Purification and characterization of polyamine oxidase from Ascaris suum

Abstract

The interconversion of polyamines in the parasite nematode Ascaris suum by a novel type of polyamine oxidase was demonstrated. The nematode enzyme was clearly distinguishable from monoamine and diamine oxidases as well as from the mammalian polyamine oxidase, as shown by the use of the specific inhibitors pargyline, aminoguanidine and MDL 72527 respectively. All three inhibitors had no effect on the parasite polyamine oxidase, and the enzyme did not accept diamines such as putrescine, cadaverine or histamine as substrates. The parasite polyamine oxidase selectively oxidizes spermine and spermidine but not N-acetylated polyamines, whereas the mammalian tissue-type polyamine oxidase shows preference for the N-acetylated polyamines. These results suggest a regulatory function of the nematode polyamine oxidase in the degradation and interconversion of polyamines in parasite nematodes. The enzyme was purified to homogeneity by gel filtration, preparative isoelectric focusing and subsequent affinity chromatography on spermine-and berenil-Sepharose 4B. With respect to reaction type, the prosthetic group FAD, the molecular mass (66 kDa) and the contents of thiol and carbonyl groups, the polyamine oxidase from A. suum is similar to the isofunctional enzyme of mammalian tissue.

Polyamine oxidases — enzymes of unknown function?

“…Thus, under these circumstances, the induction of polyamine oxidases is an adaptation to the environment [16] and this may also be the case in some helminths [23] and other parasites [24,25]. …”

Effect of haloallylamines on polyamine oxidase activity and spermine levels in Ascaris suum

“…Although free-living nematodes possess ODC (Macrae et al 1995;von Besser et al 1995), parasitic nematodes appear to lack ODC activity and, thus, are dependent on polyamine uptake and polyamine interconversion for their supply of polyamines (Wittich et al 1987;Walter 1988;Sharma et al 1991). In Ascaris suum, a convenient model for filarial parasites, polyamine oxidase is the rate-limiting enzyme of polyamine interconversion (Müller and Walter 1992). Using MDL 72527 [N,N′-2, 3(bisbutadienyl)-1, 4 butanediamine], a specific inhibitor of mammalian tissue polyamine oxidase, Seiler (1987) has shown that polyamine oxidase has no regulatory function in mammalian cells.…”

“…Polyamine oxidase from A. suum was isolated as previously described (Müller and Walter 1992), and for inhibition studies the enzyme was pre-incubated with different concentrations of inhibitors for 10 min before aliquots were assayed for polyamine oxidase activity in a standard assay mixture. The results are summarized in Table 1.…”

“…It has been suggested that inhibition of MAO A and MAO B by MDL 72145 depends on reduction of the flavin cofactor of the enzymes (Bey 1989). Since polyamine oxidase from A. suum is also a flavin-dependent oxidase (Müller and Walter 1992), it is possible that the inhibitor acts on the parasite enzyme in a similar way. Furthermore, the substrate specificities of MAO A, MAO B and semicarbazidesensitive amine oxidases as well as that of the parasite polyamine oxidase are similar (Callingham and Barrand 1987).…”

“…1 Inhibition of polyamine oxidase from Ascaris suum by MDL 72145. Polyamine oxidase was purified according to Müller and Walter (1992). For inhibition studies the enzyme was pre-incubated for 10 min at 37°C without MDL 72145 (black circles) and with the addition of 50 nM (white circles), 100 nM (black squares), 250 nM (white squares), 500 nM (black triangles) and 1,500 nM (white triangles) MDL 72145; 100-µl aliquots were removed at 2, 5, 7 and 10 min and the residual polyamine oxidase activity was determined by the standard assay (see the legend to Table 1).…”