Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P50583 (asymmetrical)
 12,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. We have studied the block by intracellular Mg2+ (0.08-4mM) of ATP-dependent potassium channels (KATP channels) from rat skeletal muscle using inside-out excised sarcolemmal patches. The block is voltage dependent, is relieved by extracellular potassium and has rapid kinetics, allowing the use of amplitude distribution analysis to estimate on and off rates. 2. To gain insight into the pore properties necessary to produce such a block, we have used an energy barrier model based on Eyring rate theory. The model has two energy wells and three barriers for K+ within the pore, while intracellular Mg2+ has access only to the inner well. We fitted the model to unitary current-voltage relations in different [Mg2+], to on and off rates, and to dissociation constants for Mg2+ block. 3. The voltage dependence of block was almost entirely due to the rate constant for unblocking. This implies that the inner energy barrier is asymmetrical, so that Mg2+ entry senses little of the voltage field, but Mg2+ exit senses about 20% of the voltage field. Best fits were obtained by placing the barrier and binding site 0.01 and 0.22, respectively, of the electrical distance through the pore from the inside. 4. The relief of block by [K+]o resulted from an increase in the unblocking rate for Mg2+, implying ionic repulsion between ions in the pore.
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PMID:A rate theory model for Mg2+ block of ATP-dependent potassium channels of rat skeletal muscle. 868 79

Current models for the action of linear biological motors may be grouped in two main categories. The conventional "bind and bend" models rely for their power stroke upon a structural change in the myosin headgroup (S1 fragment) which follows the binding of myosin to the F-actin filament. The more recent ratchet models demonstrate that directional motion of a particle along an asymmetrical ratchet is possible with a symmetrical but time-correlated stochastic drive. In this paper a new type of model is introduced which is deterministic like the "bind and bend" model but it requires no molecular structural changes to power the stroke. Like the ratchet models the motor is driven along the linear stator by tangential forces at the interface but the forces are electrostatic and controlled by the hydrolysis of ATP to ADP.
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PMID:Linear electric traction as an alternative model of the actin/myosin motor. 873 31

1. Potassium currents were measured in the extensor digitorum longus muscle of normal and mdx mice, which lack the protein dystrophin, using the cell-attached and inside-out patch clamp techniques, in the presence of asymmetrical K+ concentrations (3 mM in the pipette, 160 mM in the bath). 2. In cell-attached patches, the delayed rectifier was the most commonly found potassium channel, with a density of roughly 8 channels microns-2. Outward macroscopic currents were activated in macropatches depolarized to potentials positive to -60 mV. The probability of opening reached half-maximal values around -40 mV for control patches and -31 mV for patches from mdx mice. 3. Tail currents were linear in the range between -60 and +20 mV, reversing close to -100 mV. The single channel current at 0 mV, estimated from non-stationary analysis of variance, was used in conjunction with the slope of the linear part of the tail current to calculate the single channel conductance, yielding a value of 19 +/- 1 pS. 4. At 0 mV, the delayed rectifier inactivated with two time constants, of 70 +/- 20 ms and 600 +/- 200 ms. Prepulses of 500 ms duration to different potentials produced incomplete inactivation with inactivation reaching 50% of its maximum at -50 mV. 5. Single channel activity was recorded using small pipettes. Both single channel conductance and kinetic behaviour were in agreement with the macroscopic current data. 6. In excised patches, the delayed rectifier current ran down, unmasking other K+ channels. A Ca(2+)-dependent K+ channel of 186 pS (BK-like channel) was found frequently in patches bathed in solutions containing appropriate concentrations of calcium, especially at stronger depolarizations. A K+ channel of 63 pS was unmasked in control excised patches bathed in solutions devoid of ATP. This channel was not observed in patches excised from mdx fibers.
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PMID:A patch-clamp study of delayed rectifier currents in skeletal muscle of control and mdx mice. 873 98

Dinucleoside 5',5"'-P1,P4-tetraphosphate hydrolase (EC 3.6.1.17) has been purified to homogeneity from tomato (Lycopersicon esculentum) cells grown in suspension. The purification procedure comprised ammonium sulphate fractionation following five standard chromatography steps and a final chromatography on Ap4A-Sepharose. The homogeneous hydrolase has a molecular mass of 20 kDa and an isoelectric point of 4.5. The enzyme hydrolyses diadenosine tetraphosphate (Ap4A) asymmetrically to AMP and ATP. Among other naturally occurring dinucleoside oligophosphates, Ap5A and Ap6A are substrates whereas Ap3A is not. Of various phosphonate analogues tested, the Ap5A analogue, AppCH2pCH2ppA, was not cleaved and the Ap3A analogue, ApCH2CH2ppA, was a very poor substrate. Enzyme activity is stimulated by 5 mM Mg2+ and inhibited by fluoride anion; I50 = 6.25 microM. The K(m) value for Ap4A is 0.8 microM. The enzyme exhibits a broad pH optimum from pH 6.5 to 9.0. In order to analyze the protein at the molecular level an internal peptide sequence from the homogeneous enzyme was identified. Within the sequence of 17 amino acids a kinase II motif as a general part of a conserved sequence of nucleotide binding sites was found. Against the internal peptide sequence a polyclonal antiserum was raised. By investigating the intracellular level of Ap4A hydrolase under different kinds of environmental stress, no changes occurred in response to heat shock. But, heavy metal stress and phosphate deprivation lead to a decrease in Ap4A hydrolase.
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PMID:Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) hydrolase from tomato (Lycopersicon esculentum cv. Lukullus)--purification, biochemical properties and behaviour during stress. 881 90

Exposure of cultured neonatal rat heart cells to simulated ischaemia results in a cessation of the spontaneous contractile activity and changes at both the level of sarcolemmal phospholipid topology and the ultrastructural level. Reperfusion at a timepoint before irreversible cell damage develops leads to a recovery of contractile activity. Furthermore, the shift in transbilayer distribution of sarcolemmal phosphatidylethanolamine in favour of the outer membrane leaflet, due to the ischaemic period, is reversed during subsequent reperfusion. Also the morphological changes (mitochondrial oedema, reorganization of the mitochondrial cristae and the formation of extrusions at the sarcolemma) are reversible. At the same time total intracellular ATP levels are restored to 80% of control. The role of cellular ATP content on sarcolemmal phospholipid topology was further studied by the use of the calcium antagonist verapamil (10 microM), which preserved cellular ATP content by inhibiting cell contractility before the onset of ischaemia. After 120 min of ischaemia, cell ATP content was still 63% of control in the presence of verapamil, versus 20% of control in untreated cells. Verapamil treatment also prevented the loss of the asymmetrical distribution of phosphatidylethanolamine and sarcolemmal disruption, the latter occurring during 120 min of ischaemia in untreated cells. It is proposed that maintenance of phospholipid asymmetry of the sarcolemma of the myocytes depends on the cellular ATP concentrations, indicating the involvement of an ATP dependent aminophospholipid translocase.
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PMID:Sarcolemmal phosphatidylethanolamine reorganization during simulated ischaemia and reperfusion: reversibility and ATP dependency. 890 44

Two distinct Drosophila melanogaster P-glycoprotein (Pgp) gene homologues of different chromosomal origin, MDR49 and MDR65, have been previously identified (38). Most Pgps are implicated in the development of the multidrug-resistance phenotype. Despite intense efforts to identify the molecular mechanism(s) associated with Pgp function, the endogenous substrate(s) of these transport molecules is largely unknown. Recent studies from our laboratory indicate that a murine Pgp homologue (E. H. Abraham, A. G. Prat, L. Gerweck, T. Seneveratne, R. J. Arceci, R. Kramer, G. Guidotti, and H. F. Cantiello. Proc. Natl. Acad. Sci. USA 90: 312-316, 1993) and a related protein, the cystic fibrosis transmembrane conductance regulator (CFTR; I. L. Reisin, A. Prat, E. H. Abraham, J. F. Amara, R. J. Gregory, D. A. Ausiello, and H. F. Cantiello. J. Biol. Chem. 269: 20584-20591, 1994), are novel ATP-permeable ion channels. The common feature of these two proteins is the conserved ATP-binding cassettes (ABC); thus molecules structurally linked to the ABC transporter family may be also functionally associated with ATP channel activity. In this study, MDR65 and MDR49 Pgps were functionally expressed in Sf9 cells, and patch-clamp techniques were applied to assess the role of these proteins in the electrodiffusional movement of ATP. In the presence of intracellular ATP and external NaCl, expression of MDR65 was associated with a linear electrodiffusional pathway that was permeable to both ATP and Cl-. Under symmetrical ATP conditions, only voltage depolarization activated a MDR65-mediated ATP-conductive pathway. Expression of MDR49 was also associated with a voltage-activated ATP conductance in symmetrical ATP, but no apparent permeability to either Cl- or ATP was observed under asymmetrical conditions. The different functional properties of MDR65 and MDR49 may be indicative of distinct physiological roles in this organism. The study indicates, however, that the two Drosophila Pgp homologues share strong functional similarities with their mammalian relatives Pgp and CFTR.
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PMID:Expression of Drosophila melanogaster P-glycoproteins is associated with ATP channel activity. 894 36

1. ATP-dependent K+ (KATP) channels were studied in fibres isolated from flexor digitorum brevis and interosseal skeletal muscles of normal and mutant mdx mice using the patch clamp technique in the presence of asymmetrical K+ concentrations (5 mM K+ in the pipette and in vivo intracellular [K+] or 145 mM K+ at the cytoplasmic face). 2. In cell-attached patches from mdx muscle fibres bathed in K(+)-rich solution, cell poisoning with fluorodinitrobenzene induced partially reversible opening of channels carrying an outward current of an amplitude of 1.2 pA at 0 mV. Exposure of fibres to the K+ channel opener cromakalim led to opening of the same type of channel. These channels were assumed to be KATP channels. 3. On excision of inside-out patches from mdx muscle fibres, in the absence of intracellular ATP, KATP channels were active: they carried a unitary outward current of 1.6 pA at 0 mV and were inhibited by intracellular ATP and glibenclamide. The number of KATP channels per patch was not significantly different in muscles from normal and mdx mice. 4. In inside-out patches, in the presence of 1 mM intracellular Mg2+, slope conductances of 21 and 20.3 pS were found for KATP channels in normal and mdx muscle, respectively. In the absence of Mg2+, slope conductances of KATP channels were 31.3 and 32 pS in normal and mdx muscle, respectively and KATP channel activity was augmented in mdx muscle in the same way as in normal muscle. Activity of the same KATP channel was observed in extensor digitorum longus muscle from normal and mdx mice. 5. In inside-out patches held at 0 mV, the relationship between KATP channel activity and intracellular ATP was described by a Hill equation: Ki values were 23 and 21 microM and Hill coefficients were 1.8 and 1.9 in normal and mdx muscle, respectively. 6. These results indicate that the distribution, the conductance properties and ATP sensitivity of KATP channels do not differ in normal and in mdx mouse skeletal muscle.
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PMID:Similarity of ATP-dependent K+ channels in skeletal muscle fibres from normal and mutant mdx mice. 903 81

Phosphorescence and fluorescence energy transfer measurements have been used to locate the epsilon-subunit within the know structural frame of the mitochondrial soluble part of F-type H(+)-ATPase complex (F1). The fluorescence probe 2'-O-(trinitrophenyl)adenosine-5'-triphosphate was bound to the nucleotide binding sites of the enzyme, whereas the probe 7-diethylamino-3'-(4'-maleimidylphenyl)-4-methylcoumarin was attached to the single sulfhydryl residue of isolated oligomycin sensitivity-conferring protein (OSCP), which was then reconstituted with F1. Fluorescence and phosphorescence resonance energy transfer yields from the lone tryptophan residue of F1 present in the epsilon-polypeptide and the fluorescence labels attached to the F1 complex established that tryptophan is separated by 3.7 nm from Cys-118 of OSCP in the reconstituted OSCP-F1 complex, by 4.9 nm from its closest catalytic site and by more than 6.4 nm from the two other catalytic sites, including the lowest affinity ATP site. These separations together with the crystallographic coordinates of the F1 complex (Abrahams, J.P., A. G. W. Leslie, R. Lutter, and J.E. Walker. 1994. Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature. 370:621-628) place the epsilon-subunit in the stem region of the F1 molecule in a unique asymmetrical position relative to the catalytic sites of the enzyme.
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PMID:Structural mapping of the epsilon-subunit of mitochondrial H(+)-ATPase complex (F1). 908 86

2',3'-Dideoxynucleosides (ddN) and their derivatives are currently used as antiretroviral compounds. Their active agents are the corresponding 2',3'-dideoxynucleoside triphosphates (ddNTPs) generated inside the cell by host kinases. Dinucleoside tetraphosphates (Np4Ns) are molecules of interest in metabolic regulation; their synthesis in vitro can be catalyzed by firefly luciferase. The relative synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate or adenosine(5')tetraphospho(5')adenosine (Ap4A) from ATP is about 100-fold faster than that of di-2',3'-dideoxyadenosine 5',5'''-P1,P4-tetraphosphate or 2',3'-dideoxyadenosine (5')tetraphospho (5')-2',3'-dideoxyadenosine (ddAp4ddA) from ddATP. In the presence of ATPgammaS and ddATP the yield of adenosine(5')tetraphospo(5')-2',3'-dideoxyadenosine (Ap4ddA) was similar to that attained for Ap4A in the presence of ATP. The findings of this work indicate that the presence of a 3'-hydroxyl group is essential for the formation of the luciferase-luciferin-AMP complex, and explains the very low yield of ddAp4ddA in the presence of luciferase, luciferin and ddATP. The absence of 3'-hydroxyl groups in ddAp4ddA greatly hindered their hydrolysis by snake venom phosphodiesterase, asymmetrical dinucleoside tetraphosphatase and by a purified membrane preparation from rat liver. The possibility of using di-2',3'-dideoxynucleoside tetraphosphate (ddNp4ddN) or nucleoside(5')tetraphospho(5')-2',3'-dideoxynucleoside (Np4ddN) as a source of the active retroviral agent ddNTP, for example in HIV infection, is outlined.
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PMID:2',3'-dideoxynucleoside triphosphates (ddNTP) and di-2',3'-dideoxynucleoside tetraphosphates (ddNp4ddN) behave differently to the corresponding NTP and Np4N counterparts as substrates of firefly luciferase, dinucleoside tetraphosphatase and phosphodiesterases. 910 13

Energy metabolism and glycolysis of normal human term placental trophoblast in two-sided culture was investigated during differentiation from cytotrophoblast to syncytiotrophoblast, because glycogen metabolism is abnormal in several trophoblast related pregnancy diseases, including pre-eclampsia. After initial recovery of energy and cytoplasmic NADH/NAD+ redox by 24 h of culture, measures of cellular energy state, [ATP], [ADP], [ATP]/[ADP] ratio, ([ATP] + [ADP] + [AMP]), [ATP]/([ATP] + [ADP] + [AMP]) and energy charge remained essentially constant until 72 h, despite periods of increased energy turnover. At 24 h there was a burst of glycogenolysis, and glycolysis indicated by increased lactate production, which coincided with formation of syncytium. Subsequently, there was no resynthesis nor further breakdown of glycogen. At 48 h, oxygen consumption temporarily increased substantially, without increased glycolysis, during functional differentiation of the syncytiotrophoblast. Glucose uptake was constant and largely from the basal (in vivo fetal facing) side. Lactate output into the basal fetal medium was twice as fast as that into the microvillous (maternal) medium, and oxygen uptake was also asymmetrical. The results show that before and after differentiation substantial relatively constant aerobic glycolysis occurs, but that during increased energy demand cytotrophoblast depends on both glycolytic and aerobic energy production whereas syncytiotrophoblast relies on aerobic metabolism.
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PMID:Energy metabolism and glycolysis in human placental trophoblast cells during differentiation. 913 Oct 49


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