Kinetic isotope effects in Ras-catalyzed GTP hydrolysis: evidence for a loose transition state.

A remote labeling method has been developed to determine (18)O kinetic isotope effects (KIEs) in Ras-catalyzed GTP hydrolysis. Substrate mixtures consist of (13)C-depleted GTP and [(18)O,(13)C]GTP that contains (18)O at phosphoryl positions of mechanistic interest and (13)C at all carbon positions of the guanosine moiety. Isotope ratios of the nonvolatile substrates and products are measured by using a chemical reaction interface/isotope ratio mass spectrometer.

In vitro performance of hemodialysis membranes after repeated processing.

Background: Dialyzers reprocessed with chlorine-based solutions have been associated with increases in ultrafiltration coefficient and middle-molecule removal. Increased pore size has been hypothesized as the mechanism for the latter phenomenon. Dialyzers exposed to Amukin-D (Amuchina Int Inc, Gaithersburg, MD), a chlorine-based reprocessing agent, were evaluated for changes in molecular weight (MW) cutoff and ultrafiltration properties.

Size-exclusion chromatography in multidimensional separation schemes for proteome analysis.

Size-exclusion chromatography (SEC) is a separation technique with a relatively low resolving power, compared to those usually utilized in proteomics. Therefore, it is often overlooked in experimental protocols, when the main goal is resolving complex biological mixtures. In this report, we introduce innovative multidimensional schemes for proteomics analysis, in which SEC plays a practical role.

3H-thymidine is a defective tool with which to measure rates of DNA synthesis.

Metabolic incorporation of 3H-thymidine into cellular DNA is a widely used protocol to monitor rates of DNA synthesis and cell proliferation. However, this radiochemical has also been reported to induce cell-cycle arrest and apoptosis in addition to DNA damage. Using stable isotope-labeled thymidine, we demonstrate that 3H-thymidine induces dose-dependent inhibition of the rate of DNA synthesis.