delta-Aminolaevulinate synthase in human HepG2 hepatoma cells. Repression by haemin and induction by chemicals.

delta-Aminolaevulinate (ALA) synthase, the rate-limiting enzyme in haem biosynthesis in the normal liver, was examined in human HepG2 hepatoma cells. Haemin, up to 100 microM, had no effect on ALA synthase activity in vitro; it did, however, exhibit a dose-dependent inhibitory action when added to cells growing in culture (half-maximal inhibition at 1 microM). The half-life of ALA synthase activity after haemin treatment was 2 h, which was similar to that found after treatment with cycloheximide. Cells treated with actinomycin D showed a longer half-life of the enzyme activity, i.e. 4 h, compared with haemin or cycloheximide treatment. Treatment of cells with succinylacetone markedly inhibited the activity of ALA dehydratase and 59Fe incorporation into haem, but in increased ALA synthase activity. Both the haemin-induced repression and the succinylacetone-mediated de-repression of ALA synthase activity were reversible within 4 h after replacing the medium with fresh medium without the chemical. In addition to succinylacetone, dimethyl sulphoxide and 3-methylcholanthrene induced the enzyme. Induction of ALA synthase by these chemicals was also suppressed by treatment of cells with haemin. These findings indicate that the level of ALA synthase in HepG2 cells is maintained by both synthesis and degradation of the enzyme, and that the synthesis of the enzyme is regulated by the concentration of regulatory free haem in the cell.     

STUDIES ON HAEM BIOSYNTHESIS IN RAT BRAIN

Abstract— Abnormalities involving haem biosynthesis have been postulated as underlying mechanisms in the aetiology of the neural manifestations of acute porphyria and of lead poisoning. This paper reports a study of the enzymes of the haem biosynthetic pathway and their control in mammalian brain. The activity of rat brain 6-aminolaevulinate synthetase (ALA synthetase), 6-aminolaevulinate dehydratase (ALA dehydratase), uroporphyrinogen I synthetase, uroporphyrinogen decarboxylase and ferrochelatase were found to be between 12.5 and 0.002% of the corresponding values for liver. This accords with the lower concentrations of total haem and cytochrome P₄₅₀ found in brain and with the slower rate of incorporation of [4-¹⁴C]ALA into brain haem in vivo . The subcellular distribution of radioactivity following intraventricular injection of [4-¹⁴C]ALA confirmed that the bulk of brain haemoproteins are intramitochondrial in contrast to liver where the major portion is microsomal. Brain haem biosynthesis was apparently unaffected by factors known to influence this pathway in liver, including starvation and treatment with allylisopropylacetamide or phenobarbitone. These findings suggest that brain haem requirements are considerably less than those of liver and are not subject to significant fluctuations under normal circumstances. Apparent non-inducibility of ALA synthetase suggests that deficient haem and consequently haemoprotein production could result where other enzymes in the pathway become rate-limiting due to genetic defects or inhibition by exogenous agents such as lead.     

Iron targeting to mitochondria in erythroid cells

Immature erythroid cells have an exceptionally high capacity to synthesize haem that is, at least in part, the result of the unique control of iron metabolism in these cells. In erythroid cells the vast majority of Fe released from endosomes must cross both the outer and the inner mitochondrial membranes to reach ferrochelatase, which inserts Fe into protoporphyrin IX. Based on the fact that Fe is specifically targeted into erythroid mitochondria, we have proposed that a transient mitochondria-endosome interaction is involved in Fe transfer to ferrochelatase [Ponka (1997) Blood 89, 1-25]. In this study, we examined whether the inhibition of endosome mobility within erythroid cells would decrease the rate of (59)Fe incorporation into haem. We found that, in reticulocytes, the myosin light-chain kinase inhibitor, wortmannin, and the calmodulin antagonist, W-7, caused significant inhibition of (59)Fe incorporation from (59)Fe-transferrin-labelled endosomes into haem. These results, together with confocal microscopy studies using transferrin and mitochondria labelled by distinct fluorescent markers, suggest that, in erythroid cells, endosome mobility, and perhaps their contact with mitochondria, plays an important role in a highly efficient utilization of iron for haem synthesis.     

PufQ regulates porphyrin flux at the haem/bacteriochlorophyll branchpoint of tetrapyrrole biosynthesis via interactions with ferrochelatase

Facultative phototrophs such as Rhodobacter sphaeroides can switch between heterotrophic and photosynthetic growth. This transition is governed by oxygen tension and involves the large‐scale production of bacteriochlorophyll, which shares a biosynthetic pathway with haem up to protoporphyrin IX. Here, the pathways diverge with the insertion of Fe²⁺ or Mg²⁺ into protoporphyrin by ferrochelatase or magnesium chelatase, respectively. Tight regulation of this branchpoint is essential, but the mechanisms for switching between respiratory and photosynthetic growth are poorly understood. We show that PufQ governs the haem/bacteriochlorophyll switch; pufQ is found within the oxygen‐regulated pufQBALMX operon encoding the reaction centre–light‐harvesting photosystem complex. A pufQ deletion strain synthesises low levels of bacteriochlorophyll and accumulates the biosynthetic precursor coproporphyrinogen III; a suppressor mutant of this strain harbours a mutation in the hemH gene encoding ferrochelatase, substantially reducing ferrochelatase activity and increasing cellular bacteriochlorophyll levels. FLAG‐immunoprecipitation experiments retrieve a ferrochelatase‐PufQ‐carotenoid complex, proposed to regulate the haem/bacteriochlorophyll branchpoint by directing porphyrin flux toward bacteriochlorophyll production under oxygen‐limiting conditions. The co‐location of pufQ and the photosystem genes in the same operon ensures that switching of tetrapyrrole metabolism toward bacteriochlorophyll is coordinated with the production of reaction centre and light‐harvesting polypeptides.     

Control of delta-aminolaevulinate synthase and haem oxygenase in chronic-iron-overloaded rats.

The hepatic porphyrias are inborn errors of porphyrin and haem biosynthesis characterized biochemically by excessive excretion of delta-aminolaevulinate (ALA), porphobilinogen and other intermediates in haem synthesis. Clinical evidence has implicated iron in the pathogenesis of several types of genetically transmitted diseases. We investigated the role of iron in haem metabolism as well as its relationship to drug-mediated induction of ALA synthase and haem oxygenase in acute and chronic iron overload. Acute iron overload in rats resulted in a marked increase in hepatic haem oxygenase that was associated with a decrease in cytochrome P-450 and an increase in ALA synthase activity. Aminopyrine N-demethylase and aniline hydroxylase activities, which are dependent on the concentration of cytochrome P-450, were also decreased. In contrast, in chronic-iron-overloaded rats, there was an adaptive increase in haem oxygenase activity and an increase in ALA synthase that was associated with normal concentrations of microsomal haem and cytochrome P-450. The induction of ALA synthase in chronic iron overload was enhanced by phenobarbital and allylisopropylacetamide, in spite of the fact that these agents did not increase haem oxygenase activity. Small doses of Co2+ were potent inducers of the haem oxygenase in chronic-iron-overloaded, but not in control, animals. We conclude that increased hepatic cellular iron may predispose certain enzymes of haem synthesis to induction by exogenous agents and thereby affect drug-metabolizing enzyme activities.     

The ferrochelatase gene structure and molecular defects associated with erythropoietic protoporphyria

Ferrochelatase [heme synthase, protoheme ferrolyase (EC 4.99.1.1)], the terminal enzyme of the heme biosynthetic pathway, catalyzes the incorporation of ferrous ion into protoporphyrin IX to form protoheme IX. The genes and cDNAs for ferrochelatase from mammals and microorganisms have been isolated. The gene for human ferrochelatase has been mapped to chromosome 18q 21.3 and consists of 11 exons with a size of about 45 kilodaltons. The induction of ferrochelatase expression occurs during erythroid differentiation, and can be attributed to the existence of the promoter sequences of erythroid-related genes. Analysis of the ferrochelatase gene in patients with erythropoietic protoporphyria, an inherited disease caused by ferrochelatase defects, revealed that molecular anomalies of ferrochelatase from 11 patients were found in 9 patients as autosomal dominant type, and 2 patients as recessive type. Diversity of the mutations of the ferrochelatase gene is also briefly described.     

Distribution of δ-aminolevulinic acid biosynthetic pathways among phototrophic bacterial groups

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, -aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO₂. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNA^Glu, ATP, Mg²⁺, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[³H]glutamate or 1-[¹⁴C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[¹⁴C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the subgroup of purple bacteria.Abbreviations ALA -aminolevulinic acid - DTT dithiothreitol - PALP pyridoxal phosphate - SDS sodium dodecyl sulfate - tricine N-tris-(hydroxymethyl)methylglycine     

Tetrapyrrole biosynthesis in greening etiolated barley seedlings

Allyl isopropylacetamide (AIA) does not stimulate porphyrin biosynthesis in greening barley; AIA inhibits the synthesis of 5-aminolaevulinate (ALA) in plants and does not overcome the repression of ALA-synthetase. This indicates that the ALA synthesis system of green plants is regulated differently from ALA synthetase of mammalian systems. Laevulinic acid (LA) inhibited the biosynthesis of tetrapyrrole pigments in greening barley and diminished the insertion of ⁵⁵Fe into extractable protohaem, confirming that haem was synthesized at a time of little net increase in protohaem. ALA feeding increased iron incorporation into protohaem without increasing either extractable protohaem or cytochromes b and f. Since ALA feeding greatly increased the protochlorophyllide content of darkgrown plants and subsequent chlorophyll levels in the light, the regulation of haem pigment synthesis in plants occurs after protoporphyrin and protohaem synthesis and is likely to involve the turnover of protohaem produced in excess of haem protein requirements.     

CELL CLASSIFICATION AND KINETIC ASPECTS OF NORMOBLASTIC AND MEGALOBLASTIC ERYTHROPOIESIS

Mitotic indices and ³H-thymidine flash labelling indices have been determined in a total of 6000 erythroid cells from patients with megaloblastic anaemia (vitamin B₁₂ deficiency) and 4000 erythroid cells from patients with increased, normoblastic erythropoiesis. In the anaemic states there is a lack of mitoses in the more mature erythroid compartments relative to the number of mitoses in the early erythroid precursors; this must reflect skipped division and/or cell death in the later precursors. In order to further locate the deficit of mitoses, erythropoietic cells were subdivided in a way which aimed at stratifying them according to cell generations. It appears that there are four consecutive generations of recognizable proliferating red cell precursors. Balance considerations of mitotic figures suggest that in stressed normopoiesis all cells which enter generation III divide, whereas only about one-half of cells leaving generation III divide again in generation IV. In megaloblastic erythropoiesis it appears that only about one of three cells which leave generation III divide in generation IV. The data suggest that in megaloblastic anaemia, skipped division and/or cell death to a large extent take place in generation IV or at the transition from III to IV. In normoblastic erythropoiesis, the ratio labelled cells/mitotic cells is rather independent of cell maturation. In contrast, this ratio varies considerably in megaloblastic erythropoiesis, from 25:1 in early forms to 4-5:1 in late forms. As an explanation of the lack of mitoses, relative to cells in DNA-synthesis, in the early stages and the relative surplus of mitoses in the late stages it is proposed that cell cycle and cytological boundaries do not coincide in all cells. The present observations can be accounted for if a significant fraction of cells change their morphology (from basophilia to polychromasia) between their DNA-synthesis phase and the subsequent mitosis. It cannot be decided whether in addition there is a death function between DNA-synthesis and mitosis in the large basophilic megaloblasts, megaloblastic system could absorb a direct entry from the large basophilic cells amounting to perhaps about one-half of the flux through the S-pool of the large basophilic cells without being more dominated by very large cells than is actually the case; still, in large measure this will depend on the time from entry into the polychromatic pool until the subsequent mitosis or possible cell death. The alternative is a significant death function between S-phase and mitosis at the level of the large basophilic E1-E2 cells (generation I + II).     

Mechanism of aluminium-induced porphyrin synthesis in bacteria

In previous studies, aluminium was found to retard bacterial growth and enhance porphyrin formation in Arthrobacter aurescens RS-2. The aim of this study was to establish the mechanism of action of aluminium which leads to increased porphyrin production. Cultures of Arthrobacter aurescens RS-2 were incubated in the absence and presence of 0.74 mm aluminium. After 6 and 24 h of incubation, various parameters of the haem biosynthetic pathway were determined. After 6 h of incubation with aluminium, the activities of the enzymes aminolevulinate synthase (ALAS), aminolevulinate dehydratase (ALAD), porphobilinogen deaminase (PBGD) and uroporphyrinogen decarboxylase (UROD) were increased by 120, 170, 190 and 203%, respectively, while that of ferrochelatase (FC) was found to be unchanged. However, after 24 h of incubation, no change in the activities of ALAS and ALAD was noted, while an about 2-fold increase in PBGD and UROD activities were observed. FC activity was decreased by 63%. It was concluded that aluminium exerts its effect by inducing the enzymes PBGD and UROD rather than by a direct or indirect effect on ALAS. Its effect on the final step in the haem biosynthetic pathway is discussed.     

Increased 5-aminolevulinic acid (ALA) in heavy metal exposed bivalve molluscs,Cerastoderma edule: An effect due to inhibition of porphobilinogen synthase?

1. The effect of cadmium, lead, and mercury on 5-aminolevulinic acid (ALA), porphobilinogen (PBG), and PBG synthase was determined in hepatopancreas of the bivalve, Cerastoderma edule (L.).2. Cd and Hg exposure induced increased ALA content, and thus an initial doubling of ALA within 24 hr.3. Using ALA in excess (8 mmoll⁻¹) as substrate, no PBG synthase (ALA dehydratase, EC 4.2.1.24) activity was detectable in freshly prepared hepatopancreas homogenates.4. Increased ALA in metal exposed bivalves is not a simple effect due to metal inhibition of PBG synthase.5. The observed lack of PBG synthase suggests an alternative to the general pathway where two ALA molecules condense to one PBG.     

Inhibition of ferrochelatase during differentiation of murine erythroleukaemia cells.

During dimethyl sulphoxide-induced differentiation of DS-19 murine erythroleukaemia (MEL) cells, the activity of the terminal enzyme of the haem-biosynthetic pathway, ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), is thought to be the rate-limiting step for haem production. Differentiation of induced MEL cells in the presence of exogeneously supplied protoporphyrin IX showed that total haem production was affected by added porphyrin only after 48 h. These data suggest that iron insertion, the terminal step, is rate-limiting during the first 48 h of differentiation. Addition of low levels of diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine to differentiating cultures resulted in decreased haem production and decreased ferrochelatase activity. N-Methylprotoporphyrin at nanomolar concentrations also strongly inhibited ferrochelatase activity, but had no inhibitory effect on cellular haem production. The bivalent cations Co2+, Cd2+ and Mn2+ were tested for their effect on haem production and ferrochelatase activity. All three metals were found to inhibit both haem formation and ferrochelatase activity, with Mn2+ being the strongest effector. These data, together with those previously published, suggest that the terminal step in haem biosynthesis is rate-limiting during the early stages of differentiation in MEL cells.     

Expression of coproporphyrinogen oxidase and synthesis of hemoglobin in human erythroleukemia K562 cells.

Coproporphyrinogen oxidase (CPOX), the sixth enzyme in the heme-biosynthetic pathway, catalyzes oxidative decarboxylation of coproporphyrinogen to protoporphyrinogen and is located in the intermembrane space of mitochondria. To clarify the importance of CPOX in the regulation of heme biosynthesis in erythroid cells, we established human erythroleukemia K562 cells stably expressing mouse CPOX. The CPOX cDNA-transfected cells had sevenfold higher CPOX activity than cells transfected with vector only. Expression of ferrochelatase and heme content in the transfected cells increased slightly compared with the control. When K562 cells overexpressing CPOX were treated with delta-aminolevulinic acid (ALA), most became benzidine-positive without induction of the expression of CPOX or ferrochelatase, and the heme content was about twofold higher than that in ALA-treated control cells. Increases in cellular heme concomitant with a marked induction of the expression of heme-biosynthetic enzymes, including CPOX, ferrochelatase and erythroid-specific delta-aminolevulinic acid synthase, as well as of alpha-globin synthesis, were observed when cells were treated with transforming growth factor (TGF)beta 1. These increases in the transfected cells were twice those in control cells, indicating that overexpression of CPOX enhanced induction of the differentiation of K562 cells mediated by TGF beta 1 or ALA. Conversely, the transfection of antisense oligonucleotide to human CPOX mRNA into untreated and TGF beta 1-treated K562 cells led to a decrease in heme production compared with sense oligonucleotide-transfected cells. These results suggest that CPOX plays an important role in the regulation of heme biosynthesis during erythroid differentiation.     

Chromosomal protein synthesis during erythropoiesis in the mouse spleen

Induced erythropoiesis in the mouse spleen was employed to study chromosomal protein synthesis during erythroid cell development. Splenic erythropoiesis occurring after phenylhydrazine induced hemolysis can be divided into an early phase during which nuclear RNA polymerase activity and RNA production are maximal and a late phase in which hemoglobin synthesis and DNA accumulation are maximal. Chromatin was isolated from splenic tissue during both the early and late phases of erythropoiesis as well as from non-anemic animals. The total protein content of chromatin from the early erythroid phase was greater than that of chromatin from the late erythroid phase or from non-anemic controls. The increase was due to a coordinate increase in the concentration of both histone and nonhistone proteins. During late erythropoiesis, the concentration of each returned to pre-anemic levels. Total histone synthesis increased 2.6-fold during early erythropoiesis as compared with the pre-anemic state and remained elevated in late erythropoiesis. The increase in histone synthesis was due to an increase in the synthesis of all five major histone proteins. Nonhistone protein synthesis was more active than that of histones in the pre-anemic spleen and rose only slightly during early erythropoiesis, returning to preanemic levels during late erythropoiesis. Fractionation of nonhistone proteins on SDS-urea polyacrylamide gels revealed complex patterns with significant differences between the pattern of erythroid spleen non-histone proteins and that of the pre-anemic spleen. Analysis of the incorporation of ³H-valine into the non-histone proteins indicated that during early erythropoiesis there was a generalized increase in nonhistone protein synthesis. During the late erythroid phase, the decline in non-histone protein synthesis was most marked for the higher molecular weight proteins.     

Mechanism of hexachlorobenzene-induced porphyria in rats. Effect of phenobarbitone pretreatment.

The effect of a pretreatment with phenobarbitone (PB) on the porphyrinogenic action exerted by hexachlorobenzene (HCB) was examined in female rats. Kinetic studies of enzyme function after HCB poisoning showed that porphyrinogen carboxy-lyase was the only enzyme of haem biosynthesis that markedly lowered its activity. Both stages of uroporphyrinogen (UPG) III decarboxylation were decreased. This enzyme, together with UPG I synthase (increased levels) were the first enzymes altered. Subsequently, an increase in delta-aminolaevulinate (AmLev) synthase and ferrochelatase was detected; AmLev dehydratase was the last to increase. On long-term exposure, PB alone did not modify the basal values of haem intermediates; only the content of cytochrome P-450 increased. All the enzyme activities studied showed no significant changes, except ferrochelatase, which increased. With both drugs the metabolic impairment promoted by HCB was accelerated and enhanced by prior PB treatment leading to the onset of an earlier and stronger porphyria. A more noticeable accumulation and excretion of higher carboxylated porphyrins and precursors was more promptly observed as a consequence of the early porphyrinogen carboxy-lyase blockade and the concomitant induction of AmLev synthase. Although the enzymic activities of both AmLev dehydratase and ferrochelatase were enhanced, this response differed in time. For UPG I synthase this pretreatment elicited lower values than those found in the HCB group. Cytochrome P-450 contents were immediately and slightly enhanced by all the drugs, but the values for the combined treatment were the lowest. Of the several hypotheses that could explain the action of HCB on the haem pathway, our results would suggest that the porphyrinogenic action of HCB is mediated by some of its metabolic products.     

Nuclear-cytoplasmic ratio and shape of erythroid cells of the reindeer embryo

With the beginning of normoblastic erythropoiesis the nuclear-cytoplasmic ratio (NCR) in the erythroid cells increases. From the chromatophilic erythroblasts with NCR from 1.0 and more erythrocytes possessing the concavo-concave form are produced as a result of the nucleus elimination.