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851.
ATPase-defective derivatives of Escherichia coli DnaK that behave differently with respect to ATP-induced conformational change and peptide release. 下载免费PDF全文
We have characterized the effects of the T199S, T199A, and K70A mutations on the biochemical activity and in vivo functioning of Escherichia coli DnaK. Threonine-199 is the site of autophosphorylation of DnaK, and the lysine residue of bovine Hsc70 corresponding to K70 of DnaK has been shown to be essential for the hydrolysis of ATP. The dnaK alleles T199A and K70A are completely unable, and the T199S allele is only partially able, to complement the defects of a DeltadnaK mutant. The ATPase activities of the DnaK T199A and DnaK K70A proteins are nearly abolished, while the ATPase activity of the DnaK T199S protein has a steady-state rate similar to that of wild-type DnaK. The DnaK T199S protein also retains approximately 13% of the autophosphorylation activity of wild-type DnaK, while the autophosphorylation activities of the T199A and K70A derivatives are completely abolished. All four DnaK proteins bind a model peptide substrate, and the wild-type, T199A, and T199S DnaK proteins release the peptide with similar kinetics upon the addition of ATP. The DnaK K70A protein, in contrast, does not release the peptide upon the addition of ATP. ATP induces a conformational change in the wild-type, T199A, and T199S DnaK proteins but not in the DnaK K70A protein. The T199A and K70A mutations both disrupt the ATPase activity of DnaK but have profoundly different effects on the ATP-induced conformational change and peptide release activities of DnaK, implying that the two mutations affect different steps in the functional cycle of DnaK. The DnaK T199S protein represents a new class of DnaK mutant, one which has near-normal levels of ATPase activity and undergoes an ATP-induced conformational change that results in the release of peptide but which is not able to fully complement loss of DnaK function in the cell. 相似文献
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With delineation of the photochemical events occurring in the skin after ultraviolet exposure, there has been increased interest in the skin's role in the vitamin D-3-endocrine system. We provide here in vitro conditions for the generation of both labelled (from [3H]acetate) and unlabelled vitamin D-3 in cultures of human keratinocytes and fibroblasts. Sterol precursors and photoproducts in irradiated and non-irradiated cultures are identified by co-chromatography, ultraviolet absorbance spectra, thermal conversion characteristics of previtamin D-3 and mass spectrometry. Because the conversion of 7-dehydrocholesterol to cholesterol is more efficient in vitro than in vivo, the specific delta 7 inhibitor, AY-9944, was added in non-toxic doses to modulate 7-dehydrocholesterol content. Both cell types were equally capable of generating photoproducts, depending on the amount of 7-dehydrocholesterol present. The 290 +/- 5 and 295 nm filters were much more efficient than the 305 nm filter for generating previtamin D-3 and vitamin D-3 in fibroblasts. In contrast, the 305 nm filter was as efficient as the 290 +/- 5 and 295 nm filters in keratinocytes, where it yielded previtamin D-3, with much less lumisterol and tachysterol than appeared with the shorter-wavelength filters. The amount of lumisterol and tachysterol versus previtamin D-3 formed in both cell types was dependent on the total energy applied, with lower energies (less then 1 J/cm2) favoring previtamin D-3 over the other photoproducts. The use of cultured cells provides a system whereby the regulation of vitamin D-3 synthesis by extracutaneous factors can be studied in a homogeneous setting. 相似文献
855.
V K Shumny 《Génome》1989,31(2):900-904
Two periods of the development of genetic research in the USSR with reference to its current trends of plant and animal genetics, cytogenetics, and molecular genetics are reviewed. A short list of priority areas is established: the maintenance and use of unique gene pools of plants and animals; the domestication of animals and cultivation of new plants; the development of programmes for mathematical treatment of genetic data banks. It is suggested to consider them within the framework of international projects. The idea is to promote the collaborative efforts of scientists on an international scale. 相似文献
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We are investigating the mechanisms for producing or avoiding errors during DNA synthesis catalyzed by DNA replication and repair proteins purified from eukaryotic sources. Using assays that monitor the fidelity of a single round of DNA synthesis in vitro, we have defined the error frequency and mutational specificity of the four classes of animal cell DNA polymerases (alpha, beta, delta, gamma), and the fidelity of an SV40 origin-dependent DNA replication complex in extracts of HeLa cells. 相似文献
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