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991.
992.
Bacterial calcium transport 总被引:7,自引:0,他引:7
B P Rosen 《Biochimica et biophysica acta》1987,906(1):101-110
993.
Hui Dong Wenzhong Xu Jitesh K. Pillai Charles Packianathan Barry P. Rosen 《Analytical biochemistry》2015
Arsenic is a naturally existing toxin and carcinogen. As(III) S-adenosylmethionine methyltransferases (AS3MT in mammals and ArsM in microbes) methylate As(III) three times in consecutive steps and play a central role in arsenic metabolism from bacteria to humans. Current assays for arsenic methylation are slow, laborious, and expensive. Here we report the development of two in vitro assays for AS3MT activity that are rapid, sensitive, convenient, and relatively inexpensive and can be adapted for high-throughput assays. The first assay measures As(III) binding by the quenching of the protein fluorescence of a single-tryptophan derivative of an AS3MT ortholog. The second assay utilizes time-resolved fluorescence resonance energy transfer to directly measure the conversion of the AS3MT substrate, S-adenosylmethionine, to S-adenosylhomocysteine catalyzed by AS3MT. These two assays are complementary, one measuring substrate binding and the other catalysis, making them useful tools for functional studies and future development of drugs to prevent arsenic-related diseases. 相似文献
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J R Murrell R G Schoner J J Liepnieks H N Rosen A C Moses M D Benson 《The Journal of biological chemistry》1992,267(23):16595-16600
The most common form of hereditary systemic amyloidosis is familial amyloidotic polyneuropathy associated with single amino acid changes in the plasma protein, transthyretin. In addition, there are two variants of transthyretin (Ser6 and Thr109) not associated with familial amyloidotic polyneuropathy but with familial euthyroid hyperthyroxinemia, also an autosomal dominant disorder. In these autosomal dominant diseases, most affected individuals are heterozygous and therefore have hybrid forms of the tetrameric plasma transthyretin. In order to study the structure/function relationships of homozygous variant transthyretins, normal human transthyretin and five variant transthyretins (Gly6----Ser, Leu58----His, Thr60----Ala, Ile84----Ser, and Ala109----Thr) were produced in Escherichia coli using the expression vector, pCZ11, and site-directed mutagenesis. These recombinant transthyretin (r-TTR) proteins showed the correct size (14 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis and self-associated into tetramers as determined by size exclusion chromatography. Recombinant normal, Ser6, and Ala60 r-TTRs had an affinity for thyroxine indistinguishable from normal human TTR purified from plasma, whereas His58 and Ser84 r-TTRs had significantly reduced affinity. On the other hand, Thr109 r-TTR had a much higher affinity, probably due to its position within the thyroxine-binding pocket. Expression of mutant transthyretins in E. coli provides the opportunity to study structure/function relationships and amyloid-forming capabilities induced by single amino acid substitutions in the transthyretin molecule. 相似文献
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1000.
A multichamber equilibrium dialysis apparatus 总被引:7,自引:0,他引:7
A method for inexpensively producing large quantities of equilibrium dialysis cells as well as two types of cell rotators is described. The method of production is simple enough that several hundred chambers can be produced in a single day. The assay procedure is flexible enough that any number of assays from one to several hundred may be completed in a short time. The chambers have proved extremely useful in the isolation and kinetic characterization of proteins which appear to be associated with membrane transport systems. They will also suffice for any of the other many uses for equilibrium dialysis. The large number of chambers available also provides an ideal means for determining the proper conditions for the growth of protein crystals by dialysis against the crystallizing agent. Microgram quantities of protein are easily crystallized by this technique. 相似文献