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Reaction of 5S RNA with chlorocetaldehyde leads to the conversion of unpaired adenines to the fluorescent 1,N6-etheno-adenine derivatives. Up to 16 of the 23 adenines in free 5S RNA can be modified, the fastest reacting are A29, A34, A57-59. Partial modification of adenines in this area results in a 20% reduction in the efficiency of 5S RNA incorporation into 50S subunits during reconstitution and a 15% reduction in the activity of these subunits in peptide synthesis. Fluorescence from 1,N6-etheno-adenine is quenched in free 5S RNA and is not detectably further influenced by the binding of proteins E-L5, E-L18 and E-L25, nor by the first stage of the two step E. coli 50S subunit reconstitution procedure. However, the fluorescence is further reduced to near zero after the second step of the reconstitution. Thus, 5S RNS free in solution contains 16 unpaired adenines, those in the region between A29 and A59 particularly accessible to modification by chlorocetaldehyde. This portion of the 5S RNA molecule appears to undergo either a conformational change or interacts with other ribosomal components in the last stage of subunit reassembly. 相似文献
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Different stable forms of Escherichia coli and rat liver 5S rRNA have been probed by Pb(II)-induced hydrolysis. In the native A forms of 5S rRNA, Pb2+ reveal single-stranded RNA stretches and regions of increased conformational flexibility or distorted by the presence of bulged nucleotides. Hydrolysis of urea/EDTA-treated E. coli 5S rRNA (B form) shows the presence of two strong helical domains; helix A retained from the A form and a helix composed of RNA regions G33-C42 and G79-C88. Other RNA regions resistant to hydrolysis may be involved in alternative base pairing, causing conformational heterogeneity of that form. Pb(II)-induced hydrolysis distinguishes two different forms of rat liver 5S rRNA; the native A form and the form obtained by renaturation of 5S rRNA in the presence of EDTA. Pb(II)-hydrolysis data suggest that both forms are highly structured. In the latter form, the orientation of the bulged C66 is changed with respect to helix B. At the same time, a new helical segment is possibly formed, composed of nucleotides from helix C and loop c on one side and from helix E and loop d' on the other. 相似文献
56.
Peroxisomal multifunctional beta-oxidation protein of Saccharomyces cerevisiae. Molecular analysis of the fox2 gene and gene product. 总被引:6,自引:0,他引:6
J K Hiltunen B Wenzel A Beyer R Erdmann A Foss? W H Kunau 《The Journal of biological chemistry》1992,267(10):6646-6653
The gene encoding the multifunctional protein (MFP) of peroxisomal beta-oxidation in Saccharomyces cerevisiae was isolated from a genomic library via functional complementation of a fox2 mutant strain. The open reading frame consists of 2700 base pairs encoding a protein of 900 amino acids. The predicted molecular weight (98,759) is in close agreement with that of the isolated polypeptide (96,000). Analysis of the deduced amino acid sequence revealed similarity to the MFPs of two other fungi but not to that of rat peroxisomes or the multifunctional subunit of the Escherichia coli beta-oxidation complex. The FOX2 gene was overexpressed from a multicopy vector (YEp352) in S. cerevisiae and the gene product purified to apparent homogeneity. A truncated version of MFP lacking 271 carboxyl-terminal amino acids was also overexpressed and purified. Experiments to study the enzymatic properties of the wild-type MFP demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA). The truncated form contained only the D-3-hydroxyacyl-CoA dehydrogenase activity. These results clearly demonstrate that the beta-oxidation of fatty acids in S. cerevisiae follows a previously unknown stereochemical course, namely it occurs via a D-3-hydroxyacyl-CoA intermediate. 相似文献
57.
Lead ions have been applied to the structural analysis of 5S rRNA from Thermus thermophilus, Bacillus stearothermophilus and Escherichia coli. Based on the distribution of Pb(II)-induced cleavages, some minor modifications of the consensus secondary structure model of 5S rRNA are proposed. They include the possible base pairing between nucleotides at positions 11 and 109, as well as changes in secondary interactions within the helix B region. The 'prokaryotic arm' region is completely resistant to hydrolysis in the three RNA species, suggesting that it is a relatively stable, highly ordered structure. Hydrolysis of E. coli 5S rRNA complexed with ribosomal protein L18 shows, besides the shielding effect of the bound protein, a highly enhanced cleavage between A108 and A109. It supports the concept that the major L18-induced conformational change involves the junction of helices A, B and D. 相似文献
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Experiments were performed to elucidate the role of cyclic guanosine monophosphate (cGMP) on platelet activation induced by protein kinase C (PKC) activators and calcium ionophore. Human platelets were pretreated with acetylsalicylic acid and with hirudin and apyrase. Aggregation and ATP secretion in response to the PKC activators 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl 2-acetylglycerol (OAG) were inhibited by the nitrovasodilator sodium nitroprusside (SNP), an activator of guanylate cyclase, and by 8-bromo-cyclic GMP (8-Br-cGMP). The experiments were performed in the presence of M&B 22948, an inhibitor of cGMP phosphodiesterase. SNP and 8-Br-cGMP also inhibited platelet aggregation and secretion evoked by the ionophore ionomycin. In fura-2 loaded platelets SNP did not affect basal cytosolic Ca2+ level nor the rise induced by low concentrations of ionomycin, both in the presence and absence of extracellular Ca2+. The phosphorylation of the 47 and 20 kDa protein induced by ionomycin or PMA were not significantly decreased by SNP or 8-Br-cGMP. The present results suggest that cGMP is able to inhibit both the PKC and the Ca(2+)-dependent pathways leading to platelet activation by interfering, similarly to cAMP, with processes following protein phosphorylation, close to the effector systems. 相似文献
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Summary Mouse morulae are known to undergo cavitation as soon as some external cells have entered the sixth cell cycle (Garbutt et al. 1987). Since the early cytological features of cavitation are still unclear, we undertook a careful ultrastructural analysis of late morulae-nascent blastocysts. In addition, since maturation of lysosomes might be involved in the first step of cavity formation, we focused our attention on these organelles by means of the cytochemical localization of trimetaphosphatase activity and by the study of the effects of chloroquine on precavitation embryos. Our results suggest that cavitation starts in a few external cells (presumably competent cells entering the sixth cell cycle), by the chloroquine-sensitive formation of degradative autophagic vacuoles engulfing lipid droplets and vacuoles containing osmiophilic material. These complex structures enlarge (as a result of lipid metabolism?) and so transform into intrablastomeric cavities which, by means of a membrane fusion process, very rapidly become extracellular cavities that coalesce. The abembryonic pole of the blastocyst is determined in this way. Moreover, we suggest that the juxtacoelic cytoplasmic processes covering the inner cell mass (ICM) cells, which are known to restrict the expression of their totipotency during early cavitation (Fleming et al. 1984), are the latest remnants of the walls of the growing intrablastomeric cavities. 相似文献
60.