PURIFICATION AND CHARACTERIZATION OF DIAMINE OXIDASE FROM PORCINE KIDNEY AND INTESTINE

Diamine oxidase, the enzyme catalysing the oxidative deamination of hi stamine and other diamines, was purified from porcine kidney and porci ne intestine. During all purification steps the enzymes from both tiss ues showed identical binding and elution characteristics. The native e nzymes 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 mig rate to the same positions, respectively, on two-dimensional isoelectr ic focussing/SDS polyacrylamide gels. The sequences of the 21 N-termin al amino acids of both proteins are identical. A polyclonal antibody r aised against the kidney enzyme binds equally well to diamine oxidase from both kidney and intestine, inhibits the enzymatic activity, and p recipitates 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 K-m values for histamine, putrescine, and spermid ine are 0.02 mM, 0.35 mM, and 3.3 mM, respectively. Spermine, aliphati c monoamines, and aromatic mono- and diamines are poor substrates. In conclusion, the diamine oxidase proteins from porcine kidney and intes tine are very likely identical and constitute the only diamine oxidase activity present in these tissues. The structural identity implies id entical functions of the proteins in these organs, namely the protecti on of the organism against high concentrations of diamines.