Characterization of bovine aromatic L-amino acid decarboxylase expressed in a mouse cell line: comparison with native enzyme.
Bovine aromatic L-amino acid decarboxylase (AADC) was expressed in a mouse cell line, using a bovine papilloma virus-derived expression vector containing the full coding region of bovine AADC. The recombinant bovine AADC was characterized biochemically and immunochemically and compared with the native bovine AADC. The specific activity of crude recombinant bovine AADC was 30-fold higher than that of crude native AADC. With regard to optimal pH, effects of pyridoxal phosphate concentration and Km for 3,4-dihydroxyphenylalanine as a substrate, both native and recombinant enzymes were essentially identical. Rabbit polyclonal antiserum directed against bovine adrenal AADC recognized on Western blot a single protein band (molecular mass = 55,000 Dalton) in both native and recombinant bovine AADC crude extracts. Furthermore, double immunodiffusion analysis showed a single precipitin line of confluence with both enzyme preparations, indicating immunological identity of native and recombinant bovine AADC. Northern blot analysis identified a single mRNA species (2.2 kb) from native and recombinant bovine AADC preparations. The recombinant bovine AADC has two charge isozymes corresponding to those of the native bovine enzyme, although their relative abundances are different between native and recombinant enzymes. Taken together, our results show that recombinant bovine AADC, expressed from bovine AADC cDNA in a mouse cell line is not only enzymatically active, but also shares many biochemical and immunochemical common features with native bovine AADC.
Park
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Laboratory of Molecular Neurobiology Cornell University Medical College, White Plains, NY.
Kim
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Choi
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MU
Samanta
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Joh
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TH
D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D015152:Blotting, Northern; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D005815:Genetic Code / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D051379:Mice; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D007527:Isoenzymes; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D004247:DNA; D001142:Aromatic-L-Amino-Acid Decarboxylases