In vitro culture of murine bone marrow has proved to be a useful system for defining the haem biosynthetic and degradative enzymatic pathways during erythroid colony (CFU-E) growth and development. Previous attempts to elucidate these pathways during erythropoiesis have been limited by the amount of biological material available as well as the sensitivity of specific enzyme assays. These conditions were overcome in the present study. In order to obtain an enrichment of CFU-E, nonadherent mouse bone-marrow cells were cultured in special culture plates using a modified methyl-cellulose medium with erythropoietin. CFU-E yields were increased approximately threefold. Utilizing the sensitive radiochemical assay, direct measurement of ALAS activity was determined in very early developing CFU-E cultures as well as mature cultures. ALAS activity was found to reach a peak after 60 h of culture growth and then started to decline in activity. Cellular synthesis of haem was determined (with 14C ALA) and minor modifications of standard assays were also made in order to determine spectrophotometrically S-aminolaevulinic acid dehydratase (ALAD) and haem oxygenase activity in developing CFU-E cultures. These assays were reproducible with as few as four or five culture plates. It was found that ALAD activity rose progressively after 36 h of culture growth and reached a plateau at about 60 h of growth. A continuous increase in I4C ALA incorporation into haem was seen at later hours of culture growth, suggesting that the induction of other haem enzymes beyond ALAS is still rate limiting in haem synthesis. Conversely, haem oxygenase activity declined up to 60 h of growth and was elevated at later culture periods without a subsequent increase in ALAS and ALAD. The observed increase in haem enzymes brought about by haemin was completely suppressed by addition of cycloheximide to the cultures. These results suggest that

Hemin treatment of the Philadelphia chromosome positive leukemia cell line, K562, accentuates a number of erythroid phenotypic characteristics. The nature of this hemin effect was investigated by examining heme production and heme biosynthetic and catabolic enzyme activity in untreated and 0.05 mM hemin-treated cells. Activities of -aminolevulinic acid synthetase (ALAS). the rate limiting heme synthetic enzyme, and #{244}-aminolevulinic dehydratase (ALAD) were detectable in both uninduced and induced K562 cells. However. cells treated with hemin showed a significant increase in ALAS and ALAD activity by 2.5 days of treatment compared to untreated cells, and both enzyme activities further increased over the remainder of the incubation period. Incorporation of 3H-ALA into heme was also monitored. 3H-ALA was incorporated into heme in both untreated and treated cultures. but this incorporation was significantly greater in hemin-treated cells. Incorporation of 3H-ALA into heme indicates that cellular heme biosynthesis accounts for a portion of the heme utilized for hemoglobin production and that hemin stimulates heme synthesis by increasing enzyme activities distal to ALAS in the heme biosynthetic pathway. Heme oxygenase. the rate-limiting enzyme in mammalian heme degradation. was also studied and found to be present in large amounts in treated and untreated K562 cells. However.

Rats were chronically iron-overloaded by intraperitonel injections of iron-dextran. Electron microscopy revealed that the excess iron was deposited in ferritin-like particles packed in lysosomes and scattered in hepatic cytoplasm. No mitochondrial iron deposition or damage was seen. Furthermore, mitochondrial preparations from chronically iron-overloaded animals were found to be contaminated with lysosomes, which could explain previously reported increases in mitochondrial iron by chemical analysis. Mitochondrial function, as measured by cytochromes a-a3, b and c concentrations as well as activity of the rate-limiting enzyme of haem synthesis, delta-aminolaevulinate synthetase, was not diminished by chronic iron-overloading. Microsomal haem was decreased by 30% at the time that haem oxygenase, the rate-limiting enzyme of haem degradation, was increased approx. 3-fold. Animals were given a single intraperitoneal injection of iron-dextran and the activities of delta-aminolaevulinate synthetase and haem oxygenase were measured over 24 h. delta-Aminolaevulinate synthetase activity increased approx. 2-fold in these acutely iron-overloaded rat livers, but at a time after the increase in haem oxygenase. These results suggest that an early consequence of excess iron in liver is acceleration of the rate of haem degradation, possible by haem oxygenase.

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