Interaction between free fatty acids and Ca2+-dependent ATPase from sarcoplasmic reticulum membranes

The interaction between free fatty acids and Ca2+-dependent ATPase, an intrinsic protein of sarcoplasmic reticulum membranes, was studied with relevance to the changes in membrane permeability induced by free fatty acids. It was found that only unsaturated fatty acids increase the permeability of reticulum membranes for Ca2+, this effect being completely reversible. The increase in the membrane permeability by fatty acids is coupled to a generation of a channel for Ca2+ efflux under effect of Ca2+-dependent ATPase. The interaction between fatty acids and Ca2+-dependent ATPase was demonstrated by the protein fluorescence and electron paramagnetic resonance methods, using spin-labelled fatty acid derivatives. A model demonstrating the increase of sarcoplasmic reticulum membrane permeability for Ca2+ in the presence of the fatty acid-Ca2+-dependent ATPase complex is proposed.     

Vitamin E protection of membranes of the sarcoplasmic reticulum against the damaging effect of free fatty acids

Feeding rats the diet enriched with vitamin E or addition of alpha-tocopherol to the suspension of sarcoplasmic reticular membranes of rat and rabbit skeletal muscles protects Ca2+-dependent ATPase against thermal inactivation aggravated by the action of free fatty acids.     

The effect of electromagnetic radiation on the membranes of the sarcoplasmic reticulum

On the sarcoplasmic reticulum membranes has been shown that at temperature of Ca2(+)-ATPase activity change of dependence in the Arrhenius plot the microwaves (2450 MHz, specific absorption rate 12 w/kg) inhibit the ATP-hydrolase and Ca2(+)-transporting activity of Ca2(+)-ATPase. The effect of radiation exhibits within the narrow temperature range (approximately 1 degree C) and quantitatively corresponds to the decrease of Ca2(+)-ATPase activity caused by the decrease of temperature by 1.6 degrees C from 18 degrees C. The fluorescence intensity of naphthalene sulfonic probes reduces under the influence of microwaves at 18 degrees C.     

Localization of the amino phospholipids in sarcoplasmic reticulum membranes revealed by trinitrobenzene-sulfonate and fluorodinitrobenzene

The chemical probes for amino compounds 2,4,6-trinitrobenzenesulfonate (TNBS) and 1-fluoro-2,4-dinitrobenzene (FDNB) were utilized to determine the localization of the amino phospholipids in the sarcoplasmic reticulum membranes. At low concentrations (<1 mM), TNBS does not penetrate the sarcoplasmic reticulum membrane, while FDNB readily penetrates it. The results show that about 70% of the total phosphatidylethanolamine is located on the external surface of the membrane, about 20% is on the internal surface and 10% is probably strongly interacting with the proteins since it is not accessible to the probes. In contrast, most of the phosphatidylserine is located on the inner surface of the membrane. This molecular distribution of the amino phospholipids supports a structural assymmetry of the sarcoplasmic reticulum membrane.     

Structural changes in the sarcoplasmic reticulum membrane of skeletal muscles at the early stage of x-ray irradiation]

A single X-ray irradiation of the rabbit hindlimbs in a dose of 0.24 C/kg evokes a decrease in fluorescence of the ANS probe bound with membranes of the sarcoplasmatic reticulum as a result of the decrease of binding sites, binding constant as well as the quantum output of the probe. A decrease in fluorescence of tryptophan residues of Ca-ATPase localized in membranes and attenuation of interaction of its SH-group with dithionitrobenzoic acid has been also observed at early postradiation terms (1 and 24 h). The obtained results evidence for structural rearrangements occurring in membranes of the sarcoplasmic reticulum under the effect of ionizing radiation. Changes in conformation of CA-ATPase molecules contribute much to this process.     

Two populations of phospholipids exist in sarcoplasmic reticulum and in recombined membranes containing Ca-ATPase

Phosphorus nuclear magnetic resonance spectra of sarcoplasmic reticulum membranes from rabbit muscle and of recombined membranes containing the calcium-dependent adenosinetriphosphatase (Ca-ATPase) of sarcoplasmic reticulum reveal two distinguishable, overlapping resonances. One resonance resembles a normal phospholipid bilayer resonance, and the other is much broader. The broader component is not seen in protein-free phospholipid vesicles. In recombined membranes of the Ca-ATPase, the intensity found in the broad component was proportional to the concentration of protein in the vesicles. The two-component spectra are interpreted to arise from at least two different domains of phospholipids, one of which is motionally restricted by the Ca-ATPase. Phospholipids exchange between these two domains at a rate less than 10(3) s-1. A model for protein-lipid interactions in membranes containing the Ca-ATPase is proposed in which some of the phospholipid head groups of the membrane interact directly with the protein.     

Incorporation of synthetic phospholipids into the membranes of the sarcoplasmic reticulum from the rabbit muscle

The hydrophobic spin label used in ESR showed that the iminoxyl radical rotation in the native membrane of sarcoplasmatic reticulum (SR) occurred much faster than in the membranes, modified by a synthetic lipid. Such effect was observed throughout the whole temperature range (7-40 degrees). Experimental technique for the modification of the SR membrane and the lipid by ultrasonic treatment has been developed. Synthetic lipids without ultrasonic treatment did not inhibit the activity of Ca2+-ATPase. The change in both the enzyme activity and its ability to transport the Ca2+ ions through the membrane vesicules was observed after the phospholipids incorporation into the SR membrane. The investigation of the temperature dependence (in Arrhenius coordinates) of native and modified by lecithin Ca2+-ATPase after ultrasonic treatment and also of a "pure enzyme" showed the presence of two sharp breaks at 20 degrees and 40-42 degrees. It was shown tha the break of an Arrhenius anamorphosis was caused by a lipid environment of ATPase, "melting" of a phospholipid bilayer. The break at 20-22 degrees was observed in all cases and even after the incorporation of all the lipids into the SR membrane. This phenomenon can be explained by the distortion of the protein-lipid interaction, affecting the conformation mobility of protein and the geometry of its catalytically active center.     

Accumulation of polyunsaturated free fatty acids coincident with the fusion of rough endoplasmic reticulum membranes.

The accumulation of polyunsaturated free fatty acids (PUFAs) was observed coincident with GTP-dependent fusion of liver rough microsomes. Whereas 0.5 mM NADPH led to a parallel reduction (greater than 50%) in membrane fusion and PUFA accumulation, indomethacin (50 microM) either had little effect or slightly augmented both processes. CTP was observed to stimulate accumulation of PUFAs and diacylglycerol (DAG). Therefore PUFAs may be relevant for GTP-dependent membrane fusion and together with DAG may play a role in fusion stimulated in the presence of CTP.     

Structural and related functional changes in sarcoplasmic reticulum induced by long-chain fatty acids

The effect of palmitic and oleic acids on Ca²⁺-ATPase activity in coupled preparations of sarcoplasmic reticulum isolated from rabbit hind leg muscle have been compared with their effects on vesicles uncoupled with Ca²⁺ ionophore, A23187. Palmitate at 2 µM · mg protein^–1 has no significant effect on enzyme activity and does not uncouple catalytic activity from calcium accumulation within the vesicles. Oleic acid at 1 µM · mg protein^–1 uncouples the vesicles, whereas 2 µM · mg protein^–1 completely inhibits Ca²⁺-ATPase activity. Fluorescence anisotropy of diphenylhexatriene is not significantly altered by palmitate, but a large transient increase in motion of the probe is observed with addition of oleic acid. The effects of oleic acid on enzyme activity are not mediated via an effect on the bulk properties of the hydrophobic domain of the membrane lipids.     

Sarcoplasmic reticulum. IX. The permeability of sarcoplasmic reticulum membranes

Fragmented sarcoplasmic reticulum (FSR) membranes isolated from rabbit skeletal muscle are impermeable to inulin-¹⁴C (mol wt 5,000), and dextran-¹⁴C (mol wt 15,000–90,000) at pH 7.0–9.0, yielding an excluded space of 4–5 µl/mg microsomal protein. In the same pH range urea and sucrose readily penetrate the FSR membrane. EDTA or EGTA (1 mM) increased the permeability of microsomes to inulin-¹⁴C or dextran-¹⁴C at pH 8–9, parallel with the lowering of the FSR-bound Ca⁺⁺ content from initial levels of 20 nmoles/mg protein to 1–3 nmoles/mg protein. EGTA was as effective as EDTA, although causing little change in the Mg⁺⁺ content of FSR. The permeability increase caused by chelating agents results from the combined effects of high pH and cation depletion. As inulin began to penetrate the membrane there was an abrupt fall in the rate of Ca⁺⁺ uptake and a simultaneous rise in ATPase activity. At 40°C inulin penetration occurred at pH 7.0 with 1 mM EDTA and at pH 9.0 without EDTA, suggesting increased permeability of FSR membranes. This accords with the higher rate of Ca⁺⁺ release from FSR at temperatures over 30°C. The penetration of microsomal membranes by anions is markedly influenced by charge effects. At low ionic strength and alkaline pH acetate and Cl are partially excluded from microsomes when applied in concentrations not exceeding 1 mM, presumably due to the Donnan effect. Penetration of microsomal water space by acetate and Cl occurs at ionic strengths sufficiently high to minimize charge repulsions.     

Interaction of fatty acids with the calcium-magnesium ion dependent adenosinetriphosphatase from sarcoplasmic reticulum

The fluorescence emission spectrum of dansylundecanoic acid is sensitive to the environment and appears at a lower wavelength when the fatty acid is bound to protein than when it is bound to phospholipid. When bound to the (Ca2+-Mg2+)-ATPase of sarcoplasmic reticulum, the emission spectrum can be resolved into separate components assigned to fatty acid bound to protein and to lipid. Efficiency of energy transfer from the tryptophan residues of the ATPase to dansylundecanoic is higher for protein-bound probe than for lipid-bound probe. Fluorescence titrations are consistent with three fatty acid binding sites per ATPase with a Kd of 7 microM, and these sites are postulated to occur at the protein-protein interface in ATPase oligomers. Fatty acid incorporated into the lipid component of the membrane appears to be bound outside the lipid annulus around the protein.     

The channel function of the hydrophobic fragment of Ca ATPase in the sarcoplasmic reticulum of rabbit skeletal muscles in the early period after x-ray radiation exposure

At early times (1 and 24 h) following local single exposure of rabbit hindlimbs to 0.21 C/kg X-radiation Ca ATPase was isolated from sarcoplasmic reticulum of skeletal muscles and inserted in liposomes the treatment of which with trypsin resulted in the formation of proteoliposomes with a Ca ATPase hydrophobic fragment (Mr 45-50 kDa). It was shown that X-radiation increased the permeability of univalent and bivalent cations through a channel formed by the hydrophobic fragment of Ca ATPase and diminished ion selectivity of this channel.