ing the nontransferrin-bound iron uptake system, it is most The uptake of nontransferrin-bound iron by hepatolikely that steps involved in Fe 3/ reduction and transport of cytes is known to occur and may contribute to the depo-Fe 2/ across the lipid bilayer both require specific iron-binding sition of iron and resulting injury during hemochromaproteins.
tosis. To examine the proteins that may function in the
Several groups have attempted to isolate membrane-assotransport of nontransferrin-bound iron, the properties ciated iron-binding proteins potentially involved with the of FeNTA-binding to rat liver basolateral plasma memmembrane transport of iron. Iron-binding moieties present branes were characterized. The binding of 55 FeNTA to in the small intestine have been identified using two different purified liver basolateral plasma membranes was meachromatographic approaches. Teichmann and Stremmel 27 sured using a simple centrifugation assay. The binding used immobilized metal affinity chromatography (IMAC) and activity could be solubilized with 0.1% octylglucoside; isolated a homotrimeric protein composed of 54 kd subunits. apparent molecular weight M app Ç 210 kd for the binding Interestingly, Western blot analysis of different tissues complex was determined by gel filtration chromatograshowed the presence of some cross-reactivity with liver hophy. Immobilized metal affinity chromatography was mogenate, suggesting the presence of a similar species in the used to further purify binding protein(s) from rat liver hepatocytes. Conrad et al. 28 used radioactive iron as a tracer plasma membranes and at least six polypeptides were to isolate a membrane-associated complex of 90 kd and 150 identified by silver staining. If associated in a stoichio-kDa recognized by antibodies against b 3 -integrin and have metric complex, the molecular mass of these proteins proposed a mechanism for dietary iron absorption involving would predict a size of Ç227 kd in fairly close agreement both integrins and a soluble iron-binding factor called mobilwith the gel filtration experiments. The characferrin. 29 More recently, Li et al. 30 have presented data to terization of FeNTA-binding proteins associated with suggest that the proton pump of reticulocyte endosomes is basolateral membranes is the first step towards underinvolved in the intracellular transport of iron; in particular, standing elements responsible for the uptake of noniron-binding activity was found to be associated with a 17.5 transferrin-bound iron by the liver. (HEPATOLOGY 1996; kd H / -ATPase subunit. 31 Despite these advances in our 24:934-938.)
knowledge of the intestinal iron-binding proteins which may play a role in dietary iron absorption, as well as iron-binding complexes present within cellular endosomal compartments Iron homeostasis is strictly regulated to prevent cellular damage caused by iron-induced lipid peroxidation. Under to mediate transfer of iron from transferrin, little information is available concerning binding activities associated with the physiological conditions, the uptake of iron is typically mediated through endocytosis of transferrin with the transferrin plasma membrane of hepatocytes, the major depot for nontransferrin-bound iron. We have recently characterized the receptor. 1 However, even though transferrin-mediated uptake is down-regulated in presence of iron overload, 2-5 iron uptake of iron by hepatocytes when presented as a 1:4 molar complex in FeNTA. 13 As a first step towards the identification accumulation still occurs in patients with hemochromatosis. 2 In conditions of iron overload, the liver serves as the major of the complex responsible for nontransferrin-bound iron uptake by hepatocytes, our study characterizes FeNTA-binding site of iron deposition and injury resulting from lipid peroxidation leads to liver fibrosis, cirrhosis, and increased hepatic proteins associated with purified rat liver basolateral plasma membranes. carcinoma. 6 Uptake of nontransferrin-bound iron has been shown in
Methods
perfused liver, 7-9 isolated hepatocytes, 10-13 the human hepatoma HepG2 cell line, 14,15 as well as many other types of
Preparation of Liver Basolateral Plasma Membranes. Female
Abbreviations: IMAC, immobilized metal affinity chromatography; NTA, nitrilotriacetic acid; Mapp, apparent molecular weight; EDTA, ethylenediaminetetraacetic acid.
umes of 1 mmol/L NaHCO 3 (pH 7.5) using a Dounce homogenizer.