Antibodies to a segment of tyrosine hydroxylase phosphorylated at serine 40.

Goldstein M, Lee KY, Lew JY, Harada K, Wu J, Haycock JW, Hokfelt T, Deutch AY
J Neurochem. 1995 64 (5): 2281-7

PMID: 7722513 · DOI:10.1046/j.1471-4159.1995.64052281.x

A synthetic peptide corresponding to residues 32-47 of rat tyrosine hydroxylase (TH) was phosphorylated by protein kinase A at Ser40 and used to generate antibodies in rabbits. Reactivity of the anti-pTH32-47 antibodies with phospho- and dephospho-Ser40 forms of TH protein and peptide TH32-47 was compared with reactivity of antibodies to nonphosphorylated peptide and to native TH protein. In antibody-capture ELISAs, anti-pTH32-47 was more reactive with the phospho-TH than with the dephospho-TH forms. Conversely, antibodies against the nonphosphorylated peptide reacted preferentially with the dephospho-TH forms. In western blots, labeling of the approximately 60-kDa TH band by anti-pTH32-47 was readily detectable in lanes containing protein kinase A-phosphorylated native TH at 10-100 ng/lane. In blots of supernatants prepared from striatal synaptosomes, addition of a phosphatase inhibitor was necessary to discern labeling of the TH band with anti-pTH32-47. Similarly, anti-pTH32-47 failed to immunoprecipitate TH activity from supernatants prepared from untreated tissues, whereas prior treatment with either 8-bromoadenosine 3',5'-cyclic monophosphate or forskolin enabled removal of TH activity by anti-pTH32-47. Lastly, in immunohistochemical studies, anti-pTH32-47 selectively labeled catecholaminergic cells in tissue sections from perfusion-fixed rat brain.

MeSH Terms (20)

8-Bromo Cyclic Adenosine Monophosphate Animals Antibodies Antibody Specificity Colforsin Corpus Striatum Cyclic AMP-Dependent Protein Kinases Enzyme-Linked Immunosorbent Assay Immunoblotting Immunohistochemistry Immunosorbent Techniques Male PC12 Cells Phosphorylation Phosphoserine Rats Rats, Sprague-Dawley Recombinant Proteins Synaptosomes Tyrosine 3-Monooxygenase

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