Biomedical subjects

L Zolla

Publications and source records attributed to L Zolla.

At least 19 recordsLinked to original sources

PubMed2003

Identification of a furazolidone metabolite responsible for the inhibition of amino oxidases.

1 Furazolidone, a drug widely used in human and veterinary medicine, exhibits inhibition of monoamine oxidase activity, as observed in the tissues of a number of different animal species, including man. The aim of the current study was to determine which of the two possible metabolites, 3-amino-2-oxazolidone (AOZ) or beta-hydroxyethylhydrazine (HEH), a well-known carcinogenic compound, is involved in the toxicological effects reported. 2 A new spectrometric method was set up to differentiate intracellular HEH from AOZ inside cells. This method works well at low pH where both AOZ and HEH are free in solution and available to react with the chemical chromophore (DAB). 3 The results confirm that furazolidone has to be metabolized in the intact cell in order to exhibit mitochondrial monoamine oxidase inhibition, whereas AOZ itself is able to exert a reversible monoamine oxidase inhibition. AOZ also inhibits bovine serum amino oxidase. On the contrary, HEH gives irreversible inhibition of both enzymes. However, the reversible nature of the AOZ inhibition with respect to HEH suggests that the two metabolites act by different mechanisms which do not require the biotransformation of AOZ to HEH. 4 Cell lysates, previously incubated with AOZ, were directly analysed and the formation of HEH from AOZ was not detected, supporting the conclusion that the amino oxidase inhibition observed on treatment with furazolidone was attributable to AOZ and not to HEH.

Animals↗

PubMed2001

Isoforms of photosystem II antenna proteins in different plant species revealed by liquid chromatography-electrospray ionization mass spectrometry.

The high selectivity offered by reversed-phase high-performance liquid chromatography on-line coupled to electrospray ionization mass spectrometry has been utilized to characterize the major and minor light-harvesting proteins of photosystem II (Lhcb). Isomeric forms of the proteins, revealed either on the basis of different hydrophobicity enabling their chromatographic separation or on the basis of different molecular masses identified within one single chromatographic peak, were readily identified in a number of monocot and dicot species. The presence of several Lhcb1 isoforms (preferably in dicots) can explain the tendency of dicot Lhcb1 to form trimeric aggregates. The Lhcb1 molecular masses ranged from 24,680 to 25,014 among different species, whereas within the same species, the isoforms differed by 14-280 mass units. All Lhcb1 proteins appear to be highly conserved among different species such that they belong to a single gene group that has several different gene family members. In all species examined, the number of isoforms corresponded more or less to the genes cloned previously. Two isoforms of Lhcb3 were found in petunia and tomato. For Lhcb6, the most divergent of all light-harvesting proteins, the greatest number of isoforms was found in petunia, tobacco, tomato, and rice. Lhcb2, Lhcb4, and Lhcb5 were present in only one form. The isoforms are assumed to play an important role in the adaptation of plants to environmental changes.

Chromatography, High Pressure Liquid↗

PubMed2001

High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies.

The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.40.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.

Chromatography, High Pressure Liquid↗

PubMed2001

High-performance liquid chromatography coupled on-line with electrospray ionization mass spectrometry for the simultaneous separation and identification of the Synechocystis PCC 6803 phycobilisome proteins.

The complete resolution of the protein components of phycobilisome from cyanobacterium Synechocystis 6803, together with their detection and determination of molecular mass, has successfully been obtained by the combined use of HPLC coupled on-line with electrospray ionization mass spectrometry. The method proposed consists of the isolation of the light-harvesting apparatus of cyanobacterium, by simply breaking cells in low-ionic-strength buffer, and subsequent injection of the total mixture of phycobilisomes into a C4 reversed-phase column. Identification of proteins was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the samples collected from HPLC or by measuring the protein molecular mass coupling HPLC with mass spectrometry. The latter method allows the simultaneous separation of the phycobiliproteins, phycocyanin and allophycocyanin, from linker proteins and their identification, which due to their similar amino acid sequence and their similar hydrophobicity, might not be detected by denaturing SDS-PAGE. Under the experimental conditions used, the pigment phycobilin is not removed from the polypeptide backbone, determining the hydrophobicity of the phycoproteins and hence their interaction with the reversed-phase column as well as in determining the protein-protein interaction into the phycobilisome aggregation. Removal of the pigment, in fact, abolishes HPLC separation, emphasizing the essential role that the pigments play in maintaining the unusual tertiary structure of these proteins.

Bacterial Proteins↗

PubMed2000

High performance liquid chromatography-electrospray mass spectrometry for the simultaneous resolution and identification of intrinsic thylakoid membrane proteins.

In higher plants, both photosystem I (PSI) and II (PSII) consist of membrane-embedded proteins that contain more than one transmembrane alpha helix. PSI is a multiprotein complex consisting of a core complex of thirteen proteins surrounded by four different types of light harvesting antenna proteins. Up to now, the protein components of both photosystems have been characterized by SDS-PAGE and/or immunoblotting and, therefore, identification made only on the basis of electrophoretic mobility, which is sometimes not sufficient to discriminate between individual membrane proteins. This is also complicated by the fact that some proteins, such as the antenna proteins, have almost identical molecular mass and amino acid sequence, making it difficult to identify and ascertain the relative stoichiometry of the proteins. In this paper, we report the complete resolution of the antenna proteins and most of the core components of PSI from spinach, together with the identification of proteins by molecular mass, successfully deduced by the combined use of HPLC coupled on-line with a mass spectrometer equipped with an electrospray ion source (ESI-MS). The proposed RP-HPLC-ESI-MS method holds several advantages over SDS-PAGE, including better protein separation, especially for antenna proteins, mass accuracy, speed, efficiency, and the potential to reveal isomeric forms. Moreover, the molecular masses determined by HPLC-ESI-MS are in good agreement with the molecular masses of the individual components calculated on the basis of their nucleotide-derived amino acid sequences, indicating an absence of post-translational modifications in these proteins. It follows that if the method proposed is useful for these highly hydrophobic proteins, it may be of general use for any membrane proteins, where the presence of detergent for solubilization may compromise their characterization.

Chromatography, High Pressure Liquid↗

PubMed2000

Resolution and identification of the protein components of the photosystem II antenna system of higher plants by reversed-phase liquid chromatography with electrospray-mass spectrometric detection.

Reversed-phase liquid chromatography (RPLC) was interfaced to mass spectrometry (MS) with an electrospray ion (ESI) source for the separation and accurate molecular mass determination of the individual intrinsic membrane proteins that comprise the photosystem II (PS II) major light-harvesting complex (LHC II) and minor (CP24, CP26 and CP29) antenna system, whose molecular masses range between 22,000 and 29,000. PS II is a supramolecular complex intrinsic of the thylacoid membrane, which plays the important role in photosynthesis of capturing solar energy, and transferring it to photochemical reaction centers where energy conversion occurs. The protein components of the PS II major and minor antenna systems were extracted from spinach thylacoid membranes and separated using a butyl-silica column eluted by an acetonitrile gradient in 0.05% (v/v) aqueous trifluoroacetic acid. On-line electrospray MS allowed accurate molecular mass determination and identification of the protein components of PS II major and minor antenna system. The proposed RPLC-ESI-MS method holds several advantages over sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the conventional technique for studying membrane proteins, including a better protein separation, mass accuracy, speed and efficiency.

Chromatography, Liquid↗

PubMed1997

Effect of mercuric ions on human erythrocytes. Relationships between hypotonic swelling and cell aggregation.

This study was undertaken to verify the hypothesis that the haemolytic effect of mercuric ions on human erythrocytes is strongly decreased under swelling conditions (relative to isotonic suspensions). In fact, interaction of Hg2+ with swollen erythrocytes yields a rapid and cooperative cell aggregation, a phenomenon that appears to prevent penetration of mercuric ions into the cells and, accordingly, to avoid any haemolytic effect induced by the Hg2+ entrance. Since in vivo erythrocytes undergo big shape changes (swelling being a kind of shape modification) related to mechanical or (in some animals) osmotic stresses, the reported observations turn out to be also of some relevance for the understanding of certain toxicological effects of mercuric ions.

4-Chloromercuribenzenesulfonate↗

PubMed1997

Role of phospholipase C and D signalling pathways in vasopressin-dependent myogenic differentiation.

Arg8-vasopressin (AVP) is a potent inducer of myogenic differentiation stimulating the expression of myogenic regulatory factors. To understand the mechanism of its effect on myogenesis, we investigated the early signals induced by AVP in myogenic target cells. In the rat skeletal muscle cell line L6, AVP selectively stimulates phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho) breakdown, through the activation of phospholipases C and D (PLC, PLD), as shown by the generation of Ins(1,4,5)P3 and phosphatidylethanol (PtdEtOH), respectively. AVP induces the biphasic increase of sn-1,2-diacylglycerol (DAG) consisting in a rapid peak followed by a sustained phase, and the monophasic generation of phosphatidic acid (PA). Propranolol (a PA phosphatase inhibitor) and Zn2+ (a PLD inhibitor), abolish the sustained phase of DAG generation. Our data indicate that PtdIns-PLC activity is mainly responsible for the rapid phase of AVP-dependent DAG generation, whereas the sustained phase is dependent upon PtdCho-PLD activity and PA dephosphorylation, ruling out any significant role of DAG kinase. Modifications of PA level correlate with parallel changes of PLC activity, indicating a possible cross-talk between the two signal transduction pathways in the intact cell. PLD activation is elicited at AVP concentrations two orders of magnitude lower than those required for PLC activation. The differentiation of L6 myoblasts into multinucleated fibers is stimulated significantly by AVP at concentrations at which PLD, but not PLC, is activated. These data provide the first evidence for an important role of PLD in the mechanism of AVP-induced muscle differentiation.

Animals↗

PubMed1996

Random amplified polymorphic DNA (RAPD) interpretation requires a sensitive method for the detection of amplified DNA.

The random amplified polymorphic DNA technique (RAPD) has found wide use in molecular genetics because of its speed and ease of use. For various reasons, with this method the amplified DNA fragments are produced at different concentrations between genotypes and even between polymerase chain reaction (PCR) runs. Since the detection of the multiple amplified fragments is performed routinely by agarose gel, and seldom by acrylamide gel electrophoresis, we have found that by capillary zone electrophoresis (CZE), which is more sensitive and accurate than gel electrophoresis, it is possible to unequivocally detect amplified fragments even at low concentration, avoiding polymorphism misinterpretation. CZE is also useful to make more potentially polymorphic fragments evident per random primer used, with obvious economical benefits.

Animals↗

PubMed1996

Capillary electrophoresis of closely related intrinsic thylakoid membrane proteins of the photosystem II light-harvesting complex (LHC II).

The electrophoretic migration behavior of three closely related hydrophobic intrinsic membrane proteins of the photosystem II light-harvesting complex (LHC II) was investigated in free solution capillary electrophoresis at pH 8.0-10 with running electrolyte solutions containing either anionic, zwitter-ionic or nonionic detergents. The complete and repeatable separation of these proteins was accomplished with a running electrolyte solution of 25 mM Tris/192 mM glycine, pH 8.8, containing either sodium dodecyl sulfate or n-octyl beta-D-glucopyranoside at concentration up to 5.0 and 7.0 mM, respectively. Migration times and resolution of the individual LHC II intrinsic membrane proteins were sensitive to the type of detergent. The effect of detergent concentration on the electrophoretic behavior of the LHC II proteins was also investigated. Electroelution of the LHC II components separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to isolate these intrinsic membrane proteins, which were then injected onto the capillary electrophoresis system for peak identification.

Chloroplasts↗

PubMed1992

Entrapment of protein protease inhibitors in red blood cells.

Aprotinin and alpha 1-proteinase inhibitor have been encapsulated in human red blood cells (RBC) by a dialysis technique that involves transient hypotonic haemolysis followed by isotonic resealing. Both protease inhibitors can be encapsulated to a considerable extent. These molecules are released only by haemolysis of the cells and that excludes the possibility of using loaded erythrocytes for a slow release of the inhibitor(s) in the blood stream. However, the stability of the two inhibitors, the evidence for the binding of aprotinin to RBC components, and the results showing inhibition of endogenous proteolytic activity indicate that the inhibitors may be valuable in blocking, at least partially, undesired intraerythrocytic proteolytic reactions.

Aprotinin↗

PubMed1991

Red blood cells as carriers for delivering of proteins.

The potential use of red blood cells as a carrier system for transport and delivery of pharmacological substances is well documented. Various methods have been attempted for encapsulation of drugs; in this review we evaluate critically all the procedures illustrating their advantages and disadvantages. Moreover, we present kinetic studies of protein encapsulation by moderate hypotonic dialysis, which allows entrapment of molecules with MW less than 50,000 Da with negligible stress of the erythrocyte membrane. Furthermore data reveal that the resealing procedure commonly used is insufficient to completely seal pores of loaded erythrocytes, allowing entrapped proteins with MW less than 12-14,000 Da to escape. However, only 20-30% of the entrapped material is released, depending on the final cytocrit, while the remaining is associated to the inner membrane or to cytosolic components. Although the method of hypotonic dialysis is known to be the one that minimally affects the biophysical and immunological properties of the red blood cell membrane, the interaction of encapsulated material with cell costituents would need to be further assessed when considering red cells as macromolecular carriers.

Cell Membrane Permeability↗

PubMed1990

Encapsulation of proteins into human erythrocytes: a kinetic investigation.

Moderate osmotic shocks of human erythrocytes by hypotonic dialysis (0.06 mosmol/kg) induce cell swelling and formation of pores, without causing apparent lysis. Using 125I-labeled macromolecules of different molecular weight and net charge, we followed the kinetics and efficiency of their encapsulation into erythrocytes. After a 20-30 min period of cell dialysis, macromolecules of up to 50 kDa begin diffusing into the swollen cells by a process which can be described by a first-order two-compartment kinetics. Adsorption to the external cell surface was insignificant, while adsorption to the inner membrane surface was substantial (15-20%) only for positively charged proteins, at physiological pH. After resealing, pores of a 12-14 kDa cut-off might remain open allowing some release of entrapped material (20-30%), depending on the final cytocrit, while the remaining might be associated with inner membrane or cytosolic components. Although the method of hypotonic dialysis is known to affect minimally the biophysical and immunological properties of red blood cell membranes, the interaction of encapsulated material with cell constituents would need to be further assessed when considering red cells as macromolecular carriers.

Cell Membrane Permeability↗

PubMed1986

Discrimination of tertiary and quaternary Bohr effect in the O2 binding of Helix pomatia beta-hemocyanin.

Simultaneous determination of proton uptake and oxygen binding has been carried out on Helix pomatia beta-hemocyanin under equilibrium conditions in the absence of buffer and at different initial pH values. Oxygen-binding isotherms of unbuffered H. pomatia beta-hemocyanin, in the presence of phenol red as pH indicator, have been determined employing a thin-layer apparatus. Application of this very accurate technique allows monitoring of proton uptake (or release) coupled to O2-binding also at extremes of saturation which are often difficult to explore and analyze. The data have been analyzed within the framework of the cooperon model (M. Brunori, M. Coletta and E. Di Cera, Biophys. Chem. 23 (1986) 215) and compared with those obtained in the presence of buffer. Comparison of pH changes with ligand binding of the T state over all the saturation range has allowed us to discriminate and obtain quantitative estimates of the Bohr protons associated with both oxygenation of the T state and quaternary allosteric transition; no protons are taken up or released during oxygenation of the R state. These results differ quantitatively from those obtained in the presence of buffer, which alters significantly the T state contribution to the overall Bohr effect.

Journal Article↗

PubMed1985

Heterogeneous binding of oxygen and carbon monoxide to dissociated molluscan hemocyanin.

Functional heterogeneity in O2 or CO binding of sites of dissociated molluscan hemocyanin polypeptide chains (Helix pomatia and Octopus vulgaris) has been estimated by an analysis of accurate noncooperative binding curves. Three types of experiments were performed: pure O2 or CO binding, competitive displacement of one ligand by the other, and simultaneous removal of both gases from protein partially saturated with O2 and CO. The data were analyzed in terms of a model which has two fractions of sites with different properties for O2 and CO. The relative proportion of the different binding sites and their affinity constant values were found by the combined use of the three different procedures. All species show a marked functional heterogeneity of sites for O2 binding, while for CO binding it has been observed only in the case of H. pomatia beta-hemocyanin. Moreover, in all three molluscan hemocyanins examined, the two classes of O2-binding sites, although present in different proportions within the polypeptide chains, display similar affinity constant values. The data reported show a good consistency with results obtained using digested and isolated domains, providing confidence in the analytical procedure used. From comparison of the O2/CO affinity ratios (KO2, KCO) of each class it may be suggested that the difference in O2 affinity of two kinds of binding sites is related to a different local structure of the active sites. The results, moreover, unequivocally confirm that binding and displacement of two gaseous ligands to hemocyanin occur by a simple competitive mechanism, although the binding site is structurally complex and the two ligands are bound with different geometries.

Journal Article↗

PubMed1984

Distribution of copper atoms and binding of carbon monoxide in partially copper-depleted hemocyanin.

Binding of CO to single cuprous (half-apo) or cupric (met-apo) copper of hemocyanin is investigated by a new method which allows estimation of the total amount of CO bound to hemocyanin. Pure half-apo preparations could not be obtained with the molluscan hemocyanins from Helix pomatia and Octopus vulgaris, and a residual fraction of sites with coupled copper is always present. However, the determination of CO bound to the protein before and after addition of H2O2, used to oxidize selectively single copper sites, reveals that CO binds to half-apo Cu(I), and is released upon oxidation of copper to met-apo Cu(II). Binding of CO to half-apo is not associated to luminescence, proving that luminescence of native carboxyhemocyanin demands the presence of a second cuprous copper in the site. In addition, analysis of data indicates that the residual amount of coupled copper sites in partially copper-depleted hemocyanin is underestimated by the residual O2-copper band measured at 340 nm in air, while faithfully quantitated by the residual luminescence in the presence of CO. A distribution of the copper left in the site of three partially copper-depleted hemocyanins is depicted.

Animals↗

PubMed1983

A spectrophotometric method to determine the amount of CO bound to hemocyanin.

The amount of CO bound to hemocyanin has been determined by titration with human hemoglobin (Hb). The spectrophotometric method makes use of the high affinity of Hb for CO, which (at pH 9) allows a complete transfer of the ligand from hemocyanin to hemoglobin. Taking advantage of the large spectral changes at the level of the heme, this method allows the rapid determination of the CO bound to hemocyanin, using relatively small amounts of protein (less than 5 mg). Application of this method shows (i) that CO binds to hemocyanins with a stoichiometry of 1:1, in agreement with one earlier observation with 14C-labeled carbon monoxide (1), and (ii) that decrease of the CO-copper luminescence of hemocyanin observed upon addition of KCN is quantitatively related to the displacement of CO from the active site.

Animals↗

PubMed1983

Kinetic and thermodynamic parameters for oxygen binding to the allosteric states of Panulirus interruptus hemocyanin.

The temperature dependence of the oxygen binding equilibria and kinetics of Panulirus interruptus hemocyanin has been analyzed within the context of the two-state allosteric model. Oxygenation of the T-state is characterized by a more negative value of DeltaH than that of the R-state; therefore, cooperative effects in oxygen binding to P. interruptus hemocyanin are thermodynamically governed by favorable entropy changes. The allosteric transition in the unliganded derivative shows an enthalpy-entropy compensation effect. The activation enthalpies for oxygenation and deoxygenation of the T-state are larger than those for the R-state, while the activation entropies are favorable for the T-state and unfavorable for the R-state. Thus, the activation free energies for oxygen binding to the T- and R-states are similar, while for the deoxygenation reaction DeltaG++ is smaller for the T-state. The analysis reported confirms the applicability of the Monod-Wyman-Changeux two-state allosteric model to P. interruptus hemocyanin and yields a complete thermodynamic characterization of oxygen binding under both equilibrium and dynamic regimes.

Journal Article↗