Quantum biology is the study of quantum effects on biochemical mechanisms and biological function... more Quantum biology is the study of quantum effects on biochemical mechanisms and biological function. We show that the biological production of reactive oxygen species (ROS) in live cells can be influenced by coherent electron spin dynamics, providing a new example of quantum biology in cellular regulation. ROS partitioning appears to be mediated during the activation of molecular oxygen (O2) by reduced flavoenzymes, forming spin-correlated radical pairs (RPs). We find that oscillating magnetic fields at Zeeman resonance alter relative yields of cellular superoxide (O2•−) and hydrogen peroxide (H2O2) ROS products, indicating coherent singlet-triplet mixing at the point of ROS formation. Furthermore, the orientation-dependence of magnetic stimulation, which leads to specific changes in ROS levels, increases either mitochondrial respiration and glycolysis rates. Our results reveal quantum effects in live cell cultures that bridge atomic and cellular levels by connecting ROS partitioning ...
2016 Progress in Electromagnetic Research Symposium (PIERS), 2016
Summary form only given. This study presents experimental data and related theoretical calculatio... more Summary form only given. This study presents experimental data and related theoretical calculations for the effects of static and radio frequency magnetic fields in the biological production of reactive oxygen species (ROS). This paper discusses a mechanism of spin-biochemistry providing a fundamental relation between bioenergetics and ROS product channeling. Intracellular superoxide (O<sub>2</sub><sup>.-</sup>) and extracellular hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) were investigated in vitro along with respiration and glycolysis with primary human umbilical vein endothelial cells (HUVECs). Theoretical analysis considers RF magnetic field effects in a one-proton radical pair model. HUVECs were exposed to either 50 μT static magnetic fields (SMF), or to static magnetic fields combined with 1.4 or 7MHz RF magnetic fields in parallel or perpendicular corresponding to Zeeman and hyperfine interactions, respectively. We observe differential changes in bioenergetics and in consumption of O<sub>2</sub><sup>.-</sup> and production H<sub>2</sub>O<sub>2</sub> as a function of angle between SMF and RF magnetic fields. There are several notable results from the 1.4MHz (Zeeman) preliminary data. In perpendicular mode, both ROS are decreased but not in the same proportion - less O<sub>2</sub><sup>.-</sup> than H<sub>2</sub>O<sub>2</sub>, respiration is unchanged, whereas glycolytic activity is increased. The decrease in ROS production with enhanced glycolytic activity may be due to effective scavenging of ROS by pyruvate. In parallel mode, a decrease in O<sub>2</sub><sup>.-</sup> and an increase in H<sub>2</sub>O<sub>2</sub> is observed, respiration is increased, whereas glycolysis is unchanged. The orientation effects that lead to specific ROS product distribution is consistent with the spin biochemistry model; however, we do not know the specific spin biochemistry targets or signaling channels that lead to changes in bioenergetics. We propose that O<sub>2</sub><sup>.-</sup> and H<sub>2</sub>O<sub>2</sub> production in metabolic processes occur through singlet-triplet modulation of semiquinone flavin (FADH<sup>.</sup>) enzymes and O<sub>2</sub><sup>.-</sup> spin-correlated radical pairs. Spin-radical pair products are modulated by the RF magnetic fields that presumably decouple flavin hyperfine interactions during spin coherence. RF flavin hyperfine decoupling results in changes of H<sub>2</sub>O<sub>2</sub> singlet state products, which creates cellular oxidative stress and acts as a secondary messenger that affects bioenergetics. This study demonstrates the interplay between O<sub>2</sub><sup>.-</sup> and H<sub>2</sub>O<sub>2</sub> production when influenced by RF magnetic fields and underscores the subtle effects of low-frequency magnetic fields on oxidative metabolism, ROS signaling, and cellular growth.
In recent decades, the use of pulsed electromagnetic fields (PEMF) in therapeutics has been one o... more In recent decades, the use of pulsed electromagnetic fields (PEMF) in therapeutics has been one of the main fields of activity in the bioelectromagnetics arena. Nevertheless, progress in this area has been hindered by the lack of consensus on a biophysical mechanism of interaction that can satisfactorily explain how low-level, non-thermal electromagnetic fields would be able to sufficiently affect chemistry as to elicit biological effects in living organisms. This specifically applies in cases where the induced electric fields are too small to generate a biological response of any consequence. A growing body of experimental observations that would explain the nature of these effects speaks strongly about the involvement of a theory known as the radical pair mechanism (RPM). This mechanism explains how a pair of reactive oxygen species with distinct chemical fate can be influenced by a low-level external magnetic field through Zeeman and hyperfine interactions. So far, a study of the effects of complex spatiotemporal signals within the context of the RPM has not been performed. Here, we present a computational investigation of such effects by utilizing a generic PEMF test signal and RPM models of different complexity. Surprisingly, our results show how substantially different chemical results can be obtained within ranges that depend on the specific orientation of the PEMF test signal with respect to the background static magnetic field, its waveform, and both of their amplitudes. These results provide a basis for explaining the distinctive biological relevance of PEMF signals on radical pair chemical reactions.
Thermal stability of plasma membrane Ca 2+ pump was systematically studied in three micellar syst... more Thermal stability of plasma membrane Ca 2+ pump was systematically studied in three micellar systems of different composition, and related with the interactions amphiphile-protein measured by fluorescence resonance energy transfer. Thermal denaturation was characterized as an irreversible process that is well described by a first order kinetic with an activation energy of 222 ± 12 kJ/mol in the range 33-45°C. Upon increasing the mole fraction of phospholipid in the mixed micelles where the Ca 2+ pump was reconstituted, the kinetic coefficient for the inactivation process diminished until it reached a constant value, different for each phospholipid species. We propose a model in which thermal stability of the pump depends on the composition of the amphiphile monolayer directly in contact with the transmembrane protein surface. Application of this model shows that the maximal pump stability is attained when 80% of this surface is covered by phospholipids. This analysis provides an indirect measure of the relative affinity phospholipid/detergent for the hydrophobic transmembrane surface of the protein (K LD) showing that those phospholipids with higher affinity provide greater stability to the Ca 2+ pump. We developed a method for directly measure K LD by using fluorescence resonance energy transfer from the membrane protein tryptophan residues to a pyrene-labeled phospholipid. K LD values obtained by this procedure agree with those obtained from the model, providing a strong evidence to support its validity.
We have previously demonstrated (Diabetes 39:707-711, 1990) that in vitro glycation of the red ce... more We have previously demonstrated (Diabetes 39:707-711, 1990) that in vitro glycation of the red cell Ca(2+) pump diminishes the Ca(2+)-ATPase activity of the enzyme up to 50%. Such effect is due to the reaction of glucose with lysine residues of the Ca(2+) pump (Biochem. J. 293:369-375, 1993). The aim of this work was to determine whether the effect of glucose is due to a full inactivation of a fraction of the total population of Ca(2+) pump, or to a partial inactivation of all the molecules. Glycation decreased the V(max) for the ATPase activity leaving unaffected the apparent affinities for Ca(2+), calmodulin or ATP. The apparent turnover was identical in both, the glycated and the native enzyme. Glycation decreased the V(max) for the ATP-dependent but not for the calmodulin-activated phosphatase activities. Concomitantly with the inhibition, up to 6.5% of the lysine residues were randomly glycated. The probabilistic analysis of the relation between the enzyme activity and the fraction of nonmodified residues indicates that only one Lys residue is responsible for the inhibition. We suggest that glucose decreases the Ca(2+)-ATPase activity by reacting with one essential Lys residue probably located in the vicinity of the catalytic site, which results in the full inactivation of the enzyme. Thus, Ca(2+)-ATPase activity measured in erythrocyte membranes or purified enzyme preparations preincubated with glucose depends on the remaining enzyme molecules in which the essential Lys residue stays unglycated.
Bordetella pertussis virulence-associated 30-, 32-, 90-and 95-kDa outer membrane proteins were pu... more Bordetella pertussis virulence-associated 30-, 32-, 90-and 95-kDa outer membrane proteins were purified and their N-terminal amino acid sequences were determined. The 30-and 32-kDa outer membrane proteins showed identity to the C-terminal region of the precursors of the serum resistance protein (BrkA) and the tracheal colonization factor, respectively. We confirmed the cleavage site of these precursors after N731 for BrkA and after N393 for tracheal colonization factor. Associated with the 32-kDa outer membrane protein, we found a new group of 36-kDa virulence-associated peptides. The 95-kDa outer membrane protein showed identity to Vag8. The 90-kDa outer membrane protein did not show homology with the described proteins. We report the N-termini sequence of Vir-90, a novel potential virulence factor.
Ca 2+ pump dimerization was studied by using a combined approach of thermal denaturation and fluo... more Ca 2+ pump dimerization was studied by using a combined approach of thermal denaturation and fluorescence resonance energy transfer. The measurement of calcium pump ability to dimerize after the unfolding of individual functional domains of the enzyme demonstrated the existence of two different regions involved in the self-association process. One of these regions is highly susceptible to thermal unfolding and was identified as the calmodulin (CaM)-binding domain. The other region whose thermal stability is higher than those of the catalytic and CaM-binding domains could be related with the previously found C28W-binding regions.
Here we undertook a comparative study of the composition of the lipid annulus of three ATPases pe... more Here we undertook a comparative study of the composition of the lipid annulus of three ATPases pertaining to the P-type family: plasma membrane calcium pump (PMCA), sarcoplasmic reticulum calcium pump (SERCA) and Na,K-ATPase. The photoactivatable phosphatidylcholine analogue [ 125 I]TID-PC/16 was incorporated into mixtures of dimyristoyl phosphatidylcholine (DMPC) and each enzyme with the aid of the nonionic detergent C 12 E 10. After photolysis, the extent of the labeling reaction was assessed to determine the lipid:protein stoichiometry: 17 for PMCA, 18 for SERCA, 24 for the Na, K-ATPase (a-subunit) and 5.6 mol PC/mol protein for the Na,K-ATPase (b-subunit).
The functions of membrane proteins are highly dependent on their phospholipid environment. In thi... more The functions of membrane proteins are highly dependent on their phospholipid environment. In this article, we have used a hydrophobic photolabeling method to study the noncovalent interactions between plasma membrane calcium pump (PMCA) and surrounding phospholipids. With this approach, we determined (1) the number of lipid molecules in close contact with the transmembrane surface, i.e., the lipid-protein stoichiometry, and (2) the distribution of lipid molecules among different regions of the protein. PMCA was photolabeled in mixed micelles containing detergent, the phosphatidylcholine photoactivatable analog 1-palmitoyl-2-[9-[2′-[ 125 I]iodo-4′-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine, and different amounts of dimyristoyl phosphatidylcholine (PC). The stoichiometry was estimated after the extent of the labeling reaction had been independently assessed. We determined a maximum number of 17 ± 1 molecules of PC in close contact with the transmembrane surface per PMCA molecule. In addition, a semiquantitative description of the phospholipid environment around different regions of PMCA was carried out after limited proteolysis of the photolabeled protein. The distribution of labels among the N-terminal (1-322), the central (323-660), and the C-terminal (661-1205) regions was 26, 36, and 38%, respectively. Index Entries: Membrane proteins; plasma membrane calcium pump; photoactivatable phospholipids; lipid-protein interactions.
The oligomerization of the plasma membrane calcium pump (PMCA) in phospholipid/detergent micelles... more The oligomerization of the plasma membrane calcium pump (PMCA) in phospholipid/detergent micelles was evaluated using a combined spectroscopic and kinetic approach and related to the enzyme stability. Energy transfer between fluorescein-5Ј-isothiocyanate and eosin-5Ј-isothiocyanate attached to different PMCA molecules was used to determine the dissociation constant of dimeric PMCA (140 Ϯ 50 nM at 25°C) and characterize the time course of dimerization. The enzyme thermal stability at different dimer/monomer ratios was evaluated, quantifying the kinetic coefficient of thermal inactivation. This coefficient decreases with PMCA concentration, becoming approximately constant beyond 300 nM. Thermal treatment leads to the formation of inactive monomers that associate only with native monomers. These mixed dimers are formed with a kinetic coefficient that is half that determined for the native dimers. We proposed a model for PMCA thermal inactivation that considers the equilibria among dimers, monomers, and mixed dimers, and the inactivation of the last two species through irreversible steps. The numerical resolution of the differential equations describing this model fitted to the experimental data allowed the determination of the model coefficients. This analysis shows that thermal inactivation occurs through the denaturation of the monomer, which lifetime is 25 min at 44°C. The obtained results suggest that PMCA dimerization constitutes a mechanism of self protection against spontaneous denaturation.
Non-enzymatic glycation of biomolecules has been implicated in the pathophysiology of aging and d... more Non-enzymatic glycation of biomolecules has been implicated in the pathophysiology of aging and diabetes. Among the potential targets for glycation are biological membranes, characterized by a complex organization of lipids and proteins interacting and forming domains of different size and stability. In the present study, we analyse the effects of glycation on the interactions between membrane proteins and lipids. The phospholipid affinity for the transmembrane surface of the PMCA (plasma-membrane Ca2+-ATPase) was determined after incubating the protein or the phospholipids with glucose. Results show that the affinity between PMCA and the surrounding phospholipids decreases significantly after phosphospholipid glycation, but remains unmodified after glycation of the protein. Furthermore, phosphatidylethanolamine glycation decreases by ∼30% the stability of PMCA against thermal denaturation, suggesting that glycated aminophospholipids induce a structural rearrangement in the protein ...
Biochemical and Biophysical Research Communications, 1994
The membrane-associated regions of the human erythrocyte Ca2+ pump were investigated by hydrophob... more The membrane-associated regions of the human erythrocyte Ca2+ pump were investigated by hydrophobic photolabeling. Purified Ca2+ pump was reconstituted in asolectin vesicles loaded with [3H]DIPETPD, a photochemical probe designed to label deeply into the hydrophobic core of the lipid bilayer (Delfino et al. J. Am. Chem. Soc. 115, 3458-3474, 1993). After photolysis and SDS-PAGE analysis, a significant light-dependent labeling of the Ca2+ pump was found. Controlled proteolysis of the photoadduct with trypsin or protease V8 followed by SDS-PAGE and immunoblotting yielded individual labeled fragments. The labeling pattern indicated the existence of three sequential clusters of transmembrane regions, consistent with the current model for the topography of this enzyme.
ABSTRACT Not Available Bibtex entry for this abstract Preferred format for this abstract (see Pre... more ABSTRACT Not Available Bibtex entry for this abstract Preferred format for this abstract (see Preferences) Find Similar Abstracts: Use: Authors Title Return: Query Results Return items starting with number Query Form Database: Astronomy Physics arXiv e-prints
This work describes a simple method for determining the association constant of amphiphiles to me... more This work describes a simple method for determining the association constant of amphiphiles to membrane proteins. The method uses a fluorescent phospholipid probe, which senses the competition among unlabeled amphiphiles for positions on the transmembrane surface of the protein. The contact between the probe and the protein surface is detected through resonance energy transfer. We have analyzed theoretically this process deriving a general equation for the dependence of the energy transfer efficiency on the composition of the micelles/bilayers in which the protein is inserted. This equation includes an exchange constant for each amphiphile, which gives a measure of its affinity for the protein with respect to that of an amphiphile set as the reference. We applied this method to determine the exchange constant of different phospholipids for the plasma membrane calcium pump.
A systematic study of the membrane-associated regions in the plasma membrane Ca2+ pump of erythro... more A systematic study of the membrane-associated regions in the plasma membrane Ca2+ pump of erythrocytes has been performed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3-(trifluoromethyl)-3-(rn-[ '251]iodophenyl)diazirine ([ '251]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane-bound probe, [3H]bis-phosphatidylethanolamine (trifluoromethyl) phenyldiazirine. A significant light-dependent labeling of an M, 135,000-140,000 peptide, corresponding to the full Ca2+ pump, was observed with both probes. After proteolysis of the pump labeled with each probe and isolation of fragments by SDS-PAGE, a common pattern of labeled peptides was observed. Similarly, labeling of the Ca2+ pump with ['251]TID, either in isolated red blood cell membranes or after the enzyme was purified, yields a similar pattern of labeled peptides. Taken together, these results validate the use of either probe to study the lipid interface of the membrane-embedded region of this protein, and sustain the notion that the conformation of the pump is maintained throughout the procedures of solubilization, affinity purification, and reconstitution into proteoliposomes. In this work, we put special emphasis on a detailed analysis of the N-terminal domain of the CaZ+ pump. A labeled peptide of M, 40,000 belonging to this region was purified and further digested with V8 protease. The specific incorporation of [1251]TID to proteolytic fragments pertaining to the amino-terminal region indicates the existence of two transmembrane stretches in this domain. A theoretical analysis based on the amino acid sequence 1-322 predicts two segments with high probability of membrane insertion, in agreement with the experimental data. Each segment shows a periodicity pattern of hydrophobicity and variability compatible with a-helical structure. These results strongly suggest the existence of a transmembrane helical hairpin motif near the N-terminus of the Ca2+ pump.
Quantum biology is the study of quantum effects on biochemical mechanisms and biological function... more Quantum biology is the study of quantum effects on biochemical mechanisms and biological function. We show that the biological production of reactive oxygen species (ROS) in live cells can be influenced by coherent electron spin dynamics, providing a new example of quantum biology in cellular regulation. ROS partitioning appears to be mediated during the activation of molecular oxygen (O2) by reduced flavoenzymes, forming spin-correlated radical pairs (RPs). We find that oscillating magnetic fields at Zeeman resonance alter relative yields of cellular superoxide (O2•−) and hydrogen peroxide (H2O2) ROS products, indicating coherent singlet-triplet mixing at the point of ROS formation. Furthermore, the orientation-dependence of magnetic stimulation, which leads to specific changes in ROS levels, increases either mitochondrial respiration and glycolysis rates. Our results reveal quantum effects in live cell cultures that bridge atomic and cellular levels by connecting ROS partitioning ...
2016 Progress in Electromagnetic Research Symposium (PIERS), 2016
Summary form only given. This study presents experimental data and related theoretical calculatio... more Summary form only given. This study presents experimental data and related theoretical calculations for the effects of static and radio frequency magnetic fields in the biological production of reactive oxygen species (ROS). This paper discusses a mechanism of spin-biochemistry providing a fundamental relation between bioenergetics and ROS product channeling. Intracellular superoxide (O<sub>2</sub><sup>.-</sup>) and extracellular hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) were investigated in vitro along with respiration and glycolysis with primary human umbilical vein endothelial cells (HUVECs). Theoretical analysis considers RF magnetic field effects in a one-proton radical pair model. HUVECs were exposed to either 50 μT static magnetic fields (SMF), or to static magnetic fields combined with 1.4 or 7MHz RF magnetic fields in parallel or perpendicular corresponding to Zeeman and hyperfine interactions, respectively. We observe differential changes in bioenergetics and in consumption of O<sub>2</sub><sup>.-</sup> and production H<sub>2</sub>O<sub>2</sub> as a function of angle between SMF and RF magnetic fields. There are several notable results from the 1.4MHz (Zeeman) preliminary data. In perpendicular mode, both ROS are decreased but not in the same proportion - less O<sub>2</sub><sup>.-</sup> than H<sub>2</sub>O<sub>2</sub>, respiration is unchanged, whereas glycolytic activity is increased. The decrease in ROS production with enhanced glycolytic activity may be due to effective scavenging of ROS by pyruvate. In parallel mode, a decrease in O<sub>2</sub><sup>.-</sup> and an increase in H<sub>2</sub>O<sub>2</sub> is observed, respiration is increased, whereas glycolysis is unchanged. The orientation effects that lead to specific ROS product distribution is consistent with the spin biochemistry model; however, we do not know the specific spin biochemistry targets or signaling channels that lead to changes in bioenergetics. We propose that O<sub>2</sub><sup>.-</sup> and H<sub>2</sub>O<sub>2</sub> production in metabolic processes occur through singlet-triplet modulation of semiquinone flavin (FADH<sup>.</sup>) enzymes and O<sub>2</sub><sup>.-</sup> spin-correlated radical pairs. Spin-radical pair products are modulated by the RF magnetic fields that presumably decouple flavin hyperfine interactions during spin coherence. RF flavin hyperfine decoupling results in changes of H<sub>2</sub>O<sub>2</sub> singlet state products, which creates cellular oxidative stress and acts as a secondary messenger that affects bioenergetics. This study demonstrates the interplay between O<sub>2</sub><sup>.-</sup> and H<sub>2</sub>O<sub>2</sub> production when influenced by RF magnetic fields and underscores the subtle effects of low-frequency magnetic fields on oxidative metabolism, ROS signaling, and cellular growth.
In recent decades, the use of pulsed electromagnetic fields (PEMF) in therapeutics has been one o... more In recent decades, the use of pulsed electromagnetic fields (PEMF) in therapeutics has been one of the main fields of activity in the bioelectromagnetics arena. Nevertheless, progress in this area has been hindered by the lack of consensus on a biophysical mechanism of interaction that can satisfactorily explain how low-level, non-thermal electromagnetic fields would be able to sufficiently affect chemistry as to elicit biological effects in living organisms. This specifically applies in cases where the induced electric fields are too small to generate a biological response of any consequence. A growing body of experimental observations that would explain the nature of these effects speaks strongly about the involvement of a theory known as the radical pair mechanism (RPM). This mechanism explains how a pair of reactive oxygen species with distinct chemical fate can be influenced by a low-level external magnetic field through Zeeman and hyperfine interactions. So far, a study of the effects of complex spatiotemporal signals within the context of the RPM has not been performed. Here, we present a computational investigation of such effects by utilizing a generic PEMF test signal and RPM models of different complexity. Surprisingly, our results show how substantially different chemical results can be obtained within ranges that depend on the specific orientation of the PEMF test signal with respect to the background static magnetic field, its waveform, and both of their amplitudes. These results provide a basis for explaining the distinctive biological relevance of PEMF signals on radical pair chemical reactions.
Thermal stability of plasma membrane Ca 2+ pump was systematically studied in three micellar syst... more Thermal stability of plasma membrane Ca 2+ pump was systematically studied in three micellar systems of different composition, and related with the interactions amphiphile-protein measured by fluorescence resonance energy transfer. Thermal denaturation was characterized as an irreversible process that is well described by a first order kinetic with an activation energy of 222 ± 12 kJ/mol in the range 33-45°C. Upon increasing the mole fraction of phospholipid in the mixed micelles where the Ca 2+ pump was reconstituted, the kinetic coefficient for the inactivation process diminished until it reached a constant value, different for each phospholipid species. We propose a model in which thermal stability of the pump depends on the composition of the amphiphile monolayer directly in contact with the transmembrane protein surface. Application of this model shows that the maximal pump stability is attained when 80% of this surface is covered by phospholipids. This analysis provides an indirect measure of the relative affinity phospholipid/detergent for the hydrophobic transmembrane surface of the protein (K LD) showing that those phospholipids with higher affinity provide greater stability to the Ca 2+ pump. We developed a method for directly measure K LD by using fluorescence resonance energy transfer from the membrane protein tryptophan residues to a pyrene-labeled phospholipid. K LD values obtained by this procedure agree with those obtained from the model, providing a strong evidence to support its validity.
We have previously demonstrated (Diabetes 39:707-711, 1990) that in vitro glycation of the red ce... more We have previously demonstrated (Diabetes 39:707-711, 1990) that in vitro glycation of the red cell Ca(2+) pump diminishes the Ca(2+)-ATPase activity of the enzyme up to 50%. Such effect is due to the reaction of glucose with lysine residues of the Ca(2+) pump (Biochem. J. 293:369-375, 1993). The aim of this work was to determine whether the effect of glucose is due to a full inactivation of a fraction of the total population of Ca(2+) pump, or to a partial inactivation of all the molecules. Glycation decreased the V(max) for the ATPase activity leaving unaffected the apparent affinities for Ca(2+), calmodulin or ATP. The apparent turnover was identical in both, the glycated and the native enzyme. Glycation decreased the V(max) for the ATP-dependent but not for the calmodulin-activated phosphatase activities. Concomitantly with the inhibition, up to 6.5% of the lysine residues were randomly glycated. The probabilistic analysis of the relation between the enzyme activity and the fraction of nonmodified residues indicates that only one Lys residue is responsible for the inhibition. We suggest that glucose decreases the Ca(2+)-ATPase activity by reacting with one essential Lys residue probably located in the vicinity of the catalytic site, which results in the full inactivation of the enzyme. Thus, Ca(2+)-ATPase activity measured in erythrocyte membranes or purified enzyme preparations preincubated with glucose depends on the remaining enzyme molecules in which the essential Lys residue stays unglycated.
Bordetella pertussis virulence-associated 30-, 32-, 90-and 95-kDa outer membrane proteins were pu... more Bordetella pertussis virulence-associated 30-, 32-, 90-and 95-kDa outer membrane proteins were purified and their N-terminal amino acid sequences were determined. The 30-and 32-kDa outer membrane proteins showed identity to the C-terminal region of the precursors of the serum resistance protein (BrkA) and the tracheal colonization factor, respectively. We confirmed the cleavage site of these precursors after N731 for BrkA and after N393 for tracheal colonization factor. Associated with the 32-kDa outer membrane protein, we found a new group of 36-kDa virulence-associated peptides. The 95-kDa outer membrane protein showed identity to Vag8. The 90-kDa outer membrane protein did not show homology with the described proteins. We report the N-termini sequence of Vir-90, a novel potential virulence factor.
Ca 2+ pump dimerization was studied by using a combined approach of thermal denaturation and fluo... more Ca 2+ pump dimerization was studied by using a combined approach of thermal denaturation and fluorescence resonance energy transfer. The measurement of calcium pump ability to dimerize after the unfolding of individual functional domains of the enzyme demonstrated the existence of two different regions involved in the self-association process. One of these regions is highly susceptible to thermal unfolding and was identified as the calmodulin (CaM)-binding domain. The other region whose thermal stability is higher than those of the catalytic and CaM-binding domains could be related with the previously found C28W-binding regions.
Here we undertook a comparative study of the composition of the lipid annulus of three ATPases pe... more Here we undertook a comparative study of the composition of the lipid annulus of three ATPases pertaining to the P-type family: plasma membrane calcium pump (PMCA), sarcoplasmic reticulum calcium pump (SERCA) and Na,K-ATPase. The photoactivatable phosphatidylcholine analogue [ 125 I]TID-PC/16 was incorporated into mixtures of dimyristoyl phosphatidylcholine (DMPC) and each enzyme with the aid of the nonionic detergent C 12 E 10. After photolysis, the extent of the labeling reaction was assessed to determine the lipid:protein stoichiometry: 17 for PMCA, 18 for SERCA, 24 for the Na, K-ATPase (a-subunit) and 5.6 mol PC/mol protein for the Na,K-ATPase (b-subunit).
The functions of membrane proteins are highly dependent on their phospholipid environment. In thi... more The functions of membrane proteins are highly dependent on their phospholipid environment. In this article, we have used a hydrophobic photolabeling method to study the noncovalent interactions between plasma membrane calcium pump (PMCA) and surrounding phospholipids. With this approach, we determined (1) the number of lipid molecules in close contact with the transmembrane surface, i.e., the lipid-protein stoichiometry, and (2) the distribution of lipid molecules among different regions of the protein. PMCA was photolabeled in mixed micelles containing detergent, the phosphatidylcholine photoactivatable analog 1-palmitoyl-2-[9-[2′-[ 125 I]iodo-4′-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine, and different amounts of dimyristoyl phosphatidylcholine (PC). The stoichiometry was estimated after the extent of the labeling reaction had been independently assessed. We determined a maximum number of 17 ± 1 molecules of PC in close contact with the transmembrane surface per PMCA molecule. In addition, a semiquantitative description of the phospholipid environment around different regions of PMCA was carried out after limited proteolysis of the photolabeled protein. The distribution of labels among the N-terminal (1-322), the central (323-660), and the C-terminal (661-1205) regions was 26, 36, and 38%, respectively. Index Entries: Membrane proteins; plasma membrane calcium pump; photoactivatable phospholipids; lipid-protein interactions.
The oligomerization of the plasma membrane calcium pump (PMCA) in phospholipid/detergent micelles... more The oligomerization of the plasma membrane calcium pump (PMCA) in phospholipid/detergent micelles was evaluated using a combined spectroscopic and kinetic approach and related to the enzyme stability. Energy transfer between fluorescein-5Ј-isothiocyanate and eosin-5Ј-isothiocyanate attached to different PMCA molecules was used to determine the dissociation constant of dimeric PMCA (140 Ϯ 50 nM at 25°C) and characterize the time course of dimerization. The enzyme thermal stability at different dimer/monomer ratios was evaluated, quantifying the kinetic coefficient of thermal inactivation. This coefficient decreases with PMCA concentration, becoming approximately constant beyond 300 nM. Thermal treatment leads to the formation of inactive monomers that associate only with native monomers. These mixed dimers are formed with a kinetic coefficient that is half that determined for the native dimers. We proposed a model for PMCA thermal inactivation that considers the equilibria among dimers, monomers, and mixed dimers, and the inactivation of the last two species through irreversible steps. The numerical resolution of the differential equations describing this model fitted to the experimental data allowed the determination of the model coefficients. This analysis shows that thermal inactivation occurs through the denaturation of the monomer, which lifetime is 25 min at 44°C. The obtained results suggest that PMCA dimerization constitutes a mechanism of self protection against spontaneous denaturation.
Non-enzymatic glycation of biomolecules has been implicated in the pathophysiology of aging and d... more Non-enzymatic glycation of biomolecules has been implicated in the pathophysiology of aging and diabetes. Among the potential targets for glycation are biological membranes, characterized by a complex organization of lipids and proteins interacting and forming domains of different size and stability. In the present study, we analyse the effects of glycation on the interactions between membrane proteins and lipids. The phospholipid affinity for the transmembrane surface of the PMCA (plasma-membrane Ca2+-ATPase) was determined after incubating the protein or the phospholipids with glucose. Results show that the affinity between PMCA and the surrounding phospholipids decreases significantly after phosphospholipid glycation, but remains unmodified after glycation of the protein. Furthermore, phosphatidylethanolamine glycation decreases by ∼30% the stability of PMCA against thermal denaturation, suggesting that glycated aminophospholipids induce a structural rearrangement in the protein ...
Biochemical and Biophysical Research Communications, 1994
The membrane-associated regions of the human erythrocyte Ca2+ pump were investigated by hydrophob... more The membrane-associated regions of the human erythrocyte Ca2+ pump were investigated by hydrophobic photolabeling. Purified Ca2+ pump was reconstituted in asolectin vesicles loaded with [3H]DIPETPD, a photochemical probe designed to label deeply into the hydrophobic core of the lipid bilayer (Delfino et al. J. Am. Chem. Soc. 115, 3458-3474, 1993). After photolysis and SDS-PAGE analysis, a significant light-dependent labeling of the Ca2+ pump was found. Controlled proteolysis of the photoadduct with trypsin or protease V8 followed by SDS-PAGE and immunoblotting yielded individual labeled fragments. The labeling pattern indicated the existence of three sequential clusters of transmembrane regions, consistent with the current model for the topography of this enzyme.
ABSTRACT Not Available Bibtex entry for this abstract Preferred format for this abstract (see Pre... more ABSTRACT Not Available Bibtex entry for this abstract Preferred format for this abstract (see Preferences) Find Similar Abstracts: Use: Authors Title Return: Query Results Return items starting with number Query Form Database: Astronomy Physics arXiv e-prints
This work describes a simple method for determining the association constant of amphiphiles to me... more This work describes a simple method for determining the association constant of amphiphiles to membrane proteins. The method uses a fluorescent phospholipid probe, which senses the competition among unlabeled amphiphiles for positions on the transmembrane surface of the protein. The contact between the probe and the protein surface is detected through resonance energy transfer. We have analyzed theoretically this process deriving a general equation for the dependence of the energy transfer efficiency on the composition of the micelles/bilayers in which the protein is inserted. This equation includes an exchange constant for each amphiphile, which gives a measure of its affinity for the protein with respect to that of an amphiphile set as the reference. We applied this method to determine the exchange constant of different phospholipids for the plasma membrane calcium pump.
A systematic study of the membrane-associated regions in the plasma membrane Ca2+ pump of erythro... more A systematic study of the membrane-associated regions in the plasma membrane Ca2+ pump of erythrocytes has been performed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3-(trifluoromethyl)-3-(rn-[ '251]iodophenyl)diazirine ([ '251]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane-bound probe, [3H]bis-phosphatidylethanolamine (trifluoromethyl) phenyldiazirine. A significant light-dependent labeling of an M, 135,000-140,000 peptide, corresponding to the full Ca2+ pump, was observed with both probes. After proteolysis of the pump labeled with each probe and isolation of fragments by SDS-PAGE, a common pattern of labeled peptides was observed. Similarly, labeling of the Ca2+ pump with ['251]TID, either in isolated red blood cell membranes or after the enzyme was purified, yields a similar pattern of labeled peptides. Taken together, these results validate the use of either probe to study the lipid interface of the membrane-embedded region of this protein, and sustain the notion that the conformation of the pump is maintained throughout the procedures of solubilization, affinity purification, and reconstitution into proteoliposomes. In this work, we put special emphasis on a detailed analysis of the N-terminal domain of the CaZ+ pump. A labeled peptide of M, 40,000 belonging to this region was purified and further digested with V8 protease. The specific incorporation of [1251]TID to proteolytic fragments pertaining to the amino-terminal region indicates the existence of two transmembrane stretches in this domain. A theoretical analysis based on the amino acid sequence 1-322 predicts two segments with high probability of membrane insertion, in agreement with the experimental data. Each segment shows a periodicity pattern of hydrophobicity and variability compatible with a-helical structure. These results strongly suggest the existence of a transmembrane helical hairpin motif near the N-terminus of the Ca2+ pump.
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Papers by Pablo Castello