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

1991 ◽  
Vol 275 (2) ◽  
pp. 321-326 ◽  
Author(s):  
L H Conder ◽  
S I Woodard ◽  
H A Dailey
Keyword(s):  
De Novo ◽  
Culture Media ◽  
Reductase Activity ◽  
Erythroid Cell ◽  
Erythroid Precursor ◽  
Protoporphyrinogen Oxidase ◽  
Coproporphyrinogen Oxidase ◽  
Haem Biosynthesis ◽  
Erythroid Cell Differentiation ◽  
Iron Reductase

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.

Terminal steps of haem biosynthesis

2002 ◽  
Vol 30 (4) ◽  
pp. 590-595 ◽  
Author(s):  
H. A. Dailey
Keyword(s):  
Crystal Structure ◽  
Bacillus Subtilis ◽  
Ferrous Iron ◽  
Protoporphyrin Ix ◽  
Oxidative Decarboxylation ◽  
Protoporphyrinogen Oxidase ◽  
Coproporphyrinogen Oxidase ◽  
Cellular Location ◽  
Haem Biosynthesis ◽  
Small Set

The terminal three steps in haem biosynthesis are the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX, followed by the six-electron oxidation of protoporphyrinogen to protoporphyrin IX, and finally the insertion of ferrous iron to form haem. Interestingly, Nature has evolved distinct enzymic machinery to deal with the antepenultimate (co-proporphyrinogen oxidase) and penultimate (protoporphyrinogen oxidase) steps for aerobic compared with anaerobic organisms. The terminal step is catalysed by the enzyme ferrochelatase. This enzyme is clearly conserved with regard to a small set of essential catalytic residues, but varies significantly with regard to size, subunit composition, cellular location and the presence or absence of a [2Fe-2S] cluster. Coproporphyrinogen oxidase and protoporphyrinogen oxidase are reviewed with regard to their enzymic and physical characteristics. Ferrochelatase, which is the best characterized of these three enzymes, will be described with particular emphasis paid to what has been learned from the crystal structure of the Bacillus subtilis and human enzymes.

scholarly journals Examination of mitochondrial protein targeting of haem synthetic enzymes: in vivo identification of three functional haem-responsive motifs in 5-aminolaevulinate synthase

2005 ◽  
Vol 386 (2) ◽  
pp. 381-386 ◽  
Author(s):  
Tamara A. DAILEY ◽  
John H. WOODRUFF ◽  
Harry A. DAILEY
Keyword(s):  
Mammalian Cells ◽  
Protein Targeting ◽  
Mitochondrial Protein ◽  
Leader Sequence ◽  
Erythroid Cell ◽  
Mitochondrial Targeting ◽  
Erythroid Precursor ◽  
Binding Motifs ◽  
Coproporphyrinogen Oxidase

The initial and the terminal three enzymes of the mammalian haem biosynthetic pathway are nuclear encoded, cytoplasmically synthesized and post-translationally translocated into the mitochondrion. The first enzyme, ALAS (5-aminolaevulinate synthase), occurs as an isoenzyme encoded on different chromosomes and is synthesized either as a housekeeping protein (ALAS-1) in all non-erythroid cell types, or only in differentiating erythroid precursor cells (ALAS-2). Both ALAS proteins possess mitochondrial targeting sequences that have putative haem-binding motifs. In the present study, evidence is presented demonstrating that two haem-binding motifs in the leader sequence, as well as one present in the N-terminus of the mature ALAS-1 function in vivo in the haem-regulated translocation of ALAS-1. Coproporphyrinogen oxidase, the antepenultimate pathway enzyme, possesses a leader sequence that is approx. 120 residues long. In contrast with an earlier report suggesting that only 30 residues were required for translocation of the coproporphyrinogen oxidase, we report that the complete leader is necessary for translocation and that this process is not haem-sensitive in vivo. PPO (protoporphyrinogen oxidase) lacks a typical mitochondrial targeting leader sequence and was found to be effectively targeted by just 17 N-terminal residues. Bacillus subtilis PPO, which is very similar to human PPO at its N-terminal end, is not targeted to the mitochondrion when expressed in mammalian cells, demonstrating that the translocation is highly specific with regard to both the length and spacing of charged residues in this targeting region. Ferrochelatase, the terminal enzyme, possesses a typical N-terminal leader sequence and no evidence of a role for the C-terminus was found in mitochondrial targeting.

scholarly journals Changes in globin messenger RNA content during erythroid cell differentiation.

1975 ◽  
Vol 250 (15) ◽  
pp. 6054-6058
Author(s):  
F Ramirez ◽  
R Gambino ◽  
G M Maniatis ◽  
R A Rifkind ◽  
P A Marks ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Messenger Rna ◽  
Erythroid Cell ◽  
Rna Content ◽  
Erythroid Cell Differentiation

scholarly journals Expression of ferrochelatase mRNA in erythroid and non-erythroid cells

1993 ◽  
Vol 292 (2) ◽  
pp. 343-349 ◽  
Author(s):  
R Y Y Chan ◽  
H M Schulman ◽  
P Ponka
Keyword(s):  
Sequence Similarity ◽  
Erythroid Differentiation ◽  
Polyadenylation Signal ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Erythroleukemia Cells ◽  
Haem Biosynthesis ◽  
Polyadenylation Sites ◽  
Dna Fragment ◽  
Polyadenylation Signals

Ferrochelatase, which catalyses the last step in haem biosynthesis, i.e. the insertion of Fe(II) into protophorphyrin IX, is present in all cells, but is particularly abundant in erythroid cells during haemoglobinization. Using mouse ferrochelatase cDNA as a probe two ferrochelatase transcripts, having lengths of 2.9 kb and 2.2 kb, were found in extracts of mouse liver, kidney, brain, muscle and spleen, the 2.9 kb transcript being more abundant in the non-erythroid tissues and the 2.2 kb transcript more predominant in spleen. In mouse erythroleukemia cells the 2.9 kb ferrochelatase transcript is also more abundant; however, following induction of erythroid differentiation by dimethyl sulphoxide there is a preferential increase in the 2.2 kb transcript, which eventually predominates. With mouse reticulocytes, the purest immature erythroid cell population available, over 90% of the total ferrochelatase mRNA is present as the 2.2 kb transcript. Since there is probably only one mouse ferrochelatase gene, the occurrence of two ferrochelatase transcripts could arise from the use of two putative polyadenylation signals in the 3′ region of ferrochelatase DNA. This possibility was explored by using a 389 bp DNA fragment produced by PCR with synthetic oligoprimers having sequence similarity with a region between the polyadenylation sites. This fragment hybridized only to the 2.9 kb ferrochelatase transcript, indicating that the two transcripts differ at their 3′ ends and suggesting that the 2.2 kb transcript results from the utilization of the upstream polyadenylation signal. The preferential utilization of the upstream polyadenylation signal may be an erythroid-specific characteristic of ferrochelatase gene expression.

scholarly journals Visualizing Cancer Cell Metabolic Dynamics Regulated With Aromatic Amino Acids Using DO-SRS and 2PEF Microscopy

2021 ◽  
Vol 8 ◽  
Author(s):  
Pegah Bagheri ◽  
Khang Hoang ◽  
Anthony A. Fung ◽  
Sahran Hussain ◽  
Lingyan Shi
Keyword(s):  
Oxidative Stress ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Spatial Distribution ◽  
Lipid Droplets ◽  
De Novo ◽  
Culture Media ◽  
Aromatic Amino Acids ◽  
Unsaturated Lipids ◽  
Metabolic Activities

Oxidative imbalance plays an essential role in the progression of many diseases that include cancer and neurodegenerative diseases. Aromatic amino acids (AAA) such as phenylalanine and tryptophan have the capability of escalating oxidative stress because of their involvement in the production of Reactive Oxygen Species (ROS). Here, we use D2O (heavy water) probed stimulated Raman scattering microscopy (DO-SRS) and two Photon Excitation Fluorescence (2PEF) microscopy as a multimodal imaging approach to visualize metabolic changes in HeLa cells under excess AAA such as phenylalanine or trytophan in culture media. The cellular spatial distribution of de novo lipogenesis, new protein synthesis, NADH, Flavin, unsaturated lipids, and saturated lipids were all imaged and quantified in this experiment. Our studies reveal ∼10% increase in de novo lipogenesis and the ratio of NADH to flavin, and ∼50% increase of the ratio of unsaturated lipids to saturated lipid in cells treated with excess phenylalanine or trytophan. In contrast, these cells exhibited a decrease in the protein synthesis rate by ∼10% under these AAA treatments. The cellular metabolic activities of these biomolecules are indicators of elevated oxidative stress and mitochondrial dysfunction. Furthermore, 3D reconstruction images of lipid droplets were acquired and quantified to observe their spatial distribution around cells’ nuceli under different AAA culture media. We observed a higher number of lipid droplets in excess AAA conditions. Our study showcases that DO-SRS imaging can be used to quantitatively study how excess AAA regulates metabolic activities of cells with subcellular resolution in situ.

Effects of 2,4-D and Amino Acids on Field Bindweed in Vitro

Weed Science ◽  
1973 ◽  
Vol 21 (2) ◽  
pp. 135-138 ◽  
Author(s):  
R. G. Harvey ◽  
T. J. Muzik
Keyword(s):  
Amino Acids ◽  
Glutamic Acid ◽  
Culture Media ◽  
Reductase Activity ◽  
Convolvulus Arvensis ◽  
Field Bindweed ◽  
Agar Media ◽  
Differential Binding ◽  
Dichlorophenoxy Acetic Acid

Two clones of field bindweed (Convolvulus arvensisL.) which differed in their susceptibility to (2,4-dichlorophenoxy)acetic acid (2,4-D) under field and greenhouse conditions also exhibited similar differences when stem cells were cultured in liquid and agar media. Amino acids added to the culture media altered the response to 2,4-D. Glutamic acid increased the tolerance of the susceptible (S) clone, but reduced the tolerance of the resistant (R) clone. Glutamine increased the susceptibility of the S clone to a much greater degree than it did the R clone. No significant differences were noted in the rates of absorption of metabolism of 2,4-D by the two clones. Glutamine increased and glutamic acid decreased 2,4-D absorption by both clones. Levels of nitrate reductase activity (NRA), soluble protein (SP), and gross RNA (GR) increased in the S tissues but decreased or remained constant in the R tissues exposed to 4.5 × 10−5M 2,4-D. Correlations between 2,4-D susceptibility and NRA demonstrated a relationship between the effects of 2,4-D and nitrogen metabolism. Differential binding of 2,4-D within the cells appears to be the most likely explanation for the differences in response to 2,4-D.

scholarly journals Lipid synthesis in human erythroid cells: the effect of sickling

Blood ◽  
1976 ◽  
Vol 47 (2) ◽  
pp. 189-195 ◽  
Author(s):  
TA Lane ◽  
SK Ballas ◽  
ER Burka
Keyword(s):  
Cell Membrane ◽  
Neutral Lipid ◽  
Sickle Cell Anemia ◽  
De Novo ◽  
Membrane Lipids ◽  
Membrane Damage ◽  
Lipid Synthesis ◽  
Membrane Lipid ◽  
Erythroid Cell ◽  
Cell Membrane Damage

Abstract Human reticulocytes are capable of synthesizing membrane lipids from 14C-glycerol de novo. In both sickle and nonsickle reticulocytes the majority of 14C-glycerol was incorporated into phospholipids, primarily phosphatidylserine and phosphatidylcholine. Incorporation into sphingomyelin was minimal. The most abundant neutral lipid synthesized was triglyceride. In the absence of sickling, the rate of lipid synthesis in sickle reticulocytes was similar to that of nonsickle reticulocytes. With the induction of sickling under anoxic conditions sickle reticulocytes showed a prompt increase in the rate of lipid synthesis to an average of 69% above control values, while nonsickle reticulocytes under similar conditions decreased the rate of lipid synthesis. An increase in the rate of membrane lipid synthesis is associated in the mammalian erythroid cell with cell membrane damage. The findings further confirm that lesions of the erythroid cell membrane in sickle cell anemia are secondary to the sickling process itself.

Control of morphogenetic pathways in thin cell layers of tobacco by pH

1989 ◽  
Vol 67 (3) ◽  
pp. 650-654 ◽  
Author(s):  
A. Cousson ◽  
P. Toubart ◽  
K. Tran Thanh Van
Keyword(s):  
Butyric Acid ◽  
De Novo ◽  
Culture Media ◽  
Callus Formation ◽  
Medium Ph ◽  
Vegetative Buds ◽  
Cell Layers ◽  
Initial Medium ◽  
Vegetative Bud

Thin cell layer explants of tobacco were floated in vitro on the surface of liquid culture media. The initial exogenous concentrations of indolyl-3-butyric acid, and kinetin, the initial medium pH, and the explant density were varied. Various patterns of de novo and direct differentiation without any intermediate callus (flower, vegetative bud, root) as well as the absence of morphogenesis and callus formation without any subsequent organogenesis were separately controlled on 100% of the explants. On the same exogenous combination of glucose, indolyl-3-butyric acid, and kinetin, changes in initial medium pH changed the pattern of morphogenesis. For a given initial exogenous indolyl-3-butyric acid concentration, vegetative buds were obtained at either pH 6.1 or 7.8, whereas a mixture of flowers and vegetative buds was obtained at pH 6.8. Furthermore, changes in explant density changed the morphogenetic response. It is suggested that the effects of the initial medium pH and explant density on morphogenesis may be related partially to modifications of the physicochemical properties of the cell wall and (or) plasmalemma.

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