Purification of L-dopa decarboxylase from rat liver and production of polyclonal and monoclonal antibodies against it.
L-DOPA decarboxylase [DDC, aromatic-L-amino acid carboxyl-lyase, EC 4.1.1.28] was purified 800-fold from rat liver by several column chromatographic steps. The enzyme (specific activity, about 6 mumol/min X mg protein) had a molecular weight of 100,000 and gave a single band with a molecular weight of 50,000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was pH 5.7. The absorption spectrum in the visible region of the purified DDC showed maxima at 330 and 420 nm. Polyclonal and monoclonal antibodies against DDC were produced by using this purified protein as an antigen. Polyclonal anti-DDC serum immunoprecipitated the DDC activities of rat, guinea-pig and rabbit livers (about 1, 10, and more than 100 microliter of antiserum, respectively, were required for 50% precipitation of 2 nmol/min of activity of these enzymes). The monoclonal antibody, named MA-1, belonged to the IgG1 subclass and immunoprecipitated the DDC activities of rat and guinea-pig livers to the same extent (about 0.5 micrograms of IgG was required to immunoprecipitate 2 nmol/min activity of each enzyme), but it did not affect the rabbit enzyme. The antibody MA-1 detected DDC molecules of both the purified enzyme and crude homogenate of rat liver blotted onto a nitrocellulose sheet. Immunohistochemically this antibody also stained specific neurons in the substantia nigra, raphe nucleus and locus coeruleus of rat brain.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000917:Antibody Formation; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000302:isolation & purification*; D002845:Chromatography / Q000379:methods; D004296:Dopa Decarboxylase / Q000276:immunology / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006825:Hybridomas / Q000276:immunology; D007120:Immunochemistry; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains
D000911:Antibodies, Monoclonal; D004296:Dopa Decarboxylase; D001142:Aromatic-L-Amino-Acid Decarboxylases