Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver.
Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.
Bertocci
B
B
Pharma Research, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Garotta
G
G
Da Prada
M
M
Lahm
H W
HW
Zürcher
G
G
Virgallita
G
G
Miggiano
V
V
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002394:Catechol O-Methyltransferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D013552:Swine; D014357:Trypsin
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D002394:Catechol O-Methyltransferase; D014357:Trypsin