Purification and characterization of diamine oxidase from porcine kidney and intestine.
Diamine oxidase, the enzyme catalyzing the oxidative deamination of histamine and other diamines, was purified from porcine kidney and porcine intestine. During all purification steps the enzymes from both tissues showed identical binding and elution characteristics. The native enzymes are homodimeric glycoproteins with an apparent molecular weight of 186 kDa. Under reducing conditions the subunits migrate at 104 kDa on SDS polyacrylamide gels and the deglycosylated subunits migrate at 93 kDa which corresponds to a carbohydrate content of 11%. The native and deglycosylated forms of kidney and intestinal diamine oxidase migrate to the same positions, respectively, on two-dimensional isoelectric focussing/SDS polyacrylamide gels. The sequences of the 21 N-terminal amino acids of both proteins are identical. A polyclonal antibody raised against the kidney enzyme binds equally well to diamine oxidase from both kidney and intestine, inhibits the enzymatic activity, and precipitates all diamine oxidase activity from tissue homogenates. The kidney and intestinal enzymes have identical substrate specificities, efficiently converting aliphatic diamines, histamine, and spermidine. For both enzymes the Km values for histamine, putrescine, and spermidine are 0.02 mM, 0.35 mM, and 3.3 mM, respectively. Spermine, aliphatic monoamines, and aromatic mono- and diamines are poor substrates. In conclusion, the diamine oxidase proteins from porcine kidney and intestine are very likely identical and constitute the only diamine oxidase activity present in these tissues. The structural identity implies identical functions of the proteins in these organs, namely the protection of the organism against high concentrations of diamines.
Schwelberger
H G
HG
Labor für Theoretische Chirurgie, II. Univ.-Klinik für Chirurgie, Universität Innsbruck, Austria. hubert.schwelberger@uibk.ac.at
Bodner
E
E
D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003959:Diamines / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006632:Histamine / Q000378:metabolism; D007163:Immunosorbent Techniques; D007422:Intestines / Q000201:enzymology*; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011489:Protein Denaturation; D017421:Sequence Analysis; D013095:Spermidine / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D003959:Diamines; D006632:Histamine; D006631:Amine Oxidase (Copper-Containing); D013095:Spermidine