THE PHOSPHORYLATION OF BRAIN MICROTUBULAR PROTEINS IN SITU AND IN VITRO

Abstract

The phosphorylation of microtubular proteins isolated by reassembly in vitro from slices of guinea pig cerebral cortex labeled with [32P]orthophosphate was investigated. Under the conditions tested, both the .alpha. and .beta. forms of tubulin contained metabolically-active P which accounted for about 1/3 of the total 32P incorporated into protein; the remaining protein-bound 32P was associated with 3-4 minor high molecular weight components co-purifying with tubulin during 2 cycles of assembly-disassembly. Microtubular protein prepared in this way contained approximately 0.8 mol of alkali-labile P/mol of tubulin dimer (MW 110,000). In vitro studies showed that reassembled microtubular protein preparations catalyzed the incorporation of up to 0.55 mol of P/mol of tubulin dimer during incubation with Mg2+ and [.gamma.32P]ATP. The reaction was linear during the first 30 min of incubation at 37.degree. C. Cyclic[c]AMP (10 .mu.M, final concentration) caused a transient increase in the initial rates of tubulin phosphorylation. Little label was incorporated into the minor high molecular weight components under these conditions. The in vitro phosphorylation of microtubular protein increased in a non-linear manner with respect to protein concentration; this was in contrast to earlier experiments showing linear kinetics when chromatographically isolated tubulin was tested for intrinsic kinase activity. Isolated microtubular protein preparations bound [3H]GTP, [3H]ATP and to a lesser extent, [3H]cAMP and exhibited Ca2+-ATPase activity (up to 60 pmol Pi released min/mg protein at 37.degree. C).