Determination of diffusion coefficients by spreading from thin layers

Two new methods for the measurement of diffusion coefficients were introduced. In the first method the spreading is measured from a. thin layer in a cylindrical tube containing a suitable gel. To avoid convection in the gel the tube is rotated horizontally round its long axis. In the second method the diffusion spreading is measured from an initial thin layer under a column of solvent in a Tiselius electrophoresis cell or a micro cuvet of a spectrophotometer. The great advantage is the economy on the material investigated.     

The mechanism of adenosine to inosine conversion by the double-stranded RNA unwinding/modifying activity: a high-performance liquid chromatography-mass spectrometry analysis.

We have used directly combined high-performance liquid chromatography-mass spectrometry (LC/MS) to examine the mechanism of the reaction catalyzed by the double-stranded RNA unwinding/modifying activity [Bass & Weintraub (1988) Cell 55, 1089-1098]. A double-stranded RNA substrate in which all adenosines were uniformly labeled with 13C was synthesized. An LC/MS analysis of the nucleoside products from the modified, labeled substrate confirmed that adenosine is modified to inosine during the unwinding/modifying reaction. Most importantly, we found that no carbons are exchanged during the reaction. By including H2(18)O in the reaction, we showed that water serves efficiently as the oxygen donor in vitro. These results are consistent with a hydrolytic deamination mechanism and rule out a base replacement mechanism. Although the double-stranded RNA unwinding/modifying activity appears to utilize a catalytic mechanism similar to that of adenosine deaminase, coformycin, a transition-state analogue, will not inhibit the unwinding/modifying activity.