The role of haem biosynthetic and degradative enzymes in erythroid colony development: the effect of haemin

Abstract

Summary. 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 δ‐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 ¹⁴C 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 ALASA 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