A homologous series of affinity labeling reagents and their use in the study of antibody binding sites.
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Biomedical subjects
Publications and source records attributed to M Wilchek.
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Biotinyl-p-nitrophenyl ester (BNP), an active-ester derivative of biotin, irreversibly inactivates biotin transport in the yeast Saccharomyces cerevisiae. Transport inactivation is progressive with time and occurs at concentrations of the ester as low as 10(-7) M. In the presence of sodium azide, a reagent known to block biotin accumulation in yeast, the derivative is still effective. The specificity of inactivation by the ester is revealed by the following findings: (a) Biotinyl-p-nitroanilide and acetyl-p-nitrophenyl ester do not affect biotin transport; (b) the nitrophenyl ester does not affect the transport of lysine and aspartic acid, or that of L-sorbose; (c) inactivation of biotin transport by the ester is partially prevented when the cells are incubated with it in the presence of relatively high concentrations of biotin.
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The dissociation constants and standard free energies of complex formation determined with staphylococcal nuclease and a series of 5'-phosphoryloligothymidyl derivatives of increasing chain length suggest that maximum stability is reached with an oligonucleotide containing three nucleotide units. A proposed model of the active site that contains other knowledge of the specificity and the catalytic mechanism of this enzyme postulates the existence of three nonequivalent phosphate binding subsites and a closely related phosphodiester hydrolytic subsite.
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