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91.
Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of the mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase.
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The neutral metalloprotease elastase is one of the major proteins secreted into the culture medium by many Pseudomonas aeruginosa strains. Encoded by the lasB gene, the 33-kDa elastase is initially synthesized as a 53-kDa preproenzyme which is processed to the mature form via a 51-kDa proelastase intermediate. To facilitate studies on proteolytic processing of elastase precursors and on secretion, we developed systems for overexpression of lasB in Escherichia coli under the control of the inducible T7 and tac promoters. Although the 51-kDa proelastase form was detectable in E. coli under inducible conditions, most of the elastase produced under these conditions was found in an enzymatically active 33-kDa form. The amino-terminal sequence of the first 15 amino acid residues of this 33-kDa elastase species was identical to that of the mature P. aeruginosa enzyme, suggesting that processing was autocatalytic. To test this possibility, the codon in lasB encoding His-223, a presumed active-site residue, was changed to encode Asp-223 (lasB1) and Tyr-223 (lasB2). The effects of these mutations on enzyme activity and processing were examined. No proteolytic or elastolytic activities were detected in extracts of E. coli cells containing the lasB mutant alleles. Overexpression of the mutated lasB genes in E. coli resulted in the accumulation of the corresponding 51-kDa proelastase species. These were processed in vitro to the respective 33-kDa forms by incubation with exogenous purified elastase, without an increase in proteolytic activity. Molecular modeling studies suggest that the mutations have little or no effect on the conformation of the mutant elastases. In addition, wild-type elastase and the mutant proelastases were localized to the periplasm of E. coli. The present results confirm that His-223 is essential for elastase activity and provide evidence for autoproteolytic processing of proelastase. 相似文献
92.
Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth 总被引:13,自引:0,他引:13
Parmender P. Mehta Agnes Hotz-Wagenblatt Birgit Rose David Shalloway Warner R. Loewenstein 《The Journal of membrane biology》1991,124(3):207-225
Summary Incorporation of the gene for connexin 43, a cell-cell channel protein of gap junction, into the genome of communication-deficient transformed mouse 10T1/2 cells restored junctional communication and inhibited growth. Growth was slowed, saturation density reduced and focus formation suppressed, and these effects were contingent on overexpression of the exogenous gene and the consequent enhancement of communication. In coculture with normal cells the growth of the connexin overexpressors was completely arrested, as these cells established strong communication with the normal ones. Thus, in culture by themselves or in coculture, the connexin overexpressor cells grew like normal cells. These results demonstrate that the cell-cell channel is instrumental in growth control; they are the expected behavior if the channel transmits cytoplasmic growth-regulatory signals. 相似文献
93.
Carmen Michán Birgit Kessler Victor de Lorenzo Kenneth N. Timmis Juan L. Ramos 《Molecular & general genetics : MGG》1992,235(2-3):406-412
Summary The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida. This protein is activated by a variety of benzoate analogues. To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to -galactosidase were constructed but all are inactive. In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions. The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein. The intraallelic dominance of the Ile229 (Ser229 Ile) and Val274 (Asp274 Val) substitutions over the N-terminal His4l (Arg4l His) substitution, and the intraallelic dominance of Thr45 (Arg45 Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally. Combination of the Leu88 (Trp88 Leu) and Arg256 (Pro256 Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates. In contrast, the Leu88 semiconstitutive phenotype was suppressed by Va1288 (Asp288 Val), and the double mutant was susceptible to activation by benzoates. The results suggest that intramolecular interactions between the C- and N-terminal regions of XylS are critical for activation of the regulator by the effector. 相似文献
94.
Birgit Dräger Andreas Portsteffen Angela Schaal Peter H. McCabe Abigael C. J. Peerless Richard J. Robins 《Planta》1992,188(4):581-586
The nortropane sulphur analogues 8-thiabicyclo[3.2.1] octan-3-one, 8-thiabicyclo[3.2.1]octan-3a-ol and 8-thiabicyclo[3.2.1]octan-3-ol have been found to have differential effects in vitro on the activities of tropinone reductase I and tropinone reductase II from Datura stramonium L. It has been demonstrated that only tropinone reductase I is able to metabolise 8-thiabicyclo[3.2.1]octan-3-one and that only this enzyme is inhibited by 8-thiabicyclo[3.2.1]octan-3-ol and 8-thiabicyclo[3.2.1]octan-3-ol. A K
m of 0.035 mM was determined for 8-thiabicyclo[3.2.1]octan-3-one and I50 values of 0.081 mM and 0.021 mM for 8-thiabicyclo[3.2.1]octan-3-ol and 8-thiabicyclo[3.2.1]octan-3-ol, respectively. The influence that these differential interactions might have on metabolism was investigated in transformed root cultures of D. stramonium. It was found that when these cultures were grown in the presence of either 8-thiabicyclo[3.2.1]octan-3-one or 8-thiabicyclo[3.2.1]octan-3-ol the spectrum of alkaloids that accumulated was altered from that found in control roots in the manner predicted from the observed effects of these inhibitors on the isolated reductases. The effect could be mimicked by feeding pseudotropine, the product of tropinone reductase II. It is concluded that the relative levels of activity of the two tropinone reductases might play an important role in regulating the balance of tropan-3-ols to tropan-3-ols seen in the spectrum of tropane-alkaloid-producing plants.Abbreviations GC/MS
gas chromatography/mass spectrometry;
- I50
concentration of inhibitor required to reduce the rate of reaction to half the maximal value;
- -TBOL
8-thiabicyclo[3.2.1]octan-3-ol;
- -TBOL
8-thiabicyclo[3.2.1]octan-3-ol;
- TBON
8-thiabicyclo[3.2.1]octan-3-one;
- TR
tropinone reductase
We are most grateful to J. Eagles (I.F.R., Norwich) for GC/MS analysis, to colleagues at I.P.B.P. and I.F.R. for helpful discussions, to the technical staff (Chemistry, Glasgow) and to W. Millar (Chemistry, Glasgow) for assistance with the reduction of TBON. This work was, in part, supported by a grant to B Dräger from the Deutsche Forschungsgemeinschaft (Dr227/I-I). The research reported here was supported by an Academic Research Collaboration Cooperative Award (project No. 215) from the British Council and the Deutscher Akademischer Austauschdienst to R.J. Robins and B. Dräger. 相似文献
95.
Birgit Sklorz und Tanja Volz 《Journal of Ornithology》1990,131(2):157-160
Zusammenfassung Frisch geschlüpften Hühnerküken wurden schwarz-gelb geringelte und grüne Attrappen sowie Mehlwürmer angeboten, um zu testen, ob eine angeborene Hemmung gegenüber warnfarbiger Beute besteht. Unsere Küken zeigten sowohl eine eindeutige Präferenz für schwarzgelb geringelte Attrappen als auch für Mehlwürmer. Daraus schließen wir, daß es keine genetisch fixierte Hemmung gegenüber schwarz-gelber Ringelung, d. h. auffälliger Beute gibt.
Innate and learned preferences in food selection of chicken (Gallus domesticus)
Summary In reference to the experiment ofSchuler &Hesse (1985), recently hatched chicken were offered a choise between warningly coloured black and yellow and nonwarningly coloured green prey. We can not support their hypothesis of a genetically fixed pecking inhibition towards prey with warning colouration. The chicken directed their first peck at the warningly coloured ones and they ate them at a significantly higher rate. These results support the idea ofGittleman &Harvey (1980), that chicken prefer conspicuous prey.相似文献
96.
Changes in macromolecule syntheses, especially RNA synthesis, and the energy providing system were investigated in seeds ofAgrostemma githago aged for different periods. In embryos of aged seeds all macromolecule syntheses start later and reach a lower level than
young ones. It was found that the synthesis of rRNA in embryos of aged seeds is reduced whereas the synthesis of poly (A+) RNA in relation to the total RNA synthesis is highly increased as well as the amount of this RNA species with long poly
(A) chains. The results are discussed in connection with the decreased protein synthesis and the reduced ATP content and ATP
formation ability in embryos during the long time storage of seeds. 相似文献
97.
R Vieth M J Kessler K P Pritzker 《Canadian journal of physiology and pharmacology》1990,68(10):1368-1371
The specific binding of 25-hydroxyvitamin D3 to its binding protein was studied in serum of the human, rhesus monkey, cow, horse, and rat. The free fraction of 25-hydroxyvitamin D3 in the rat was 0.34 +/- 0.15 pmol free/nmol total (+/- SD) and this was lower than in any of the other species (p less than 0.01). In the human, the free fraction was 1.5 +/- 0.32 pmol free/nmol total, which was higher than in any of the other species (p less than 0.001). The differences in the free fraction were mainly due to differences in dissociation constant. The relative levels of free 25-hydroxyvitamin D should be taken into account when extrapolating findings about vitamin D metabolism in animals to the human. A technical outcome of this study is that of the species tested, vitamin D binding protein from rat serum is the most suitable as a reagent component for methods used to measure total 25-hydroxyvitamin D by competitive protein binding assay. 相似文献
98.
The chicken urokinase-type plasminogen activator gene 总被引:3,自引:0,他引:3
N D Leslie C A Kessler S M Bell J L Degen 《The Journal of biological chemistry》1990,265(3):1339-1344
99.
Photosynthetic c-type cytochromes isolated from various pro- and eukaryotic algae have been compared by an immunochemical method. Thereby the extent of cross-reactivity of several cytochromes with antisera to cytochrome c from Spirulina platensis, Bumilleriopsis filiformis, and Scenedesmus acutus was quantitatively determined by antigen-binding tests. When immunological relationship is taken as a measure of structural relationship, the following conclusions can be drawn: (1) c-type cytochromes from Anabaena variabilis, Nostoc muscorum, Calothrix membranacea, and Spirulina platensis show large differences in cross-reactivity. (2) The acidic Spirulina cytochrome c is fairly closely related to the two eukaryotic cytochromes assayed here.Abbreviations SAUG
Sammlung von Algenkulturen am Pflanzenphysiologischen Institut der Universität Göttingen, FRG
- PCC
Pasteur Culture Collection 相似文献
100.
The coupling of choline transport to acetylcholine synthesis has been investigated by measurement of the isotopic dilution of a pulse of [3H]choline during its incorporation into the recently synthesised acetylcholine of cerebral cortex synaptosomes. Recently synthesised acetylcholine was identified as that containing 14C-labelled precursors introduced by a preincubation before the pulse. When [14C]glucose was used to label acetyl-CoA coupling ratios (calculated as the inverse of the dilution of extracellular [3H]choline during its incorporation into [3H]acetylcholine) of about 0.05-0.2 were found at a choline concentration of 1 microM, rising to 0.5 at choline concentrations of 10-50 microM. Experiments using [14C]choline as a precursor gave similar results, and it was shown that the isotopic dilution did not occur extrasynaptosomally and was not affected by low glucose concentrations. Coupling ratios were always less than unity and rose as the choline concentration increased. It is concluded that choline transported into the nerve terminal has no privileged access to choline acetyltransferase. The results can be explained by a rate-controlling transport of choline into the terminal followed by its rapid acetylation rather than any linkage or coupling of the two processes. 相似文献