Biomedical subjects

H A Dailey

Publications and source records attributed to H A Dailey.

At least 55 records · Page 3Linked to original sources

PubMed1994

Human ferrochelatase is an iron-sulfur protein.

Recombinant human ferrochelatase has been expressed in Escherichia coli and purified to homogeneity. Metal analyses revealed approximately 2 mol of non-heme Fe per mol of the purified enzyme (M(r) = 40,000). The UV-visible absorption spectrum of the purified enzyme consists of a protein absorption at 278 nm (epsilon approximately 90,000 M-1 cm-1) and bands at 330 nm (epsilon approximately 24,000 M-1 cm-1), 460 nm (shoulder, epsilon approximately 11,000 M-1 cm-1), and 550 nm (shoulder, epsilon approximately 9000 M-1 cm-1) that are indicative of a [2Fe-2S]2+ cluster. The spectra show an additional band at 415 nm that varied in intensity for different preparations and is attributed, at least in part, to a minor component of enzyme-associated high-spin Fe(III) heme. The presence of a single [2Fe-2S]2+,+ cluster as a redox active component of human ferrochelatase was confirmed by variable-temperature MCD and EPR studies of the dithionite-reduced enzyme which showed the presence of a S = 1/2 [2Fe-2S]+ cluster in addition to residual high spin Fe(II) heme. The reduced enzyme exhibits a S = 1/2 EPR signal, g = 2.00, 1.94, 1.91 accounting for 0.75 +/- 0.25 spins/molecule, that readily saturates at low microwave powers below 10 K but is observable without significant broadening at temperatures up to 100 K. The Fe-S cluster is labile and gradually disappears over period of 24 h, with concomitant loss of enzyme activity, when the enzyme is stored aerobically at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Expression of a cloned protoporphyrinogen oxidase.

The previously cloned hem Y gene of Bacillus subtilis (Hansson, M., and Hederstedt, L. (1992) J. Bacteriol. 174, 8081-8093) has been expressed in Escherichia coli. The expressed protein has been shown to be the penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (EC 1.3.3.4) and, thus, the gene designation should be hem G. This represents the first report of the expression of a cloned protoporphyrinogen oxidase from any source. The enzyme is present in the soluble cytoplasmic fraction and is, thus, unlike all previously reported eukaryotic or prokaryotic protoporphyrinogen oxidases, which are membrane-bound. It utilizes molecular oxygen as a terminal electron acceptor, and protoporphyrinogen IX, mesoporphyrinogen IX, and coproporphyrinogen III serve as substrates. The diphenyl ether herbicide acifluorfen, which is a strong inhibitor of the eukaryotic enzyme, is only weakly inhibitory. The enzyme has a predicted molecular weight of 51,200, which corresponds well with molecular weight determination via high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis. In addition the enzyme contains a putative dinucleotide binding region at the amino terminus, which is consistent with the previously demonstrated presence of a flavin moiety in the characterized mammalian enzymes.

Amino Acid Sequence↗

PubMed1994

Mammalian ferrochelatase. Expression and characterization of normal and two human protoporphyric ferrochelatases.

Ferrochelatase (EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX. Herein we report the expression, purification, and characterization of the mature processed form of human and mouse ferrochelatase in Escherichia coli JM109. Metal analysis of the recombinant normal human ferrochelatase reveals that there are approximately 2 iron atoms/molecule of enzyme. This, along with the presence of spectral absorbance near 320 nm, is strongly suggestive that recombinant mammalian ferrochelatase as expressed in E. coli may contain an iron sulfur cluster. Two human protoporphyric ferrochelatases, F417S and M267I, were also expressed and characterized. The M267I mutant possesses the same Km and Vmax as the normal enzyme but exhibits increased thermolability when compared with normal human ferrochelatase. The F417S mutant has less than 2% of the normal activity. Since the Phe-->Ser substitution in this mutation is both chemically and structurally significant, three single amino acid substitutions (Lys, Tyr, and Trp) were engineered and characterized. None of these resulted in a protein with wild type activity. Additionally the carboxyl-terminal 10-amino acid segment, which contains Phe-417, from the yeast sequence was substituted, but this construct had no activity. Elimination of the carboxyl-terminal 30 amino acid residues (which include Phe-417) results in a protein the same length as the bacterial ferrochelatases, but it is an inactive enzyme.

Amino Acid Sequence↗

PubMed1993

Expression of mammalian 5-aminolevulinate synthase in Escherichia coli. Overproduction, purification, and characterization.

5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in nonplant higher eukaryotes. A cDNA encoding for the mouse erythroid 5-aminolevulinate synthase (Schoenhaut, D. S., and Curtis, P.J. (1986) Gene (Amst.) 48, 55-63) has been expressed in Escherichia coli, using the alkaline phosphatase promoter, to a level of 50-60% of the total bacterial protein. Aminolevulinate synthase was overexpressed in an active form and, therefore, was able to rescue hemA mutants, which are unable to grow in the absence of 5-aminolevulinate. A simple purification from the aminolevulinate synthase-overproducing bacterial strain yielded approximately 50 mg of protein, in a high state of purity, per liter of bacterial culture. Moreover, the expressed aminolevulinate synthase could be easily concentrated up to 6-8 mg/ml. Significantly, recombinant aminolevulinate synthase retained physical and catalytic properties identical to those of natural sources. These include the dimeric structure, subunit molecular mass, and pyridoxal 5'-phosphate as an essential cofactor. Removal of the pyridoxal 5'-phosphate led to complete loss of activity. However, the apoenzyme could be readily reconstituted by incubation with 20 microM 5'-pyridoxal phosphate. The Km values are 51 mM for glycine and 55 microM for succinyl-CoA, in the same range of the Km values determined for the nonrecombinant enzyme. This report describes the overexpression of a mammalian 5-aminolevulinate synthase in E. coli and its purification from an overproducing strain. The ready availability of the pure, cloned, sequenced erythroid 5-aminolevulinate synthase makes it possible now for questions pertinent to the enzyme's structure, mechanism, and regulation to be addressed.

5-Aminolevulinate Synthetase↗

PubMed1993

In situ conversion of coproporphyrinogen to heme by murine mitochondria: terminal steps of the heme biosynthetic pathway.

Coproporphyrinogen oxidase (EC 1.3.3.3), protoporphyrinogen oxidase (EC 1.3.3.4), and ferrochelatase (EC 4.99.1.1) catalyze the terminal three steps of the heme biosynthetic pathway. All three are either bound to or associated with the inner mitochondrial membrane in higher eukaryotic cells. A current model proposes that these three enzymes may participate in some form of multienzyme complex with attendant substrate channeling (Grand-champ, B., Phung, N., & Nordmann, Y., 1978, Biochem. J. 176, 97-102; Ferreira, G.C., et al., 1988, J. Biol. Chem. 263, 3835-3839). In the present study we have examined this question in isolated mouse mitochondria using two experimental approaches: one that samples substrate and product levels during a timed incubation, and a second that follows dilution of radiolabeled substrate by pathway intermediates. When isolated mouse mitochondria are incubated with coproporphyrinogen alone there is an accumulation of free protoporphyrin. When Zn is added as a substrate for the terminal enzyme, ferrochelatase, along with coproporphyrinogen, there is formation of Zn protoporphyrin with little accumulation of free protoporphyrin. When EDTA is added to this incubation mixture with Zn, Zn protoporphyrin formation is eliminated and protoporphyrin is formed. We have examined the fate of radiolabeled substrates in vitro to determine if exogenously supplied pathway intermediates can compete with the endogenously produced compounds. The data demonstrate that while coproporphyrinogen is efficiently converted to heme in vitro when the pathway is operating below maximal capacity, exogenous protoporphyrinogen can compete with endogenously formed protoporphyrinogen in heme production.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Heme biosynthesis in mammalian systems: evidence of a Schiff base linkage between the pyridoxal 5'-phosphate cofactor and a lysine residue in 5-aminolevulinate synthase.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in nonplant higher eukaryotes. Murine erythroid 5-aminolevulinate synthase has been purified to homogeneity from an Escherichia coli overproducing strain, and the catalytic and spectroscopic properties of this recombinant enzyme were compared with those from nonrecombinant sources (Ferreira, G.C. & Dailey, H.A., 1993, J. Biol. Chem. 268, 584-590). 5-Aminolevulinate synthase is a pyridoxal 5'-phosphate-dependent enzyme and is functional as a homodimer. The recombinant 5-aminolevulinate synthase holoenzyme was reduced with tritiated sodium borohydride and digested with trypsin. A single peptide contained the majority of the label. The tritiated peptide was isolated, and its amino acid sequence was determined; it corresponded to 15 amino acids around lysine 313, to which pyridoxal 5'-phosphate is bound. Significantly, the pyridoxyllysine peptide is conserved in all known cDNA-derived 5-aminolevulinate synthase sequences and is present in the C-terminal (catalytic) domain. Mutagenesis of the 5-aminolevulinate synthase residue, which is involved in the Schiff base linkage with pyridoxal 5'-phosphate, from lysine to alanine or histidine abolished enzyme activity in the expressed protein.

5-Aminolevulinate Synthetase↗

PubMed1993

Biphasic ordered induction of heme synthesis in differentiating murine erythroleukemia cells: role of erythroid 5-aminolevulinate synthase.

During dimethyl sulfoxide (DMSO)-stimulated differentiation of murine erythroleukemia (MEL) cells, one of the early events is the induction of the heme biosynthetic pathway. While recent reports have clearly demonstrated that GATA-1 is involved in the induction of erythroid cell-specific forms of 5-aminolevulinate synthase (ALAS-2) and porphobilinogen (PBG) deaminase and that cellular iron status plays a regulatory role for ALAS-2, little is known about regulation of the remainder of the pathway. In the current study, we have made use of a stable MEL cell mutant (MEAN-1) in which ALAS-2 enzyme activity is not induced by DMSO, hexamethylene bisacetamide (HMBA), or butyric acid. In this cell line, addition of 2% DMSO to growing cultures results in the normal induction of PBG deaminase and coproporphyrinogen oxidase but not in the induction of the terminal two enzymes, protoporphyrinogen oxidase and ferrochelatase. These DMSO-treated cells did not produce mRNA for beta-globin and do not terminally differentiate. In addition, the cellular level of ALAS activity declines rapidly after addition of DMSO, indicating that ALAS-1 must turn over rapidly at this time. Addition of 75 microM hemin alone to the cultures did not induce cells to terminally differentiate or induce any of the pathway enzymes. However, the simultaneous addition of 2% DMSO and 75 microM hemin caused the cells to carry out a normal program of terminal erythroid differentiation, including the induction of ferrochelatase and beta-globin. These data suggest that induction of the entire heme biosynthetic pathway is biphasic in nature and that induction of the terminal enzymes may be mediated by the end product of the pathway, heme. We have introduced mouse ALAS-2 cDNA into the ALAS-2 mutant cell line (MEAN-1) under the control of the mouse metallothionein promoter (MEAN-RA). When Cd and Zn are added to cultures of MEAN-RA in the absence of DMSO, ALAS-2 is induced but erythroid differentiation does not occur and cells continue to grow normally. In the presence of metallothionein inducers and DMSO, the MEAN-RA cells induce in a fashion similar to that found with the wild-type 270 MEL cells. Induction of the activities of ALAS, PBG deaminase, coproporphyrinogen oxidase, and ferrochelatase occurs. In cultures of MEAN-RA where ALAS-2 had been induced with Cd plus Zn 24 h prior to DMSO addition, onset of heme synthesis occurs more rapidly than when DMSO and Cd plus Zn are added simultaneously. This study reveals that induction of ALAS-2 alone is not sufficient to induce terminal differentiation of the MEAN-RA cells, and it does not appear that ALAS-2 alone is the rate-limiting enzyme of the heme biosynthetic pathway during MEL cell differentiation.

5-Aminolevulinate Synthetase↗

PubMed1992

Yeast ferrochelatase: expression in a baculovirus system and purification of the expression protein.

The terminal step of the heme biosynthetic pathway is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1). In eukaryotes this enzyme is bound to the inner mitochondrial membrane with its active site facing the matrix side of the membrane. Previously this laboratory has characterized this enzyme via kinetic and protein chemical modification techniques, and with the recent cloning of the enzyme from yeast, mouse, and human sources it now becomes possible to approach structure-function questions by using site-directed mutagenesis. Of primary significance to this is the development of an efficient expression vector. This is of particular significance for ferrochelatase, as it is a low-abundance protein whose DNA coding sequence has a very low codon bias. In the current work we describe the production of yeast ferrochelatase in a baculovirus system. This system is shown to be an excellent one in which to produce large quantities of active ferrochelatase. The expressed enzyme is membrane associated and is not released into the growth medium either during or after virus development and cell lysis. The expressed protein can be purified in a procedure that requires only 1 day and makes use of a Pharmacia Hi Trap blue affinity column. The measured Km's for the substrates mesoporphyrin and iron are the same as those reported previously for the yeast enzyme. To our knowledge this is the first example of a mitochondrial membrane protein that has been expressed in a baculovirus system.

Animals↗

PubMed1992

Characteristics of murine protoporphyrinogen oxidase.

Protoporphyrinogen oxidase (EC 1.3.3.4) (PPO) is the penultimate enzyme of the heme biosynthetic pathway. Mouse PPO has been purified in low yield and kinetically characterized by this laboratory previously. A new more rapid purification procedure is described herein, and with this protein we detect a noncovalently bound flavin moiety. This flavin is present at approximately stoichiometric amounts in the purified enzyme and has been identified by its fluorescence spectrum and high performance liquid chromatography as flavin mononucleotide (FMN). Fluorescence quenching studies on the flavin yielded a Stern-Volmer quenching constant of 12.08 M-1 for iodide and 1.1 M-1 for acrylamide. Quenching of enzyme tryptophan fluorescence resulted in quenching constants of 6 M-1 and 10 M-1 for iodide and acrylamide, respectively. Plasma scans performed on purified enzyme preparations did not reveal the presence of stoichiometric amounts of protein-bound metal ions, and we were unable to detect any protein-associated pyrroloquinoline quinone (PQQ). Data from circular dichroism studies predict a secondary structure of the native protein consisting of 30.5% alpha helix, 40.5% beta sheet, 13.7% turn, and 15.3% random coil. Denaturation of PPO with urea resulted in a biphasic curve when ellipticity is plotted against urea concentration, typical of amphipathic proteins.

Animals↗

PubMed1992

A molecular defect in human protoporphyria.

Protoporphyria is generally an autosomal dominant disease that is characterized clinically by photosensitivity and hepatobiliary disease and that is characterized biochemically by elevated protoporphyrin levels. The enzymatic activity of ferrochelatase, which catalyzes the last step in the heme biosynthetic pathway, is deficient in all tissues of patients with protoporphyria. In this study, sequencing of ferrochelatase cDNAs from a patient with protoporphyria revealed a single point mutation in the cDNAs resulting in the conversion of a Phe(TTC) to a Ser(TCC) in the carboxy-terminal end of the protein, F417S. Further, the human ferrochelatase gene was mapped to chromosome 18q21.3 by chromosomal in situ suppression hybridization. Finally, expression of recombinant ferrochelatase in Escherichia coli demonstrated a marked deficiency in activity of the mutant ferrochelatase protein and of mouse-human mutant ferrochelatase chimeric proteins. Therefore, a point mutation in the coding region of the ferrochelatase gene is the genetic defect in some patients with protoporphyria.

Amino Acid Sequence↗

PubMed1992

Serum-free, defined medium for the growth and differentiation of murine erythroleukemia cells.

Murine erythroleukemia (MEL) cells are frequently employed to study both cell growth and erythroid differentiation. Although these cells are easily cultured and induced to differentiate, they are routinely maintained in a medium that contains 10%-15% fetal bovine serum. Because of the variability between different lots and the cost of serum, it was desirable to define a serum-free medium in which to culture MEL cells. In the present work, a totally serum-free, defined medium is described that supports both normal cell growth and dimethyl sulfoxide induced differentiation in the two MEL cell lines examined (DS-19 and 270). A variety of hormones and biological compounds are examined in this medium to determine their effects on growth and differentiation. This medium does not support the growth of the mouse hepatoma cell line.

Animals↗

PubMed1991

Multiple mechanisms for the regulation of haem synthesis during erythroid cell differentiation. Possible role for coproporphyrinogen oxidase.

Murine erythroleukaemia (MEL) cells are virus-transformed erythroid precursor cells that, when induced to differentiate by dimethyl sulphoxide (DMSO), will initiate haem biosynthesis by the induction and synthesis de novo of all of the enzymes of the haem-biosynthetic pathway. The activities of porphobilinogen (PBG) deaminase (EC 4.3.1.8), coproporphyrinogen oxidase (EC 1.3.3.3), protoporphyrinogen oxidase (EC 1.3.3.4), ferrochelatase (EC 4.99.1.1) and NADH:ferric iron reductase, as well as the synthesis of the enzyme ferrochelatase and the levels of excreted porphyrins, were monitored during DMSO-induced differentiation of MEL cells in culture. The data demonstrate that PBG deaminase and protoporphyrinogen oxidase activities rise rapidly and early, in comparison with ferrochelatase activity, which rises more slowly, and coproporphyrinogen oxidase activity, which decreases by 60% within 24 h of induction before returning to initial levels by 72 h. NADH:ferric iron reductase activity increases slightly, but is always present at levels higher than needed for haem synthesis. Total immunoprecipitable ferrochelatase also rises slowly and parallels the increase in its activity, suggesting that it is not synthesized early in a slowly processed precursor form. Examination of culture media demonstrated that, whereas excretion of protoporphyrin and coproporphyrin occurs within 24 h of induction, coproporphyrin is excreted in amounts 4-15 times greater than protoporphyrin.

Animals↗

PubMed1989

Interaction of free porphyrins and metalloporphyrins with mouse ferrochelatase. A model for the active site of ferrochelatase.

The ability of purified mouse ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) to bind and catalytically utilize a variety of porphyrins has been examined. In all, the kd, Km or Ki values for eleven different porphyrins, the Ki values for four metalloporphyrins and the kd values for two metalloporphyrins were determined. The data obtained demonstrate that mouse ferrochelatase binds a wide variety of porphyrins and metalloporphyrins with kd values ranging from 6 nM for N-methylprotoporphyrin to 1.08 microM for coproporphyrin III. However, the enzyme shows a degree of catalytic specificity for the substituents at the 2.4 positions and utilizes only proto-, hemato-, meso-, deutero-, 2,4-monohydroxy-ethylmonovinyl- and 2,4-monohydroxymethylmonovinyl deuteroporphyrin as substrates. The data show that the magnitude of the kd is not an accurate indicator of the ability of the porphyrin to serve as a substrate or inhibitor and, with the exception of N-methylprotoporphyrin, the size of the kd is several orders of magnitude less than that of the Km or Ki. Of the metalloporphyrins examined (Fe, Co, Zn and Sn) all inhibited ferrochelatase at micromolar concentrations, although tin protoporphyrin was the least effective. These data are discussed in terms of an active site model for mammalian ferrochelatase.

Animals↗

PubMed1988

The synthesis of murine ferrochelatase in vitro and in vivo.

Ferrochelatase (protohaem ferro-lyase, EC 4.99.1.1), the terminal enzyme of the haem-biosynthetic pathway, is an integral membrane protein of the mitochondrial inner membrane. When murine erythroleukaemia cells are labelled in vivo with [35S]methionine, lysed, and the extract is immunoprecipitated with rabbit anti-(mouse ferrochelatase) antibody, a protein of Mr 40,000 is isolated. However, when isolated mouse RNA is translated in a cell-free reticulocyte extract, a protein of Mr 43,000 is isolated. Incubation of this Mr 43,000 protein with isolated mitochondria resulted in processing of the Mr 43,000 precursor to the Mr 40,000 mature-sized protein. Addition of carbonyl cyanide m-chlorophenylhydrazone and/or phenanthroline inhibits this processing. These data indicate that ferrochelatase, like most mitochondrial proteins, is synthesized in the cytoplasm as a larger precursor and is then translocated and processed to a mature-sized protein in an energy-required step.

Animals↗

PubMed1988

Organization of the terminal two enzymes of the heme biosynthetic pathway. Orientation of protoporphyrinogen oxidase and evidence for a membrane complex.

Protoporhyrinogen oxidase (EC 1.3.3.4), the penultimate enzyme of the heme biosynthetic pathway, catalyzes the removal of six hydrogens from protoporphyrinogen IX to form protoporphyrin IX. The enzyme in eukaryotes is associated with the inner mitochondrial membrane. In the present study we have examined requirements for solubilization of this enzyme and find that it behaves as an intrinsic membrane protein that is solubilized only with detergents such as sodium cholate. The in situ orientation of the enzyme with respect to the inner mitochondrial membrane places the active site on the cytosolic face of this membrane rather than the matrix side where the active site of ferrochelatase, the terminal pathway enzyme, is located. Examination of the kinetics of the two terminal enzymes in mitochondrial membranes demonstrates that substrate channeling occurs between these terminal two-pathway enzymes. However, examination of solubilized and membrane-reconstituted enzymes shows no evidence for a stable complex. Based upon these and previous data a model for the terminal three-pathway enzymes is presented.

Animals↗

PubMed1988

Mouse protoporphyrinogen oxidase. Kinetic parameters and demonstration of inhibition by bilirubin.

The penultimate step of haem biosynthesis, the oxidation of protoporphyrinogen to protoporphyrin, was examined with purified murine hepatic protoporphyrinogen oxidase (EC 1.3.3.4) in detergent solution. The kinetic parameters for the two-substrate (protoporphyrinogen and oxygen) reaction were determined. The limiting Km for protoporphyrinogen when oxygen is saturating is 6.6 microM, whereas the Km for oxygen with saturating concentrations of protoporphyrinogen is 125 microM. The kcat. for the overall reaction is 447 h-1. The ratio of kcat. to the Km for protoporphyrinogen is approx. 20-fold greater than the kcat./Km,O2 ratio. The ratio of protoporphyrin formed to dioxygen consumed is 1:3. Ubiquinone-6, ubiquinone-10 and dicoumarol stimulate protoporphyrinogen oxidase activity at low concentrations (less than 15 microM), whereas coenzyme Q0 and menadione show no activation at these concentrations. Above 30 microM, all five quinones inhibit the enzyme activity. FAD does not significantly affect the activity of the enzyme. Bilirubin, a product of haem catabolism, is shown to be a competitive inhibitor of the penultimate enzyme of the haem-biosynthetic pathway, protoporphyrinogen oxidase, with a calculated Ki of 25 microM. The terminal enzyme of haem-biosynthetic pathway, namely ferrochelatase, is not inhibited by bilirubin at concentrations over double the Ki value for the oxidase. In contrast with other enzymic systems, the toxicity of bilirubin is not reversed by binding to albumin.

Animals↗

PubMed1987

Purification and characterization of murine protoporphyrinogen oxidase.

The penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (EC 1.3.3.4), has been purified to apparent homogeneity from mouse liver mitochondria. The purification involves solubilization from mitochondrial membranes with sodium cholate followed by ammonium sulfate fractionation and gel filtration on a Sepharose CL-6B column. The eluate is adjusted to 0.67 M (NH4)2SO4 and loaded onto a phenyl-Sepharose column. After salt washes, the enzyme is eluted with 0.5% sodium cholate and 0.5% Brij 35. The final step is high-pressure ion-exchange chromatography on a DEAE-5PW column. The purified protein has a molecular weight of approximately 65,000 by gel filtration chromatography on Sepharose CL-6B in the presence of 0.5% sodium cholate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a single band corresponding to a molecular weight of 65,000. The absorption spectrum of the purified enzyme shows no evidence of a chromophoric cofactor. Purified protoporphyrinogen oxidase has a Km for protoporphyrinogen IX of 5.6 microM with a Vmax of 2300 nmol mg-1 h-1. It utilizes meso- and hematoporphyrinogen at about 10% the level of protoporphyrinogen. The pH optimum is broad with a maximum at 7.1. There is no stimulation or inhibition by any tested divalent cations, and sulfhydryl reagents have no inhibitory effect on the purified enzyme.

Animals↗

PubMed1987

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.

Animals↗