is 2.0 +/- 0.6 microM; the apparent Hill coefficient of the bound versus free ligand relationship is 1.7 +/- 0.5; half saturation of the binding sites is sufficient for 85% activation of calcium permeability. A gating mechanism is proposed.

Purification and characterisation of an odorant-binding protein from cow nasal tissue.

Cow nasal tissue contains a protein which shows specific binding activity for 'green' smelling compounds such as 2-isobutyl-3-methoxypyrazine. This protein has now been purified using anion-exchange fast protein liquid chromatography. The protein has a relative molecular mass of 40 0000-44 000, s = 3.1 +/- 0.3 S, pI = 4.7 +/- 0.1 with an absorbance maximum at 278 nm, and consists of two subunits with an identical relative molecular mass of 19 000. It is localised in the soluble fraction of cells from the olfactory mucosa and respiratory mucosa from the middle part of the maxillary and nasal turbinates, and is absent from all other tissues tested.

Cyclic GMP releases calcium from disc membranes of vertebrate photoreceptors.

Physiological concentrations of cyclic guanosine 3',6'-monophosphate (cGMP) inhibit 45Ca uptake and increase 45Ca release from vertebrate photoreceptor rod outer segment disc membranes. These effects are specific for cGMP. Several facts, including the independence of these effects from added triphosphates, suggest that cGMP diminishes the Ca-binding capacity of the disc membranes. Preliminary data show that the apparent affinity constant of the cGMP-dependent Ca-binding sites of the disc membranes is of the same (or even higher) order of magnitude as that of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. As expected, the observed cGMP effects are not dependent on the light or dark conditions of the disc membranes.

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.

Cyclic GMP and the permeability of the disks of the frog photoreceptors.

1. The diffusion of sodium, potassium and rubidium (not chloride) ions across the disk membrane is increased by cyclic guanosine monophosphate (cyclic GMP). 2. The increase is greater for sodium than for rubidium in the 0.01-0.1 mM concentration range. 3. Cyclic adenosine monophosphate (cyclic AMP) is less efficient than cyclic GMP; GMP and guanosine triphosphate are without effect. 4. The effect is present with either 1.8 mM calcium ions or 4 mM-EGTA in the perfusion fluid. 5. The presence of the cyclic GMP phosphodiesterase on the disk membranes is not needed for this effect. 6. The effect is present in both unbleached and fully bleached membranes.

Phosphodiesterase and GTPase in rod outer segments. Kinetics in vitro.

The hydrolysis of cyclic guanosine monophosphate (cyclic GMP) and of guanosine triphosphate (GTP) by the broken rods of the frog retina after a flash of light have been studied in vitro with a constant perfusion method. The activation has an onset apparently instantaneous as observed with the existing possible time resolution of 3 s. The activation is followed by a partial inactivation that does not bring the activity back to the pre-flash level. GTP or the non-hydrolysable guanyl-5'-ylimidodiphosphate (GMP-PNP) is required for the normal light-activation of the phosphodiesterase and in its absence both the speed of activation and the sensitivity are greatly reduced. The activation speed, the sensitivity (threshold at approx. 0.00004% bleaching), and the kinetic constants do not exclude a direct role in the process of excitation for the phosphodiesterase and suggest a subsidiary but as yet undefined role for the GTPase.

Light-activated hydrolysis of GTP and cyclic GMP in the rod outer segments.

1. The hydrolysis of guanosine triphosphate (GTP) and the consequent formation of guanosine diphosphate (GDP) and phosphate (P1) are activated by light in a suspension of broken retinal rods: the hydrolysis rate with GTP in the micrometer concentration range is 2.5-3.5 n-mole/min per mg of rhodopsin in the preparation. 2. The ionic composition of the medium suspending the rods is not critical: the hydrolysis is present in NaCl saline solution with MG2+ as well as in Tris-HC1 buffer solution, and with the chelating agent EDTA. 3. The ionic strength is critical: the effect is reduced when the broken rods are suspended in a low salt mannitol solution, and is altogether abolished when they are separated from the mannitol solution; it reappears when the mannitol solution is added again in the presence of salts. An element essential for the effect is thus reversibly released in the mannitol solution. No hydrolytic activity on GTP, however, is found in the mannitol soluble fraction. 4. The cyclic nucleotide phosphodiesterase is eluted from the rods in the mannitol solution, and is reaggregated to the rods in the presence of salts; once recombined with the rods, it can be activated by light. 5. The activation of the phosphodiesterase by light is present in the absence of added nucleotide triphosphates.

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.

Efflux of potassium from isolated rod outer segments: a photic effect.

1. Illumination of the isolated outer segments of rod photoreceptors loaded with (42)K or (86)Rb reduces the efflux of these ions.2. During the perfusion of the isolated rod outer segments with a solution containing only 2.36 mM-Na the effect of light is absent, and the amplitude of the photic effect is linearly related to the logarithm of the extracellular Na concentration.3. In darkness, raising the concentration of K in the fluid of perfusion gives an increase of the efflux of (86)Rb and increasing the extracellular concentration of Ca yields a retention. The efflux of (86)Rb and (42)K is greater in darkness when sucrose or choline substitute for Na.4. It is suggested that in darkness the isolated outer segments are permeable both to Na and to K. Light appears to decrease the permeability for Na ions. There is no evidence that the permeability for K ions is modified by light.

Efflux of potassium from the isolated frog retina: a study of the photic effect.

1. Illumination of the isolated frog retina reduces the efflux of (42)K (and (86)Rb).2. Perfusion with a solution containing glutamate ions does not abolish the effect, although the electroretinogram is modified and the b-wave absent in this condition.3. Perfusion with a solution with only 2.36 mM sodium abolishes the effect of light on the efflux of potassium (and rubidium).4. The amplitude of the photic effect, measured in the first 2 min of illumination, is in linear proportion with the logarithm of the concentration of extracellular sodium ions.5. A steady rate of absorption of a few quanta per rod and per second yields a just detectable effect on the efflux of potassium (and rubidium).6. Strong illuminations followed by darkness reduce the efflux of rubidium for a period of time roughly comparable to the time of dark adaptation.7. The interpretation is proposed that the photoreceptor cells (and possibly other retinal cells too) cause the effect, and that the retention of potassium induced by light is secondary to the hyperpolarization of their membrane.