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981.
The kinetic analysis of l-amino acid uptake by the green alga Chlorella revealed at least seven different uptake systems to be present in cells grown autotrophically with nitrate as nitrogen source. There is a ‘general system’ which transports most neutral and acidic amino acids, a system for short-chain neutral amino acids including proline, a system for basic amino acids including histidine, a special system for acidic amino acids, and specific systems for methionine, glutamine and threonine. The ‘general system’ is possibly the same as that which can be stimulated by incubation of cells in glucose plus ammonium (Sauer, N. (1984) Planta 161, 425–431). The incubation of Chlorella in glucose induces the increased synthesis of six amino acid uptake systems, namely the above-mentioned system for short-chain neutral amino acids, a threonine system, a methionine system, and a glutamine system. These results indicate that the uptake of l-amino acids by the green alga Chlorella is as complex as in other free-living organisms such as bacteria or yeast. The small number of amino acid uptake systems found in cells of higher plants, i.e. two or three, seems therefore to be a consequence of integration of the cells in a tissue supplying a relatively constant environment, and not a consequence of autotrophic growth on mineral carbon and mineral nitrogen. 相似文献
982.
The binding of lipophilic ions to the membrane of envelope vesicles from Halobacterium halobium was examined in the absence and presence of membrane potential. The lipophilic ions used constitute a homologous series of (Phe)3-P+-(CH2)n-CH3 (n = 0–4) and tetraphenylphosphonium (TPP+). In the absence of membrane potential, the amounts of binding were proportional to the probe concentration in the medium when the concentration is dilute. Upon illumination, interior negative membrane potential is generated which induces the uptake of phosphonium cation probe. 2 μM were employed as the initial probe concentration. The real membrane potential was evaluated by means of extrapolation to the state of no binding: The values of
for various probes are plotted against the binding coefficient. Here, Ciapp is the apparent intra-vesicular concentration of the probes which is calculated without consideration of bound probes. The ordinate intercept of the plot gives the true concentration ratio, and from this the membrane potential is evaluated. The membrane potential-dependent binding was analysed with a model: the membrane is split into two halves, outer and inner half, and the amounts of bound probes in each region are governed by the concentration in the contiguous solution. We obtained a formula which describes amounts of binding as a function of the membrane potential. 相似文献
983.
The interaction between poly(l-lysines) of varying size with cardiolipin was investigated via binding assays, X-ray diffraction, freeze-fracture electron microscopy, and 31P- and 13C-NMR. Binding of polylysines to the lipid only occurred when three or more lysine residues were present per molecule. The strength of the binding was highly dependent on the polymerization degree, suggesting a cooperative interaction of the lysines within the polymer. Upon binding, a structural reorganization of the lipids takes place, resulting in a closely packed multilamellar system in which the polylysines are sandwiched in between subsequent bilayers. Acyl chain motion is reduced in these liquid-crystalline peptide-lipid complexes. From competition experiments with Ca2+ it could be concluded that when the affinity of the polylysine for cardiolipin was much larger than that of Ca2+, a lamellar polylysine-lipid complex was formed, irrespective of whether an excess of Ca2+ was added prior to or after the polypeptide. When the affinity of the polylysine for cardiolipin was less or of the same order as that of Ca2+, the lipid was organized in the hexagonal HII phase in the presence of Ca2+. These results are discussed in the light of the peptide specificity of bilayer (de)stabilization in cardiolipin model membranes. 相似文献
984.
var1 Gene on the mitochondrial genome of Torulopsis glabrata 总被引:5,自引:0,他引:5
We have cloned and sequenced a region of the Torulopsis glabrata mitochondrial genome homologous to the Saccharomyces cerevisiae var1 gene (var1Sc). An open reading frame that could encode a protein of 339 amino acids was found with 72.7% amino acid and 85.3% nucleotide sequence homology to the S. cerevisiae var1 gene. The T. glabrata gene (var1Tg) is transcribed yielding two stable RNAs, a more abundant 13.5 S RNA and a less abundant 18 S species. We have also identified a candidate for a T. glabrata var1 protein among mitochondrial translation products labeled in isolated mitochondria. The var1Tg gene is even more A + T-rich (93%) than var1Sc (89.6%) and has conserved the strong codon bias of var1Sc. Major differences between the two sequences were found. Significant among these are that no GC clusters are found in var1Tg and the sequences surrounding each of the sites where known polymorphisms exist in var1Sc have deletions at the corresponding sites in var1Tg. These data are discussed with respect to possible origins of these var1 genes and translocation of GC clusters in S. cerevisiae mitochondrial DNA. 相似文献
985.
Promoter mutations affecting divergent transcription in the Tn10 tetracycline resistance determinant 总被引:6,自引:0,他引:6
The tetracycline resistance determinant in transposon Tn10 consists of two genes, the tetA resistance gene and the tetR repressor gene, that are transcribed from divergent overlapping promoters. We determined the levels of pulse-labeled tet messenger RNA in Escherichia coli strains with the Tn10 tet genes on a multicopy plasmid. Addition of the inducer 5a,6-anhydrotetracycline results in a 270- to 430-fold increase in tetA mRNA and a 35- to 65-fold increase in tetR mRNA. As judged by the relative molar amounts of tetA and tetR mRNA synthesized under maximally inducing conditions, the tetA promoter (tetPA) is 7 to 11 times more active than the two tetR promoters (tetPR1 and tetPR2) combined. We characterized ten mutations in tetPA, including nine single-base-pair substitutions and a 30-base-pair deletion. All of the single-base-pair changes reduce the agreement with the consensus sequence for promoters recognized by E. coli RNA polymerase. Mutations in highly conserved nucleotides result in a 200- to 600-fold reduction in tetPA activity in vivo. Unexpectedly, tetPA mutations reduce by two- to fourfold the combined activity in vivo of tetPR1 and tetPR2, in spite of their locations outside the -35 and -10 regions of tetPR1 and tetPR2. For two tetPA mutations, the negative effect on tetPR activity was also demonstrated in tetR- tetPR-lacZ operon fusion strains, thus eliminating the possibility that it is due to variations in either plasmid copy-number or induction efficiency. The pleiotropic effects of tetPA mutations are discussed in terms of the expectation that the overlapping tet promoters compete for RNA polymerase. 相似文献
986.
Genetic reconstruction and functional analysis of the repeating lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli 总被引:11,自引:0,他引:11
J R Guest H M Lewis L D Graham L C Packman R N Perham 《Journal of molecular biology》1985,185(4):743-754
The dihydrolipoamide acetyltransferase component (E2p) of the pyruvate dehydrogenase complex of Escherichia coli contains three highly homologous sequences of about 100 residues that are tandemly repeated to form the N-terminal half of the polypeptide chain. All three sequences include a lysine residue that is a site for lipoylation and they appear to form independently folded functional domains. These lipoyl domains are in turn linked to a much larger (about 300 residues) subunit-binding domain of the E2p chain that aggregates to form the octahedral inner core of the complex and also contains the acetyltransferase active site. In order to investigate whether individual lipoyl domains play different parts in the enzymic mechanism, selective deletions were made in vitro in the dihydrolipoamide acetyltransferase gene (aceF) so as to excise one or two of the repeating sequences. This was facilitated by the high degree of homology in these sequences, which allowed the creation of hybrid lipoyl domains that closely resemble the originals. Pyruvate dehydrogenase complexes incorporating these genetically reconstructed E2p components were purified and their structures were confirmed. It was found that the overall catalytic activity, the system of active site coupling, and the ability to complement pyruvate dehydrogenase complex mutants, were not significantly affected by the loss of one or even two lipoyl domains per E2p chain. No special role can be attached thus far to individual lipoyl domains. On the other hand, certain genetic deletions affecting the acetyltransferase domain caused inactivation of the complex, highlighting particularly sensitive areas of that part of the E2p chain. 相似文献
987.
988.
Summary A procedure to reconstitute CF0CF1 proteoliposomes by gel filtration through a Sephadex-column pre-equilibrated with valinomycin and potassium is described. Proteoliposomes reconstituted by this procedure catalyze an ATP-induced pH of 2.5 to 3.5 units. pH was measured with either 9-aminoacridine or with the pH indicator pyranine trapped inside the proteoliposomes. CF0CF1 proteoliposomes prepared by conventional techniques catalyzed an ATP-induced formation, but were unable to catalyze an ATP-induced pH even in the presence of valinomycin.The ATP-induced pH was sensitive to uncouplers and energy transfer inhibitors and was increased at low temperatures. It is suggested that ATP-induced pH was observed in these proteoliposomes due to the efficient removal of intravesicular ammonium introduced with the CF0CF1 preparation. The ammonium acted as an internal buffer, and thus prevented an observable pH formation. 相似文献
989.
Summary Lens fibers are coupled by communicating junctions, clusters of cell-to-cell channels composed of a 28-kD intrinsic membrane protein (MIP26). Evidence suggests that these and other cell-to-cell channels may close as a result of protein conformational change induced by activated calmodulin. To test the validity of this hypothesis, we have measured the intrinsic fluorescence emission and far-ultraviolet circular dichroism of the isolated components MIP26, calmodulin, and the MIP26-calmodulin complex, both in the absence and presence of Ca++, an uncoupling agent. MIP26 shows no change in either, fluorescence emission (primarily tryptophan and a measure of aromatic constitutivity) or in its circular dichroism spectrum. Calmodulin exhibits a 32% increase in fluorescence emission intensity with constant emission wavelength, entirely tyrosine, and a 44% increase in -helicity, changes previously described. The MIP26-calmodulin complex, on the other hand, displays fluorescence emission and circular dichroism spectra which are slightly different from the sum of the two single components, but shows marked differences in both spectra upon Ca++ addition. This indicates a change in conformation in one or both of the two components. Spectral changes include a 5-nm blue-shift, a 50% increase in tyrosine fluorescene emission, a 25% decrease in tryptophan fluorescence emission, and a 5% increase in the -helicity of the complex. These changes also occur about an isosbestic point and are fully reversible. These data provide additional evidence that activated calmodulin may modulate gating of cell-to-cell channels by affecting channel protein. 相似文献
990.