Haem is a prosthetic group for haem proteins, which play an essential role in oxygen transport, respiration, signal transduction, and detoxification. In haem biosynthesis, the haem precursor protoporphyrin IX (PP IX) must be accumulated into the mitochondrial matrix across the inner membrane, but its mechanism is largely unclear. Here we show that adenine nucleotide translocator (ANT), the inner membrane transporter, contributes to haem biosynthesis by facilitating mitochondrial accumulation of its precursors. We identified that haem and PP IX specifically bind to ANT. Mitochondrial uptake of PP IX was inhibited by ADP, a known substrate of ANT. Conversely, ADP uptake into mitochondria was competitively inhibited by haem and its precursors, suggesting that haem-related porphyrins are accumulated into mitochondria via ANT. Furthermore, disruption of the ANT genes in yeast resulted in a reduction of haem biosynthesis by blocking the translocation of haem precursors into the matrix. Our results represent a new model that ANT plays a crucial role in haem biosynthesis by facilitating accumulation of its precursors into the mitochondrial matrix.

Methotrexate (MTX) is the anticancer and antirheumatoid drug that is believed to block nucleotide synthesis and cell cycle by inhibiting dihydrofolate reductase activity. We have developed novel affinity matrices, termed SG beads, that are easy to manipulate and are compatible with surface functionalization. Using the matrices, here we present evidence that deoxycytidine kinase (dCK), an enzyme that acts in the salvage pathway of nucleotide biosynthesis, is another target of MTX. MTX modulates dCK activity differentially depending on substrate concentrations. 1-␤-D-Arabinofuranosylcytosine (ara-C), a chemotherapy agent often used in combination with MTX, is a nucleoside analog whose incorporation into chromosome requires prior phosphorylation by dCK. We show that, remarkably, MTX enhances incorporation and cytotoxicity of ara-C through regulation of dCK activity in Burkitt's lymphoma cells. Thus, this study provides new insight into the mechanisms underlying MTX actions and demonstrates the usefulness of the SG beads.

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Phthalate esters are commonly used plasticizers; however, some are suspected to cause reproductive toxicity. Administration of high doses of di-(2-ethylhexyl) phthalate (DEHP) induces germ cell death in male rodents. Mono-(2-ethylhexyl) phthalate (MEHP), a hydrolyzed metabolite of DEHP, appears to be responsible for this testicular toxicity; however, the underlying mechanism of this chemical's action remains unknown. Here, using a one-step affinity purification procedure, we identified glycogen debranching enzyme (GDE) as a phthalate-binding protein. GDE has oligo-1,4-1,4-glucanotransferase and amylo-1,6-glucosidase activities, which are responsible for the complete degradation of glycogen to glucose. Our findings demonstrate that MEHP inhibits the activity of oligo-1,4-1,4-glucanotransferase, but not of amylo-1,6-glucosidase. Among various phthalate esters tested, MEHP specifically binds to and inhibits GDE. We also show that DEHP administration affects glycogen metabolism in rat testis. Thus, inhibition of GDE by MEHP may play a role in germ cell apoptosis in the testis.

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