The phosphorylation of brain microtubular proteins in situ and in vitro.
The phosphorylation of microtubular proteins isolated by reassembly in vitro from slices of guinea-pig cerebral cortex labelled with [32P]orthophosphate was investigated. Under the conditions tested, both and the alpha and beta forms of tubulin contained metabolically-active P which accounted for about one third of the total 32P incorporated into protein; the remaining protein-bound 32P was associated with 3-4 minor high MW components co-purifying with tubulin during two cycles of assembly-disassembly. Microtubular protein prepared in this way contained approx. 0.8 mol of alkalilabile P/mol of tubulin dimer (M.W. 110,000). In vitro studies showed that reassembled microtubular protein preparations catalysed 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 degrees C. Cyclic AMP (10 microM, final concentration) caused a transient increase in the initial rates of tubulin phosphorylation. Little label was incorporated into the minor high M.W. 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]cyclic AMP, and exhibited Ca(2+)-ATPase activity (up to 60 pmol Pi released min/mg protein at 37 degrees C).
Reddington
M
M
Department of Biochemistry, Bedford College, Regent's Park, London NW1 4NS, England.
Tan
L P
LP
Lagnado
J R
JR
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry; D002118:Calcium / Q000494:pharmacology; D002540:Cerebral Cortex / Q000737:chemistry / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000502:physiology; D004795:Enzyme Stability; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008868:Microtubule Proteins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry / Q000187:drug effects; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism
D008868:Microtubule Proteins; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D014404:Tubulin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D000251:Adenosine Triphosphatases; D002118:Calcium