Intrinsic fluorescence of elongation factor Tu in its complexes with GDP and elongation factor Ts

The intrinsic fluorescence properties of elongation factor Tu (EF-Tu) in its complexes with GDP and elongation factor Ts (EF-Ts) have been investigated. The emission spectra for both complexes are dominated by the tyrosine contribution upon excitation at 280 nm whereas excitation at 300 nm leads to exclusive emission from the single tryptophan residue (Trp-184) of EF-Tu. The fluorescence lifetime of this tryptophan residue in both complexes was investigated by using a multifrequency phase fluorometer which achieves a broad range of modulation frequencies utilizing the harmonic content of a mode-locked laser. These results indicated a heterogeneous emission with major components near 4.8 ns for both complexes. Quenching experiments on both complexes indicated limited accessibility of the tryptophan residue to acrylamide and virtually no accessibility to iodide ion. The quenching patterns exhibited by EF-Tu-GDP and EF-Tu X EF-Ts were, however, different; both quenchers were more efficient at quenching the emission from the EF-Tu x EF-Ts complex. Steady-state and dynamic polarization measurements revealed limited local mobility for the tryptophan in the EF-Tu x GDP complex whereas formation of the EF-Tu x EF-Ts complex led to a dramatic increase in this local mobility.     

Quantal sarcomere-length changes in relaxed single myofibrils.

We carried out experiments on single isolated myofibrils in which thin filaments had been functionally removed, leaving the connecting (titin) filaments as the sole agent taking up the length change. With technical advances that gave sub-nanometer detectability we examined the time course of single sarcomere-length change when the myofibril was ramp-released or ramp-stretched by a motor. The sarcomere-length change was stepwise. Step sizes followed a consistent pattern: the smallest was approximately 2.3 nm, and others were integer multiples of that value. The approximately 2.3-nm step quantum is the smallest consistent biomechanical event ever demonstrated. Although the length change must involve the connecting filament, the size of the quantum is an order of magnitude smaller than anticipated from folding of Ig- or fibronectin-like domains, implying either that folding occurs in sub-domain units or that other mechanisms are involved.     

Phospholipid oxidation catalyzed by cytochrome c in liposomes

Oxidation of liposome phospholipids has been studied in the presence of cytochrome c. Sonicated vesicles of soya bean or egg-yolk lipids, or purified phospholipid preparations, were treated with oxidized cytochrome c at a 10:4 lipid/protein ratio (w/w). Lipid peroxidation was examined by oxygen polarography, gas-liquid chromatography (GLC) and the thiobarbituric acid test. Oxidized, but not reduced, cytochrome effectively catalyzes lipid oxidation under these conditions. Oxygen consumption and disappearance of unsaturated fatty acids follow closely similar patterns, the O2 consumption rate showing a maximum (1.53 mol O2/min per mol heme) shortly before fatty acid loss reaches its peak. GLC and O2 consumption data suggest that monohydroperoxides are the most abundant oxidized species in the system. The thiobarbituric acid reaction, however, appears only to be of qualitative value in peroxidation studies. In order to test the mechanism through which oxidation occurs in our system, the effect of liposome composition and the presence of antioxidants was tested, both on cytochrome c binding to bilayers and on O2 consumption. Oxidized and reduced cytochrome c bind the lipid bilayers with similar affinity, but only the oxidized form is active in autoxidation. Antioxidants do not modify either cytochrome c binding to sonicated liposomes. Lipid composition does influence considerably cytochrome binding, and O2 consumption is correspondingly altered. Studies with various antioxidants and inhibitors suggest that both free radicals and singlet oxygen may be involved in the process under study.     

Effect of acute altitude hypoxia on fatty acid proportion in the plasma of rats of different ages

Using gas chromatography, the authors studied the development of the proportion of 25 saturated and non-saturated fatty acids in the plasma of Wistar rats of their own breed aged 5, 10, 14 and 25 days and adult (both sexes), and in three age groups (5 and 14 day and adult) they determined the effect of acute altitude hypoxia. Fatty acids were determined as methylesters (FAME) (Base 1978), using the method already described by Smídová et al. (1984a), in mixed blood plasma obtained from decapitated control and experimental animals. Altitude hypoxia, which corresponded to 9 000 m, lasted 20 min. The temperature was kept stable at 25 degrees C. Marked changes occurred during ontogenesis in the proportion of total fatty acids (TFA) in the plasma; the proportion of sigma non-saturated FA fell significantly during maturation, chiefly in favour of monoene FA, with a resultant significant drop in their reciprocal index (sigma saturated/sigma monoene). The proportion of all saturated FA amounted in 5-day-old rats to 54% of the total quantity, but in adult rats to only 40%. Acute hypoxia had a demonstrable effect in all three age categories. In 5-day-old rats it led to significant changes in 7 FA and in adult rats in 10. In both 5-day-old and adult animals it produced an increase in the arachidonic acid level. The results are discussed in relation to previous findings on the effect of the same hypoxia on FA proportion in various parts of the CNS of rats of different ages.     

NMR studies of the conformational change in human N-p21ras produced by replacement of bound GDP with the GTP analog GTP gamma S.

1H-Detected 15N-edited NMR in solution was used to study the conformational differences between the GDP- and GTP gamma S-bound forms of human N-p21ras. The amide protons of 15N-labeled glycine and isoleucine were observed. Resonances were assigned to residues of particular interest, glycines-60 and -75 and isoleucines-21 and -36, by incorporating various 13C-labeled amino acids in addition to [15N]glycine and [15N]iosleucine and by replacing Mg2+ by Co2+. When GTP gamma S replaced GDP in the active site of p21ras, only 5 of the 14 glycine amide resonances show major shifts, indicating that the conformational effects are fairly localized. Responsive glycines-10, -12, -13, and -15 are in the active site. Gly-75, located at the far end of a conformationally-active loop and helix, also responds to substitution of GTP gamma S for GDP, while Gly-77 does not, supporting a role for Gly-75 as a swivel point for the conformational change. The amide proton resonances of isoleucines-36 and -21 and a third unidentified isoleucine also undergo major shifts upon replacement of GDP by GTP gamma S. Thus, the effector-binding loop containing Ile-36 is confirmed to be involved in the conformational change, and the alpha-helix containing Ile-21 is also shown to be affected.     

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