Step In Heme Synthesis Research Articles

L-Alanine: 4,5-dioxovalerate transaminase in Leishmania donovani that differs from mammalian enzyme

Leishmania protozoans are the causative agents of leishmaniasis, a major parasitic disease in humans. The parasites manifest a nutritional requirement for heme compounds since they are deficient in heme biosynthesis. In this study we have demonstrated for the first time the presence of the enzyme L-alanine: 4,5-dioxovalerate transaminase in Leishmania donovani. This enzyme catalyzes the synthesis of 5-aminolevulinic acid (ALA), the first committed step in heme synthesis. Thus the defect in heme biosynthesis pathway in Leishmania must lie at some enzymatic level subsequent to synthesis of ALA. The enzyme was found to be present in both virulent and avirulent strains of L. donovani. The virulent promastigotes showed a 41% higher specific activity as compared to the avirulent strain. The enzyme activity was found to be inhibited in the presence of heme and methylglyoxal. Immunoblot analysis revealed that L-alanine: 4,5-dioxovalerate transaminase in L. donovani was immunologically different from that in mammals.

Terminal Enzymes of Heme Biosynthesis in the Plant Plasma Membrane

Purified plasma membrane fractions from barley leaf exhibited activities of ferrochelatase and iron reductase, two of the terminal enzymes in heme biosynthesis. These activities were also present in purified barley leaf plastid and mitochondrial fractions. The plasma membrane fractions were shown to be free from contamination with plastid and mitochondrial membrane markers. Previous studies had demonstrated that barley plasma membranes exhibited activity for converting protoporphyrinogen to protoporphyrin, another late step in heme synthesis. The presence of the terminal steps of heme synthesis in the plant plasma membrane is compatible with the hypothesis that late heme precursors such as protoporphyrin or protoporphyrinogen synthesized in the chloroplast can be exported and converted to heme within the plasma membrane for subsequent incorporation into plasma membrane hemoproteins.

Anesthetic Considerations in Porphyrias

Anesthetic Considerations in Porphyrias

Hematin therapy for the neurologic crisis of tyrosinemia

Hereditary tyrosinemia (tyrosinemia type I) is an autosomal recessive disorder caused by a defect of the final enzyme in tyrosine metabolism. The disease is associated with renal tubular defects, acute and chronic liver failure, and hepatocellular carcinoma. An elevation in serum 6-aminolevulinic acid concentration is common and is due to competitive inhibition of ALA dehydrase by succinylacetone, a metabolite of tyrosine degradation. 1 A syndrome of acute neurologic crises, which has striking similarity to the acute porphyrias, is seen in as many as 40% of the children with this disorder. 2 As in the acute porphyrias, the episodes of acute neuropathy associated with tyrosinemia are best correlated with ALA excretion. 2 Hematin suppresses ALA synthase, the first committed step in heme synthesis. 3 Clinical studies in patients with porphyria have established the efficacy of hematin in decreasing ALA excretion and the improvement of clinical symptoms after intravenous administration. 4 The drug has few side effects and has been used without major complications in the treatment of numerous patients with acute porphyria. 4 We describe a case of severe neurologic crises associated with extremely high serum ALA levels in a child with tyrosinemia I. We elected to treat this patient with hematin, followed by liver transplantation. We believe this to be a rational course given the marked similarities of this disorder to the acute porphyrias.

Protoporphyrinogen oxidation, a step in heme synthesis in soybean root nodules and free-living rhizobia.

Extracts of the crude bacteroid fraction of symbiotically grown Bradyrhizobium japonicum were much more active in oxidizing protoporphyrinogen to protoporphyrin than were extracts of cells grown under free-living conditions, especially when assayed in atmospheres containing only traces of oxygen. This correlates with the higher heme content of the microaerophilic nodules. Furthermore, the high level of oxidative activity in the crude bacteroid fraction was associated with an uncharacterized membrane fraction, probably of plant origin, that was separable from the bacteroids by Percoll gradient centrifugation.

Ferrochelatase and N-alkylated porphyrins

The final step in heme synthesis is catalyzed by the mitochondrial enzyme, ferrochelatase. Characterization of this enzyme has been complicated by a number of factors including the dependence of enzyme activity on lipids. Purification of ferrochelatase from rat and bovine sources has been achieved only relatively recently using blue Sepharose CL-6B chromatography. When 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) is given to animals, it produces a hepatic porphyria resembling human variegate porphyria thus providing an experimental system in which to study this disease. DDC has been found to cause the accumulation of a green pigment, identified as N-methyl protoporphyrin IX (N-MePP), which is a potent inhibitor of ferrochelatase. The source of the N-methyl substituent of N-MePP was found to be the 4-methyl group of DDC. Considerable evidence indicates that the protoporphyrin IX moiety of N-MePP originates from the heme moiety of cytochrome P-450 and that DDC is a suicide substrate for this hemoprotein. Some studies suggest that cytochrome P-450 isozymes differ in their susceptibility to destruction by DDC and its 4-alkyl analogues. Griseofulvin has also been reported to inhibit hepatic ferrochelatase in rodents but not in the 17-day old chick embryo nor in hepatocyte culture systems. Thus, the mechanism by which griseofulvin produces an experimental porphyria in chick embryo liver cell culture is different from that for rodents.

Assay for enzymatic protoporphyrinogen oxidation, a late step in heme synthesis.

Detailed directions are given for the spectrophotometric and fluorometric assays for protoporphyrinogen oxidation in rat liver mitochondria and other tissues. Linearity of the two assays with enzyme concentration is demonstrated. Some possible assay modifications and sources of error are mentioned. The fluorometric assay appears more sensitive, while the spectrophotometric assay may be more useful for assaying a wider variety of tissues. By both methods, rat liver mitochondria exhibit enzymatic oxidation at the rate of approximately 10 nmol protoporphyrin produced per hour per milligram protein.

608 TIME SEQUENCE OF RBC PROTOPORPHYRIN (PP) ACCUMULATION IN LEAD POISONING

Lead intoxication in children deranges the last step in heme synthesis and results in the accumulation in the RBCs of protoporphyrin in the form of the Zn-chelate (ZnPP); some patients however exhibit an increased level of unchelated PP (Free PP). To evaluate the hypothesis that Free PP accumulates first and is later converted to ZnPP, the sequence of accumulation of the two PP types was followed in lead poisoned animals. Total PP, ZnPP, and Free PP were quantitated by fluorescence spectrophotometry. Three different species were used:rats, guinea pigs and rabbits. In rats and guinea pigs, increase in total PP was prompt (within 10 days) but modest (3× normal); in rabbits the increase in total PP was more marked (10× normal). Initially in all 3 species there was a rapid increase in Free PP, with a subsequent increase in ZnPP. During Pb intoxication, the relative concentration of the two types of PP was measured in RBCs separated according to age on density gradients. There was 4 times more Free PP than ZnPP in the youngest RBCs, while the oldest RBCs contained 3 times more ZnPP than Free PP. After Pb administration was discontinued the predominant PP species was ZnPP and the concentration of Free PP was minimal. These data indicate that the effect of Pb on heme synthesis results in initial accumulation of Free PP which later chelates Zn to form ZnPP. Extrapolation of these data to humans suggests that when in children both Free PP and ZnPP are present, the exposure to lead is recent.

TWO MOLECULAR SPECIES OF PROTOPORPHYRIN IN LEAD INTOXICATION AND IRON DEFICIENCY ANEMIA;

In iron deficiency anemia (FeDA) and lead intoxication (Pbl) the final step in heme synthesis is deranged in such a manner that protoporphyrin (PP) accumulates within the red blood cells. A Zinc-PP chelate (ZnPP) is the primary species of PP present in these conditions. By fluorescent spectra we have found some free (unchelated) PP in addition to ZnPP in the rbcs of children with FeDA and Pbl. In fact, in one child with acute FeDA the predominant species present was free PP, and this PP was changed to ZnPP during the course of treatment. Sequential studies of lead-poisoned rats show that PP in the peripheral blood increases rapidly (within a week of exposure) and that initially free PP is the predominant species; as exposure continues ZnPP becomes more prominent. Density gradient separation of these rbcs reveals that the youngest cells contain a large amount of PP, which is mainly free, while the older cells contain predominantly ZnPP. Therefore, in the rat the proportion of ZnPP to free PP increases in the rbc, both with chronic lead exposure and with increasing cell age. These data indicate that in Pbl and FeDA the PP produced in the rbcs is initially tree PP which subsequently chelates zinc.

Nitrate, fumarate, and oxygen as electron acceptors for a late step in microbial heme synthesis

Nitrate can serve as anaerobic electron acceptor for the oxidation of protoporphyrinogen to protoporphyrin in cell-free extracts of Escherichia coli grown anaerobically in the presence of nitrate. Two kinds of experiments indicated this: anaerobic protoporphyrin formation from protoporphyrinogen, followed spectrophotometrically, was markedly stimulated by addition of nitrate; and anaerobic protoheme formation from protoporphyrinogen, determined by extraction procedures, was markedly stimulated by addition of nitrate. In contrast, anaerobic protoheme formation from protoporphyrin was not dependent upon addition of nitrate. This was the first demonstration of the ability of nitrate to serve as electron acceptor for this late step of heme synthesis. Previous studies with mammalian and yeast mitochondria had indicated an obligatory requirement for molecular oxygen at this step. In confirmation of our previous preliminary report, fumarate was also shown to be an electron acceptor for anaerobic protoporphyrinogen oxidation in extracts of E. coli grown anaerobically on fumarate. For the first time, anaerobic protoheme formation from protoporphyrinogen, but not from protoporphyrin, was shown to be dependent upon the addition of fumarate. The importance of these findings is 2-fold. First, they establish that enzymatic protoporphyrinogen oxidation can occur in the absence of molecular oxygen, in contrast to previous observations using mammalian and yeast mitochondria. Secondly, these findings help explain the ability of some facultative and anaerobic bacteria to form very large amounts of heme compounds, such as cytochrome pigments, when grown anaerobically in the presence of nitrate or fumarate. In fact, denitrifying bacteria are known to form more cytochromes when grown anaerobically than during aerobic growth. An unexpected finding was that extracts of another bacterium, Staphylococcus epidermidis, exhibited very little ability to oxidize protoporphyrinogen to protoporphyrin as compared to E. coli extracts. This finding suggests some fundamental differences in these two organisms in this key step in heme synthesis. It is known that these two facultative organisms also differ in that E. coli synthesizes cytochrome during both aerobic and anaerobic growth, while Staphylococcus only synthesizes cytochromes when grown aerobically.

Chloramphenicol-induced erythroid suppression and bone marrow ferrochelatase activity in dogs.

Ferrochelatase activity was determined in bone marrow mitochondria of dogs receiving 100 mg. of chloramphenicol per kilogram per day and of control animals. Those animals receiving chloramphenicol for 3 weeks exhibited a 65 to 90 per cent decrease in ferrochelatase activity as compared to the controls; this enzymic activity returned to normal levels 8 weeks after discontinuation of treatment. Accompanying the drop in ferrochelatase activity was a marked elevation of the level of free erythrocyte protoporphyrin and an increase in stainable bone marrow iron, findings consistent with a block in the final step of heme synthesis. Two of the 3 dogs receiving chloramphenicol showed a significant retiwlocytopenia; the reticulocyte count returned to normal 2 weeks after cessation of chloramphenicol therapy. No significant alteration in the white cell and platelet values were observed. Whatever the mechanism involved, it is hypothesized that the suppression of ferrochelatase activity by chloramphenicol accompanied by a block in the last step of heme synthesis offers a reasonable explanation for the apparent vulnerability of the erythroid cells to the drug.

Characterization of the late steps of microbial heme synthesis: conversion of coproporphyrinogen to protoporphyrin.

Cell-free extracts of various cytochrome-containing, heterotrophic microorganisms were examined for ability to convert coproporphyrinogen to protoporphyrin. Extracts of Escherichia coli and Pseudomonas denitrificans readily accumulated large amounts of protoporphyrin when assayed under aerobic conditions. However, protoporphyrin did not accumulate under either aerobic or anaerobic conditions of assay or in the presence of various supplements in extracts of the aerobe Micrococcus lysodeikticus, the facultative anaerobe Staphylococcus aureus, or the anaerobe Vibrio succinogenes. Protoporphyrin also accumulated when extracts of E. coli and P. denitrificans were incubated aerobically with the early heme precursor, delta-amino levulinic acid (ALA). This protoporphyrin accumulation was markedly stimulated by the iron chelator, o-phenanthroline. Extracts of S. aureus and M. lysodeikticus accumulated coproporphyrin, but not protoporphyrin when incubated with ALA. The enzyme system in extracts of E. coli which converts coproporphyrinogen to protoporphyrin under aerobic conditions of assay was also partially characterized. This conversion was stimulated by the iron chelator, o-phenanthroline, the respiratory inhibitor, cyanide, and the reducing agent, thioglycolate. Dialysis of the extract did not diminish enzyme activity. Certain alternate electron acceptors and nitrite caused a marked inhibition of the conversion. These results indicate that this late step in heme synthesis, the conversion of coproporphyrinogen to protoporphyrin, can be readily demonstrated in extracts of some, but not all, cytochrome-containing bacteria and that the aerobic conversion in E. coli exhibits many characteristics similar to those demonstrated for the aerobic conversion previously studied in liver mitochondria.

Effect of drugs on heme synthesis in the liver

Several substances have been shown to affect hepatic heme synthesis in the rat liver. Phenobarbital and the polycyclic hydrocarbon, 3,4-benzpyrene, produce an induction of aminolevulinic acid synthetase (ALAS), the enzyme mediating the first step in heme synthesis. This is followed sequentially by increased incorporation of glycine into microsomal heme, increased microsomal protoheme, cytochrome P-450, and increases in activity of certain microsomal mixed function oxidate reactions. Microsomal cytochrome b 5 is not altered during such events. 3-amino-1,2,4-triazole inhibits increases in hepatic heme synthesis and drug oxidations produced by phenobarbital and 3,4-benzyprene, but has no effect on ALAS changes or cytochrome b 5. Ferrchelatase, an enzyme mediating the last step in heme synthesis, is also increased by phenobarbital treatment. This enzyme was shown to be inhibited by lead and prior administration to the animal of 3,5-diethoxycarbonyl-2,4,6-trimethylpyridine (DDC). DDC also induces ALAS activity. The combined increase in ALAS activity and inhibition of ferrochelatase by DDC could account for the profound porphyria produced by this agent.

Induction of hepatic mitochondrial ferrochelatase by phenobarbital

Ferrochelatase, the catalyst for the last step in heme synthesis, is induced by chronic phenobarbital administration and this induction is prevented by the simultaneous administration of cycloheximide. This induction is revealed when the pyridine hemochrome was measured under anaerobic conditions but cobalt chelatase activity in hepatic mitochondria was not increased by phenobarbital. Furthermore, certain metals inhibited ferrochelatase activity but had no effect on cobalt chelatase. Although both delta aminolevulinic acid synthetase and ferrochelatase are located in a similar region in the mitochondria, the time course of induction for ferrochelatase is different than that observed for delta aminolevulinic acid synthetase.

Role of oxygen and heme in heme synthesis and the development of hemoprotein activity in an anaerobically-grown staphylococcus

Abstract 1. 1. Cell suspensions of anaerobically-grown Staphylococcus epidermidis exhibit very low catalase and respiratory activity and low content of protoheme when compared to cells grown aerobically. Protoheme was determined by extraction with acid-acetone and conversion to the pyridine hemochromogen. Large amounts of coproporphyrin are formed during growth under anaerobic conditions. If added hemin is present in the medium during anaerobic growth, the resulting cells exhibit a marked increase in catalase and respiratory activity. 2. 2. When resting suspensions of cells grown anaerobically without hemin supplementation are aerated at 37°, they exhibit a rapid, 30-fold increase in catalase activity, a 4-fold increase in respiratory activity, and, in the presence of δ-amino-levulinic acid, a 5-fold increase in protoheme content. These effects of aeration do not require addition of amino acids and are not appreciably inhibited by chloramphenicol, which causes a marked inhibition of protein synthesis as indicated by [14C]leucine incorporation. 3. 3. When hemin is added under anaerobic conditions to resting suspensions of anaerobically-grown cells a marked rise in catalase and respiratory activity also occurs. This effect of hemin is not blocked by chloramphenicol. 4. 4. These results suggest that when heme synthesis is impaired by growth in the absence of oxygen, this organism forms all the enzymes required for heme synthesis, and some of the apoenzymes of the functional hemoproteins (minus the heme prosthetic group). Oxygen may be required as substrate for a terminal step in heme synthesis in this organism.

Erythrocyte δ-aminolevulinic acid dehydrase activity in thalassemia major and sickle-cell anemia

An enzyme deficiency has been postulated to account for the defect in hemoglobin synthesis in certain hereditary anemias. The enzyme necessary for the conversion of δ-aminolevulinic acid to porphobilinogen, an obligatory step in heme synthesis, was assayed. The values obtained from the circulating erythrocyte of patients with thalassemia and sickle-cell anemia were higher than normal.