Rat Brain Microtubule Protein: Purification and Determination of Covalently Bound Phosphate and Carbohydrate

Microtubule protein purified from rat brain by published procedures contains nucleic acid and carbohydrate contaminants. Microtubule protein isolated from the soluble fraction of rat brain homogenates by a new procedure contains no covalently bound amino sugars and no more than 0.2% neutral sugar, but does contain covalently bound phosphate. Total phosphate analysis of rat brain microtubule protein indicates the presence of 0.8 mol of phosphate per mol of protein dimer (110,000 g). The presence of covalently bound phosphate has been confirmed by preparation of (32)P-labeled microtubule protein from rat brain slices maintained in tissue culture in the presence of (32)Porthophosphate. Furthermore, phosphorylation is specific to the more negatively charged microtubule protein subunit. (32)P-Labeled serine phosphate has been identified after partial acid hydrolysis of labeled microtubule protein.