Distinction Between Choline and Ethanolamine Phosphorylation in Entodinium caudatum

Contrary to electrofocusing in sucrose gradients free [3H]oestradiol-17/l did not electrofocus and did not migrate significantly from the point of application. This could present difficulties in the interpretation and resolution of components if the sample is placed on the gel at a point close to the isoelectric points of the components of interest. By placing the sample 10-15mm from the cathode, acidic oestrogen receptors migrate towards the anode, whereas unbound [3H]oestradiol-17/l does not move. Fig. 1 shows the effect of focusing time on the resolution of [3H]oestradiol-17/lbinding proteins from intact mature rat uteri. The gel contained pH5-8 and pH3-10 ampholines. Migration of binding components was incomplete at 4h (Fig. la) with a marked improvement in resolution after 5.5h (Fig. lb); at 21 h (Fig. lc), a general deterioration in resolution was evident. [3H]Oestradiol-17/l-binding proteins from immature (21-day old) rat uteri were prepared and examined both by electrofocusing in a pH5-8 gradient and by ultracentrifugation through a 5-20% (w/v) sucrose density gradient (Fig. 2); a 50pl sample (100pg of protein) was electrofocused for 6h at 250V. The sucrose gradient profile (Fig. 26) indicated that a binding protein with a sedimentation coefficient of about 8s was present together with some intermediate sized aggregates. One major cytosol component having an isoelectric point of 5.8 was detected with a second component at pH5.4 (Fig. 2a). This agrees with data obtained with the sucrose-column method of focusing (De Sombre et al., 1969).