Purification and characterization of the intestinal promoter of iron(3+)-transferrin formation

Abstract

The nonceruloplasmin enzyme located in the intestinal mucosa which promotes the incorporation of iron into transferrin has been resolved into a small, heat-stable component and a heat-labile protein component. The small, heat-stable component was purified from the high-speed supernatant of intestinal mucosal homogenates by ion-exchange chromatography and gel filtration and identified as xanthine. The heat labile protein component was purified from the high-speed supernatant of intestinal mucosal homogenates by heat treatment, gel filtration, and ion-exchange chromatography. The physical, spectral, and kinetic properties of the heat-labile protein component strongly suggest that it is xanthine oxidase. By promotion of the oxidation and incorporation of iron into transferrin, intestinal xanthine oxidase could perform a similar function in iron absorption as ceruloplasmin serves in the mobilization of iron from liver stores.

Evidence for the participation of intestinal xanthine oxidase in the mucosal processing of iron

“…A non-ceruloplasmin enzyme, which promotes the oxidation and incorporation of iron into transferrin, has been isolated and purified from intestinal mucosal homogenates and has been identified as intestinal xanthine oxidase (Topham, 1978;Topham et al, 1981). It has been postulated that intestinal xanthine oxidase, by promoting the oxidation and incorporation of iron into mucosal transferrin, could peform a function in iron absorption similar to the role ceruloplasmin serves in the f From the Department of Chemistry, University of Richmond, Richmond, Virginia 23173.…”

Studies of the ferroxidase activity of native and chemically modified xanthine oxidoreductase

“…This spectrophotometric assay has been described in detail and validated in numerous previous reports (Osaki et al, 1966(Osaki et al, , 1971Johnson et al, 1967;Osaki & Johnson, 1969;Topham & Johnson, 1974). The application of this assay specifically for the analysis of the ferroxidase activity of purified xanthine oxidoreductase and xanthine oxidoreductase in tissue homogenates has been recently reported (Topham, 1978;Topham et al, 1981Topham et al, , 1982. In assays containing NBT, MB, TNBS or PMS as artificial electron acceptors, the final concentration of each of these acceptors was 50 fM.…”

“…This enzyme preparation exhibited a single protein band upon polyacrylamide-gel electrophoresis. The specific ferroxidase activity of milk xanthine oxidoreductase has been shown to be equivalent to that ofhighly purified intestinal xanthine oxidoreductase (Topham et al, 1981).…”

“…A non-caeruloplasmin enzyme that promotes the oxidative incorporation of iron into transferrin has been isolated from intestinal-mucosal homogenates (Topham, 1978). This intestinal ferroxidase has been purified and identified as xanthine oxidoreductase (Topham et al, 1981). Recent studies in vivo suggest that intestinal xanthine oxidoreductase may facilitate the transcellular transport of ionic iron in the mucosal cell by promoting its oxidative incorporation into mucosal transferrin (Topham et al, 1982).…”

Effects of dietary iron deficiency and tungsten supplementation on59Fe absorption and gastricretention from59Fe compounds in rats

“…The mucosal cells were gently scraped off using a microscope slide and homogenized in an Ika-Werk turrax homogenizer in approximately 2 ml N-(2-hydroxyethyl) piperazine-N'-2-ethanesulphonic acid (HEPES) buffer at pH 7.5 (0.05 M) for 45 s. The mucosal samples were then centrifuged at 11 5 000 g for 1 h at 4". The intestinal ferroxidase being almost exclusively contained in the supernatant fraction, the ferroxidase activity was determined at room temperature using a Unicam SPI 800 ultraviolet spectrophotometer and Unicam AR25 linear recorder according to the method of Topham et al (1981). All solutions were prepared using deionized, distilled water and all glassware was acid-treated to prevent trace Fe contamination.…”