The Stork and the Syringe: A Political History of Reproductive Medicine

Kinetic Comparison of the Specificity of the Vancomycin Resistance Kinase VanS for Two Response Regulators, VanR and PhoB

“…Detailed investigations on the interactions between twocomponent system proteins have been hindered by a number of factors including (a) difficulties in the preparation of large quantities of the purified proteins, (b) fast phosphotransfer kinetics, and (c) short-lived phosphorylated protein intermediates. As a result, investigations on the phosphorylation kinetics of the response regulator class have focused on reactions with small molecule phosphodonors [e.g., acetyl phosphate (Stock et al, 1995)] or have been limited to kinetic simulations of the overall phosphotransfer process (Lukat et al, 1991;Bray et al, 1993;Morrison & Parkinson, 1994;Hauri & Ross, 1995). (5) Our interests in the development of in ViVo models of the Enterococcus faecium vancomycin resistance two-component system (VanS/VanR) through the heterologous cross-talk interaction between the sensor kinase VanS and the E. coli response regulator PhoB (Fisher et al, 1995) led us to investigate the differences in reactivities between the cognate and the heterologous systems.…”

“…Purification of PhoB to homogeneity followed a strategy similar to that used for VanR. We used a modified version of the radioactive SDS-PAGE and phosphorimage analysis assay reported by Morrison and Parkinson (1994) developed for the CheA-CheY two-component regulatory system. In our studies, we found that exposing the radioactively labeled, wet polyacrylamide gels on phosphorimager plates at -80 °C prevented diffusion of the inorganic phosphate and allowed extended exposure times (>24 h) for signal enhancement.…”

“…The second phase of the phosphotransfer is characterized by a slow decay of both labeled proteins with a corresponding accumulation of P i . Studies on the related chemotaxis two-component system have invoked a simple model (eq 1) for describing the phosphotransfer processes between the kinase CheA and the response regulator CheY (Lukat et al, 1991;Bray et al, 1993;Morrison & Parkinson, 1994;Hauri & Ross, 1995). The model predicts a simple bimolecular reaction between the two proteins with phosphate release arising from the autodephosphorylation of the phosphorylated response regulator.…”