Some problems with the diamine oxidase (DAO) assay using putrescine as substrate in rat liver.
Determination of diamine oxidase (DAO) activity in rat liver preparations by measuring the formation of radioactive delta 1-pyrroline from 14C-putrescine is complicated by the complexity of competing metabolic pathways. This can lead to complete masking of the DAO activity present when rat liver homogenates are used as the enzyme source. However, subcellular fractionation of rat liver homogenates makes it possible to detect some putrescine oxidizing activity in the microsomal fraction when assayed at pH 8.5. When 1 mM putrescine was used as the substrate, over 90% of this activity was inhibited by 6 x 10(-4) M selegiline (deprenyl), indicating that monoamine oxidase (MAO) rather than DAO activity was being measured. The observed activity was also interfered with by agents that reduced acetylation processes and polyamine synthesis. A different picture appears when microM concentrations of putrescine are used: in these conditions all interference is strongly reduced and DAO activity can be measured in rat liver microsomes. Furthermore, kinetic studies on deaminative oxidation of 14C-putrescine at concentrations from 1 microM to 5 mM confirm the existence of two enzymes: one with a high affinity for the substrate and similar to intestinal mucosa DAO in its sensitivity to alpha-aminoguanidine, and the other one with a low affinity and selegiline-sensitive.
Ignesti
G
G
Dipartimento di Farmacologia Preclinica, University of Florence, Italy.
Banchelli
G
G
Pirisino
R
R
Raimondi
L
L
Buffoni
F
F
D006631:Amine Oxidase (Copper-Containing) / Q000032:analysis* / Q000037:antagonists & inhibitors; D000818:Animals; D006146:Guanidines / Q000494:pharmacology; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000187:drug effects / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology; D011700:Putrescine / Q000031:analogs & derivatives / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012642:Selegiline / Q000494:pharmacology; D013055:Spectrophotometry, Infrared; D013347:Subcellular Fractions / Q000187:drug effects; D013379:Substrate Specificity
D006146:Guanidines; D012642:Selegiline; D006631:Amine Oxidase (Copper-Containing); C004479:pimagedine; D011700:Putrescine