ISSN:
1471-4159
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Medicine
Notes:
Abstract: 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 ∼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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1046/j.1471-4159.1995.64052281.x
Permalink
