Characterization of recombinant bovine phenylethanolamine N-methyltransferase expressed in a mouse C127 cell line.
Bovine phenylethanolamine N-methyltransferase (PNMT) cDNA was inserted into a bovine papilloma virus-based expression vector and used to transfect a mouse C127 cell line. The resultant recombinant bovine PNMT was characterized biochemically and immunochemically. Recombinant bovine PNMT activity, like the native bovine enzyme, was enhanced by phosphate ion in a concentration-dependent manner. Their molecular weights were shown to be identical by Western blot analysis. Antibodies raised against native bovine adrenal PNMT equally immunoprecipitated the activity of the recombinant and native enzymes. In addition, double immunodiffusion analysis showed a single precipitin line of confluence with both enzyme preparations, indicating immunological identity of native and recombinant bovine PNMT. These antibodies immunostained the recombinant enzyme protein in transfected cells and in their neurite-like processes. In addition, in situ hybridization with the bovine PNMT cDNA probe resulted in a labelling pattern similar to the immunostaining. The recombinant bovine PNMT as the native bovine enzyme exist in multiple-charge forms, but only one form is predominant. Taken together, our results suggest that recombinant bovine PNMT, expressed from bovine PNMT cDNA in a mouse cell line is enzymatically active and shares many common features with native bovine adrenal PNMT.
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Laboratory of Molecular Neurobiology, Cornell University Medical College, Burke Rehabilitation Center, White Plains, NY 10605.
Wessel
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Baker
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Joh
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Samanta
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D000311:Adrenal Glands / Q000201:enzymology; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D005779:Immunodiffusion; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010625:Phenylethanolamine N-Methyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D012333:RNA, Messenger; D011994:Recombinant Proteins; D010625:Phenylethanolamine N-Methyltransferase