Reconstituted Sarcoplasmic Reticulum Research Articles

Fourier transform infrared spectroscopic studies of lipid-protein interaction in native and reconstituted sarcoplasmic reticulum

Fourier transform infrared spectroscopy has been used to monitor lipid-protein interaction and protein secondary structure in native and reconstituted sarcoplasmic reticulum vesicles. Studies of the temperature dependence of the CH 2 symmetric stretching frequency reveal no cooperative phase transitions in purified sarcoplasmic reticulum or in vesicles reconstituted with dioleoylphosphatidylcholine, although a continuous introduction of disorder into the lipid acyl chains is observed as the temperature is raised. In addition, temperature-dependent changes are observed in the Amide I and Amide II vibrations arising from protein peptide bonds. A comparison of lipid order in native sarcoplasmic reticulum and its lipid extract showed that the introduction of protein is accompanied by a slight increase in lipid order. Reconstitution of Ca 2+-ATPase from sarcoplasmic reticulum with dipalmitoylphosphatidylcholine (lipid/protein ratio 30:1), reveals a perturbed lipid melting event broadened and reduced in midpoint temperature from multilamellar lipid vesicles. The onset of melting (27–28°C) correlates well with the onset of ATPase activity and confirms a suggestion (Hesketh, T.R., Smith G.A., Houslay M.D., McGill, K.A., Birdsall, N.J.M., Metcalfe, J.C. and Warren, G.B. (1976) Biochemistry 15, 4145–4151) that a liquid crystalline environment is a requirement for optimal protein function. Finally, Ca 2+-ATPase has been reconstituted into binary lipid mixtures of DOPC and acyl-chain perdeuterated DPPC. The effect of protein on the structure and melting behavior of each lipid component was monitored. The protein appears to preferentially interact with the DOPC component.

Target size of calcium pump protein from skeletal muscle sarcoplasmic reticulum.

The oligomeric size of calcium pump protein (CPP) in fast skeletal muscle sarcoplasmic reticulum membrane was determined using target theory analysis of radiation inactivation data. There was a parallel decrease of Ca2+-ATPase and calcium pumping activities with increasing radiation dose. The loss of staining intensity of the CPP band, observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, also correlated directly with the loss of activity. The target size molecular weight of the CPP in the normal sarcoplasmic reticulum membrane ranged between 210,000 and 250,000, which is consistent with a dimeric structure. Essentially the same size is obtained for the non-phosphorylated CPP or for the phosphoenzyme form generated from either ATP (E1 state) or inorganic phosphate (E2 state). Hence, the oligomeric state of the pump does not appear to change during the catalytic cycle. Similar results were obtained with reconstituted sarcoplasmic reticulum membrane vesicles with different lipid to protein ratios. We conclude that the CPP is a dimer in both native and reconstituted sarcoplasmic reticulum membranes. The target size of the calcium-binding protein (calsequestrin) was found to be 50,000 daltons, approximating a monomer.

The determination of the separate Ca 2+ pump protein and phospholipid profile structures within reconstituted sarcoplasmic reticulum membranes via X-ray and neutron diffraction

We have previously compared the electron density profiles for several highly-functional reconstituted sarcoplasmic reticulum membranes with that for the isolated sarcoplasmic reticulum membrane (Herbette, L., Scarpa, A., Blasie, J.K., Wang, C.T., Saito, A. and Fleischer, S. (1981) Biophys. J. 36, 47–72). In this paper, we compare the separate calcium pump protein profile within these reconstituted sarcoplasmic reticulum membranes, as derived by X-ray and neutron diffraction methods, with that within isolated sarcoplasmic reticulum membranes. In addition, the time-average perturbation of the lipid bilayer by the incorporated calcium pump protein within these reconstituted sarcoplasmic reticulum membranes has been determined in some detail.

Induced Ca 2+ release in skeletal muscle sarcoplasmic reticulum by sulfhydryl reagents and chlorpromazine

Sarcoplasmic reticulum is a specialized membrane system in muscle involved in the energized uptake, storage, and release of Ca 2+. The sulfhydryl content of normal and reconstituted sarcoplasmic reticulum was measured using Ellman reagent. For both preparations, we find 17 and 26 mol sulfhydryls per mole calcium pump protein assayed in the absence and presence of sodium dodecyl sulfate. The release of Ca 2+ from sarcoplasmic reticulum, which triggers muscle contraction, likely involves the regulation of a channel. This report deals with an experimental approach to studying the Ca 2+ release in isolated sarcoplasmic reticulum. We find that sulfhydryl agents of which water-soluble mercurials were most effective induce Ca 2+ release. Chlorpromazine acts synergistically with the sulfhydryl reagents. Ca 2+ release under optimal conditions is very rapid compared with calcium leakage from preloaded but untreated sarcoplasmic reticulum. The imposed rapid release of Ca 2+ is suggestive of the opening of a channel. Ca 2+ release by mercurials is retained in reconstituted sarcoplasmic reticulum membrane vesicles.

Proton NMR T1, T2, and T1 rho relaxation studies of native and reconstituted sarcoplasmic reticulum and phospholipid vesicles

The phospholipids protons of native and reconstituted sarcoplasmic reticulum (SR) membrane vesicles yield well-resolved nuclear magnetic resonance (NMR) spectra. Resonance area measurements, guided by the line shape theory of Bloom and co-workers, imply that we are observing a large fraction of the lipid intensity and that the protein does not appear to reduce the percent of the signal that is well resolved. We have measured the spin-lattice (T1) and spin-spin (T2) relaxation rates of the choline, methylene, and terminal methyl protons at 360 MHz and the spin-lattice relaxation rate in the rotating frame (T1 rho) at 100 MHz. Both the T1 and T2 relaxation rates are single exponential processes for all of the resonances if the residual water proton signal is thoroughly eliminated by selective saturation. The T1 and T2 relaxation rates increase as the protein concentration increases, and T2 rate decrease with increasing temperature. This implies that the protein is reducing both high frequency (e.g., trans-gauche methylene isomerizations) and low frequency (e.g., large amplitude, chain wagging) lipid motions, from the center of the bilayer to the surface. It is possible that spin diffusion contributes to the effect of protein on lipid T1's although some of the protein-induced T1 change is due to motional effects. The T2 relaxation times are observed to be near 1 ms for the membranes with highest protein concentration and approximately 10 ms for the lipids devoid of protein. This result, combined with the observation that the T2 rates are monophasic, suggests that at least two lipid environments exist in the presence of protein, and that the lipids are exchanging between these environments at a rate greater than 1/T2 or 10(3) s-1. The choline resonance yields single exponential T1 rho relaxation in the presence and absence of protein, whereas the other resonances measured exhibit biexponential relaxation. Protein significantly increases the single T1 rho relaxation rate of the choline peak while primarily increasing the T1 rho relaxation rate of the more slowly relaxing component of the methylene and methyl resonances.

CA ++-ATPase-Detergent Interactions : A Good Model for Protein-Lipid Interactions

of immobilized lipid by spin-labels. Biochemistry. 18:5006-5013. 4. Jost, P. C., and 0. H. Griffith. 1978. The spin labeling technique. Methods Enzymol. 49:369-418. 5. Deese, A. J., E. A. Dratz, L. Hymel, and S. Fleischer. 1982. Proton NMR T1, T2, and Trh relaxation studies of native and reconstituted sarcoplasmic reticulum and phospholipid vesicles. Biophys. J. 37:207-216. 6. McLaughlin, A. C., L. Herbette, J. K. Blasie, C. T. Wang, L. Hymel, and S. Fleischer. 1981. 31P NMR studies of oriented multilayers formed from isolated sarcoplasmic reticulum and reconstituted sarcoplasmic reticulum. Biophys. J. 37:49-50.

31P NMR Studies of Oriented Multilayers Formed from Isolated Sarcoplasmic Reticulum and Reconstituted Sarcoplasmic Reticulum : Evidence that “Boundary-Layer” Phospholipid is not Immobilized

Studies were made of the angular dependence of the /sup 31/P NMR signal from oriented sarcoplasmic reticulum membranes. Similar spectra were obtained from oriented reconstituted sarcoplasmic reticulum membranes with lipid-to-protein ratios ranging from 42.1 to 110.1 and from oriented bilayer membranes formed from sarcoplasmic reticulum phospholipids. The dependence of the /sup 31/P NMR spectra on the alignment of the membranes with respect to the magnetic field was used to draw two conclusions about the motion of the phospholipid molecules that contribute to the observed spectra. First, the phosphate group and the two adjacent methylene groups are able to rotate rapidly (i.e., faster than 10/sup -5/ s) around the normal to the plane of the membrane. Second, calibration experiments showed that all (100 +/- 7%) of the phospholipid molecules in the membrane can be accounted for in the observed spectra. Thus, essentially all the phospholipid molecules in the sarcoplasmic reticulum and the reconstituted sarcoplasmic reticulum membranes have the same motion in the polar headgroup region as found in model bilayer membranes. (JMT)

Functional characteristics of reconstituted sarcoplasmic reticulum membranes as a function of the lipid-to-protein ratio

The ATP-induced Ca2+ accumulation efficiency and rates of Ca2+ uptake of the reconstituted sarcoplasmic reticulum (RSR) model membrane system were measured over an extended range of lipid-to-protein (L/P) molar ratios and were compared to those of isolated light sarcoplasmic reticulum (LSR). Highly purified sarcoplasmic reticulum (SR), dissociated in the presence of deoxycholate, was reconstituted for several L/P ratios, according to the same procedure, forming closed membranes vesicles composed of greater than 95% Ca2+ pump protein and SR lipids which were capable of ATP-induced Ca2+ accumulation in the absence of oxalate or other Ca2+ precipitating agents. This suggests that dissociation of SR and reconstitution to form RSR does not significantly affect the ability of the Ca2+ pump protein incorporated into the SR lipid bilayer to establish Ca2+ gradients. Electron micrographs of fixed and stained dispersions of RSR revealed a structural organization of the membrane that was dependent upon the L/P molar ratio. RSR with L/P greater than 88 were composed of closed vesicles whose membranes stained asymmetrically, similar to that observed for LSR. Closed vesicles of RSR with L/P less than 88 were composed of membrane that stained symmetrically. In addition, reconstituted SR preparations with well-defined L/P molar ratios greater than 88 possess a functional behavior similar to that of LSR (in the absence of oxalate, energy efficiencies are 60-70% and apparent initial uptake rates are 80% that of isolated LSR controls); RSR preparations with L/P less than 88 are characterized by significantly depressed values of the energy efficiencies and apparent initial uptake rates especially at low L/P ratios. Thus, we are the first to report a reconstituted SR model membrane system capable of attaining rates of Ca2+ uptake comparable to isolated LSR controls at comparable L/P ratios in the absence of oxalate or other Ca2+ precipitating agents.

Comparison of the profile structures of isolated and reconstituted sarcoplasmic reticulum membranes

The profile structures of functional reconstituted sarcoplasmic reticulum (RSR) membranes were investigated as a function of the lipid/protein (L/P) ratio via x-ray diffraction studies of hydrated oriented multilayers of these membranes to a resolution of 10-15 A, and neutron diffraction studies on these multilayers to lower resolutions. Our results at this stage of investigation indicate that reconstitution of SR with variable amounts of Ca2+ pump protein for L/P ratios greater than 88 results in closed membraneous vesicles in which the Ca2+ pump protein is distributed asymmetrically in the membrane profile; a majority of the protein density is contained primarily in the extravesicular half of the membrane profile whereas a relatively lesser portion of the protein spans the hydrocarbon core of the RSR membranes. These RSR membranes are functionally similar and resemble isolated light sarcoplasmic reticulum in both profile structure and function at a comparable L/P ratio. Reconstitution with greater amounts of Ca2+ pump protein (e. g. L/P approximately 50-60) resulted in substantially less functional membranes with a dramatically thicker profile structure.

Deuterium and phosphorus nuclear magnetic resonance and fluorescence depolarization studies of functional reconstituted sarcoplasmic reticulum membrane vesicles.

Deuterium and phosphorus nuclear magnetic resonance and fluorescence depolarization studies of functional reconstituted sarcoplasmic reticulum membrane vesicles.

Effect of phospholipid substitution on the mobility of protein-bound spin labels in sarcoplasmic reticulum.

The influence of phospholipid environment upon the mobility of spin labels covalently bound to the Ca2+-transport ATPase (ATP phosphohydrolase [EC 3.6.1.3]) was studied by electron spin resonance spectroscopy in native and reconstituted sarcoplasmic reticulum membranes. Fragmented sarcoplasmic reticulum of rabbit skeletal muscle was covalently labeled with maleimide spin-labels of different chain length or with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl, and the phospholipids were exchanged for dipalmitoylphosphatidylcholine or dioleoylphosphatidylcholine. With short-chain maleimide or iodoacetamide spin labels, the spectrum of the protein-bound label reflected the change in microenvironment caused by replacement of endogenous phospholipids with dipalmitoylphosphatidylcholine as a decrease in mobility. In contrast, after labeling with long-chain maleimide derivatives, there were no noticeable differences in the spectra before and after substitution with dipalmitophatidylcholine. Replacement of endogenous phospholipids with dioleoylphosphatidylcholine did not affect the spectra. The data indicate that increased viscosity in the environment of Ca2+-transport ATPase produced by replacement of sarcoplasmic reticulum lipids with dipalmitoylphosphatidylcholine reduces the mobility of short-chain maleimide spin labels covalently attached to the Ca2+-transport ATPase polypeptide.