The increase in viscosity and peroxidation of sarcoplasmic reticulum membrane lipids in isadrine myocarditis

The viscosity of membranes isolated from sarcoplasmic reticulum of rabbits with isadrine myocarditis was studied, using pyrene as a hydrophobic fluorescent probe. The increase in the viscosity of membranes from injured heart occurred at lower temperatures and was sharper than in the case of intact heart in both "free" and "bound" lipid domains. The increase in the lipid viscosity under myocarditis was associated with decreased Ca++, Mg++ -ATPase and cAMP-dependent protein kinase activities and with an elevated content of lipid peroxidation products.     

Effects of irradiation on fatty acid structure of membrane lipids of the sarcoplasmic reticulum]

It has been shown that single local X-irradiation (0.21 C/kg) of the rabbit hind limb in the early period of acute radiation injury (1 and 14 h) causes a decrease in saturation of sarcoplasmic reticulum membrane lipids. It is mainly connected with a decreased saturation of the total fraction of phosphatidyl serine, phosphatidyl inositol and phosphatidyl choline. The above changes can increase permeability of the sarcoplasmic reticulum membranes for Ca(2+)-ion after X-irradiation.     

The modulation of Ca-ATPase activity and protein-lipid interactions in the sarcoplasmic reticulum by ATP

The time-course of ATP hydrolysis by Ca-ATPase of purified sarcoplasmic reticulum is biphasic with an initial rate over 1 to 2 min exceeding the subsequent rate. Hydrolysis of GTP and p-nitrophenylphosphate (pNPP) occurs at a slower but constant rate. Arrhenius plots of GTP, p-nitrophenylphosphate and initial rates of ATP hydrolysis all exhibit a discontinuity at about 20-24 degrees C; no breaks are observed in plots of the slower phase of ATP hydrolysis. The effect of substrate hydrolysis on the disposition of the enzyme in the membrane was examined by monitoring the quenching of tryptophan fluorescence by pyrene present in the hydrophobic domain of the membrane. The presence of ATP, but not GTP, prevents a temperature-dependent decrease in fluorescence quenching suggesting that ATP binding causes a change in the protein domain in contact with the membrane lipids.     

Passive Ca2+ permeability of phospholipid vesicles and sarcoplasmic reticulum membranes.

During the excitation of muscle the estimated rate of Ca2+ release from sarcoplasmic reticulum may increase 10(3)- to 10(4)-fold compared with relaxed muscle or isolated sarcoplasmic reticulum in vitro, implying a major change in the calcium permeability of the sarcoplasmic reticulum membrane. As a first step in the assessment of the role of various membrane constituents in the regulation of calcium fluxes, the contribution of phospholipids to the definition of calcium permeability was studied in model systems. The rate of calcium release from vesicles prepared from pure phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositides, cardiolipin, and extracted microsomal lipids is in the range of 10(-15) to 10(18) mol of calcium/cm2/s. This rate is several orders of magnitude lower than the passive calcium outflux from isolated sarcoplasmic reticulum membranes. The permeability to Ca2+ is influenced by fatty acid composition and net charge and it is markedly increased with increasing temperature or after the addition of local anesthetics.     

Lysophospholipid-mediated alterations in the calcium transport systems of skeletal and cardiac muscle sarcoplasmic reticulum

Summary The effects of various lysophospholipids on the calcium transport activity of sarcoplasmic reticulum (SR) from rabbit skeletal and canine cardiac muscles were examined. The lipids decreased calcium transport activity in both membrane types; the effectiveness being in the order lysoPC > lsyoPS, lysoPG > lysoPE. The maximum inhibition induced by lysoPC, lysoPG and lysoPS was greater than 85% of the normal Ca²⁺-transport rate. In cardiac SR lysoPE had a maximal inhibition of about 50%. Half maximal inhibition of calcium transport by lysoPC was achieved at 110 nmoles lysoPC/mg SR. At this concentration of lysoPC, the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺-uptake activities were inhibited to the same extent (about 60%) in skeletal sarcoplasmic reticulum, while in cardiac sarcoplasmic reticulum, there was less than 20% inhibition of the Ca²⁺ + Mg²⁺-ATPase activity. Studies with EGTA-induced passive calcium efflux showed that up to 200 nmoles lysoPC/mg SR did not alter calcium permeability significantly in cardiac sarcoplasmic reticulum. In skeletal muscle membranes the lysophospholipid mediated decrease in calcium uptake correlated well with the increase in passive calcium efflux due to lysophosphatidylcholine. The difference in the lysophospholipid-induced effects on the sarcoplasmic reticulum from the two muscle types probably reflects variations in protein and other membrane components related to the respective calcium transport systems.     

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.     

The separate profile structures of the functional calcium pump protein and the phospholipid bilayer within isolated sarcoplasmic reticulum membranes determined by X-ray and neutron diffraction

The detailed profile structure of the isolated sarcoplasmic reticulum membrane was studied utilizing a combination of X-ray and neutron diffraction. The water and lipid profile structures within the sarcoplasmic reticulum membrane were determined at 28 A resolution directly by neutron diffraction and selective deuteration of the water and lipid components. The previously determined electron density profile structure of the sarcoplasmic reticulum membrane at 12 A resolution was subjected to model refinement analysis constrained by the neutron diffraction results, thereby providing unique higher resolution calculated lipid and protein profile structures. It was found that the lipid bilayer profile structure of the isolated sarcoplasmic reticulum membrane is asymmetric, primarily the result of more lipid residing in the inner versus the outer monolayer of the sarcoplasmic reticulum lipid bilayer. The asymmetry in the lipid composition was necessarily coincident with a complimentary asymmetry in the protein mass distribution between the two monolayers in order to preserve the overall cross-sectional area of lipid and protein throughout the lipid bilayer region of the sarcoplasmic reticulum membrane profile structure. Approximately 50% of the mass of the total protein was found to be localized externally to the sarcoplasmic reticulum membrane lipid bilayer protruding from the outer lipid monolayer into the extravesicular medium. The structural features of the protein protrusion appear to be rather variable depending upon the environment of the sarcoplasmic reticulum membrane. This highly asymmetric structural organization of the sarcoplasmic reticulum membrane profile is consistent with its primary function of unidirectional calcium transport.     

Changes in the structure, composition and function of sarcoplasmic-reticulum membrane during development.

The structure, chemical composition and function of the microsomal fraction, isolated by differential centrifugation and purified on sucrose gradients, from muscle of fetal, newborn and young rabbits were characterized and compared with those of sarcoplasmic reticulum vesicles from adult muscle. Negative staining shows that the microsomal vesicles isolated from muscles of embryos and newborn animals are smooth, in contrast to vesicles obtained from adult muscle which contain 4-nm particles on their surface. The particles appear first in the microsomal vesicles from muscles of 5--8-day-old rabbits. Their number increases with the age of the animals. Ca2+-pump protein, with molecular weight about 100000, accounts for 10% of the total protein content in sarcoplasmic reticulum membrane, isolated at the earliest stages of development analysed. Its amount increases continuously with the rabbit's age to the adult value of about 70% of total sarcoplasmic reticulum protein. The low amount of 100000-dalton protein and lack of 4-nm surface particles in sarcoplasmic reticulum vesicles obtained from fetal and newborn rabbits are strictly correlated with the low activity of Ca2+-dependent ATPase and the ability to take up Ca2+. These activities rise in parallel with the age of the rabbits. On the other hand, Mg2+-dependent ATPase activity is very high at the early stages of development and declines continuously to a low value in sarcoplasmic reticulum from adult muscle. The sarcoplasmic reticulum membrane from fetal and newborn rabbits contains a higher amount of lipids as compared with the membrane present in the muscle of adult animals. The ratio of both phospholipid to protein and neutral lipid to protein decreases with the age of the rabbits. The composition of sarcoplasmic reticulum phospholipids also changes during development.     

Evidence that lipid lateral phase separation induces functionally significant structural changes in the Ca+2ATPase of the sarcoplasmic reticulum.

We have studied lipid lateral phase separation (LPS) in the intact sarcoplasmic reticulum (SR) membrane and in bilayers of isolated SR membrane lipids as a function of temperature, [Mg+2], and degree of hydration. Lipid LPS was observed in both the intact membrane and in the bilayers of isolated SR lipids, and the LPS behavior of both systems was found to be qualitatively similar. Namely, lipid LPS occurs only at relatively low temperature and water content, independently of the [Mg+2], and the upper characteristic temperature (th) for lipid LPS for both the membrane and bilayers of its isolated lipids coincide to within a few degrees. However, at similar temperatures, isolated lipids show more LPS than the lipids in the intact membrane. Lipid LPS in the intact membrane and in bilayers of the isolated lipids is fully reversible, and more extensive for samples partially dehydrated at temperatures below th. Our previous x-ray diffraction studies established the existence of a temperature-induced transition in the profile structure of the sarcoplasmic reticulum Ca+2ATPase which occurs at a temperature corresponding to the [Mg+2]-dependent upper characteristic temperature for lipid LPS in the SR membrane. Furthermore, the functionality of the ATPase, and in particular the lifetime of the first phosphorylated enzyme conformation (E1 approximately P) in the Ca+2 transport cycle, were also found to be linked to the occurrence of this structural transition. The hysterisis observed in lipid LPS behavior as a function of temperature and water content provides a possible explanation for the more efficient transient trapping of the enzyme in the E1 approximately P conformation observed in SR membranes partially dehydrated at temperatures below th. The observation that LPS behavior for the intact SR membrane and bilayers of isolated SR lipids (no protein present) are qualitatively similar strongly suggests that the LPS behavior of the SR membrane lipids is responsible for the observed structural change in the Ca+2ATPase and the resulting significant increase in E1 approximately P lifetime for temperatures below th.     

Ca2+ uptake and membrane potential in sarcoplasmic reticulum vesicles

The rate of calcium uptake by sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was stimulated by inside-negative membrane potential generated by K+ gradients in the presence of valinomycin. The increase in the calcium transport rate was accompanied by a proportional increase in the rate of calcium-dependent ATP hydrolysis, without significant change in the steady state level of the phosphorylated enzyme intermediate. Changes in the sarcoplasmic reticulum membrane potential during calcium transport were monitored with the optical probe, 3,3'-diethylthiadicarbocyanine. The decrease in the absorbance of 3,3'-diethylthiadicarbocyanine at 660 nm following generation of inside-negative membrane potential was reversed during ATP-induced calcium uptake. These observations support an electrogenic mechanism for the transport of calcium by the sarcoplasmic reticulum.     

The evaluation of the age-related characteristics of the structural status of the plasma membrane lipid phase in rat fatty tissue by using the method of inductive-resonance energy transfer

Using the method of inductance-resonance energy transfer from tryptophanyl residues to fluorescent pyrene probe the structural state of plasmatic membranes from adipose tissue of different age rats has been studied. The structural heterogeneity of membrane lipid phase has been revealed. The differences in physical properties of annular and bilayer lipids don't depend on age. During aging the membrane lipid viscosity including lipids of near protein area decreases, the conformation of membrane protein components alters during aging as well. The data on various effectiveness of energy transfer from tryptophanyls to pyrene probe in young and aged animals with stable polypeptide composition of membrane proteins indicates that. The structure of membrane lipid phase is suggested to be the main factor affecting the conformational state and functional activity of membrane-bound proteins during aging.     

Ca-ATPase self-fluorescence in the sarcoplasmic reticulum of the rabbit skeletal muscle

The quenching of the intrinsic protein fluorescence of sarcoplasmic reticulum Ca-ATPase from the rabbit skeletal muscles by hydrophylic (NaI, CsCl) or hydrophobic (pyrene, fluorescamine) substances has been studied. CsCl (up to 1 M) has been shown not to affect the intrinsic protein fluorescence while NaI (250 mM) quenches it at 15%, pyrene (8 mkM) decreases the intrinsic fluorescence of Ca-ATPase at 35% and fluorescamine (up to 40 mkM)--at 80%. Possible mechanisms of the interaction of the quenchers with the intrinsic fluorescence of sarcoplasmic reticulum Ca-ATPase are being discussed.     

Cytochemical studies of myocardial adenylate cyclase after its activation and inhibition

Incubation medium, as previously described (J Histochem Cytochem 27:774, 1979), was used to demonstrate the presence of adenylate cyclase (AC) in myocardium. NaF and ouabain were used to inhibit adenosine triphosphatases (ATP) and NaF and isoproterenol were used as activators of AC. The inhibitory effect of adenosine on AC was blocked by the addition of adenosine deaminase. The addition of tetramisol blocked the influence of the alkaline phosphatases on adenylyl imidodiphosphate hydrolysis. The use of these substances resulted in specific precipitation localized in junctional sarcoplasmic reticulum and sarcolemma. The reaction product was dramatically intensified after activation of AC by NaF or isoproterenol. Preincubation in 10-100 mM of propranolol, for 30 min, blocked AC stimulation by isoproterenol and prevented the appearance of the specific precipitate. The localization of specific precipitate in junctional sarcoplasmic reticulum and subsarcolemmal cisternae corresponds to the localization of Na+, K+ ATPase and may reflect the similar role that AC and Na+, K+ ATPase play in calcium release from sarcoplasmic reticulum of internal and peripheral couplings.     

Role of interprotein interactions in the regulation of the sarcoplasmic reticulum Ca-pump

The data on the structural state of sarcoplasmic reticulum membranes in skeletal muscles of rabbit were obtained by EPR-spectroscopy, fluorescent analysis and flash-photolysis and discussed in the paper. Comparison of the functional state of Ca-pump and variations in hydrophobic volume and membrane microviscosity permits concluding that thermoinduced anomalies of the enzymic activity are due to changes in the phase state of lipids. It is shown that changes in the physiochemical state of lipids affect the interprotein interactions in the oligomeric Ca-pump structure. In this case ATP weakens the interaction between Ca-pump globules, while a decrease in the hydrophobic membrane volume intensifies it. An assumption is advanced that the modifying ATP effect on Ca-pump is based on an increase in the functional independence of Ca2+-ATPase monomers, and therefore it is under the control of the membrane lipid phase.     

Sarcoplasmic reticulum from flounder muscle having improved lipid peroxidative activity

A procedure for the isolation of sarcoplasmic reticulum from winter flounder (Pseudopleuronectes americanus) resulted in a fraction with a specific activity of lipid peroxidation two to three times that of previous preparations. In addition, good stability of the NADH-dependent lipid peroxidative activity was achieved. There appeared to be minimal contamination of the preparation with lysosomes and mitochondria. The flounder sarcoplasmic reticulum was highly active with respect to ATPase and calcium uptake. The membrane fraction contained 43% lipid and 57% protein; 60% of the lipids were phospholipids. Phosphatidylcholine was the major phospholipid present.     

Phospholipid fatty acyl chain asymmetry in the membrane bilayer of isolated skeletal muscle sarcoplasmic reticulum

We previously showed [Herbette, L. G., Blasie, J. K., DeFoor, P., Fleischer, S., Bick, R. J., Van Winkle, W. B., Tate, C. A., & Entman, M. L. (1984) Arch. Biochem. Biophys. 234, 235-242; Herbette, L. G., DeFoor, P., Fleischer, S., Pascolini, D., Scarpa, A., & Blasie, J. K. (1985) Biochim. Biophys. Acta 817, 103-122] that the phospholipid head-group distribution in the membrane bilayer of isolated sarcoplasmic reticulum is asymmetric. From these studies, both the total number of phospholipid head groups and the total lipid, as well as the head-group species for these lipids, were found to be different for each monolayer of the membrane bilayer. In this paper, we demonstrate for the first time that there is significant asymmetry in the distribution of unsaturated fatty acids between the two monolayers; i.e., the outer monolayer of the sarcoplasmic reticulum contained more unsaturated and polyunsaturated chains when compared to the inner monolayer. X-ray diffraction measurements demonstrated that the time-averaged fatty acyl chain extension for the outer monolayer was approximately 20% less than for the inner monolayer. This is consistent with the concept that the greater degree of unsaturation in the outer monolayer may provide for a decreased average fatty acyl chain extension for that layer. This architecture for the bilayer may be related to both the "resting" state mass distribution of the calcium pump protein within the membrane bilayer and possible "conformational" states of the calcium pump protein during calcium transport by the sarcoplasmic reticulum.     

Analysis of the arrangement of protein components in the sarcomplasmic reticulum of rat skeletal muscle.

The nature of the protein components and their location in the sarcoplasmic reticulum membrane were studied using sarcoplasmic reticulum vesicles isolated from rat skeletal muscle and purified by a density gradient centrifugation system. On the basis of analysis by means of sodium dodecyl sulfate gel electrophoresis, the protein components appear to be similar if not identical with those reported by others for rabbit sarcoplasmic reticulum, and the relative amount of each component is also similar to that found with rabbit sarcoplasmic reticulum. Evidence is presented that radioiodine-labeled diazotized diiodosulfanilic acid is a nonpermeant labeling agent of the protein components of sarcoplasmic reticulum vesicles; this agent minimally disturbs the functional activities of these membranes. By means of this labeling agent and perturbing agents, it is concluded that the protein components with molecular weights greater than 120,000 and the (Ca2+ + Mg2+)-adenosine triphosphatase partially or totally reside on or at the external surface of the sarcoplasmic reticulum vesicles. In the case of the adenosine triphosphatase, highly controlled trypsin treatment cleaves the molecule into two products, a 65,000 molecular weight fragment and a 56,000 molecular weight fragment. The evidence indicates that the 65,000 molecular weight component of the (Ca2+ + Mg2+)-adenosine triphosphatase is located in a more exposed fashion on the external surface of the vesicles than the 56,000 molecular weight compoenet and that some adenosine triphosphatase molecules have a more exposed position on the external surface of the vesicle than others. The protein components designated by MacLennan (MacLennan, D. H. (1975) Can. J. Biochem. 53, 251-261) as "calsequestrin" and "high affinity Ca2+ binding protein" are shown not to be on the external surface of the rat sarcoplasmic reticulum vesicle but rather to reside either within the core of the membrane or on the inside surface of the vesicle. The results of this study are in agreement with the model for the organization of the protein components of the sarcoplasmic reticulum membrene recently proposed by MacLennan (MacLennan, D. H. (1975) Can. J. Biochem. 53, 251-261).     

Localization of Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in adult rat papillary muscle

Localization of the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in rat papillary muscle was determined by indirect immunofluorescence and immunoferritin labeling of cryostat and ultracryotomy sections, respectively. The Ca2+ + Mg2+-ATPase was found to be rather uniformly distributed in the free sarcoplasmic reticulum membrane but to be absent from both peripheral and interior junctional sarcoplasmic reticulum membrane, transverse tubules, sarcolemma, and mitochondria. This suggests that the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum is antigenically unrelated to the Ca2+ + Mg2+-ATPase of the sarcolemma. These results are in agreement with the idea that the sites of interior and peripheral coupling between sarcoplasmic reticulum membrane and transverse tubules and between sarcoplasmic reticulum and sarcolemmal membranes play the same functional role in the excitation-contraction coupling in cardiac muscle.     

Effect of pH on calcium accumulation and release by isolated fragments of cardiac and skeletal muscle sarcoplasmic reticulum.

The ability of a sudden increase in pH to initiate a release of calcium from isolated skeletal and cardiac muscle sarcoplasmic reticulum following calcium accumulation in the absence of a precipitating anion (calcium binding) is described. In skeletal sarcoplasmic reticulum a sudden increase in pH caused a rapid release of accumulated calcium. In cardiac sarcoplasmic reticulum a sudden increase in pH before the calcium binding process was complete caused the release of a small amount of calcium at a relatively slow rate. A sudden change in pH after the completion of calcium binding failed to trigger a release of calcium. The effect of pH on oxalate supported calcium uptake and on unidirectional calcium efflux rate by cardiac sarcoplasmic reticulum was also studied. Both the rate of calcium uptake and of unidirectional calcium efflux increased as the pH was raised from 6.4 to 7.2, reflecting an increased permeability of the sarcoplasmic reticulum membrane to calcium. These results indicate that in cardiac muscle a sudden increase in pH is unlikely to be the in vivo signal for calcium release from the sarcoplasmic reticulum. However, the effect of pH on calcium uptake and efflux by cardiac sarcoplasmic reticulum may contribute to the negative inotropic effect of an acidosis on the heart.     

The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes

Ryanodine at concentrations of 0.01-10 microM increased, while greater concentrations of 10-300 microM decreased the calcium permeability of both rabbit fast twitch skeletal muscle junctional and canine cardiac sarcoplasmic reticulum membranes. Ryanodine did not alter calcium binding by either sarcoplasmic reticulum membranes or the calcium binding protein, calsequestrin. Therefore, the effects by this agent appear to involve only changes in membrane permeability, and the characteristics of the calcium permeability pathway affected by ryanodine were those of the calcium release channel. Consistent with this, the actions by ryanodine were localized to junctional sarcoplasmic reticulum membranes and were not observed with either longitudinal sarcoplasmic reticulum or transverse tubular membranes. In addition, passage of the junctional sarcoplasmic reticulum membranes through a French press did not diminish the effects of ryanodine indicating that intact triads were not required. Under the conditions used for the permeability studies, the binding of [3H]ryanodine to skeletal junctional sarcoplasmic reticulum membranes was specific and saturable, and Scatchard analyses indicated the presence of a single binding site with a Kd of 150-200 nM and a maximum capacity of 10.1-18.9 pmol/mg protein. [3H]ryanodine binding to this site and the increase in membrane calcium permeability caused by low concentrations of ryanodine had similar characteristics suggesting that actions at this site produce this effect. Depending on the assay conditions used, ryanodine (100-300 microM) could either increase or decrease ATP-dependent calcium accumulation by skeletal muscle junctional sarcoplasmic reticulum membranes indicating that the alterations of sarcoplasmic reticulum membrane calcium permeability caused by this agent can be determined in part by the experimental environment.