Accumulation of heme in mitochondria from rabbit reticulocytes with inhibited globin synthesis

Incubation of rabbit reticulocytes with cycloheximide and ⁵⁹Fe bound to transferrin in plasma induces excessive non-hemoglobin ⁵⁹Fe-labeled heme accumulation in mitochondria. During incubation of these mitochondria in vitro a part of ⁵⁹Fe-labeled heme is released into the surrounding medium. The addition of globin or bovine serum albumin to the incubation mixture essentially increases the amount of heme released from mitochondria.     

Iron utilization in rabbit reticulocytes: A study using succinylacetone as an inhibitor of heme synthesis

We have investigated the effect of succinylacetone (4,6-dioxoheptanoic acid) on hemoglobin synthesis and iron metabolism in reticulocytes. Succinylacetone, 0.1 and 1 mM, inhibited [2-¹⁴C]glycine incorporation into heme by 91.2 and 96.4%, respectively, and into globin by 85 and 90.2%, respectively. 60 μM hemin completely prevented the inhibition of globin synthesis by succinylacetone, indicating that succinylacetone inhibits specifically the synthesis of heme. Added porphobilinogen, but not δ-aminolevulinic acid, partly overcame the inhibition of ⁵⁹Fe incorporation into heme caused by succinylacetone suggesting that the drug inhibits δ-aminolevulinic acid dehydratase in reticulocytes. Succinylacetone, 10 μM, 0.1 and 1 mM, inhibited ⁵⁹Fe incorporation into heme by 50, 90 and 93%, respectively, but stimulated reticulocyte ⁵⁹Fe uptake by about 25–30%. In succinylacetone-treated cells ⁵⁹Fe accumulates in a fraction containing plasma membranes and mitochondria as well as cytosol ferritin and an unidentified low molecular weight fraction obtained by Sephacryl S-200 chromatography. Reincubation of washed succinylacetone- and ⁵⁹Fe-transferrin-pretreated reticulocytes results in the transfer of ⁵⁹Fe from the particulate fraction (plasma membrane plus mitochondria) into hemoglobin and this process is considerably stimulated by added protoporphyrin. Although the nature of the iron accumulated in the membrane-mitochondria fraction in succinylacetone-treated cells is unknown some of it is utilizable for hemoglobin synthesis, while cytosolic ferritin iron would appear to be mostly unavailable for incorporation into heme.     

Evidence against a regulatory role for heme oxygenase in hepatic heme synthesis

The role of a microsomal enzyme system, heme oxygenase, in the regulation of intracellular heme concentration and of the heme biosynthetic pathway was investigated. It was determined that alterations in heme oxygenase activity were not consistent with the observed alterations of heme biosynthesis produced by the administration of various drugs. It is concluded that heme oxygenase does not play a role in the regulation of heme biosynthesis under these circumstances.     

Accelerated heme synthesis and degradation in transformed fibroblasts

Various parameters of the heme biosynthetic pathway were studied in two cell lines, one nontransformed and the other malignantly transformed (MLV/MS), both replicating at the same rate. Using the above system enabled us to distinguish between phenomena characteristic of the malignant transformation per se and those due to accelerated growth rate. Heme synthesis and degradation as well as the activities of ALAS, ALAD, PBGD, and FC were found to be increased in the transformed cells. However, the concentration of intracellular heme was markedly reduced from 30.4 +/- 4.4 pmole/mg protein in nontransformed cells to 10.5 +/- 2.6 pmole/mg protein in transformed cells. These observations show that malignant transformation leads to changes in heme metabolism unrelated to growth rate in this cell line.     

Fenoprofen: Inhibitor of prostaglandin synthesis

Fenoprofen, an anti-inflammatory agent, is a potent inhibitor of prostaglandin synthesis at physiological substrate concentration. I₅₀'s of 1 μM to 100 μM were obtained when the concentration of arachidonate was increased from 1 μM to 82 μM. In contrast, a small change in I₅₀ was observed with indomethacin. Kinetic studies indicated that inhibition by fenoprofen was competitive while that of indomethacin was non-competitive.     

Regulation of heme synthesis in the regenerating rat liver

This investigation shows that the regulation of heme synthesis in the regenerating rat liver does not differ from the regulation in the normal liver. The heme saturation of tryptophan pyrrolase was found to be low, indicating a reduced concentration of heme in the regulatory heme pool of the regenerating rat liver. As expected, ALAS in the mitochondrial fraction was found to be elevated. It was also shown that ALAS in the regenerating rat liver can be induced by the porphyrinogenic drugs AIA and DDC and that heme reduces its activity. The decrease observed in the activity of cytosolic ALAS might be due to impaired synthesis of the enzyme but does not affect the regulation of the heme biosynthetic pathway.