Subcellular localization of recently‐absorbed iron in mouse duodenal enterocytes: Identification of a basolateral membrane iron‐binding site

Abstract

The subcellular distribution of newly absorbed iron in isolated mouse duodenal enterocytes was investigated by analytical subcellular fractionation using sucrose density gradient centifugation. Two major peaks of mucosal 59Fe activity were observed: one soluble and one particulate (density 1.18-1.20 g ml-1). The latter was increased following prior exposure of animals to chronic hypoxia. The particulate 59Fe was localized to the basolateral membranes using the marker enzyme Na+, K+ activated, Mg2+ dependent, ATPase and by washing intact enterocytes with the selective plasma membrane perturbant digitonin. The basolateral membrane can be selectively labelled by in vitro incubation of intact enterocytes at 0 degrees C with 59Fe(III)-nitrilotriacetate complex, confirming the presence of a 59Fe binding site on this membrane. No significant difference in in vitro iron binding to this site was observed between normal and chronically hypoxic animals. Iron binding to the basolateral membrane was significantly higher in disrupted, compared to intact enterocytes, indicating that this site is present on both sides of the basolateral membrane. It is therefore suggested that the increased labelling of this site in hypoxia, in vivo, is a consequence of an increase in a mucosal Fe pool which is available for binding to a membrane receptor.

Iron proteins of duodenal enterocytes isolated from mice with genetically and experimentally altered iron metabolism

“…its state of aggregation (Pietrangelo et al , 1995)], or to changes in the H‐ to L‐subunit ratio (Gerard et al , 1996) or to the specific enterocyte subpopulation within which the Ft resides (Parmley et al , 1984), or to the fact that initial rates of absorption measured in the present study are too rapid for significant quantities of iron to enter Ft during flux across the mucosa. The latter possibility is consistent with our previous studies of radioiron uptake by mucosal Ft‐during iron absorption in mice (Snape et al , 1990). The measurement of mRNA levels suggested an increase in L‐ferritin relative to H‐ferritin, consistent with the increased ferritin in hpx/hpx mice being of the storage type which takes up iron relatively slowly and therefore may not interfere with the rapid absorption of iron.…”

Kinetic analysis of59Fe movement across the intestinal wall in duodenal rat segments ex vivo

“…This finding suggests that both moieties of the Fe-NTA complex pass the mucosal barrier separately. This is in accordance with findings by Snape et al (38), who found a different subcellular distribution of [ 14 C]NTA and 59 Fe in the mouse duodenum. Therefore, 59 Fe-NTA can be used to load duodenal segments with 59 Fe.…”

“…8,17,30,39,40). A transport protein was also described at the basolateral membrane (38), and some earlier findings argue for the basolateral transfer step as being rate limiting (24,42). This would lead to an accumulation of recently absorbed iron in the intestinal mucosa.…”

Strategies for revealing lower abundance proteins in two-dimensional protein maps

“…This approach improves resolution and enables one to increase the number of protein spots that can be resolved and identified with the probability of identifying low abundant proteins associated with a particular organelle. Such techniques have been successfully applied to identify low abundant membrane protein [24]. However, such fractionation procedures require larger quantities of biological samples, which in all cases is not always possible.…”