In recent reports attention has been drawn to the extensive amino acid homology between pig heart, yeast, and Escherichia cofi aconitases (EC 4.2. In the past 10 years evidence has been obtained that intracellular iron levels are controlled by a posttranscriptional mechanism which correlates translation of mRNA for the H subunit of ferritin and stabilization of transferrin receptor mRNA. This is accomplished by the interaction ofa cytosolic protein with iron-responsive elements (IREs),-which are stem-loop structures located in the untranslated regions of the respective mRNAs (1-3). Small quantities (nanograms to micrograms) of a cytosolic protein of -100 kDa that binds to IREs (IRE binding protein, IRE-BP) have been isolated (4-6). This research took an unexpected turn when the cDNA sequence for the protein from human liver was determined and the protein sequence deduced was found to have a striking homology to the amino acid sequence of pig heart mitochondrial aconitase (m-aconitase) (7). All active-site residues identified in the aconitase crystal structure are conserved (8 MATERIALS AND METHODS m-Aconitase was prepared and enzyme activation, assay, and analysis for S2-, SO, and Fe were carried out as described (18)(19)(20). Protein was determined by a biuret method, standardized for m-or c-aconitase, respectively, by amino acid analysis.Purification of c-Aconitase. m-Aconitase is the least desirable contaminant of c-aconitase. Hence, we chose as the initial part of the purification procedure the separation of cytosol and mitochondria by a method previously used for the large-scale preparation of mitochondria from slaughterhouse tissue (21); the following modifications were incorporated: (i) 2 mM Hepes (pH 7.2) containing 2 mM citrate was used as buffer, (ii) the tissue grinding step was omitted, (iii) blending time was only 30 sec, and (iv) the homogenate was centrifuged at 1300 x g for 60 min. All manipulations were done at 0-40C and the initial ratio of liver to buffer was 1:3.5 (wt/vol). The supernatant obtained after the sedimentation of the mitochondria was made 20%o (vol/vol) 11730The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The translation of ferritin mRNA and degradation of transferrin receptor mRNA are regulated by the interaction of an RNA-binding protein, the iron-responsive element binding protein (ERE-BP), with RNA stem-oop structures known as iron-responsive elements (IREs) contained within these transcripts. IRE-BP produced in iron-replete cells has aconitase (EC 4.2.1.3) activity. The protein shows extensive sequence homology with mitochondrial aconitase, and sequences of peptides prepared from cytosolic aconitase are identical with peptides of IRE-BP. As an active aconitase, IRE-BP is expected to have an Fe-S duster, in analogy to other aconitases. This Fe-S cluster has been implicated as the region of the protein that senses intracellular iron levels and accordingly modifies the ability of the IRE-BP to interact with IREs. Expression of the IRE-BP in cultured cells has revealed that the IRE-BP functions either as an active aconitase, when the cells are iron-replete, or as an active RNA-binding protein, when the cells are iron-depleted. We compare properties of purified authentic cytosolic aconitase from beef liver with those of IRE-BP from tissue culture cells and establish that characteristics of the physiologically relevant form of the protein from iron-depleted cells resemble those of cytosolic aconitase apoprotein. We demonstrate that loss of the labile fourth iron atom of the Fe-S cluster results in loss of aconitase activity, but that more extensive cluster alteration is required before the IRE-BP acquires the capacity to bind RNA with the affinity seen in vivo. These results are consistent with a model in which the cubane Fe-S cluster is disassembled when intracellular iron is depleted.

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New spectrophotometric bioassay procedures have been developed for evaluating chemical toxicity, using electron transport particles isolated from bovine heart mitochondria, based on the ability of many toxic chemicals to interfere with the integrated function of electron transport enzymes. The sensitivity of the mitochondrial assay is compared to published sensitivities of other in vivo and in vitro toxicity testing methods. Regression analysis of logarithmically transformed toxicity values for 42 chemicals, including 8 pesticides, 5 drugs, 6 metals, 8 alcohols, 5 respiratory inhibitors, 4 phenols, and 2 phthalates, indicates excellent correlation between the sensitivity of the new assays and the sensitivity of mammalian cytotoxicity studies (r2 = 0.86). Data from aquatic exposure toxicity tests conducted in fish are also highly correlated with the mitochondrial assay results (r2 = 0.79). However, correlation of data from these methods with median lethal dose studies conducted in rats is not as good because of the inability of in vitro and aquatic exposure analyses to account for the gastrointestinal absorption, hepatic metabolism, and excretion processes which modify toxic responses following oral administration.

Partition studies have established that phospholipids generally have the capabilities to mediate the transmembrane transport of the full range of ions and solutes that physiologically cross biological membranes. The list of transportable species includes cations, anions, amino acids, citric acid cycle intermediates, nucleotides, and sugars. Phospholipid-mediated transport can be readily modulated by altering the phospholipid mixture or by addition of detergents, nucleotides, divalent metals, proteins, peptides, or ring compounds. Containment of phospholipid within channels in protein appears to be the precondition for the formation of the micellar structure requisite for solute transport. Phospholipid-mediated transport is postulated to be a central feature of energy coupling, membrane-spanning systems, and membrane-bound, phospholipid-requiring enzymes.

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