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41.
To investigate the relationship between thin filament Ca2+ binding and activation of the MgATPase rate of myosin subfragment 1, native cardiac thin filaments were isolated and characterized. Direct measurements of 45Ca binding to the thin filament were consistent with non-cooperative binding to two high affinity sites (Ka 7.3 +/- 0.8 x 10(6) M-1) and either cooperative or non-cooperative binding to one low affinity site (Ka 4 +/- 2 x 10(5) M-1) per troponin at 25 degrees C, 30 mM ionic strength, pH 7.06. Addition of a low concentration of myosin subfragment 1 to the native thin filaments produced a Ca2+-regulated MgATPase activity with Kapp (2.5 +/- 1.3 x 10(5) M-1), matching the low affinity Ca2+ site. The MgATPase rate was cooperatively activated by Ca2+ (Hill coefficient 1.8). To determine whether Ca2+ binding to the low affinity sites was cooperative, native thin filament troponin was exchanged with troponin labeled on troponin C with 2-(4'-iodoacetamidanilo)naphthalene-6-sulfonic acid. From the Ca2+-sensitive fluorescence of this complex, Ca2+ binding was cooperative with a Hill coefficient of 1.7-2.0. Using the troponin-exchanged thin filaments, myosin subfragment 1 MgATPase rate activation was also cooperative and closely proportional to Ca2+ thin filament binding. Reconstitution of the thin filament from its components raised the Ca2+ affinity by a factor of 2 (compared with native thin filaments) and incorporation of fluorescently modified troponin raised the Ca2+ affinity by another factor of 2. Stoichiometrically reconstituted thin filaments produced non-cooperative MgATPase rate activation, contrasting with cooperative activation with native thin filaments, troponin-exchanged thin filaments and thin filaments reconstituted with a stoichiometric excess of troponin. The Ca2+-induced fluorescence transition of stoichiometrically reconstituted thin filaments was non-cooperative. These results suggest that Ca2+ binds cooperatively to the regulatory sites of the cardiac thin filament, even in the absence of myosin, and even though cardiac troponin C has only one Ca2+-specific binding site. A theoretical model for these observations is described and related to the experimental data. Well-known interactions between neighboring troponin-tropomyosin complexes are the proposed source of cooperativity and also influence the overall Ka. The data indicate that Ca2+ is four times more likely to elongate a sequence of troponin-tropomyosin units already binding Ca2+ than to bind to a site interior to a sequence of units without Ca2+. 相似文献
42.
Hereditary spherocytosis of man. Altered binding of cytoskeletal components to the erythrocyte membrane 总被引:2,自引:1,他引:1
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John S. Hill William H. Sawyer Geoffrey J. Howlett James S. Wiley 《The Biochemical journal》1982,201(2):259-266
The endogenous respiration of the rumen ciliate Dasytricha ruminantium maintained under an O2 tension of 2kPa (approximately 0.02 atm) was partially inhibited by KCN (40% inhibition) and NaN3 (58% inhibition). The organisms lack cytochromes, and sensitivity of respiration to KCN, NaN3, chloroquine and quercetin suggest that the operation of flavoprotein-iron-sulphur-mediated electron transport. As in Tritrichomonas foetus, hydrogenosomal respiration can be stimulated by the addition of CoA in the presence of 0.025% Triton X-100; stimulation by ADP was not detected. Stimulation of pyruvate-supported O2 uptake by Pi suggests that acetate is produced via acetyl phosphate. 相似文献
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L. Sawyer C.L. Jones A.M. Damas M.M. Harding R.O. Gould R.P. Ambler 《Journal of molecular biology》1981,153(3):831-835
The dihaem cytochrome c4 from Pseudomonas aeruginosa has been crystallized in space group P6522 with cell dimensions , and one molecule per asymmetric unit. Two heavy-atom derivatives, UO2(NO3)2 and K2Pt(NO2)4, which substitute at one and three sites, respectively, have allowed a low-resolution electron density map to be obtained. This shows clearly the two domains of the molecule. 相似文献
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The rotational diffusion of actin was studied with the technique of time-resolved phosphorescence anisotropy using actin labeled at Cys-374 with erythrosin iodoacetamide. Immediately after the polymerization of actin was initiated, the correlation time increased sharply, passing through a maximum at 5 min and then declined to low values. F-Actin at equilibrium showed no anisotropy decay. The results were interpreted as indicating the initial formation of short mobile filaments which became increasingly immobile as elongation proceeded, leaving a decay which was dominated by shorter filaments. Some of these short filaments could have arisen by fragmentation of longer filaments. Eventually, the shorter filaments themselves became immobilized by entanglement within the gel matrix. The infinite-time anisotropy increased during polymerization, reflecting a smaller range of angular motion of the probe brought about by restricted torsional motion on the submicrosecond time scale. The results were compared with the length distribution of actin filaments revealed by electron microscopy [Kawamura, M., & Maruyama, K. (1970) J. Biochem. (Tokyo) 67, 437-457]. Polymerization in the presence of 1 microM cytochalasin B abolished the maximum in the correlation time profile and tended to prevent the immobilization of filaments by favoring shorter capped filaments which retained considerable rotational freedom. Addition of spectrin dimer to F-actin caused an increase in the time-invariant anisotropy. Subsequent additions of spectrin-binding proteins (erythrocyte bands 2.1 and 4.1) caused further increases in the anisotropy in a concentration-dependent manner, suggesting additional restriction of submicrosecond torsional motions. The results suggest that actin filaments within nonmuscle cells are rotationally immobile particularly if they are cross-linked by actin-binding proteins. 相似文献
50.
The influence of ionic strength or the concentration of K+ ([K+]) of the aqueous phase on the spontaneous transfer of cholesterol between negatively charged bilayer vesicles composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) (1:1, mole:mole) was studied using a pyrene-labelled cholesterol analogue, 1-pyrenemethyl-3 beta-hydroxy-22,23-bisnor-5-cholenate (PMC), as the probe. The decrease in PMC excimer fluorescence was best fitted to a bi-exponential function. Increasing [K+] from 0.1 M to 0.3 M had little effect on the shorter half-time (1.4 +/- 0.2 min) but increased the longer half-time from 16.3 +/- 1.9 min to 26.7 +/- 2.1 min. Fluorescence quenching and titration of an interface-located fluorophore, 1-anilinonaphthalene-8-sulfonic acid (ANS) revealed an increase in interfacial hydrophobicity upon increasing in ionic strength. The physical state of the acyl chains was not affected by ionic strength as indicated by a constant PMC excimer:monomer fluorescence intensity ratio. However, an increase in enthalpy change of the lipid phase transition from 15.7 kJ/mol ([K+] = 0.1 M) to 21.3 kJ/mol ([K+] = 0.3 M), together with a slight increase in the transition temperature, implies that interactions between adjacent molecules in the charged lipid bilayer vesicles became stronger at higher ionic strength. Our results suggest that the van der Waals attraction between PMC and phospholipid molecules could be affected by conformation changes in the charged head group region brought about by changes of ionic strength in the aqueous phase, with consequent effects on the desorption of cholesterol from the bilayer surface. 相似文献