DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions. II. Purification and characterization of the phosphoprotein from bovine caudate nucleus.
DARPP-32 is a neuronal phosphoprotein of Mr = 32,000, originally identified in rat brain (Walaas, S.I., D.W. Aswad, and P. Greengard (1983) Nature 301: 69-72). This protein has now been identified in bovine caudate nucleus cytosol and purified 435-fold to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification procedure involved acid extraction at pH 2, CM-cellulose chromatography, DEAE-cellulose chromatography, hydroxylapatite chromatography, and gel filtration on Ultrogel AcA 44. The purified catalytic subunit of cAMP-dependent protein kinase catalyzed the incorporation of 0.96 mol of phosphate/mol of purified DARPP-32. Phosphorylation occurred exclusively on threonine. The isoelectric point of dephospho-DARPP-32 was 4.7, and that of phospho-DARPP-32 was 4.6. The amino acid composition showed a high content of glutamate/glutamine and proline, and a low content of hydrophobic amino acids. DARPP-32 was found to have a Stokes radius of 34 A and a sedimentation coefficient of 2.05 S, indicating that it exists as an elongated monomer.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002421:Caudate Nucleus / Q000187:drug effects / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology*; D004298:Dopamine / Q000502:physiology*; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification* / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation
D000596:Amino Acids; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D000242:Cyclic AMP; D004298:Dopamine