Nuclear magnetic resonance procedures
Follicular oocytes were prepared as described above and continuously superfused (1 ml/min) with aerated Ringer's solution at 20°C in a 10 mm NMR tube as illustrated in Figure 8. 31P NMR experiments were carried out using a Varian VXR 500 spectrometer operating at 202 MHz with a spectral width of 20 KHz. Assignment of 31P-peaks were determined as described by Nuccitelli et al. [45]. Saturation transfer experiments were performed by application of a low power radio frequency (RF) pulse for a time >3T1 to either the γATP or PCr resonance [12,46]. Control spectra were obtained by positioning the saturating pulse off-resonance on the other side of PCr or γATP, respectively, at the same distance from the observed resonance. Both control and appropriately saturated spectra were accumulated in alternate blocks of 100 scans for a total of 1000 scans each. Pseudo first order rate constants were calculated from the extent of the saturation transfer effect measured in the difference spectrum. NMR measures the pseudo first order rate constant kf = k1[ADP], where k1 is the true second order rate constant for the creatine kinase reaction. Quantitation of phosphometabolites was accomplished from 31P NMR of oocytes by comparing the areas of the various fully relaxed resonances with that of a standard phosphate sample, taking into account the fraction of the NMR-sensitive window occupied by intracellular H20 [6].