Mass spectrometry techniques for detection of ligand-dependent changes in the conformational flexibility of cellular retinol-binding protein type I localized by hydrogen/deuterium exchange.

Hydrogen/deuterium exchange, measured by electrospray ionization with orthogonal quadrupole time-of-flight mass spectrometry (ESI-Q-TOFMS) and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), was used as a means to probe and map differences in conformational flexibility between the ligand-free and ligand-bound forms of cellular retinol-binding protein type I. Labelled fragments were obtained by digestion of the protein with pepsin. The differences in space-resolved time courses of deuterium incorporation identified regions that exhibit a remarkably higher degree of flexibility in the apo-protein than in the holo-protein. These segments encompass residues that are thought, on the basis of structural homology of the retinol carrier with other members of the intracellular lipid-binding proteins family, to belong to the dynamic portal through which all-trans retinol can access its high-affinity, solvent-shielded, binding site.

Structure of S35C flavodoxin mutant from Desulfovibrio vulgaris in the semiquinone state.

The crystallographic structure of an engineered flavodoxin mutant from Desulfovibrio vulgaris has been analysed. Site-directed mutagenesis was used to substitute serine 35 with a cysteine to provide a possible covalent linkage. The crystal structure of the semiquinone form of this mutant is similar to the corresponding oxidation state of the wild-type flavodoxin. Analysis of the structural changes reveals the interaction between N(5)H of the flavin and the carbonyl O atom of Gly61 to be critical for modulation of the electrochemical properties of the protein.

Non-covalent binding of endogenous ligands to recombinant cellular retinol-binding proteins studied by mass spectrometric techniques.

Recent developments in mass spectrometry have demonstrated the capability of this technique to transfer non-covalent protein complexes, involving low and high molecular weight ligands, from a condensed state to the gas phase. In this work, electrospray mass spectrometry with a quadrupole analyzer (ES-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) were used to analyze the non-covalent association between recombinant rat cellular retinol-binding protein type-I (CRBP) with its specific ligand, all-trans retinol (vitamin A), and with fatty acids. Under denaturing conditions, MALDI-TOFMS and ES-MS techniques allowed determination of the molecular weight of apo-CRBP with good accuracy (<0.01%) and to identify a protein fraction ( approximately 20%) retaining the initial methionine. By adding saturating amounts of vitamin A, ES-MS studies on the protein in the holo-form under native conditions allowed detection of retinol bound within the cavity together with water molecules, as expected from its crystal structure. ES mass spectra of CRBP in the native state were also recorded under non-denaturing conditions, with the aim to study non-covalent interactions between CRBP and non-specific ligands such as fatty acids, bound to the protein as a result of expression in various strains of E. coli grown in different media. Since ES mass spectra do not elucidate which species interact with the protein, in order to investigate the ligands possibly retained in the active site of recombinant CRBP, liquid chromatography/ES-tandem mass spectrometry was used. In particular, this technique was applied to identify and quantify fatty acids bound to CRBP. Quantitative data indicated the presence of a few fatty acids at a total concentration lower than 2% of that of the protein. Similar findings were observed for the homolog rat cellular retinol-binding protein type-II, demonstrating the high degree of purity and homogeneity of apo-CRBP preparations derived from gene expression.

Retinoic acid synthesis in the somatic cells of rat seminiferous tubules.

At physiological plasma concentrations, retinoic acid (RA) cannot cross the blood-testis barrier formed by Sertoli and peritubular cells, and it is thought to be mainly synthesized in situ through the oxidation of retinol. We have thus examined the in vitro RA biosynthetic capacity of cultured Sertoli and peritubular cells isolated from the seminiferous tubules of prepubertal rats, using holo-cellular retinol binding protein (CRBP) as a substrate. Although both somatic cell types contain CRBP and retinoic acid nuclear receptors, RA synthesis was only detected with Sertoli cell subcellular fractions. Most of the RA synthesizing activity of these cells is contributed by a microsomal-cytosolic system that shares many functional similarities with a RA biosynthetic pathway originally identified in rat liver. RA synthesis is maximal at a time of postnatal life (20 days) preceding meiotic cell accumulation and remains nearly constant thereafter. The unique ability of Sertoli cell subcellular fractions to support RA formation from holoCRBP, along with the observed age-dependent modulation of this activity, indicate that Sertoli cells represent the main site of intratubular RA production and that they may play a key role in controlling RA-dependent processes within the seminiferous tubule.

An estimation of the left ventricular diastolic function from the spectral analysis of the fourth heart sound. A Doppler validated study in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a typical primary cardiac disease characterized by diastolic abnormal function due to both prolonged relaxation and decreased compliance (Sanderson et al., 1977; Spirito & Maron, 1990). Since the contribution of the atrial systole to ventricular filling is usually increased, the appearance of a fourth heart sound (S4) is a common finding in HCM. This sound is related to the ventricular compliance and to the atrial contractility. It is generated during the rapid setting into vibration of the left ventricular walls that results from the rapid rush of the blood due to the atrial contraction (Nishimura et al., 1989; Tavel, 1978). The aim of this study is to look for relationships between the frequency peak of S4 and various mono- and two-dimensional echocardiographic parameters in order to identify those cardiac structures involved in its genesis and obtaining an estimation of the stiffness of the acoustic vibrating system with a simply vibratory model (Baracca et al., 1991).

Effect of acute and long-term administration of ramipril on circadian rhythm of blood pressure in essential hypertension.

Ambulatory monitoring was used to evaluate the antihypertensive efficacy and effect on circadian rhythms of blood pressure and heart rate of a single dose and long-term administration of ramipril in 20 patients with mild to moderate essential hypertension. Patients initially were randomized to receive either placebo or a single 5-mg dose of ramipril, followed 1 week later by 5 mg of ramipril daily for 6 months. Systolic (SBP) and diastolic blood pressure (DBP) and heart rate were measured every 20 minutes for 24 hours. Single-dose ramipril reduced both SBP and DBP (P < .001) without affecting heart rate. Long-term treatment produced a small additional antihypertensive effect, again without modifying heart rate. Cosinor analysis demonstrated that both administrations of ramipril effectively lowered SBP and DBP mesors (P < .001), compared to placebo; circadian rhythms remained undisturbed. Heart rate also was not modified on any circadian parameter. A significant reduction (P < .001) of blood pressure amplitude, however, occurred after long-term treatment and may have importance in terms of preventing cardiac damage.

Retinol bound to cellular retinol-binding protein is a substrate for cytosolic retinoic acid synthesis.

Retinol bound to cellular retinol-binding protein (CRBP) was found to be oxidized to retinoic acid by a soluble activity from calf liver. Cytosolic retinoic acid synthesis from retinol-CRBP was strictly dependent on the exogenous supply of either NAD or NADP. NAD-supported reactions carried out in the presence or in the absence of dimethyl sulfoxide yielded apparent Km and Vmax values for the retinol-CRBP complex of 3.5 +/- 0.6 microM, 611 +/- 49 pmol h-1 (mg of protein)-1, and 0.84 +/- 0.12 microM, 601 +/- 38 pmol h-1 (mg of protein)-1, respectively. The corresponding values for the oxidation of free retinol, dissolved in dimethyl sulfoxide, were 7.1 +/- 0.3 microM and 948 +/- 47 pmol h-1 (mg of protein)-1. Since the dissociation constant of the bovine retinol-CRBP complex is less than 10(-8) M, whereas the Km for retinol-CRBP is of the same order as the Km for free retinol, synthesis of retinoic acid from retinol-CRBP does not rely on prior dissociation of retinol. ApoCRBP proved to be a specific inhibitor of retinoic acid synthesis from CRBP-bound retinol. Its inhibitory effect was indistinguishable from the dilution of the radioactive retinol-CRBP substrate that was obtained by the addition of unlabeled holoCRBP. In contrast, the oxidation of CRBP-bound retinol was not inhibited by the addition of other retinoid binding proteins nor by the addition of either free retinol or retinol complexed with proteins distinct from CRBP. These results indicate that the protein moiety of holoCRBP is specifically recognized by the cytosolic enzyme system that catalyzes retinoic acid synthesis from CRBP-bound retinol.