Abstract
Several techniques were devised to estimate the phosphorylation capacity of spectrin (band 2 according to Fairbank's numbering) using intact erythrocytes. 32P incorporation from inorganic phosphate into erythrocytes was accelerated by eliminating chloride ions from the incubation media. The simultaneous addition of adenine and inosine accelerated the 32P incor-poration into ATP and effectively maintained the level of ATP in the cell as well as the specific radioactivity of ATP labile phosphate. Incubation was carried out using citrate medium or calcium-sucrose medium with 32P, adenine, and inosine. After incubation for 2, 6, or 20 h, the radioactivity of labile P of ATP in the cell was assayed by acid extraction, absorption on plastic-coated charcoal, elution from the charcoal, removal of the solvent, hydrolysis in 1 N HCl and extraction with organic solvent as a phosphate-molybdate complex. Membrane proteins in the hemolysate precipitate were subjected to slab sodium dodecyl sulfate gel elec-trophoresis according to Laemmli and Fairbanks. After staining with Coomassie Blue, the gels were dried on transparent sheets. Radioactivity was counted through a specifically devised transparent slit, and after densitometry a radioautogram was taken on X-ray film. The amount of phosphate incorporated into band 2 was calculated from the cpm/mol value for band 2 (not band 1+2) and the specific radioactivity of ATP labile phosphate.
The amount of ATP in the cell and the specific radioactivity of ATP labile P remained nearly constant for 6 and 20 h, respectively. The maximum 32P incorporation into band 2 of normal intact cells was 5.03±1.94 mol/mol of band 2 (citrate medium) (n=7) and 8.25±1.73 (Ca-sucrose medium) (n=4). These are larger than the values previously reported. The value for hereditary spherocytotic cells was over 22.6+5.75 (control 7.4±2.86) in 3 cases in the presence of calcium in the medium and 6.65±1.42 (control 3.9±1.07) in the citrate medium (4 cases). These results are quite different from the observations of other workers. 32P incorporation into cell membranes, which was determined by hemolysis, was below 1, which is similar to values given in other reports.
32P incorporation into other membrane proteins was also observed by taking radioauto-grams. All radioactive bands other than 2, 21, 23, 25, 29, and hemoglobin were different from the Coomassie Blue-stained bands. The incorporation was greatest at band 2. The bands of 21, 23, 25, and 29 also showed high specific activity, but the amounts of these proteins were not great. The radioactivities of all these bands seemed to be a little higher in spherocytic cells.
