In situ substrate specificity and ultrastructural localization of polyamine oxidase activity in unfixed rat tissues.

Abstract

Data concerning the substrate specificity and the exact intracellular localization of the polyamine-catabolizing enzyme polyamine oxidase are conflicting. Biochemical studies have shown that N1-acetylation of spermine and spermidine dramatically increases the specificity of these compounds for peroxisomal polyamine oxidase to produce spermidine and putrescine, respectively. On the other hand, polyamine oxidase activity was demonstrated histochemically both in peroxisomes and in cytoplasm of several tissues, using spermidine and/or spermine as substrate. To elucidate the in situ substrate specificity of polyamine oxidase and the localization of its activity, enzyme activity was detected in rat liver, kidney, and duodenum at the light and electron microscopic levels. For this purpose, unfixed cryostat sections were applied to avoid changes in enzyme activity owing to chemical fixation. Spermine, spermidine, their N1-acetylated forms, and putrescine were used as substrates, and cerium ions as capturing agent for H2O2. Control reactions were performed in the absence of substrate or in the presence of substrate and specific oxidase inhibitors. At the light microscopic level, final reaction product specifically generated by polyamine oxidase activity was found exclusively in a granular form in hepatocytes, epithelial cells of proximal tubules of the kidney, and epithelial cells of duodenal villi with N1-acetylspermidine or N1-acetylspermine as substrates. Final reaction product was not observed in any of the tissues after incubation in the presence of putrescine, spermidine, or spermine. Formation of specific final reaction product was prevented by incubation in the presence of a specific polyamine oxidase inhibitor, but it was not affected by a diamine oxidase inhibitor. Ultrastructural studies revealed that polyamine oxidase activity is localized exclusively to the matrix of peroxisomes of kidney and liver and to microperoxisomes of the duodenum. The localization patterns obtained with unfixed tissues are in agreement with biochemical data. Strong intraperoxisomal, interperoxisomal, and intercellular heterogeneity in polyamine oxidase activity was found in all tissues investigated.

In situ heterogeneity of peroxisomal oxidase activities: An update

“…but not in the core, and that different peroxisomes within a single cell contained different amounts of reaction product (Van den Munckhof et al, 1995a) (Table 4).…”

“…Since spermine, spermidine, and putrescine are degraded by the cytoplasmic cuproprotein diamine oxidase to form different aldehydes, polyamine oxidase may have been confused with diamine oxidase (Robinson, 1991;Nakos & Gossrau, 1994). Histochemically, this hypothesis is supported by studies showing that peroxisomal polyamine oxidase is not inhibited by a specific inhibitor of diamine oxidase, aminoguanidine, whereas it is completely inhibited by MDL 72527, a specific polyamine oxidase inhibitor Van den Munckhof et al, 1995a). Histochemically, this hypothesis is supported by studies showing that peroxisomal polyamine oxidase is not inhibited by a specific inhibitor of diamine oxidase, aminoguanidine, whereas it is completely inhibited by MDL 72527, a specific polyamine oxidase inhibitor Van den Munckhof et al, 1995a).…”

“…Reduction of H202 by catalase and a concomitant oxidation of DAB leads to an insoluble DAB-polymer, which is coloured and osmiophilic. It became clear that an aldehyde fixation is a prerequisite for the histochemical localization of catalase activity since use of unfixed sections gave rise to hardly any reaction product, whereas fixation resulted in partial inhibition of peroxidase activity (Fahimi, 1974;Herzog & Fahimi, 1974aRoels et al, 1975;Fahimi et al, 1976;Frederiks et al, 1995a). In order to discriminate between catalase and peroxidase, and to increase the validity of the method,, this technique has frequently been optimized since its introduction in 1968 (Goldfischer & Essner, 1969;Novikoff & Goldfischer, 1969;Novikoff et al, 1972;Fahimi, 1973;Herzog & Fahimi, 1973Roels et al, 1975Roels et al, , 1987LeHir et al, 1979;Roels & Goldfischer, 1979;Anger-m~iller & Fahimi, 1981;Frederiks et aI., 1995a).…”

Gender-related differences in polyamine oxidase activity in rat tissues

“…By using a polarographic method, Pavlov et al (1991) showed a relatively high level, but a different subcellular distribution, of PAO activity in liver and kidney of female rats. Further investigations could take into consideration this different subcellular distribution of the enzyme in various tissues (Pavlov et al, 1991;Van den Munckhof et al, 1995), and also the different level of its activity in male and female rats. For this purpose, the contrasting results previously obtained in PAO activity after partial hepatectomy or carbon tetrachloride by HOlttfi (1977) and Hayashi et al (1989) might be due not only to the difference in the enzyme preparations, but also to the different sex of the rats used.…”

Differential Induction of Polyamine Oxidase Activity in Liver and Heart of Iron-Overloaded Rats

“…Histochemical studies have shown that a substantial amount of PA0 activity i n the liver i s localized in peroxisomes ( Van den Munckhof et al, 1995). The macrophages contain high PA0 activity (Morgan et al, 1980), and the polyamine degradation products are thought to play a role i n the regulation of inflammatory responses (Ferrante,1 985).…”