RESUMO
Plasma membrane Ca2+-ATPase (PMCA) transports Ca2+ by a reaction cycle including phosphorylated intermediates. Calmodulin binding to the C-terminal tail disrupts autoinhibitory interactions, activating the pump. To assess the conformational changes during the reaction cycle, we studied the structure of different PMCA states using a fluorescent probe, hydrophobic photolabeling, controlled proteolysis and Ca2+-ATPase activity. Our results show that calmodulin binds to E2P-like states, and during dephosphorylation, the hydrophobicity in the nucleotide-binding pocket decreases and the Ca2+ binding site becomes inaccessible to the extracellular medium. Autoinhibitory interactions are disrupted in E1Ca and in the E2P ground state whereas they are stabilized in the E2·Pi product state. Finally, we propose a model that describes the conformational changes during the Ca2+ transport of PMCA.
Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Membrana Celular/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Sítios de Ligação , Calmodulina/genética , Humanos , Cinética , Fosforilação , Ligação Proteica , Conformação ProteicaRESUMO
In the recent years, the toxicity of certain divalent cations has been associated with the alteration of intracellular Ca2+ homeostasis. Among other mechanisms, these cations may affect the functionality of certain Ca2+-binding proteins and/or Ca2+ pumps. The plasma membrane calcium pump (PMCA) maintains Ca2+ homeostasis in eukaryotic cells by mediating the efflux of this cation in a process coupled to ATP hydrolysis. The aim of this work was to investigate both in vitro and in cultured cells if other divalent cations (Sr2+, Ba2+, Co2+, Cd2+, Pb2+ or Be2+) could be transported by PMCA. Current results indicate that both purified and intact cell PMCA transported Sr2+ with kinetic parameters close to those of Ca2+ transport. The transport of Pb2+ and Co2+ by purified PMCA was, respectively, 50 and 75% lower than that of Ca2+, but only Co2+ was extruded by intact cells and to a very low extent. In contrast, purified PMCA-but not intact cell PMCA-transported Ba2+ at low rates and only when activated by limited proteolysis or by phosphatidylserine addition. Finally, purified PMCA did not transport Cd2+ or Be2+, although minor Be2+ transport was measured in intact cells. Moreover, Cd2+ impaired the transport of Ca2+ through various mechanisms, suggesting that PMCA may be a potential target of Cd2+-mediated toxicity. The differential capacity of PMCA to transport these divalent cations may have a key role in their detoxification, limiting their noxious effects on cell homeostasis.
Assuntos
Cátions/farmacocinética , Metais/farmacocinética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Transporte Biológico , Cálcio/farmacocinética , Calmodulina/química , Calmodulina/metabolismo , Cátions/toxicidade , Células Cultivadas , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Células HEK293 , Humanos , Inativação Metabólica , Metais/toxicidade , Fosfatidilserinas/metabolismo , Fosfatidilserinas/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Domínios ProteicosRESUMO
The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca(2+) with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. To assess the conformational behavior of the Ca(2+) binding domain, we also studied the occlusion of Ca(2+), both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca(2+) and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only.
Assuntos
Trifosfato de Adenosina/química , Membrana Eritrocítica/enzimologia , Modelos Químicos , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Trifosfato de Adenosina/metabolismo , Membrana Eritrocítica/química , Humanos , Transporte de Íons/fisiologia , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Estrutura Terciária de ProteínaRESUMO
As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca(2+) with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca(2+)-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca(2+)-ATPase activity was related to an increase in the apparent affinity for Ca(2+) and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca(2+) homeostasis.
Assuntos
Actinas/química , Cálcio/química , Membrana Eritrocítica/química , Homeostase/fisiologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Multimerização Proteica/fisiologia , Actinas/isolamento & purificação , Actinas/metabolismo , Animais , Cálcio/metabolismo , Membrana Eritrocítica/metabolismo , Humanos , Transporte de Íons/fisiologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , CoelhosRESUMO
Lipid-protein interactions play an essential role in the regulation of biological function of integral membrane proteins; however, the underlying molecular mechanisms are not fully understood. Here we explore the modulation by phospholipids of the enzymatic activity of the plasma membrane calcium pump reconstituted in detergent-phospholipid mixed micelles of variable composition. The presence of increasing quantities of phospholipids in the micelles produced a cooperative increase in the ATPase activity of the enzyme. This activation effect was reversible and depended on the phospholipid/detergent ratio and not on the total lipid concentration. Enzyme activation was accompanied by a small structural change at the transmembrane domain reported by 1-aniline-8-naphtalenesulfonate fluorescence. In addition, the composition of the amphipilic environment sensed by the protein was evaluated by measuring the relative affinity of the assayed phospholipid for the transmembrane surface of the protein. The obtained results allow us to postulate a two-stage mechanistic model explaining the modulation of protein activity based on the exchange among non-structural amphiphiles at the hydrophobic transmembrane surface, and a lipid-induced conformational change. The model allowed to obtain a cooperativity coefficient reporting on the efficiency of the transduction step between lipid adsorption and catalytic site activation. This model can be easily applied to other phospholipid/detergent mixtures as well to other membrane proteins. The systematic quantitative evaluation of these systems could contribute to gain insight into the structure-activity relationships between proteins and lipids in biological membranes.
Assuntos
Proteínas de Membrana/química , Modelos Moleculares , Fosfolipídeos/química , Algoritmos , Ativação Enzimática , Humanos , Proteínas de Membrana/metabolismo , Micelas , Fosfolipídeos/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/químicaRESUMO
The autoinhibition/activation of the PMCA (plasma membrane Ca2+-ATPase) involves conformational changes in the membrane region of the protein that affect the amount of lipids directly associated with the transmembrane domain. The lipid-protein-dependence of PMCA isoforms 2 and 4 expressed and obtained in purified form from Saccharomyces cerevisiae was investigated using the phosphatidylcholine analogue [125I]TID-PC/16 {l-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromemyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine}, which was incorporated into mixtures of dimyristoylphosphatidylcholine and the non-ionic detergent C12E10 [deca(ethylene glycol) dodecyl ether]. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca2+/calmodulin of PMCA2 showed 30-fold higher affinity than PMCA4. PMCA2 exhibited a lower level of labelling in the autoinhibited conformation relative to PMCA4, indicating that the lower autoinhibition was correlated with a lower exposure to lipids in the autoinhibited state. Analysis of the lipid-protein stoichiometry showed that the lipid annulus of PMCA varies: (i) in accordance to the conformational state of the enzyme; and (ii) depending on the different isoforms of PMCA. PMCA2 during Ca2+ transport changes its conformation to a lesser extent than PMCA4, an isoform more sensitive to modulation by calmodulin and acidic phospholipids. This is the first demonstration of a dynamic behaviour of annular lipids and PMCA.
Assuntos
Ativação Enzimática , Fosfolipídeos/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Animais , Calmodulina/química , Cromatografia de Afinidade , Eritrócitos/enzimologia , Humanos , Isoenzimas/biossíntese , Isoenzimas/química , Isoenzimas/isolamento & purificação , ATPases Transportadoras de Cálcio da Membrana Plasmática/biossíntese , ATPases Transportadoras de Cálcio da Membrana Plasmática/isolamento & purificação , Ligação Proteica , Conformação Proteica , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae , Coloração e Rotulagem , TitulometriaRESUMO
We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca(2+)-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300µg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50µg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300µg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca(2+) transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50µg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.
Assuntos
Cálcio/metabolismo , Membrana Celular/enzimologia , Lipídeos de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Encéfalo/metabolismo , Química Encefálica/fisiologia , Membrana Celular/química , Transporte de Íons/fisiologia , Lipídeos de Membrana/química , Proteínas do Tecido Nervoso/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Ratos , Tubulina (Proteína)/química , Fosfolipases Tipo C/químicaRESUMO
The exposure of the plasma membrane calcium pump (PMCA) to the surrounding phospholipids was assessed by measuring the incorporation of the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16 to the protein. In the presence of Ca(2+) both calmodulin (CaM) and phosphatidic acid (PA) greatly decreased the incorporation of [(125)I]TID-PC/16 to PMCA. Proteolysis of PMCA with V8 protease results in three main fragments: N, which includes transmembrane segments M1 and M2; M, which includes M3 and M4; and C, which includes M5 to M10. CaM decreased the level of incorporation of [(125)I]TID-PC/16 to fragments M and C, whereas phosphatidic acid decreased the incorporation of [(125)I]TID-PC/16 to fragments N and M. This suggests that the conformational changes induced by binding of CaM or PA extend to the adjacent transmembrane domains. Interestingly, this result also denotes differences between the active conformations produced by CaM and PA. To verify this point, we measured resonance energy transfer between PMCA labeled with eosin isothiocyanate at the ATP-binding site and the phospholipid RhoPE included in PMCA micelles. CaM decreased the efficiency of the energy transfer between these two probes, whereas PA did not. This result indicates that activation by CaM increases the distance between the ATP-binding site and the membrane, but PA does not affect this distance. Our results disclose main differences between PMCA conformations induced by CaM or PA and show that those differences involve transmembrane regions.
Assuntos
Calmodulina/metabolismo , Membrana Eritrocítica/enzimologia , Ácidos Fosfatídicos/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Calmodulina/química , Ativação Enzimática , Humanos , Interações Hidrofóbicas e Hidrofílicas , Micelas , Ácidos Fosfatídicos/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/isolamento & purificação , Estrutura Terciária de ProteínaRESUMO
The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca(2+)-pump (PMCA) in the membrane region upon interaction with Ca(2+), calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16, measuring the shift of conformation E(2) to the auto-inhibited conformation E(1)I and to the activated E(1)A state, titrating the effect of Ca(2+) under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca(2+) regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca(2+) transport but does not modify the affinity for Ca(2+) at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E(1)I to the activated E(1)A conformation and thus modulating the transport of Ca(2+). This is reflected in the different apparent constants for Ca(2+) in the absence of CaM (calculated by Ca(2+)-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca(2+) site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca(2+) and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca(2+) binding and activation.
Assuntos
Azirinas/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Membrana Celular/enzimologia , Sondas Moleculares/metabolismo , Fosfatidilcolinas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Membrana Celular/efeitos dos fármacos , Quimotripsina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Cinética , Ácido Oleico/farmacologia , Ácidos Fosfatídicos/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estrutura Terciária de Proteína , Deleção de Sequência/efeitos dos fármacos , Titulometria , Tosilina Clorometil Cetona/farmacologiaRESUMO
The plasma membrane Ca(2+)-ATPase (PMCA) plays a key role in the regulation of the intracellular Ca(2+) concentration. Ethanol stimulates this Ca(2+) pump in an isoform-specific manner. On search for a physiological molecule that could mimic the effect of ethanol, we have previously demonstrated that some sphingolipids containing free "hydroxyl" groups, like ceramide, are able to stimulate the PMCA. Since diacylglycerol (DAG) structurally shares some characteristics with ceramide, we evaluate its effect on the PMCA. We demonstrated that DAG is a potent stimulator of this enzyme. The activation induced is additive to that produced by calmodulin, protein-kinase C and ethanol, which implies that DAG interacts with the PMCA through a different mechanism. Additionally, by different fluorescent approaches, we demonstrated a direct binding between PMCA and DAG. The results obtained in this work strongly suggest that DAG is a novel effector of the PMCA, acting by a direct interaction.
Assuntos
Cálcio/química , Diglicerídeos/química , Membrana Eritrocítica/enzimologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Cálcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Ceramidas/química , Ceramidas/metabolismo , Diglicerídeos/metabolismo , Ativação Enzimática/fisiologia , Etanol/química , Etanol/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Ligação Proteica/fisiologia , Proteína Quinase C/química , Proteína Quinase C/metabolismoRESUMO
The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca(2+) pumps. We have assessed changes in the overall exposure of these proteins to surrounding lipids by quantifying the extent of protein labeling by a photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2'-[(125)I]iodo-4'-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([(125)I]TID-PC/16) under different conditions. We determined the following. 1) Incorporation of [(125)I]TID-PC/16 to SERCA decreases 25% when labeling is performed in the presence of Ca(2+). This decrease in labeling matches qualitatively the decrease in transmembrane surface exposed to the solvent calculated from crystallographic data for SERCA structures. 2) Labeling of PMCA incubated with Ca(2+) and calmodulin decreases by approximately the same amount. However, incubation with Ca(2+) alone increases labeling by more than 50%. Addition of C28, a peptide that prevents activation of PMCA by calmodulin, yields similar results. C28 has also been shown to inhibit ATPase SERCA activity. Interestingly, incubation of SERCA with C28 also increases [(125)I]TID-PC/16 incorporation to the protein. These results suggest that in both proteins there are two different E(1) conformations as follows: one that is auto-inhibited and is in contact with a higher amount of lipids (Ca(2+) + C28 for SERCA and Ca(2+) alone for PMCA), and one in which the enzyme is fully active (Ca(2+) for SERCA and Ca(2+)-calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments.
Assuntos
Membrana Celular/enzimologia , Sondas Moleculares/química , Fosforilcolina/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , Animais , Cálcio/química , Cálcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Humanos , Fosforilcolina/análogos & derivados , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Estrutura Terciária de Proteína/fisiologia , Coelhos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
It is known that human syncytiotrophoblast (hSCT) actively transports more than 80% of the Ca2+ that goes from maternal to fetal circulation. Transepithelial transport of Ca2+ is carried out through channels, transporters and exchangers located in both microvillous (MVM) and basal (BM) plasma membranes. The plasma membrane Ca-ATPase (PMCA) is the most important mechanism of Ca2+ homeostasis control in the human placenta. In this work, we reexamined the distribution of PMCA in isolated hSCT of term placenta. The PMCA activity was determined in isolated hSCT plasma membranes. A partial characterization of the PMCA activity was performed, including an evaluation of the sensitivity of this enzyme to an in vitro induced lipid peroxidation. Expression of the PMCA in hSCT plasma membranes and tissue sections was investigated using Western blots and immunohistochemistry, respectively. Our study demonstrates, for the first time, a correlation between the activity and structural distribution of PMCA in both MVM and BM of hSCT. It also demonstrates a higher PMCA activity and expression in MVM as compared to BM. Finally, PMCA4 seems to be preferentially distributed in both hSCT plasma membranes, while PMCA1 is shown to be present in the hSCT homogenate. However, the membrane fractions did not show any PMCA1 labeling. Our results must be taken into account in order to propose a new model for the transport of calcium across the hSCT.
Assuntos
Membrana Celular/metabolismo , Vilosidades Coriônicas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Nascimento a Termo/metabolismo , Trofoblastos/metabolismo , Transporte Biológico/fisiologia , Cálcio/metabolismo , Separação Celular , Vilosidades Coriônicas/enzimologia , Vilosidades Coriônicas/ultraestrutura , Feminino , Humanos , Isoenzimas/metabolismo , Microvilosidades/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/fisiologia , Gravidez , Distribuição Tecidual , Extratos de Tecidos/química , Extratos de Tecidos/metabolismo , Trofoblastos/enzimologia , Trofoblastos/ultraestruturaRESUMO
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 Ca(2+)-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 approximately 30% the stability of PMCA against thermal denaturation, suggesting that glycated aminophospholipids induce a structural rearrangement in the protein that makes it more sensitive to thermal unfolding. We also verified that lipid glycation decreases the affinity of lipids for two other membrane proteins, suggesting that this effect might be common to membrane proteins. Extending these results to the in vivo situation, we can hypothesize that, under hyperglycaemic conditions, glycation of membrane lipids may cause a significant change in the structure and stability of membrane proteins, which may affect the normal functioning of membranes and therefore of cells.
Assuntos
Proteínas de Membrana/química , Fosfatidiletanolaminas/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Animais , Proteína 1 de Troca de Ânion do Eritrócito/química , Detergentes/química , Dimiristoilfosfatidilcolina/química , Estabilidade Enzimática , Eritrócitos/enzimologia , Glucose/química , Produtos Finais de Glicação Avançada/química , Glicosilação , Humanos , Micelas , ATPases Transportadoras de Cálcio da Membrana Plasmática/sangue , Polietilenoglicóis/química , Desnaturação Proteica/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/química , SuínosRESUMO
The plasma membrane calcium ATPase (PMCA) reacts with ATP to form acid-stable phosphorylated intermediates (EP) that can be measured using (gamma-32P)ATP. However, the steady-state level of EP at [ATP] higher than 100 microM has not yet been studied due to methodological problems. Using a microscale method and a purified preparation of PMCA from human red blood cells, we measured the steady-state concentration of EP as a function of [ATP] up to 2 mM at different concentrations of Mg2+, both at 4 and 25 degrees C. We have measured the Ca2+-ATPase activity (v) under the same conditions as those used for phosphorylation experiments. While the curves of ATPase activity vs [ATP] were well described by the Michaelis-Menten equation, the corresponding curves of EP required more complex fitting equations, exhibiting at least a high- and a low-affinity component. Mg2+ increases the apparent affinity for ATP of this latter component, but it shows no significant effect on its high-affinity one or on the Ca2+-ATPase activity. We calculated the turnover of EP (k(pEP)) as the ratio v/EP. At 1 mM Mg2+, k(pEP) increases hyperbolically with [ATP], while at 8 microM Mg2+, it exhibits a behavior that cannot be explained by the currently accepted mechanism for ATP hydrolysis. These results, together with measurements of the rate of dephosphorylation at 4 degrees C, suggest that ATP is acting in additional steps involving the interconversion of phosphorylated intermediates during the hydrolysis of the nucleotide.