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Antibodies to a Segment of Tyrosine Hydroxylase Phosphorylated at Serine 40
Author(s) -
Goldstein M.,
Lee K. Y.,
Lew J. Y.,
Harada K.,
Wu J.,
Haycock J. W.,
Hokfelt T.,
Deutch A. Y.
Publication year - 1995
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1995.64052281.x
Subject(s) - antibody , tyrosine hydroxylase , peptide , microbiology and biotechnology , phosphorylation , phosphatase , biochemistry , protein tyrosine phosphatase , serine , chemistry , alkaline phosphatase , protein kinase a , blot , biology , enzyme , immunology , gene
A synthetic peptide corresponding to residues 32–47 of rat tyrosine hydroxylase (TH) was phosphorylated by protein kinase A at Ser 40 and used to generate antibodies in rabbits. Reactivity of the anti‐pTH 32–47 antibodies with phospho‐ and dephospho‐Ser 40 forms of TH protein and peptide TH 32–47 was compared with reactivity of antibodies to nonphosphorylated peptide and to native TH protein. In antibody‐capture ELISAs, anti‐pTH 32–47 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 ∼60‐kDa TH band by anti‐pTH 32–47 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‐pTH 32–47 . Similarly, anti‐pTH 32–47 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‐pTH 32–47 . Lastly, in immunohistochemical studies, anti‐pTH 32–47 selectively labeled catecholaminergic cells in tissue sections from perfusion‐fixed rat brain.