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Gene encoding the 37,000-dalton minor sigma factor of Bacillus subtilis RNA polymerase: isolation, nucleotide sequence, chromosomal locus, and cryptic function. 总被引:23,自引:19,他引:4
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We began an analysis of rpoF, the gene encoding the cryptic, 37,000-dalton minor sigma factor (sigma-37) of Bacillus subtilis RNA polymerase. Using antibody raised against sigma-37 holoenzyme to probe a lambda gt11 expression vector library, we isolated a 901-base-pair EcoRI fragment that expressed the COOH-terminal half of sigma-37 fused to lacZ. We used this fragment as a hybridization probe to isolate the entire rpoF gene and additional flanking sequences. Identity of the cloned gene was confirmed by the size and immunological reaction of its product expressed in Escherichia coli and, after DNA sequencing, by the homology of its predicted product (264 residues; 30,143 daltons) with other sigma factors. The DNA sequence also suggested that rpoF may lie in a gene cluster. Upstream of rpoF was an open reading frame that would encode a protein of 17,992 daltons; this frame overlapped the rpoF-coding sequence by 41 base pairs. Immediately following rpoF was a reading frame that would encode a protein of at least 20,000 daltons; expression of this region may be translationally coupled to that of rpoF. By plasmid integration and PBS1 transduction, we found the chromosomal locus of rpoF linked to ddl and dal at 40 degrees on the B. subtilis map and near no known lesions affecting growth regulation or development. Further, an rpoF null mutation resulting from gene disruption had no effect on cell growth or sporulation in rich medium, suggesting that sigma-37 may partly control a regulon not directly involved in the sporulation process. 相似文献
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K B Nazaryan F Climent S Simonian P Tompa J Batke 《Archives of biochemistry and biophysics》1992,296(2):650-653
The interaction of rabbit skeletal muscle enolase and 3-phosphoglycerate mutase was detected by an ELISA test, a batch gel-filtration technique, and fluorescence anisotropy measurements, and the activity of enolase was determined to be a function of mutase concentration. The apparent dissociation constant of this enzyme complex is approximately 1 microM. This value seems to be independent of the presence (in fluorescence anisotropy measurements) or the absence (in activity as well as in ELISA experiments) of fluorescein isothiocyanate used widely as a label for determining the complex formation between enzymes in fluorescence anisotropy measurements. 相似文献
68.
Effect of Potassium on the Release of [3 H]Noradrenaline from Rabbit and Human Pulmonary Artery 总被引:1,自引:0,他引:1
The release of [3H]noradrenaline ( [3H]NA) from rabbit and human isolated pulmonary artery has been measured. Removal of external potassium ions enhanced both the resting and stimulated release of [3H]NA from the strips. On adding K+ to tissues which had been suspended in K+-free Krebs solution, the release of [3H]NA was reduced in both stimulated and unstimulated tissues. Selective inhibition of presynaptic alpha 2-adrenoceptors by yohimbine significantly potentiated the release of [3H]NA evoked by stimulation in K+-free solution. The presynaptic inhibitory effect of NA was much less pronounced when the release was enhanced by the removal of external K+. Since the activity of NA, K-ATPase may be affected by removing K+ or by adding it to tissue previously kept in K+-free solution, the results may indicate involvement of the sodium pump in NA release. 相似文献
69.
A Simoncsits M L Tj?rnhammar M Kalman I Cserpan G Gafvelin T Bartfai 《European journal of biochemistry》1988,178(2):343-350
Synthetic genes coding for elongated precursors of the vasoactive intestinal polypeptide (VIP) were synthesized and cloned in a highly efficient Escherichia coli expression vector. The synthetic genes code for VIP with its methionine (at position 17) replaced by leucine and elongated at the C-terminus by Gly (vasoactive intestinal polypeptide-Gly, i.e. VIPa) or by Gly-Lys-Arg (vasoactive intestinal polypeptide-Gly-Lys-Arg, i.e. VIPb). The synthetic genes fused to the N-terminal part of the E. coli beta-galactosidase gene were expressed to yield high amounts of fusion proteins reaching upon induction at least 60% of the total cellular protein. The fusion proteins of 314 and 316 amino acids carrying in their C-terminal portion either the 29 or 31 amino acids long VIP precursor polypeptide were shown to be immunoreactive with VIP antisera and were further purified and cleaved by CNBr. The resulting purified peptide precursors (VIPa and VIPb) were recognized by VIP receptors in rat liver plasma membranes and by antibodies to porcine VIP in a radioimmunoassay. Both precursors activated adenylate cyclase in rat liver membranes and stimulated pancreatic secretion in the cat. The affinity and potency of the cloned precursors is close to that of VIP purified from porcine intestine, suggesting that the elongated VIP precursors may act even without being converted into the C-terminal amide form of the peptide. The elongated VIP precursors expressed in E. coli may provide a cheap, large-scale source of experimental material for studies on VIP actions. 相似文献
70.
Transient-time analysis of substrate-channelling in interacting enzyme systems. 总被引:1,自引:0,他引:1
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The kinetics of dynamically interacting enzyme systems is examined, in the light of increasing evidence attesting to the widespread occurrence of this mode of organization in vivo. The transient time, a key phenomenological parameter for the coupled reaction, is expressed as a function of the lifetime of the intermediate substrate. The relationships between the transient time and the pseudo-first-order rate constants for the coupled reaction by the complexed and uncomplexed enzyme species are indicative of the mechanism of intermediate transfer ('channelling'). In a dynamically interacting enzyme system these kinetic parameters are composite functions of those for the processes catalysed by the complex and by the separated enzymes. The mathematical paradigm can be extended to a linear sequence of N coupled reactions catalysed by dynamically (pair-wise) interacting enzymes. 相似文献