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1.
Using alamethicin, permitting the measurement of genuine catalytic enzyme activity, hypercholesterolemia was shown to cause a 10-30% reduction of specific Ca-ATPase activity registered at 37 degrees C and the shift of Arrhenius plot in 20-30 degrees C temperature range. Reconstruction of delipidated Ca-ATPase of sarcoplasmic reticulum membranes by egg lecithin in animals with hypercholesterolemia does not lead to the recovery of Arrhenius plot. The data obtained demonstrate that modification of temperature-dependent Ca-ATPase activity in hypercholesterolemia is associated with the changes in the polypeptide with a catalytic function and is not induced by the changes in phospholipid enzyme surroundings. 相似文献
2.
The amount and the reaction capacity of the thiol groups in the sarcoplasmic reticulum containing up to 86% of Ca-ATPase were determined using 7-chloro-4-nitrobenzo-2-hydroxo-1,3-diazole (NBD-chloride). The total amount of SH-groups interacting with NBD-chloride is about 9 moles/10(5) g of protein as determined in the excess of NBD-chloride (750 micrometers). With respect to their sensitivity to NBD-chloride the SH-groups may be divided into two classes: slow and fast ones (5,3 and 3,5 moles/10(5) g of protein, respectively). The modification constants for the fast and slow SH-groups are 0,16 and 0,015min-1. ATP (30 micrometers) decreases the number of fast groups by 1 mole/10(5) g of protein. At higher concentrations of ATP (1--3 mM) the amount of fast SH-groups is decreased by 3 moles/10(5) g of protein, their modification rate constant being decreased 2-fold. ATP at concentration of 1 mM, decreases the rate constant for the Ca-ATPase inactivation by NBD-chloride from 0.68 down to 0,073 min-1, which coincides with the modification rate constant for fast SH-groups (0,071 min-1) under the same conditions. Ca2+ at concentration of 10(-4) M increases the amount of fast thiol groups by 1 mole/10(5) g of protein, the rate constant of their modification by NBD-chloride being increased 2-fold. A half-maximal effect was observed in the presence of 5.10(-7) M Ca2+ . Mg2+ did not affect the total amount of fast thiol groups; however, it decreased their modification rate constant. 相似文献
3.
I L Kuz'mina A A Timofeev S V Germanov L V Levchenko M A Kulish 《Biulleten' eksperimental'no? biologii i meditsiny》1986,101(2):163-165
The effect of antioxidant--alpha-tocopherol--on Ca2+-transporting system in sarcoplasmic reticulum (SR) of the rabbit skeletal muscles was studied in hypercholesterolemia (HC). alpha-tocopherol administration to animals with HC produced a break on the curve of temperature dependence of Ca-ATPase activity at about 20 degrees C, that disappeared in HC, increased the rate of "rapid" SH-group binding by thiol reagents, and normalized the level of unsaturated fatty acids in SR membranes without altering phospholipid content. It is suggested that the damage of Ca-ATPase in HC is mainly due to activation of lipid peroxidation. 相似文献
4.
A A Timofeev O A Azizova G V Chernysheva 《Biulleten' eksperimental'no? biologii i meditsiny》1985,100(11):568-571
Membrane lipid phase has been studied in the sarcoplasmic reticulum (SR) of skeletal muscles, using spin probes. Hypercholesterolemia was found to increase rotation and decrease hydrophobicity of water-soluble probe medium located inside SR vesicles. This is probably indicative of the reduction in SR vesicle membranes. At the same time reduced rotation and increased hydrophobicity and regularity of fat-acid probe micro-environment are observed in hypercholesterolemia, with the differences disappearing nearer to the centre of the membrane. It is suggested that POL activation and cholesterol accumulation in SR membranes in hypercholesterolemia lead to greater density of phospholipid molecules in the membrane. 相似文献
5.
Sarcoplasmic reticulum (SR) fragments from the skeletal muscles of rabbit with marked atherosclerosis possessed decreased Ca2+-accumulating capacity. Lowering of transport efficiency, namely reduction of the Ca/ATP ratio from 1.9--normal value--to 0.9 during the experiment at 26 degrees C was accompanied by activation of Ca-ATPase and simultaneously of the rate of Ca2+ outflux from the SR. Arrhenius plots of Ca-ATPase temperature dependence characterized under normal conditions by a break at 20--21 degrees C was linearized under hypercholesterolemia. At the same time there was a rise (from 0.03 under normal conditions to 0.15 in atherosclerosis) of cholesterol/protein ratio in the SR membrane preparations. Activation energy for Ca-ATPase crude membranes under normal conditions was equal to 15.6 and 28.7 kcal/mol above and below the break point respectively; this value for Ca-ATPase of membranes with increased cholesterol level was 19 kcal/mol for all the temperatures investigated. 相似文献
6.
A A Timofeev O A Azizova G V Chernysheva 《Biulleten' eksperimental'no? biologii i meditsiny》1985,99(3):301-303
Using the method of spin labels it was shown that in hypercholesterolemia (HCh), the following parameters decreased: the velocity of maleimide spin label binding to sarcoplasmic reticulum (SR) Ca-ATPase of rabbit skeletal muscles, the accessibility of spin-labeled thiol groups of the enzyme to potassium ferricyanide and sodium ascorbate, and the mobility of the Ca-ATPase molecule fragment to which the spin label was attached. In addition, intensification of lipid peroxidation was demonstrated in SR membranes. Supplementation of the high-cholesterol diet with alpha-tocopherol resulted in the decreased rates of lipid peroxidation in SR membranes and increased values of the above parameters relative to the values found under HCh. It is concluded that the effect of alpha-tocopherol in vivo on the structure of the Ca-ATPase proteolipid complex in HCh is due mainly to antioxidant properties of the diet-supplementing substance. 相似文献
7.
Two populations of phospholipids exist in sarcoplasmic reticulum and in recombined membranes containing Ca-ATPase 总被引:1,自引:0,他引:1
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. 相似文献
8.
A A Timofeev L V Sto?da O A Azizova A G Maksina G V Chernysheva 《Biulleten' eksperimental'no? biologii i meditsiny》1983,96(11):59-62
The method of electron paramagnetic resonance with spin-labeled maleimide was used to study variation of the structure of Ca-ATPase of the sarcoplasmic reticulum (SR) in rabbit skeletal muscles under long-term hypercholesterolemia (HC). The rate of the maleimide spin label binding with Ca-ATPase of the SR was decreased in HC, which correlated with a lesser access of spin-labeled thiol groups for potassium ferricyanide and sodium ascorbate. HC led to a considerable reduction in the lability and to enhancement of hydrophobia of the spin-labeled fragment of the enzyme. It is concluded that the disordered function of the SR Ca-pump is a consequence of structural changes in the Ca-ATPase molecule in HC. 相似文献
9.
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. 相似文献
10.
A A Timofeev I L Kuz'mina O A Azizova G V Chernysheva 《Biulleten' eksperimental'no? biologii i meditsiny》1985,99(1):55-57
During prolonged hypercholesterolemia (HCh), there was a reduction in the efficacy of Ca-pump function of the sarcoplasmic reticulum (SR) of rabbit skeletal muscles, an increase in cholesterol content, and intensification of lipid peroxidation in SR membranes. Supplementation of the cholesterol-rich diet with alpha-tocopherol effectively prevented dysfunction of the SR Ca-pump and reduced the enhanced LPO level in SR membranes without having any effect on cholesterol content in blood serum and SR membranes. The increase in the LPO level seems likely to be primarily responsible for abnormalities in the SR Ca-pump during HCh. 相似文献
11.
Cooperative properties of Ca-ATPase of the sarcoplasmic reticulum (SR) of rabbit skeletal muscles were examined in health and hypercholesterolemia. As the concentration of ATP was raised (from 50-100 microM to 5 mM) the Hill ratio (Nh) for ATP increased from 0.4 to 3.2. It is assumed that increased cooperative interaction between Ca-ATPase polymers led to a rise in the efficacy of Ca-pump work. Under the conditions described the Nh for UTP increased from 0.43 to 1.0. During hypercholesterolemia (1 g/kg cholesterol for 1, 3 and 6 months), the maximal values of the Nh for ATP did not exceed 2.0, whereas those for UTP 1.0. 相似文献
12.
The role of cholesterol in the activity of reconstituted Ca-ATPase vesicles containing unsaturated phosphatidylethanolamine 总被引:4,自引:0,他引:4
The effect of cholesterol on the activity of sarcoplasmic reticulum Ca-ATPase, reconstituted in proteoliposomes containing soybean phosphatidylethanolamine (PE), egg phosphatidylcholine (PC), and cholesterol, was examined. The protein incorporation efficiency increased with PE content but appeared to be independent of cholesterol content. At low cholesterol, PE stimulated calcium uptake. The coupling efficiency of the proteoliposomes increased with an increase in cholesterol content at each PC/(PC + PE) ratio and was more pronounced for those proteoliposomes containing high PE. Dynamic fluorescence measurements of the incorporated lipophilic probe, diphenyl-1,3,5-hexatriene, revealed a decrease in the motion and an increase in the order of the phospholipid fatty acyl chains in proteoliposomes with high cholesterol content. A complementary observation was made using electron spin resonance of the spin label, 2,2-dimethyl-5-dodecyl-5-methyloxazolidine N-oxide. Freeze-fracture electron microscopy studies on proteoliposomes containing 0.20 molar ratio of PC/(PC + PE) and cholesterol revealed predominantly vesicular structures with occasional bilayer defects at high cholesterol content. It is postulated that the cholesterol-induced enhancement of the Ca-transport function of the Ca-ATPase is related to the hydration-related bilayer-destabilizing characteristic of the cholesterol molecule as revealed by 31P NMR. 相似文献
13.
The effects of temperature on reconstituted sarcoplasmic Ca-ATPase preparations from vitamin E-deficient dystrophic and control rabbits were studied. Delipidated Ca-ATPase from vitamin E-deficient sarcoplasmic reticulum (SR) reconstituted with lipid of control SR exhibited properties similar to preparations reconstituted with lipid of vitamin E-deficient SR, namely low Ca-ATPase activity and a linear Arrhenius plot of enzyme activity. On the other hand, delipidated control SR Ca-ATPase reconstituted with lipid of vitamin E-deficient SR showed a reduction in activity but retained the discontinuity in the Arrhenius plot. These results indicated that the altered property of sarcoplasmic Ca-ATPase from vitamin E-deficient dystrophic rabbit was associated with the protein and not the lipid component. 相似文献
14.
Phospholamban (PLB) is a major target of the beta-adrenergic cascade in the heart, and functions as an endogenous inhibitor of Ca-ATPase transport activity. To identify whether oligomeric interactions between PLB molecules are involved in regulating Ca-ATPase transport activity, we have investigated functional interactions between PLB and the Ca-ATPase in proteoliposomes of purified PLB functionally co-reconstituted with the SERCA2a isoform of the Ca-ATPase isolated from cardiac sarcoplasmic reticulum (SR). The calcium sensitivity of this reconstituted preparation and functional stimulation by cAMP-dependent protein kinase (PKA) are virtually identical to those of the Ca-ATPase in cardiac SR microsomes, ensuring the functional relevance of this reconstituted preparation. Interactions between PLB molecules were measured following covalent modification of the single lysine (i.e., Lys(3)) in PLB isolated from cardiac SR membranes with fluorescein isothiocyanate (FITC) prior to co-reconstitution with the Ca-ATPase. FITC modification of PLB does not interfere with the ability of PLB to inhibit the Ca-ATPase, since FITC-PLB co-reconstituted with the Ca-ATPase exhibits a similar calcium dependence of Ca-ATPase activation to that observed in native SR membranes. Thus, the functional arrangement of PLB with the Ca-ATPase is not modified by FITC modification. Using changes in the anisotropy of FITC-PLB resulting from fluorescence resonance energy transfer (FRET) between proximal PLB molecules to measure the average size and spatial arrangement of FITC chromophores, we find that PLB self-associates to form oligomers whose spatial arrangement with respect to one another is in agreement with earlier suggestions that PLB exists predominantly as a homopentamer. The inability of PKA to activate PLB following covalent modification with FITC permits functional interactions between PLB molecules associated with the Ca-ATPase activation to be identified. A second-order loss of Ca-ATPase activation by PKA is observed as a function of the fractional contribution of FITC-PLB, indicating that PKA-dependent activation of two PLB molecules within a quaternary complex containing the Ca-ATPase is necessary for activation of the Ca-ATPase. We suggest that the requirement for activation of two PLB molecules by PKA represents a physiological mechanism to ensure that activation of the Ca-ATPase following beta-adrenergic stimulation in the heart only occurs above a threshold level of PKA activation. 相似文献
15.
16.
A M Gunteski-Hamblin J Greeb G E Shull 《The Journal of biological chemistry》1988,263(29):15032-15040
17.
The physical mechanism of calcium pump regulation in the heart. 总被引:4,自引:3,他引:1
The Ca-ATPase in the cardiac sarcoplasmic reticulum membrane is regulated by an amphipathic transmembrane protein, phospholamban. We have used time-resolved phosphorescence anisotropy to detect the microsecond rotational dynamics, and thereby the self-association, of the Ca-ATPase as a function of phospholamban phosphorylation and physiologically relevant calcium levels. The phosphorylation of phospholamban increases the rotational mobility of the Ca-ATPase in the sarcoplasmic reticulum bilayer, due to a decrease in large-scale protein association, with a [Ca2+] dependence parallel to that of enzyme activation. These results support a model in which phospholamban phosphorylation or calcium free the enzyme from a kinetically unfavorable associated state. 相似文献
18.
V D Prokopjeva I V Barannik V Z Roshepkin N P Larionov 《Biochemistry international》1984,8(6):843-850
Phenothiazines--trifluoperazine, chloropromazine and ethmozine-- inhibit the sarcoplasmic reticulum Ca-ATPase from skeletal and cardiac muscles of the rabbit. The inhibition constants for both preparations are of the same order of magnitude. The experimental data suggest that the effect of phenothiazine on the sarcoplasmic reticulum Ca-ATPase is not mediated by CaM, but is directed toward the enzyme molecule. 相似文献
19.
Skeletal muscle contraction and relaxation is modulated through the reaction of sarcoplasmic reticulum (SR) protein thiols with reactive oxygen and nitrogen species. Here, we have utilized high-performance liquid chromatography-electrospray mass spectrometry and a specific thiol-labeling procedure to identify and quantify cysteine residues of the SR Ca-ATPase that are modified by exposure to nitric oxide (NO). NO and/or NO-derived species inactivate the SR Ca-ATPase and modify a broad spectrum of cysteine residues with highest reactivities towards Cys364, Cys670, and Cys471. The selectivity of NO and NO-derived species towards the SR Ca-ATPase thiols is different from that of peroxynitrite. The efficiency of NO at thiol modification is significantly higher compared with that of peroxynitrite. Hence, NO has the potential to modulate muscle contraction through chemical reaction with the SR Ca-ATPase in vivo. 相似文献
20.
A comparative study of the effect of an experimental hypercholesterolemia and in vitro induced lipid peroxidation (LPO) on the temperature dependence of the activity of sarcoplasmic reticular Ca-ATPase from rabbit skeletal muscle (SR) has been performed. A control Arrhenius plot of ATPase activity determined in the presence of alamethicin was characterized by discontinuity in the 20 degrees C area. Both in vitro induced LPO and hypercholesterolemia resulted in a shift of discontinuity to 30 degrees C area. The replacement of lipid Ca-ATPase membrane environment by egg yolk lecithin did not affect the temperature dependence of the activity in control SR and failed to restore the original nature of the Arrhenius plot for Ca-ATPase modified by hypercholesterolemia or the in vitro induced LPO. 相似文献