Numerous phosphates of microtubule-associated protein 2 in living rat brain

Microtubule-associated protein 2 (MAP 2) purified from microwave-irradiated rat head contained about 46 esterified phosphates (mole/mol), which were not bound covalently to lipids and did not assemble with microtubules. After some phosphates were released by calf intestinal alkaline phosphatase, the phosphate content of MAP-2 decreased to 16 mol of phosphate and the protein assembled in vitro. MAP-2 purified after microtubule assembly cycles and also the cytosolic heat-stable fraction without assembly cycles had 10 mol of phosphate, and both assembled with microtubules. The MAP-2 with 46 phosphates and that with 10 had different pI in isoelectric focusing, but the components, MAP-2a and -2b, were always near each other. In high-pressure liquid chromatography, MAP-2 containing 46 mol of phosphate appeared after that 10 mol of phosphate. Phosphoserine, phosphothreonine, and phosphotyrosine were recovered from tryptic digestion of MAP-2 with 46 mol of phosphate. These findings suggest that two kinds of MAP-2, one with 46 phosphates and not bound to tubulin and the other with 10-16 phosphates and bound to tubulin, are present in the living rat brain.