Biomedical subjects
Publications and source records attributed to V B Ritov.
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.
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Purified preparations of Ca2+-dependent ATPase were lipid-deleted and incorporated into egg lecithin (EL) and dipalmitoyl lecithin (DPL) liposomes. The temperature dependences of the catalytic activity and of molecular mobility of the spin label (N-1-hydroxyl-2,2,6,6-tetramethyl-4-piperidyl) maleimide linked to a highly reactive SH-group in the vicinity of the active center (15-16 A) and of the fatty acid spin probe (6-doxylpalmitate) located in the protein-lipid moiety were compared. The molecular mobility of the spin label was measured by the saturation transfer method; that of the spin probe was estimated from the maximal splitting value. It was found that the catalytic activity of DPL is correlated with the molecular mobility of the hydrophobic part of ATPase, while that of EL with the segment flexibility in the vicinity of the active center.
Chromatography of a mixture of phospholipid and Ca-dependent ATPase of sarcoplasmic reticulum solubilized with choleate in a column with anionite results in proteolyposome formation. It was shown that dilution of ATPase with phospholipids makes it possible to reduce the concentration of ATPase in the membrane. Using the bifunctional reagent difluorodinitrobenzene it was demonstrated that reduction of the ATPase concentration in the membrane leads to the dissociation of the enzyme to monomers. It was disclosed that the monomeric form of ATPase is capable of active Ca2+ transport.
The interaction between 3-acyl-2-(6-doxylpalmitoyl) phosphatidyl choline used as a hydrophobic spin probe and Ca2+-dependent ATPase from sarcoplasmic reticulum membranes of rabbit and carp white skeletal muscles was studied. The spin label incorporation into ATPase preparations was performed at initial steps of ATPase isolation by incubating reticulum membranes with the spin probe in the presence of cholic acid. A comparison of the molecular mobility of the probe incorporated into ATPase preparations and into liposomes formed from ATPase phospholipids demonstrated that the presence of the protein in the membrane produces the same effect on the probe mobility as does the decrease of temperature by 10-15 degrees C. The molecular mobility of the probe in the ATPase preparation is increased during protein thermal denaturation. The breaks on the Arrhenius plots for the probe molecular mobility are revealed at the same temperatures (25 degrees for rabbit reticulum and 16 degrees for carp reticulum) as those for the ATPase activity.
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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.
The effect of cAMP-dependent protein kinases from rabbit skeletal muscles on Ca2+ uptake by fragments of skeletal muscle sarcoplasmic reticulum was studied. It was shown that incubation of the sarcoplasmic reticulum fragments with protein kinase increases the rate of Ca2+ uptake without changing the activity of Ca2+-dependent ATPase. This phenomenon is not accompanied by phosphorus incorporation into the protein components of the reticulum membranes. The protein kinase preparation subjected to "self-phosphorylation" is also capable to increase the rate of Ca2+ uptake. Using (14C) -oleic acid, it was shown that the increase of the rate of Ca2+ transport under effects of the "self-phosphorylated" protein kinase occurs due to the binding of free fatty acids present in the sarcoplasmic reticulum membranes. It was found that the effect observed is due to phosphofructokinase (ATP : D-fructose-6-phosphate-1-phosphotransferase) present in the protein kinase preparation.
Chromatography of a mixture of phospholipids and Ca-dependent ATPase from sarcoplasmic reticulum solubilized by cholate on a column with anionite results in a formation of proteoliposomes. Using the freeze-fracture technique, it was shown that the density of intramembrane particles in fractured proteoliposomes formed is reversely proportional to the lipid--protein ratio. This is indicative of possible changes in ATPase concentration in the membrane, when the above-mentioned method was used. It was shown that dilution of ATPase with phospholipids increases its thermal stability. This is probably due to aggregation of the ATPase molecules and increase in the length of the borderline of lipids around each enzyme molecule.
The uncoupling of Ca2+ uptake and ATP hydrolysis by acetylcholine (ACh) and choline has been demonstrated on fragmented sarcoplasmic reticulum (FSR) isolated from femoral muscles of Rana ridibunda. The uncoupling depends on the content of oxalate in the incubation medium. An increase in the Ca/ATP ratio caused by serum albumin observed at low oxalate concentrations is indicative of participation of free fatty acids in the uncoupling effect of ACh and choline. The effects of ACh and choline are especially well-pronounced in the case of FSR preparations pretreated with a fatty acid. ACh and choline have no effect on FSR from femoral muscles of Rana temporaria at all oxalate concentrations studied, which may be due to the absence of free fatty acids in these preparations. A pronounced uncoupling effect of ACh is observed after FSR incubation with a fatty acid; the activity of choline is pronounced in a much lesser degree. It is concluded that a decrease in the Ca/ATP ratio in the presence of ACh is due to the enhancement of the uncoupling effects of endogenous free fatty acids.
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The dependence of the state of the hydrophobic zone of rabbit sarcoplasmic reticulum (SR) membranes on temperature of the membrane fragment suspension before rapid freezing was studied by the freeze fracturing technique. It was shown that within the temperature range of--15-- +37 degrees C the amount of intramembrane particles and their distribution in the membrane plane and between their convex and concave surfaces do not practically depend on the temperature of the SR membrane suspension. This is indicative of the lack of correlation between the physical state of the phospholipid matrix (gel -- liquid crystal) before freezing and the nature of the profile of the membrane hydrophobic zone revealed after fracturing. The disturbances in the protein -- lipid interactions in the membrane under the effects of mersalyl or aqueous solutions of diethyl ester followed by complete inactivation of Ca2+-dependent ATPase lead to a decrease in the amount of intramembrane particles, which is especially well-pronounced at 37 degrees and -15 degrees C.
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Two highly purified sarcoplasmic reticulum membrane fractiones differing in their sensitivities to the uncoupling action of caffeine were isolated from white skeletal muscles of the rabbit. The main protein component of both fractions is a catalytical polypeptide of Ca2+-dependent ATPase. Treatment of the caffeine-sensitive reticular fraction by trypsin or DTNB completely removes the effect of caffeine. It was found that similar effects on the caffeine-sensitive reticular fraction are exerted by bemegride, camphor, ethymizole and cordiamine. Isolation of Ca2+-dependent ATPase from both reticular fractions and reconstruction of Ca2+-transporting vesicles were carried out. Ca2+ transport by the vesicles enriched by ATPase from the caffeine-sensitive reticular fraction is uncoupled under the effect of caffeine; however, caffeine has no effect on the vesicles enriched by caffeine-insensitive reticular ATPase. The molecular weight of caffeine-sensitive and caffeine-insensitive ATPases determined in the presence of sedium dodecyl sulfate are found to be identical. Electrophoresis in the presence of digitonin revealed different electrophoretic behaviour of the two forms of ATPase.
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Mechanisms of the inhibitory effect of vitamin E on the process of lipid peroxidation (LPO) in biological membranes are studied. Both alpha-tocopherol and its derivatives (a-tocopherylacetate, o- and p-tocopherylquinones possess no radical scavenging activity) inhibit non-enzymatic (Fe2+ + ascorbate)-induced LPO and prevent LPO-dependent inhibition of Ca2+ transport in sarcoplasmic reticulum membranes from skeletal muscles. The protective effect of alpha-tocopherylacetate, tocopherilquinones and partially of alpha-tocopherol is due to a stabilizing effect of these compounds on sarcoplasmic reticulum membranes, registered by a decrease of fluidity of membrane lipid bilayer (probed by nitroxile radical TEMPO) and by a decrease of its passive permeability for Ca2+. Under the enzymatic NADPH-dependent LPO induction in rat liver microsomal fraction a strong inhibitory effect of tocopherylquinones is similar to the effect of other electron acceptors (methylnaphtoquinone, TEMPO) and is due to their ability to compete with LPO reaction for reducing equivalents in NADPH-dependent electron carriers wich results in the formation of hydroxy-derivatives having pronounced radical scavenging activity.