Abstract
We have sought to determine whether aromatic L-amino acid decarboxylase which functions as a neurotransmitter biosynthetic enzyme in neuronal cells can be distinguished from an enzyme with similar activity found in peripheral tissues where no neurotransmitters are synthesized. Aromatic L-amino acid decarboxylase was purified to electrophoretic homogeneity from bovine adrenal medulla, and highly specific antibodies were produced. In addition, a DNA clone complementary to aromatic L-amino acid decarboxylase mRNA was isolated by immunological screening of a lambda gt11 cDNA expression library. We have used these antibodies and cDNA probes for biochemical, immunochemical, and molecular analyses. A single form of aromatic L-amino acid decarboxylase is detected in rat and bovine tissue. Specifically, aromatic L-amino acid decarboxylase protein is biochemically and immunochemically indistinguishable in brain, liver, kidney, and adrenal medulla. Hybridization to aromatic L-amino acid decarboxylase cDNA identifies a single mRNA species of 2.3 kilobase pairs in rat tissue. Furthermore, Southern blot analysis reveals that a single gene codes for aromatic L-amino acid decarboxylase.
Highlights
EXPERIMENTAL PROCEDURESAssay of AADC-AADC assays wereperformedaccording to a modification of the COZ trapping procedure described by Lunanand Mitchell [19]
We have sought to determine whether aromatic Lamino acid decarboxylase which functions as a neurotransmitter biosynthetic enzyme in neuronal cells can be distinguished from an enzyme with similar activity found in peripheral tissues whereno neurotransmitters are synthesized
We report the purification of bovine adrenal medullary aromatic L-amino acid decarboxylase to homogeneity, the production of highly specific antibodies, and the isolation of a cDNA clone complementary to bovine adrenal AADC mRNA
Summary
Assay of AADC-AADC assays wereperformedaccording to a modification of the COZ trapping procedure described by Lunanand Mitchell [19]. The ligated linkers were digested with EcoRI (100 units) in a sonicated denatured salmon sperm DNA [49], and lo7cpm of a cDNA reaction buffer containing 50 mM Tris, pH 7.5, and 100 mM NaCl for probe labeled to 108-109cpmlpg by nick translation [50]. DNA fragments infected Escherichia coli 1090 cells by standard procedures [34] and were separated in1% agarose gels, transferred to nitrocellulose filters digested with EcoRI in 50 mM Tris, pH 7.5, 100 m M NaCl for 2 h at [52], and hybridized to cDNA probes as described for Northern blot.
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