Fluorescence study on rat epithelial cells and liposomes exposed to aromatic nitroxides

This study was performed to evaluate the effects, if any, of aromatic nitroxides, namely, indolinic nitroxides, on membrane fluidity of rat epithelial cells using steady-state fluorescence. These nitroxides are being increasingly considered as new and versatile compounds to reduce oxidative stress in biological systems. Hence, the results obtained in this study will give more insights on the interaction of these compounds with biological structures which at present is lacking, especially in view of their possible application as antioxidant therapeutic agents. The probes DPH and Laurdan which give information on the hydrophobic and hydrophilic-hydrophobic regions of the membrane bilayer, respectively, showed that nitroxide 1 (1,2-dihydro-2-methyl-3H-indole-3-one-1-oxyl) significantly increases membrane fluidity, whereas the corresponding phenylimino nitroxide derivative 2 (1,2-dihydro-2-methyl-3H-indole-3-phenylimino-1-oxyl) leads to membrane rigidification. The aliphatic nitroxide TEMPO included in this study for comparison produced no modifications. Consequently, it appears that the structure of the heterocyclic rings (aromatic or aliphatic) and the substituents may affect membrane fluidity differently.     

Effect of complexation with randomly methylated beta-cyclodextrin on the aqueous solubility, photostability and antioxidant activity of an indolinonic nitroxide radical

The interaction between the hydrophobic indolinonic nitroxide radical, 1,2-dihydro-2-methyl-2-phenyl-3H-indole-3-one-1-oxyl and hydrophilic alpha-, beta- and gamma-cyclodextrin derivatives was investigated in water by phase-solubility analysis. Among the studied cyclodextrins, random methyl-beta-cyclodextrin (RM-beta-CD) had the greatest solubilizing activity (1312-fold increase in. the intrinsic aqueous solubility). Solid complexes were prepared by the freeze-drying method and characterized by powder X-ray diffractometry and thermal analysis. Complexation of the nitroxide with RM-beta-CD was also confirmed in solution by electron paramagnetic resonance (EPR) spectroscopy. Photodegradation of the nitroxide was reduced by complexation with RM-beta-CD, this effect being more pronounced in the solid-state (the extent of degradation was 28.0% for the complex vs. 78.8% for uncomplexed nitroxide) than in solution (41.2 vs. 69.1% for uncomplexed nitroxide). The antioxidant activity of the complex was also investigated on the peroxidation of methyl linoleate micelles and on protein oxidation induced by free radical generators, and in both systems the free form of the nitroxide as well as its complex with RM-beta-CD, showed essentially the same degree of protection. Moreover, EPR experiments showed a time-dependent decrease in the EPR signal of both the complexed and uncomplexed nitroxides with the free-radical generators. Therefore, RM-beta-CD complexation of the nitroxide represents an effective strategy to improve its aqueous solubility and photostability, which is essential for certain biological applications, while it does not interfere with its radical scavenging efficiency.     

Preferential hydrolysis of peroxidized phospholipid by lysosomal phospholipase C

The susceptibility of partially peroxidized liposomes of 2-[1-14C] linoleoylphosphatidylethanolamine ([14C]PE) to hydrolysis by cellular phospholipases was examined. [14C]PE was peroxidized by exposure to air at 37 degrees C, resulting in the formation of more polar derivatives, as determined by thin-layer chromatographic analysis. Hydrolysis of these partially peroxidized liposomes by lysosomal phospholipase C associated with cardiac sarcoplasmic reticulum, and by rat liver lysosomal phospholipase C, was greater than hydrolysis of non-peroxidized liposomes. By contrast, hydrolysis of liposomes by purified human synovial fluid phospholipase A2 or bacterial phospholipase C was almost completely inhibited by partial peroxidation of PE. Lysosomal phospholipase C preferentially hydrolyzed the peroxidized component of the lipid substrate which had accumulated during autoxidation. The major product recovered under these conditions was 2-monoacylglycerol, indicating sequential degradation by phospholipase C and diacylglycerol lipase. Liposomes peroxidized at pH 7.0 were more susceptible to hydrolysis by lysosomal phospholipases C than were liposomes peroxidized at pH 5.0, in spite of greater production of polar lipid after peroxidation at pH 5.0. Sodium bisulfite, an antioxidant and an inhibitor of lysosomal phospholipases, prevented: (1) lipid autoxidation, (2) hydrolysis of both non-peroxidized and peroxidized liposomes by sarcoplasmic reticulum and (3) loss of lipid phosphorus from endogenous lipids when sarcoplasmic reticulum was incubated at pH 5.0. These studies show that lipid peroxidation may modulate the susceptibility of phospholipid to attack by specific phospholipases, and may therefore be an important determinant in membrane dysfunction during injury. Preservation of membrane structural and functional integrity by antioxidants may result from inhibition of lipid peroxidation, which in turn may modulate cellular phospholipase activity.     

Peroxide modification of skeletal muscle sarcoplasmic reticulum in antioxidant deficiency and under the effect of ionol. II. Physico- chemical properties of the sarcoplasmic reticulum membrane

Physico-chemical parameters of membranes of skeletal muscles' sarcoplasmic reticulum in antioxidant insufficiency, which was modelled by excluding alpha-tocopherol from the animals ration, and after treatment with phenol antioxidant ionol were studied. It was shown that activation of lipid peroxidation in vitamin E insufficiency results in a significant lowering of microviscosity of lipid bilayer membranes of sarcoplasmic reticulum. Using polarography significant changes in membrane protein conformation were revealed, which were characterized by lowering of integrity and by disorganization of protein globules. Treatment of animals with antioxidant insufficiency with ionol led to certain normalization of changes of physico-chemical characteristics of the learned membrane structures caused by lipid peroxidation.     

Oligomerization of integral membrane proteins under lipid peroxidation

Using polyacrylamide gel electrophoresis in the presence of Na-SDS, the oligomerization of membrane proteins of the retinal rod outer segments of the frog and the wall-eyed pollock and of rabbit skeletal muscle sarcoplasmic reticulum was studied. It was shown that under storage of the retinal rod outer segments the rhodopsin oligomerization is inhibited by the lipid peroxidation inhibitor--ionol. Similar oligomerization was observed under induction of lipid peroxidation in the membranes; the accumulation of the lipid peroxidation product--malonic dialdehyde--was accompanied by disappearance of the rhodopsin monomeric form in the outer segments. The cross-linking agent--glutaric dialdehyde--also causes oligomerization of the rhodopsins. Similar aggregation is also characteristic of the major protein of the sarcoplasmic reticulum fragments, i. e. Ca2+-dependent ATP-ase. Thus, one of the main changes in the protein content of biomembranes under lipid peroxidation is the oligomerization of integral proteins due to their interaction with bifunctional reagents, i. e. lipid peroxidation products.     

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.     

Oxygen radical-mediated lipid peroxidation and inhibition of Ca2+-ATPase activity of cardiac sarcoplasmic reticulum

Oxygen radicals have been implicated as important mediators of myocardial ischemic and reperfusion injury. A major product of oxygen radical formation is the highly reactive hydroxyl radical via a biological Fenton reaction. The sarcoplasmic reticulum is one of the major target organelles injured by this process. Using a oxygen radical generating system consisting of dihydroxyfumarate and Fe3+-ADP, we studied lipid peroxidation and Ca2+-ATPase of cardiac sarcoplasmic reticulum. Incubation of sarcoplasmic reticulum with dihydroxyfumarate plus Fe3+-ADP significantly inhibited enzyme activity. Addition of superoxide dismutase, superoxide dismutase plus catalase (15 micrograms/ml) or iron chelator, deferoxamine (1.25-1000 microM) protected Ca2+-ATPase activity. Time course studies showed that this system inhibited enzyme activity in 7.5 to 10 min. Similar exposure of sarcoplasmic reticulum to dihydroxyfumarate plus Fe3+-ADP stimulated malondialdehyde formation. This effect was inhibited by superoxide dismutase, catalase, singlet oxygen, and hydroxyl radical scavengers. EPR spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide verified production of the hydroxyl radical. The combination of dihydroxyfumarate and Fe3+-ADP resulted in a spectrum of hydroxyl radical spin trap adduct, which was abolished by ethanol, catalase, mannitol, and superoxide dismutase. The results demonstrate the role of oxygen radicals in causing inactivation of Ca2+-ATPase and inhibition of lipid peroxidation of the sarcoplasmic reticulum which could possibly be one of the important mechanisms of oxygen radical-mediated myocardial injury.     

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.     

Quinolinic Aminoxyl Protects Albumin Against Peroxyl Radical Mediated Damage

A study of peroxyl radical-mediated bovine serum albumin oxidation in the presence of the quinolinic aminoxyl 1,2-dihydro-2,2-diphenyl-4-ethoxy-quinoline-1-oxyl (QAO) was carried out in order to test its efficiency as a protein antioxidant. Albumin oxidation was induced by the tert-butylhydroperoxide/PbO₂ system. The extent of protein oxidation, measured by monitoring the formation of carbonyl groups, was considerably reduced in the presence of QAO. ESR measurements were carried out to confirm the consumption of the nitroxide during oxidation and its incorporation in the protein. The data obtained indicate that the quinolinic aminoxyl function can be used as an effective antioxidant in biological systems.     

Identification of plasmalogen as the major phospholipid constituent of cardiac sarcoplasmic reticulum

The phospholipid molecular species of canine myocardial sarcoplasmic reticulum were identified by fast atom bombardment mass spectrometry, reverse-phase high-performance liquid chromatography, and other conventional techniques. Cardiac sarcoplasmic reticulum contains 1.4 mumol of lipid Pi/mg of protein which is comprised of 53% plasmalogen. Cardiac sarcoplasmic reticulum ethanolamine glycerophospholipid contains 73% plasmalogen that is predominantly comprised of moieties with 18-carbon vinyl ethers at the sn-1 position and arachidonic acid at the sn-2 position. In contrast, canine skeletal muscle sarcoplasmic reticulum contains only 19% plasmalogen that is predominantly comprised of ethanolamine plasmalogen (78% of skeletal muscle sarcoplasmic reticulum ethanolamine glycerophospholipid) with arachidonic and docosatetraenoic acids at the sn-2 position. The possibility that tetraenoic ethanolamine plasmalogens in both cardiac and skeletal muscle sarcoplasmic reticulum facilitate calcium translocation by their propensity for adopting a hexagonal II conformation at physiologic temperatures is discussed.     

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.     

Lipoxygenase-induced lipid peroxidation of isolated cardiac microsomes modulates their calcium-transporting function

We demonstrated previously that products of linoleic and arachidonic acids, arising from enzymatic or non-enzymatic oxidation, inhibit ATP-dependent calcium accumulation into and promote release of calcium from vesicles derived from sarcoplasmic reticulum of guinea-pig heart. In the present study, direct enzymatic peroxidation of cardiac membrane lipids was performed and the effect on calcium transport was examined. Vesicles were preincubated at 37 degrees C with soybean lipoxygenase-1 (linoleate:oxygen oxidoreductase, EC 1.13.11.12) for up to 1 h prior to the initiation of calcium accumulation. The extent of membrane peroxidation was assessed by monitoring the production of malondialdehyde. Pretreatment of vesicles with lipoxygenase for 40 and 60 min markedly depressed calcium accumulation. The lipoxygenase-induced suppression of calcium transport was completely antagonized by nordihydroguaiaretic acid (1 microM), not at all by indomethacin (1 microM), and only partially by 5,8,11,14-eicosatetraynoic acid (0.3 microM). Low concentrations of calcium (10(-5)-5 X 10(-5) M) enhanced, and a high concentration (10(-3) M) inhibited lipoxygenase-induced peroxidation of membrane lipids. The calcium-accumulating ability of the vesicles was inversely related to the extent of membrane peroxidation. The vesicles which showed the highest degree of peroxidation in the presence of 5 X 10(-5) M calcium, accumulated the lowest amount of calcium. In contrast, calcium at 10(-3) M suppressed lipid peroxidation, resulting in higher calcium uptake than in vesicles peroxidized in the absence of calcium. Thus, calcium transport is depressed in microsomes undergoing lipoxygenase-induced peroxidation, a process which in turn is modulated by calcium.     

Sodium-calcium exchanger and lipid rafts in pig coronary artery smooth muscle

Pig coronary artery smooth muscle expresses, among many other proteins, Na+-Ca2+-exchanger NCX1 and sarcoplasmic reticulum Ca2+ pump SERCA2. NCX1 has been proposed to play a role in refilling the sarcoplasmic reticulum Ca2+ pool suggesting a functional linkage between the two proteins. We hypothesized that this functional linkage may require close apposition of SERCA2 and NCX1 involving regions of plasma membrane like lipid rafts. Lipid rafts are specialized membrane microdomains that appear as platforms to co-localize proteins. To determine the distribution of NCX1, SERCA2 and lipid rafts, we isolated microsomes from the smooth muscle tissue, treated them with non-ionic detergent and obtained fractions of different densities by sucrose density gradient centrifugal flotation. We examined the distribution of NCX1; SERCA2; non-lipid raft plasma membrane marker transferrin receptor protein; lipid raft markers caveolin-1, flotillin-2, prion protein, GM1-gangliosides and cholesterol; and cytoskeletal markers clathrin, actin and myosin. Distribution of markers identified two subsets of lipid rafts that differ in their components. One subset is rich in caveolin-1 and flotillin-2 and the other in GM1-gangliosides, prion protein and cholesterol. NCX1 distribution correlated strongly with SERCA2, caveolin-1 and flotillin-2, less strongly with the other membrane markers and negatively with the cytoskeletal markers. These experiments were repeated with a non-detergent method of treating microsomes with sonication at high pH and similar results were obtained. These observations are consistent with the observed functional linkage between NCX1 and SERCA2 and suggest a role for NCX1 in supplying Ca2+ for refilling the sarcoplasmic reticulum.     

Membrane localization of myocardial type II cyclic AMP-dependent protein kinase activity

Crude cardiac membrane vesicles were separated into subfractions of sarcolemma and sarcoplasmic reticulum. The subfractions were used to determine the origin and type of cyclic AMP-dependent protein kinase activity present in myocardial membranes. A cyclic AMP-binding protein of molecular weight 55,000 was covalently labeled with the photoaffinity probe 8-azido adenosine 3',5'-mono[32P]phosphate, and found to copurify with the (Na+ + K+)-ATPase activity of sarcolemma, and away from the (Ca2+ + K+)-ATPase activity of sarcoplasmic reticulum. Endogenous cyclic AMP-dependent protein kinase activity also copurified with sarcolemma. Protein substrates phosphorylated by cyclic AMP-dependent protein kinase activity had apparent molecular weights of 21,000 and 8000 and were present in both sarcolemma and sarcoplasmic reticulum. However, while addition of cyclic AMP alone resulted in phosphorylation of sarcolemma proteins, both cyclic AMP and exogenous, soluble cyclic AMP-dependent kinase were required for phosphorylation of sarcoplasmic reticulum proteins. Addition of the calcium-binding protein, calmodulin, to either sarcolemma or sarcoplasmic reticulum resulted in phosphorylation of the 21,000 and 8000-dalton proteins, as well. The results suggest that cardiac sarcolemma contains an intrinsic type II cyclic AMP-dependent protein kinase activity that is not present in sarcoplasmic reticulum. On the other hand, Ca2+- and calmodulin-dependent protein kinase activity is present in both sarcolemma and sarcoplasmic reticulum.     

Heterogeneous distribution of calmodulin- and cAMP-dependent regulation of Ca2+ uptake in cardiac sarcoplasmic reticulum subfractions

The activity of the Ca2+-pumping ATPase of cardiac sarcoplasmic reticulum is controlled by the phosphorylation level of the intrinsic membrane protein phospholamban. Phospholamban monomers contain two distinct phosphorylation sites for either the cAMP-dependent or the calmodulin-dependent kinase. The two kinases, however, preferentially phosphorylate different populations of phospholamban molecules and double phosphorylation of the same subunit by their concerted action is a phenomenon that occurs only under particular experimental conditions. This study investigates the phosphorylation pattern of phospholamban in various subfractions derived from dog cardiac sarcoplasmic reticulum. The results show that the endogenous calmodulin-dependent kinase preferentially phosphorylates the phospholamban population found in association with the cisternal compartments of the reticulum. The differential phosphorylation occurs despite the presence of sufficient amounts of the kinase in all sarcoplasmic reticulum subfractions. On the other hand, phospholamban molecules localized on the longitudinal system are preferential substrates for the cAMP-dependent kinase. Possibly, the different lipid and/or protein microenvironment of phospholamban in the various sarcoplasmic reticulum domains is responsible for the apparent heterogeneity of phosphorylation. The present findings are compatible with the concept of additive and independent action of the cAMP-dependent and calmodulin-dependent kinases on cardiac sarcoplasmic reticulum. The imply, however, that different regions of the sarcoplasmic reticulum network are controlled by the two regulatory mechanisms.     

Alkaloids from Portulaca oleracea L

Five alkaloids (oleraceins A, B, C, D and E) were isolated from Portulaca oleracea L., and their structures determined by spectroscopic methods as 5-hydroxy-1-p-coumaric acyl-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-beta-D-glucopyranoside, 5-hydroxy-1-ferulic acyl-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-beta-D-glucopyranoside, 5-hydroxy-1-(p-coumaric acyl-7'-O-beta-D-glucopyranose)-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-beta-D-glucopyranoside, 5-hydroxy-1-(ferulic acyl-7'-O-beta-D-glucopyranose)-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-beta-D-glucopyranoside and 8,9-dihydroxy-1,5,6,10b-tetrahydro-2H-pyrrolo[2,1-a]isoquinolin-3-one, respectively.     

Function of the membrane lipids of the heart sarcoplasmic network in izadrin myocarditis studied by using a pyrene probe

The fluorescent hydrophobic pyrene probe was employed to study the viscosity of membrane lipids of rat heart sarcoplasmic reticulum in isoproterenol myocarditis. During pyrene incorporation into the reticulum obtained from the affected myocardium, the increase in the microviscosity occurred at lower temperatures and more rapidly both in "bound" and "free" membrane lipids as compared with normal. The increase of the viscosity of the reticulum membranes in isoproterenol myocarditis was accompanied by a lowering of the activity of Ca, Mg-ATPase of the sarcoplasmic reticulum coupled with an elevation of the content of lipid peroxidation products.     

Age-dependent effect of oxidative stress on cardiac sarcoplasmic reticulum vesicles

The oxidative stress hypothesis of aging suggests that accumulation of oxidative damage is a key factor of the alterations in physiological function during aging. We studied age-related sensitivity to oxidative modifications of proteins and lipids of cardiac sarcoplasmic reticulum (SR) isolated from 6-, 15- and 26-month-old rats. Oxidative stress was generated in vitro by exposing SR vesicles to 0.1 mmol/l FeSO4/EDTA + 1 mmol/l H2O2 at 37 degrees C for 60 min. In all groups, oxidative stress was associated with decreased membrane surface hydrophobicity, as detected by 1-anilino-8-naphthalenesulfonate as a probe. Structural changes in SR membranes were accompanied by degradation of tryptophan and significant accumulation of protein dityrosines, protein conjugates with lipid peroxidation products, conjugated dienes and thiobarbituric acid reactive substances. The sensitivity to oxidative damage was most pronounced in SR of 26-month-old rat. Our results indicate that aging and oxidative stress are associated with accumulation of oxidatively damaged proteins and lipids and these changes could contribute to cardiovascular injury.     

Effects of selenium deficiency on Ca transport function of sarcoplasmic reticulum and lipid peroxidation in rat myocardium

Two groups of weanling Sprague-Dawley rats were fed a low-selenium basal diet (Se 0.009 mg/kg) and the same diet supplemented with sodium selenite (Se 0.25 mg/kg), respectively, for 1, 2, and 3 months. At each feeding time, the Ca²⁺-ATPase activity, Ca²⁺ uptake rate and the capacity of Ca²⁺ uptake in isolated cardiac sacroplasmic reticulum from the Se-deficient rats were decreased significantly compared to those from the Se-supplemented rats, the contents of lipid peroxide in postmitochondrial supernatant and isolated sarcoplasmic reticulum from the Se-deficient rats were significantly higher than that from Se-supplemented rats. Compared to the Se-supplemented rats, the cytosolic glutathione peroxidase activity in Se-deficient rats decreased significantly. In addition, significant linear negative correlations of lipid peroxide in postmitochondrial supernatant to sarcoplasmic reticular Ca²⁺-ATPase activity, Ca²⁺ uptake rate and to whole blood selenium concentration were observed. The results suggest that the enhancement of lipid peroxidation via the depressed glutathione peroxidase activity might be responsible for the decrease of Ca²⁺-ATPase and Ca²⁺ uptake activities in sarcoplasmic reticulum in Se-deficient animals.     

Separation of vesicles of cardiac sarcolemma from vesicles of cardiac sarcoplasmic reticulum. Comparative biochemical analysis of component activities.

Sarcolemmal and sarcoplasmic reticulum membrane vesicle fractions were isolated from cardiac microsomes. Separation of sarcolemmal and sarcoplasmic reticulum membrane markers was documented by a combination of correlative assay and centrifugation techniques. To facilitate the separation, the crude microsomes were incubated in the presence of ATP, Ca2+, and oxalate to increase the density of the sarcoplasmic reticulum vesicles. After sucrose gradient centrifugation, the densest subfraction (sarcoplasmic reticulum) contained the highest (K+,Ca2+)-ATPase activity and virtually no (Na2+,K+)-ATPase activity, even when latent (Na+,K+)-ATPase activity was unmasked. In addition, the sarcoplasmic reticulum fraction contained no significant sialic acid, beta receptor binding activity, or adenylate cyclase activity. Sarcolemmal membrane fractions were of low buoyant density. Preparations most enriched in sarcolemmal vesicles contained the highest level of all the other parameters and only about 10% of the (K+,Ca2+)-ATPase activity of the sarcoplasmic reticulum fraction. The results suggest that (Na+,K+)-ATPase, sialic acid, beta-adrenergic receptors, and adenylate cyclase can be entirely accounted for by the sarcolemmal content of cardiac microsomes. Gel electrophoresis of the sarcolemmal and sarcoplasmic reticulum membrane fractions showed distinct bands. Membrane proteins exclusive to each of the fractions were also demonstrated by phosphorylation. Cyclic AMP stimulated phosphorylation by [gamma-32P]ATP of two proteins of apparent Mr = 20,000 and 7,000 that were concentrated in sarcoplasmic reticulum, but the stimulation was markedly dependent on the presence of added soluble cyclic AMP-dependent protein kinase. Cyclic AMP also stimulated phosphorylation of membrane proteins in sarcolemma, but this phosphorylation was mediated by an endogenous protein kinase activity. The apparent molecular weights of these phosphorylated proteins were 165,000, 90,000, 56,000, 24,000, and 11,000. The results suggest that sarcolemma may contain an integral enzyme complex, not present in sarcoplasmic reticulum, that contains beta-adrenergic receptors, adenylate cyclase, cyclic AMP-dependent protein kinase, and several substrates of the protein kinase.