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121.
Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB 总被引:6,自引:1,他引:5
Eva Koronakis Colin Hughes Irina Milisav Vassilis Koronakis 《Molecular microbiology》1995,16(1):87-96
The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding casette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete less of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change. Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity. 相似文献
122.
Gerd Maulthaup Hans Mechler Colin L. Masters 《Journal of molecular recognition : JMR》1995,8(4):247-257
The Alzheimer's disease βA4 amyloid precursor protein (APP) has been shown to be involved in a diverse set of biological protein precursor-like proteins (APLP1 and APLP2) belong to a superfamily of proteins that are probably functionally related. In order to characterize the cell adhesion properties of APP the brain specific isoform APP695 was purified and used to assess the binding to herparin, a structural and functional analogue of the glycosaminoglycan heparan sulfate. We show that APP binds in a time dependent and saturable manner to heparin. The salt concentration of 620 mM at which APP elutes from heparin Sepharose is greater than physiological. Tha apparent equilibrium constant for dissociation was determined to be 300 pM for APP binding to heparin Sepharose. A high affinity heparin binding site was identified within a region conversed in rodent and human APP, APLP1 and APLP2. This binding site was located between residues 316-337 of APP695 which is within the carbohydrate domain of APP. We also demonstrate an interaction between this heparin binding site and the zinc(II) binding site which is conserved in all members of the APP superfamily. We show by using an automated surface plasmon resonance biosensor (BIAcore, Pharmacia) that the affinity for heparin is increased two- to four-fold in the presence of micromolar zinc(II). The identification of zinc-enhanced binding of APP to heparin sulfate side chains of proteoglycans offers a molecular link between zinc(II), as a putative environmental toxin for Alzheimer's disease, and aggregation of amyloid βA4 protein. 相似文献
123.
Summary
Ankistrodesmus braunii and Chlorella vulgaris were cultured heterotrophically under various operating conditions. The maximum rate of biomass production was 900 and 900 mg L-1 d-1 by C. vulgaris and 1000 and 700 mg L-1 d-1 by A. braunii in the light and dark, respectively. This indicates that these algae could produce in excess of 1530 dry weight tonnes ha-1 y-1 which is 10–20 times higher than the maximum production levels in the literature. 相似文献
124.
We present a method of synchronizing cells in G1-, S-, and G2M-phases employing sequential centrifugal elutriation and viable flow cytometric cell sorting of Hoechst-33342 stained Chinese hamster ovary cells. G1- and S-phase cells can be separated to greater than 99% homogeneity and G2-M to 70% purity. Most of the 30% contamination in the G2-M fraction was due to S-phase cells, whose reproductive integrity could be eliminated through the use of high specific activity 3H-TdR. There were minimal toxic effects or perturbations to growth following the selection procedures. The most significant limitation of this technique appears to be the rate of cell sorting, which, with current equipment, is approximately 3,000 cells per second. 相似文献
125.
Radical-pair decay kinetics and molecular triplet quantum yields at various magnetic fields are reported for quinone-depleted reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides R26. The radical-pair decay is observed by picosecond absorption spectroscopy to be a single exponential to within the experimental uncertainty at all fields. The decay time increases from 13 ns at zero field to 17 ns at 1 kG, and decreases to 9 ns at 50 kG. The orientation averaged quantum yield of formation of the molecular triplet of the primary electron donor, 3P, drops to 47% of its zero-field value at 1 kG and rises to 126% at 50 kG. Combined analysis of these data gives a singlet radical-pair decay rate constant of 5 · 107s?1, a lower limit for the triplet radical-pair decay rate constant of 1 · 108s?1 and a lower limit for the quantum yield of radical-pair decay by the triplet channel of 38% at zero field. The upper limit of the quantum yield of 3P formation at zero field is measured to be 32%. In order to explain this apparent discrepancy, decay of the radical pair by the triplet channel must lead to some rapid ground state formation as well as some 3P formation. It is proposed that the triplet radical pair decays to a triplet charge-transfer state which is strongly coupled to the ground state by spin-orbit interactions. Several possibilities for this charge-transfer state are discussed. 相似文献
126.
Matthew J. Friedman Ante M. Krstulović Henri Colin Georges Guiochon Kathleen Pajer 《Analytical biochemistry》1984,142(2):480-486
Described in this paper is a rapid, isocratic assay for serum indole-3-acetic acid (IAA). The sample preparation involves only protein precipitation using sulfosalicylic acid, and the sensitivity of amperometric detection is in the picogram range. The chromatographic analysis time is approximately 4 min. The devised method was used for a longitudinal study of IAA levels in serum samples from control subjects and newly abstinent alcoholics. Dietary variations were eliminated by administering a 2.0-g loading dose of L-Trp to all subjects investigated. The results are presented in the form of cumulative frequency polygons. Preliminary data indicate no differences in IAA levels between newly abstinent alcoholics and control subjects. 相似文献
127.
Saroja Narasimhan Stephen Allen R Colin Hughes Harry Schachter 《Glycoconjugate journal》1984,1(1):51-61
The ricin-resistant mutant baby hamster kidney (BHK) cell line RICR21 is unable to make the sialylated bi- or triantennary complexN-glycans found in wild type cells and accumulates instead non-bisected hybrid structures containing three Man residues and one or two sialylated antennae (Hugheset al 1983, Carbohydr Res 120215-34). Specific assays forN-acetylglucosaminyltransferases I, II, III and IV were applied to Triton X-100 extracts of wild type BHK, RICR14 and RICR21 cells. It was shown that RICR21 cell extracts had a decreasedN-acetylglucosaminyltransferase II specific activity (17 to 27% of wild type values). It is suggested that in wild type cellsN-acetylglucosaminyltransferase II action proceeds quickly, leading to complexN-glycan synthesis, while in RICR21 cells potential substrates forN-acetylglucosaminyltransferase II move into the trans-Golgi compartment before the transferase can act, thereby leading to hybrid structures. 相似文献
128.
Synchronized cells of the Harding Passey melanoma grown in culture were given a heat shock treatment of 44° C for 36 min. Thymidine incorporation was measured at frequent intervals after heat shock to determine the time of onset of the next DNA synthetic period. If the heat shock was given at the end of G1, the following S was delayed by 20 hr. Heating at other times in the cell cycle resulted in an even longer interval before the onset of S. the end of G1 was also the most resistant to hyperthermic killing and to the effect of heat on the magnitude of thymidine incorporation in the following S. Heating the cells a second time did not repeat the effect of the first treatment unless the second heat shock treatment was at a considerably higher temperature. Thus thermotolerance to heat shock killing also applies to cell-cycle delay. 相似文献
129.
In a continuation of studies on protein intake and aflatoxin B1 (AFB1) metabolism, weanling rats were fed semipurified diets containing either 20% casein or 5% casein for two weeks to determine the effect of dietary protein level on hepatic microsomal epoxide hydrase activity and AFB1 metabolism in an effort to evaluate the role of protein intake on the formation and degradation of the reactive metabolite of AFB1. Styrene oxide was used as substrate for epoxide hydrase since the hypothetical AFB1 2,3-epoxide (AFB-epox) cannot be synthesized because of its lability. Two groups of animals were fed 20% casein diets; one was fed ad libitum and the second was pair fed to the 5% casein group in order to control the effects of total feed intake. The depression of epoxide hydrase activities caused by the 5% casein diets was approximately equivalent to that previously seen with hepatic microsomal mixed function oxidase (MFO) activities with the identical protocol. Similarly, the metabolism of AFB1 to AFQ1 and AFM1 was depressed by the 5% casein diets, with an increase in the production of chromatographically more polar material. The relationship of the MFO and epoxide hydrase activities to AFB1 metabolism and formation of macromolecular adducts is discussed. 相似文献
130.
Thyrotopin releasing hormone (TRH) produces “wet dog shakes” in rats similar to those observed during morphine withdrawal. The shaking behavior precipitated by morphine abstinence can be exacerbated by TRH administration while the other components of the morphine withdrawal syndrome remain unchanged. Morphine, chlorpromazine, apomorphine, and Δ9-tetrahydrocannabinol effectively block shakes induced by either TRH administration or morphine withdrawal. These results suggest the possibility that endogenous TRH may be associated with the “wet dog shakes” observed as a portion of morphine's abstinence syndrome in rats. However, TRH is unable to alter the stereospecific binding of morphine or , and naloxone fails to potentiate the number of TRH-induced shakes. TRH has no antinociceptive properties, and it cannot alter those of morphine. These data suggest that more than one neuromechanism may be responsible for shaking behavior in rats. 相似文献