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91.
92.
D Marsh A Watts W Maschke P F Knowles 《Biochemical and biophysical research communications》1978,81(2):397-402
Cytochrome oxidase-dimyristoyl phosphatidylcholine complexes have been prepared at defined lipid:protein ratios to study the effects of protein packing density on the lipid fluidity. All the complexes reveal a two-component ESR spectrum from an incorporated phosphatidylcholine spin label, corresponding to both an immobilized lipid boundary layer and fluid bilayer regions. Difference spectra, obtained by subtracting the same immobilized spectrum from the spectra of the various complexes, demonstrate a strong perturbation of the lipid bilayer fluidity which is quite distinct from the immobilized boundary layer formation. 相似文献
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Titration of elongation factor G (EF-G) with the thiol reagents 5,5'-dithiobis(2-nitrobenzoate) (DNTB), p-hydroxymercuribenzoate (HMB), and N-ethylmaleimide and analysis of cysteic acid after performic acid oxidation revealed a total of four sulfhydryl groups per EF-G molecule. One of these is exposed in the native state and could be used to distinguish between two different conformations of EF-G in our preparations according to its rate of reaction with DTNB and HMB. No evidence for disulfide bridges was obtained. Among the different nucleotides tested, GTP, GDP, and GMP were able to protect the native sulfhydryl group against reaction with DTNB in the absence of ribosomes. Their Kd values with the faster reacting EF-G were 3.4 x 10(-4) M, 0.3 X 10(-4)M, and 2.0 x 10(-4) M, respectively. Because of the specificity of protection by guanine nucleotides and the correspondence of the Kd values with Ki values for GDP and GMP in the ribosome-EF-G GTPase reaction, their binding site on EF-G should be closely related to the active center for ribosome-dependent GTP hydrolysis. Blockage of the native sulfhydryl group of EF-G with a variety of irreversible thiol reagents reduced its activity from one to two-thirds in ribosome-dependent complex formation, GTP hydrolysis, and poly(U)-directed poly(phenylalanine) synthesis. A test of the N-ethylmaleimide-treated EF-G showed both the Km and Vmax of the GTPase reaction to be affected. Thus, the native sulfhydryl group, although important, appears not to be located in the GTPase active center. Denaturation of EF-G with guanidine-HCl and random blockage of any of the three masked sulfhydryl groups caused inactivation, likely due to steric interference with proper chain folding upon renaturation. Treatment of ribosomes or ribosomal subunits with six different thiol reagents at a concentration of 0.27 mM had little or no effect on the ribosome-EF-G GTPase, except for the case with HMB which inactivated the 30 S subunit. An interaction of EF-G with the 30 S subunit in addition to that known to occur with the 50 S subunit is suggested by a rapid and preferential exchange of HMB from the native sulfhydryl group of EF-G to the 30 S subunit of 70 S ribosomes. 相似文献
97.
The structure of the chromaffin granule membrane has been probed using a number of different spin labels. Both the effect of temperature and high levels of calcium have been studied. 1. The results from three positional isomers of the stearic acid spin label demonstrate that a substantial part of the membrane lipid (that is sensed by the probe) is in a bilayer structure which undergoes a structural transition at 32-36 degrees C, characterized by an increase in the population of gauche isomers in the lipid chains. A possible mechanism for this transition would be the preferential segregation of cholesterol. 2. The covalently bound iodoacetamide spin label reveals a transition within the protein component of the membrane or its immediate lipid environment at 32 degrees C. This transition corresponds to an increased degree of motional freedom of the spin label above the transition temperature. 3. The lipid-soluble spin label 2,2,6,6-tetramethyl-piperidine-1-oxyl exhibits a break at 34 degrees C in the temperature-dependence of its partitioning into the membrane. This could correspond to the onset of a lateral separation in the membrane lipid, again possible involving a re-distribution of cholesterol. 4. Calcium abolishes, diminishes or shifts the transition observed by the spin label and decreases the amplitude of motion of the stearic acid spin labels, again possibly involving a redistribution of cholesterol and also lysolecithin. The temperatures of the structural transition agree well with the changes in the enzymic activity of the membrane ATPase and NADH oxidase functions and also with the results from fluorescent probes [Bashford et al., Eur. J. Biochem. 67, 105-114(1976)]. It is possible that triggering of the transition either by calcium or some other stimulus may play a role in catecholamine release and membrane fusion. 相似文献
98.
Ovulation is a complex phenomenon, involving a series of biochemical events within the ovary, leading to the rupture of the follicle. This paper summarizes recent studies in our laboratory of some of these biochemical changes using the rabbit as an experimental model. It has been shown in our laboratory that isolated Graafian follicles obtained from oestrous rabbits synthesize steroids and cyclic AMP when incubated in vitro. Luteinizing hormone added to the incubation medium increased steroidogenesis and cyclic AMP synthesis many fold. When follicles were isolated from rabbits at different times following the ovulatory stimulus (mating or HCG injection) it was found that the in vitro response to LH in terms of steroidogenesis and cylcic AMP synthesis was lost as ovulation approached. In contrast, when prostaglandins (PGF and PGE) were measured in rabbit Graafian follicles it was found that the PGF and PGE levels increased as ovulation approached. From these data and from reports in the literature, we have developed a hypothetical model for ovulation in the rabbit which may help in a better understanding of the ovulatory process. 相似文献
99.
Resting fibrinolytic activity and the response of the fibrinolytic system to venous occlusion were studied in a group of healthy middle-aged men. All subjects showed increased fibrinolytic activity but of varying degrees. There was a linear relationship between resting and occluded levels of fibrinolytic activity but many subjects with lower levels of fibrinolytic activity showed an anomalous response. Responses over the expected level were more common than unexpectedly low levels of response. Fibrinogen and plasminogen concentrations were inversely correlated with fibrinolytic activity. 相似文献
100.
Joseph A. Marsh 《Journal of molecular biology》2009,391(2):359-318
Obtaining detailed structural models of disordered states of proteins under nondenaturing conditions is important for a better understanding of both functional intrinsically disordered proteins and unfolded states of folded proteins. Extensive experimental characterization of the drk N-terminal SH3 domain unfolded state has shown that, although it appears to be highly disordered, it possesses significant nonrandom secondary and tertiary structure. In our previous attempts to generate structural models of the unfolded state using the program ENSEMBLE, we were limited by insufficient experimental restraints and conformational sampling. In this study, we have vastly expanded our experimental restraint set to include 1H-15N residual dipolar couplings, small-angle X-ray scattering measurements, nitroxide paramagnetic relaxation enhancements, O2-induced 13C paramagnetic shifts, hydrogen-exchange protection factors, and 15N R2 data, in addition to the previously used nuclear Overhauser effects, amino terminal Cu2+-Ni2+ binding paramagnetic relaxation enhancements, J-couplings, chemical shifts, hydrodynamic radius, and solvent accessibility restraints. We have also implemented a new ensemble calculation methodology that uses iterative conformational sampling and seeks to calculate the simplest possible ensemble models. As a result, we can now generate ensembles that are consistent with much larger experimental data sets than was previously possible. Although highly heterogeneous and having broad molecular size distributions, the calculated drk N-terminal SH3 domain unfolded-state ensembles have very different properties than expected for random or statistical coils and possess significant nonnative α-helical structure and both native-like and nonnative tertiary structure. 相似文献