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A Migala

Publications and source records attributed to A Migala.

At least 19 recordsLinked to original sources
PubMed1998

Cations and anions as modifiers of ryanodine binding to the skeletal muscle calcium release channel.

Rate and equilibrium measurements of ryanodine binding to terminal cysternae fractions of heavy sarcoplasmic reticulum vesicles demonstrate that its activation by high concentrations of monovalent salts is based on neither elevated osmolarity nor ionic strength. The effect of the ions specifically depends on their chemical nature following the Hofmeister ion series for cations (Li+ < NH+4 < K- approximately Cs+ </= Na+) and anions (gluconate- < Cl- < NO3- approximately ClO4- approximately SCN-) respectively, indicating that both are involved in the formation of the salt-protein complex that can react with ryanodine. Activation by rising salt concentrations exhibits saturation kinetics with different dissociation constants (25-11 m) and different degrees of cooperativity (n = 1.5-4.0) for the respective salts. Maximal second order binding rates between 40,000 and 80,000 (m-1 x sec-1) were obtained for chlorides and nitrates of 1a group alkali ions with the exception of lithium supporting only rates of maximally 10,000 (M-1 x sec-1). The nitrogen bases, NH+4 and Tris+, in combination with chloride or nitrate, behave divergently. High maximal binding rates were achieved only with NH4NO3. The dissociation constants for the ryanodine-protein complexes obtained by measurements at equilibrium proved to depend differently on salt concentration, yet, converging to 1-3 nm for the applied salts at saturating concentrations. The salts do not affect dissociation of the ryanodine protein complex proving that the effect of salts on the protein's affinity for ryanodine is determined by their effect on the on-rate of ryanodine binding. ATP and its analogues modify salt action resulting in elevated maximal binding rates and reduction or abolition of binding cooperativity. Linear relations have been obtained by comparing the rates of ryanodine binding at different salt concentrations with the rates or the initial amplitudes (15 sec) of salt induced calcium release from actively loaded heavy vesicles indicating that the various salts promote specifically and concentration dependently channel opening and its reaction with ryanodine.

Adenine Nucleotides↗
PubMed1997

Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein.

Biosynthetic transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is mediated by secretory vesicles. We analyzed secretory vesicle transport in real time using a GFP-tagged secretory protein, hCgB-GFP, consisting of human chromogranin B (hCgB) and green fluorescent protein (GFP). The fusion protein was expressed transiently in Vero cells or in a stable clone after induction with butyrate. After arrest of the biosynthetic protein transport at 20 degrees C, fluorescent hCgB-GFP colocalized with TGN38, a marker of the TGN. Subsequent release of the secretion block at 37 degrees C led to the formation of green fluorescent vesicles. Confocal analysis revealed that these vesicles were devoid of TGN38 and of Texas Red-coupled transferrin and cathepsin D, markers of the endosomal/lysosomal pathway. As determined by fluorometry and metabolic labelling hCgB-GFP was secreted from the TGN to the PM with a t(1/2) of 20-30 minutes. Video-microscope analysis of green fluorescent vesicles showed brief periods of rapid directed movement with maximal velocities of 1 microm/second. Vesicle movement occurred in all directions, centrifugal, centripetal and circumferential, and 50% of the vesicles analyzed reversed their direction of movement at least once within an observation period of 45 seconds. In the presence of nocodazole the movement of fluorescent vesicles ceased. Concomitantly, secretion of hCgB-GFP was slowed but not completely blocked. We suggest that microtubules (MT) facilitate the delivery of secretory vesicles to the PM by a stochastic transport, thereby increasing the probability for a vesicle/target membrane encounter.

Animals↗
PubMed1990

Blockage of a pump-related calcium-efflux pathway in light sarcoplasmic reticulum vesicles by Mops.

Mops, used as a proton buffer, specifically enhances the accumulation of calcium or strontium by light sarcoplasmic reticulum vesicles driven by ATP or dinitrophenylphosphate as energy-yielding substrates when calcium-precipitating agents are absent. The enhancement of ion uptake by Mops is much greater for strontium than for calcium and is further increased when potassium is replaced by sodium as the dominant monovalent cation. Mops affects neither the activity of the calcium- or strontium-activated transport enzyme nor the active accumulation of calcium in the presence of oxalate, i.e. when the pump runs unidirectionally forward. Passive calcium and strontium efflux rates of approximately 40-50 nmol.mg-1.min-1 are considerably reduced when histidine/glycerophosphate or Tris/maleate are exchanged for Mops. The observed passive efflux rates and their modulation by Mops are too small, in relation to the rate of ion influx, to account for either the relatively small calcium and strontium load in the absence of precipitating agents or for its modulation by Mops. The results imply that the pump itself mediates ion efflux dependent on pump activity and the different degree of saturation of lumenal ion-binding sites by calcium and strontium, as well as their susceptibility to Mops.

Adenosine Triphosphate↗
PubMed1989

The discrepancy between ryanodine binding and its effects on the calcium releasing system of the sarcoplasmic reticulum.

Heavy sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle were reacted in high ionic strength solutions with ryanodine. The effect of this reaction on ATP - and dinitrophenyl phosphate supported calcium uptake and caffeine induced calcium release were studied. At pH 7.0 calcium uptake and caffeine induced calcium release are simultaneous affected by the occupation of 0.5 pmol ryanodine binding sites/mg protein, having an affinity of 0.33 nM-1.

Alkaloids↗
PubMed1987

Activation and inhibition of the calcium gate of sarcoplasmic reticulum by high-affinity ryanodine binding.

The occupancy of high-affinity ryanodine-binding sites of isolated heavy sarcoplasmic reticulum vesicles occurring in concentrated salt solutions affects ATP-dependent calcium accumulation and caffeine-induced calcium release. The initial suppression of calcium uptake is followed by a marked uptake activation resulting in a reduction of the final calcium level in the medium. Simultaneously, caffeine-induced calcium release is blocked. The dependence of inhibition of calcium uptake and caffeine-induced calcium release observed in assay media containing physiological concentrations of magnesium and ATP on the concentration of ryanodine corresponds to the drug's effectiveness in living muscles.

Adenosine Triphosphate↗
PubMed1983

Inactivation of the calcium-transport ATPase in the sarcoplasmic reticulum by the combined effect of lasolocid and Triton X-100.

The calcium-transport ATPase of the sarcoplasmic reticulum membranes is irreversibly inactivated by the combined action of Lasolocid and Triton X-100 at concentrations which separately do not interfere with the enzyme's activity. In the presence of Lasolocid the enzyme is most susceptible to inactivation when the Triton X-100 concentration just exceeds its critical micellar concentration, approximately, 0.2 mg X ml-1. Lasolocid becomes effective at a concentration of 10 microM and produces rapid inactivation at 100 microM. Phosphoprotein formation is less affected than phosphate liberation. The influence of the ATPase protein on the fluorescence intensity of Lasolocid passes a distinct maximum at the most effective Triton X-100 concentration of 0.2 mg X ml-1.

Adenosine Triphosphatases↗
PubMed1981

Magnesium dependence of sarcoplasmic reticulum calcium transport.

The activity of the calcium transport systems in the sarcoplasmic reticulum membranes operating in the forward and reverse modes depends on the presence of magnesium ions. When the system operates as an NTP-driven calcium pump, magnesium enters the reaction chain chelated with NTP. Magnesium is a component of the NDP-sensitive phosphoprotein species involved in the NDP-NTP exchange reaction. When the system operates in the reverse mode as a calcium efflux-driven NDP synthetase, magnesium together with inorganic phosphate rapidly forms the initial phosphorylated intermediate. The stability of the magnesium-phosphoprotein complex is considerably enhanced by an existing calcium gradient. Magnesium as well as calcium affects the partitioning of the energy in the reaction sequence.

Adenosine Diphosphate↗
PubMed1979

Comparison between ATP-supported and GTP-supported phosphate turnover of the calcium-transporting sarcoplasmic reticulum membranes.

The study deals with the interrelationship of the phosphate-transferring activities of the calcium-transporting sarcoplasmic reticulum membrane vesicles: the phosphate exchange between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP) (NTP-NDP exchange), the calcium-dependent NTase, and the phosphorylation of NDP by inorganic phosphate in the presence of NTP (NTP-Pi exchange). Different nucleotides were used as phosphate donors and acceptors. It is demonstrated for the phosphate transfer from ITP to GDP that the NTP-NDP exchange exhibits ping-pong kinetics with Mg-ITP and unliganded GDP as substrates. The apparent affinities of the enzyme for the nucleoside diphosphate and triphosphate species are deduced according to this mechanism. The enzyme's affinity for the nucleoside triphosphates and diphosphates depends on its functional state being considerably lower under conditions of NTP-NDP exchange than during NTP splitting or NTP synthesis. ATP and GTP are split with the same low rates when calcium-activated NTPase is inhibited by high internal calcium concentrations after calcium transport has reached steady state. The rates of the NTP-NDP exchange reactions, however, differ by a factor of about 10 being approximately equal to 3 mumol . mg-1 . min-1 for ATP-ADP and only approximately equal to 0.3 mumol . mg-1 . min-1 (22 degrees C) for GTP-GDP. When the sarcoplasmic reticulum vesicles are made calcium-permeable, the calcium transport ATPase is turned on and the rates of GTP and ATP splitting increase about tenfold. Yet, while the rate of ATP-ADP exchange is little reduced, the rate of GTP-GDP exchange drops by approximately 50%. The persisting exchange activity of calcium-permeable vesicles demonstrates that high internal calcium concentrations are not required for the transfer of the protein-bound phosphoryl group to NDP during NTP-NDP exchange.

Adenosine Diphosphate↗
PubMedDate not supplied

The location of the calcium precipitating protein in the sarcoplasmic membrane.

Fluram, a fast reacting reagent for primary amino groups does not label proteins enclosed in reconstituted sarcoplasmic vesicles (SR vesicles) when applied at low reagent/protein ratios. Hence, Fluram does not penetrate through SR membranes under these conditions. Likewise, the membrane of erythrocytes prevent the reagent from reacting with hemoglobin. However, at high reagent/protein ratios the membranes of the SR vesicles disintegrate. In contrast, liposomal membranes prepared from SR lipids are not affected even at a high degree of labeling. Disintegration of SR membranes starts to occur when the phosphatidylethanolamine fraction is completely substituted by Fluram and the transport protein is labeled with 3-4 mol of Fluram per 100000 d. When closed SR vesicles are labeled at low Fluram/protein ratios, the calcium precipitating protein is four times more intensely labeled than the transport protein. The labeling of the calcium transport protein in closed vesicles excludes its location in the intravesicular space. In SR vesicles solubilized with deoxycholate the relative degree of labeling of the calcium precipitating protein remains unchanged while the transport ATPase is more intensely labeled at the expense of the labeling of the amino lipids. The relative degree of labeling of the protein components depends not only on the number of labable groups but also on the rates with which these groups react with Fluram. Therefore, the experimental data do not give quantitative information concerning the distribution of the protein components in the SR membranes.

Adenosine Triphosphatases↗
PubMedDate not supplied

Arrangement of proteins and lipids in the sarcoplasmic membrane.

The number of amino residues present in the proteins of the sarcoplasmic reticulum which can react with Fluram has been determined in native and sonicated SR vesicles. Sonication increases the number of amino groups accessible to Fluram from 0.57 to 0.87 mumol-mg prot.-1. This increase indicates that 66% of the amino residues are present in the external and 34% in the internal membrane leaflet. The distribution of the amino phospholipids is computed from the distribution of Fluram in the membrane proteins in comjunction with the relative distribution of Fluram between protein and lipid in native and sonicated vesicles. The distribution of the calcium transport protein has been approximated under different assumptions concerning the distribution of the residual protein and taking into account that 15% of the membranes of the SR vesicles might have changed their sideness during preparation.

Amines↗
PubMedDate not supplied

Calcium gradient dependent pyrophosphate formation by sarcoplasmic vesicles.

The vesicles of the sarcoplasmic membranes synthesize pyrophosphate from inorganic phosphate. Pyrophosphate synthesis proceeds as long as a calcium gradient is maintained across the vesicular membranes. Pyrophosphate synthesis is inhibited by low concentrations of nucleoside triphosphates.

Adenine Nucleotides↗
PubMedDate not supplied

The inhibition of the calcium transport ATPase of the sarcomplasmic reticulum by fluorescamine: evidence for an oligomeric functional unit of the calcium transport system.

The labeling of the protein moiety of the sarcoplasmic calcium transport ATPase by fluorescamine suppresses calcium transport, calcium dependent ATPase activity, protein phosphorylation by [gamma-32P]ATP and [32P]phosphate at different extent of amino group substitution. For the hydrolysis of para nitrophenylphosphate by the calcium transport ATPase, it is shown that the relationship between the extent of amino group labelling can considerably be altered by the temperature and the presence of ethyleneglycol. It is shown that the amino residues of the phosphatidylethanolamine moiety do not contribute to the inhibiting effect of fluorescamine labelling. The observations suggest that the different functions of the calcium transport system are based on the cooperation of a varying number of calcium transport ATPase molecules.

4-Nitrophenylphosphatase↗
PubMedDate not supplied

Inactivation of the sarcoplasmic reticulum calcium-transport-ATPase by Lasolocid in combination with Triton X-100.

The calcium-transport-ATPase of the sarcoplasmic reticulum membranes is irreversibly inactivated by the combined action of Lasolocid and Triton X-100 at concentrations which separately do not interfere with the enzyme's activity. In the presence of Lasolocid the enzyme is most susceptible to inactivation when the Triton X-100 concentration just exceeds its critical micellar concentration, approximately 0.2 mg . ml-1. Lasolocid becomes effective at a concentration of 10 microM and produces rapid inactivation at 100 microM. The enzyme is more rapidly inactivated in the active than in the inactive state.

Animals↗
PubMedDate not supplied

A conformational transition of the sarcoplasmic reticulum calcium transport ATPase induced by vanadate.

Vanadate binding to sarcoplasmic reticulum vesicles results in the loss of the externally located high affinity calcium binding sites of the calcium transport ATPase. Conversely the occupation by calcium of the internally located low affinity sites in the vanadate enzyme complex leads to the release of vanadate. Since the total number of calcium binding sites is not diminished by vanadate binding but slightly increases we conclude that vanadate binding induces a transition of the enzymes external high to internal low affinity calcium binding sites. The transposition of external to internal calcium binding sites is accompanied by a definite change in the structure of the sarcoplasmic reticulum membranes. On vanadate binding the asymmetrically arranged electron dense protein particles become symmetrically distributed.

Animals↗