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At least 19 recordsLinked to original sources
PubMed1978

Control of protein synthesis by hemin. An association between the formation of the hemin-controlled translational repressor and the phosphorylation of a 100 000 molecular weight protein.

The control of protein synthesis by hemin in rabbit reticulocytes is mediated by the formation of a high molecular weight protein inhibitor of polypeptide chain initiation, termed the hemin-controlled translational repressor, from a presynthesized prorepressor. The prorepressor, purified approx. 600-fold, was used to study the mechanism of hemin-controlled translational repressor formation. When the prorepressor is converted to the hemin-controlled translational repressor, either by prolonged warming in the absence of hemin or by incubation with N-ethylmaleimide for 5 min, and then incubated briefly with [gamma-32P]-ATP and Mg2+, a protein that migrates as a 100 000 molecular weight component on sodium dodecyl sulfate-polyacrylamide gels becomes phosphorylated. The extent of phosphorylation of this component is directly proportional to the amount of prorepressor converted to the hemin-controlled translational repressor. In addition, the 100 000 molecular weight protein is not labeled when phosphorylation is attempted with the prorepressor or prorepressor warmed in the presence of hemin, indicating that the protein kinase responsible is probably the hemin-controlled translational repressor. Since the 100 000 molecular protein copurifies with the prorepressor and since the phosphorylation reaction is very rapid (50% complete within 30 s at 34 degrees C), relatively insensitive to dilution, and behaves like an intramolecular reaction, the data suggest that the hemin-controlled translational repressor, once activated, may autophosphorylate a 100 000 molecular weight subunit of itself. Approx. 5 mol phosphate are incorporated per mol of 100 000 molecular weight protein, when the prorepressor is completely converted to the hemin-controlled translational repressor by N-ethylmaleimide. Neither the rate of conversion of prorepressor to the hemin-controlled translational repressor nor the subsequent phosphorylation of the 100 000 molecular weight protein is enhanced by cyclic AMP or reduced by incubation with 3':5'-cyclic nucleotide phosphodiesterase, indicating that cyclic AMP plays no role in hemin-controlled translational repressor formation.

Animals↗
PubMed1978

Regulation of protein synthesis by hemin: effect of dithiothreitol on the formation and activity of the hemin-controlled translational repressor.

Previous studies have demonstrated that the hemin-controlled translational repressor (HCR), a high molecular weight protein inhibitor of polypeptide chain initiation in rabbit reticulocyte lysate, is formed from a presynthesized prorepressor over a period of 12--18 h in three stages denoted reversible, intermediate, and irreversible. The prorepressor can, however, be completely converted to irreversible HCR within 2 min by incubation with such sulfhydryl reagents as N-ethylmaleimide. The results in this report demonstrate that dithiothreitol, which stabilizes thiol groups, will, like hemin, prevent the conversion of the prorepressor to HCR and will inactivate reversible HCR. Unlike hemin, dithiothreitol also inactives the intermediate form of HCR. Neither dithiothreitol nor hemin has any effect on the activity of irreversible HCR. Since the prorepressor used in these experiments had been separated from the supernatant factor (a soluble protein that reverses the inhibition of protein synthesis due to HCR), the effect of dithiothreitol and of hemin is independent of this factor and may be mediated by direct interaction with the prorepressor and HCR. Dithioerythritol, the erythro isomer of dithiothreitol, is as effective as dithiothreitol in preventing the formation of HCR, whereas glutathione and beta-mercaptoethanol have little or no effect.

Animals↗
PubMed1992

Effect of porphyrins and host iron transport proteins on outer membrane protein expression in Porphyromonas (Bacteroides) gingivalis: identification of a novel 26 kDa hemin-repressible surface protein.

Porphyromonas gingivalis is capable of in vitro growth when iron sources are either complexed to hemin or host iron transport proteins, or exist in an inorganic form. This study examined the effect of these iron sources on outer membrane protein (OMP) expression in P. gingivalis W50. Hemin (iron) starved P. gingivalis was transferred into growth medium containing hemin, hemoglobin, hemin-saturated human serum albumin, hemin-free human serum albumin, transferrin, lactoferrin, or inorganic iron. Surface proteins were identified by 125I-labeling and resolved by SDS-PAGE and autoradiography. When grown under hemin starved conditions, P. gingivalis W50 and related strains expressed a major 26 kDa OMP, as revealed by 125I-autoradiography. Autoradiographic analysis demonstrated the absence of this 26 kDa OMP from the P. gingivalis surface in hemin-containing environments. Growth of P. gingivalis W50 in the presence of host iron transport proteins (hemin-free) or inorganic iron resulted in surface expression of a 26 kDa OMP. The presence of protoporphyrin IX or substitution of hemin-associated iron with zinc, resulted in continued surface expression of the 26 kDa OMP, indicating that repressibility of this OMP required the coordination of iron to the protoporphyrin IX molecule (i.e. hemin). A survey of 125I-labeled OMPs from several hemin starved P. gingivalis and related strains, demonstrated that a hemin-repressible 26 kDa OMP occurred only in P. gingivalis. We report here a newly described 26 kDa hemin-regulated surface protein occurring in several strains of P. gingivalis which is expressed on the cell surface in hemin starved conditions and is lost from the cell surface in response to an environment containing iron coordinated specifically to protoporphyrin IX (i.e. hemin).

Bacterial Outer Membrane Proteins↗
PubMed1976

The transport of hemin and protoporphyrin across the plasma membrane of chick embryo liver cells in culture.

Primary monolayer cultures of chick embryo hepatocytes can be cultured in a chemically defined medium (Ham F-12) containing insulin. The absence of serum from the medium permitted a study of the effects of added serum proteins on the transport of hemin and protoporphyrin across the plasma membrane of the hepatocyte. As the criterion of hemin uptake we used its unique and selective activity in repressing the induced synthesis of delta-aminolevulinate synthetase by various chemicals. Movement of hemin into the cells is rapid and does not require added serum proteins. Hemin represses the induced synthesis. The repression by hemin is decreased 50% when the molar ratio of hemin to human serum albumin (6.5 muM) is 1 :2, i.e., where the calculated concentration of dissociated hemin is 10(-8) M. Apparently serum albumin does not enter the cells; it decreases entry of hemin into the cells by virtue of its high affinity for hemin. Compared to human serum albumin, bovine serum albumin and chicken serum albumin, under the same conditions, have a much lower affinity for hemin and scarcely influence the repression effect by hemin. Protoporphyrin can be specifically caused to accumulate in the cytosol, and uroporphyrin in the nucleus of the hepatocytes by the use of different inducers of delta-aminolevulinate synthetase. Protoporphyrin, but not uroporphyrin, is released rapidly from the cells when the moles of human serum albumin added to the medium are 5 times that of porphyrin. This culture system may provide a useful model for studying the mechanism of transport of organic anions across the hepatocyte plasma membrane.

5-Aminolevulinate Synthetase↗
PubMed1978

Hemin control of heme biosynthesis in mouse Friend virus-transformed erythroleukemia cells in culture.

Hemin treatment of mouse Friend virus-transformed cells in cultured caused a dose-dependent increase in hemoglobin synthesis. By the addition of radioactively labeled hemin and by the analysis of the radioactive heme in hemoglobin, only 60 to 70% of heme in the newly synthesized hemoglobin was accounted for by the exogenously added hemin. In keeping with this finding, hemin treatment increased the activity of two enzymes in the heme biosynthetic activity, i.e. delta-aminolevulinate (ALA) dehydratase and uroporphyrinogen-I (URO) synthase in these cells. Incorporation of [2(-14C)]glycine, [14C]ALA, and 59Fe into heme was also significantly increased in the cells treated with hemin, suggesting that essentially all enzyme activities in the heme biosynethetic pathway were increased after hemin treatment. These results indicate that heme in the newly synthesized hemoglobin in hemin-treated Friend cells derives both from hemin added to the culture and from heme synthesized intracellularly. In addition, these results suggest that the stimulation of heme biosynthesis by hemin in Friend virus-transformed cells is in contrast to the hemin repression of heme biosynthesis in liver cells.

Animals↗
PubMed1978

Translational control by hemin is due to binding to cyclic AMP-dependent protein kinase.

Our previous work [Proc. Natl, Acad. Sci. USA (1977) 74, 1463-1467, 3326-3329] is consistent with the view that (a) the hemin-controlled inhibitor of protein synthesis in reticulocyte lysates (active eIF-2 kinase) is formed by phosphorylation of proinhibitor (inactive eIF-2 kinase) catalyzed by cyclic AMP-dependent protein kinase (ATP-protein phosphotransferase; EC 2.7.1.37), and (b) hemin prevents this conversion by blocking the interaction of cyclic AMP with the kinase's regulation subunit, thereby rendering the enzyme inactive. We now show that hemin blocks cyclic AMP binding because it itself binds specifically to the regulatory subunit. This binding is noncompetitive with respect to cyclic AMP. Whereas unlabeled hemin can displace bound [3H]hemin as well as cyclic [3H]AMP, unlabeled cyclic AMP can displace bound cyclic [3H]AMP but not [3H]hemin. This suggests that cyclic AMP and hemin bind to different sites on the protein and that hemin binding affects cyclic AMP binding in an allosteric manner.

Allosteric Regulation↗
PubMed1992

Hemin stimulation of cAMP production in human lymphocytes.

Hemin stimulates cAMP production in human peripheral blood mononuclear cells (PBMC). The kinetics are similar to that of hormone-induced cAMP generation, namely a rapid effect followed by a desensitization phase. Several experimental findings suggest that prostaglandins do not mediate this effect. First, macrophage depleted T and B cells purified by erythrocyte-rosetting were as responsive as unfractionated PBMC to hemin. Second, indomethacin, an inhibitor of prostaglandin synthesis, and meclofenamate, a prostaglandin E2 receptor antagonist, had no effect on hemin stimulated cAMP production. In addition, propranolol, a beta-adrenergic receptor antagonist, had no effect on hemin-stimulated cAMP production. We also examined structural analogues of hemin. Among the metalloporphyrins (Fe, Ni, Co, Zn and Sn) and protoporphyrin IX tested only hemin (Fe-protoporphyrin) was active in stimulating cAMP production. No correlation was found between the ability of metalloporphyrins to stimulate cAMP production and their ability to generate H2O2. The data indicate that hemin stimulates cAMP production by directly affecting lymphocytes and that prostaglandins do not mediate this effect.

Animals↗
PubMed1979

Effect of hemin and Protoporphyrin IX on the protein-synthesizing activity of human granulocytes, lymphocytes and platelets.

The hemin effect on protein synthesis of human granulocytes, lymphocytes and platelets was examined. Hemin added to culture media without serum caused a dose-dependent inhibition of protein synthesis in all three cell types. A cell-specific enhancement of protein-synthesizing capability was observed in 24-hour cultures in the presence of hemin and serum. A marked increase of protein synthesis was found in granulocytes, unchanged in lymphocytes and decreased in platelets. Lymphocytes from patients with chronic lymphatic leukemia (CLL) were moderately inhibited by hemin when incubated in media containing serum, the effect being more pronounced in the presence of freshly disolved doses of hemin. Addition of protoporphyrin IX to cells cultures resulted in a marked suppression of protein synthesis by the three cell types, in all experiments. These results confirm the importance of serum proteins in preventing the inhibitory effects of free hemin and protoporphyrin IX on blood cell protein synthesis. On the other hand, they show a cell-specific enhancement of the protein-synthesizing capacity mediated by hemin.

Blood Platelets↗
PubMed2026

Cryo-EM provides insight into how the Staphylococcus aureus IsdH receptor removes hemin from the hemoglobin:haptoglobin complex.

Staphylococcus aureus extracts hemin from human hemoglobin (Hb) to overcome host-imposed iron limitation. How it recovers Hb-bound hemin from the hemoglobin:haptoglobin (Hb:Hp) complex, the major circulating form of Hb outside red blood cells, remains unclear. Here we use cryo-electron microscopy, biophysical measurements, and solution kinetics to define how the S. aureus IsdH surface receptor extracts hemin from Hb:Hp. A 3.1 Å cryo-EM structure of Hb:Hp bound by full-length IsdH reveals that its N-terminal NEAT domain (N1) anchors it to αHb, whereas its downstream N2N3 extraction unit engages βHb to remove its hemin. The receptor engages Hb:Hp differently than isolated Hb, because N-linked glycans on haptoglobin bias the extraction unit toward βHb, sterically occluding its access to αHb while still permitting engagement by N1. Kinetic assays show that IsdH actively accelerates hemin release from Hb:Hp. Three-dimensional variability analysis indicates that this likely occurs via a dynamic interface in which receptor motions reposition the extraction unit relative to βHb, collectively supporting a model in which IsdH transiently perturbs the F-helix to promote hemin extraction. Alignment of that model with a previously determined CD163:Hb:Hp structure shows how IsdH may disrupt Hb:Hp recognition by macrophage and monocyte CD163 receptors, helping to explain how it may hinder clearance of Hb:Hp from circulation. In aggregate, these results help define the structural basis for hemin extraction from Hb:Hp and how IsdH may subvert receptor-mediated clearance of the Hb:Hp complex.

Journal Article↗
PubMed1976

Induction of globin mRNA accumulation by hemin in cultured erythroleukemic cells.

The role of heme in erythroid development is investigated in erythroleukemic (Friend) cells. Exogenous hemin induces the accumulation of globin mRNA and globin protein in T3-C12 erythroleukemia cells to levels comparable to those induced by polar solvents, such as dimethylsulfoxide (DMSO). The hemin concentration required for maximal induction (10(-4) M) is the same as that which stimulates globin message translation in reticulocytes or cell-free reticulocyte lysates. Hemin and DMSO together cause T3-C12 cells to accumulate 8-9 fold more globin mRNA than either inducer individually. The kinetics of globin mRNA induction in hemin as compared to DMSO are very different: globin message accumulation begins 4 hr after hemin addition, but not until 30--40 hr after DMSO addition. Biliverdin induces 20--40 fold less hemoglobin than hemin; delta-aminolevulinic acid and porphobilinogen do not induce.

Aminolevulinic Acid↗
PubMed1975

Mechanism of hemin inhibition of erythroid cytoplasmic DNA polymerase.

Hemin, which has an important role in the regulation of hemoglobin synthesis, also regulates the activity of cytoplasmic DNA polymerase from erythroid hyperplastic bone marrow cells and reticulocytes. Hemin inhibits DNA synthesis by binding reversibly to the enzyme. Binding assays demonstrated that hemin prevents association and causes dissociation of the DNA-enzyme complex. This is in contrast to inhibitory compounds that specifically interact with DNA such as ethidium bromide and daunomycin which have little or no effect on the DNA polymerase-template complex. Kinetic analysis reveals that hemin inhibition of DNA synthesis is competitive with respect to template and noncompetitive with respect to substrate. The inhibitory effect of hemin can be reversed by subsequent addition of globin, indicating that the inhibition of DNA synthesis by hemin is not due to irreversible inactivation of the enzyme.

Bone Marrow↗
PubMed1979

Studies on rat liver catalase. X. Effect of hemin and an inhibitor on the translation of catalase messenger RNA1.

Rat liver catalase mRNA was translated in a rabbit reticulocyte lysates and wheat germ cell-free system in the presence or absence of hemin and/or a translational inhibitor prepared from reticulocytes, liver cells, and wheat germs. Failure to add hemin to the lysates, or the addition of a hemin-regulated translational inhibitor (HRI) to the hemin-supplemented lysates caused a repressed translation. A preparation of inhibitor from rat liver showed activity similar to that of HRI for this translating system. The translation repression by rat liver inhibitor was reversed by eIF-2 (initiation factor) or GTP, but ATP enhanced the repression. The translation of catalase mRNA in the wheat germ system was not affected by the addition of hemin. An inhibitor prepared from wheat germ extracts, as well as the rat liver inhibitor, markedly decreased the rate of translation. eIF-2, GTP, and ATP behaved in the manner described above. Catalase synthesis in a cell-free system derived from rat liver (using endogenous mRNA) was not influenced by either hemin or the inhibitor. The possibilities are discussed that the synthesis of catalase in liver cells is controlled by a translational inhibitor at the level of chain initiation, and that the formation of the inhibitor from its inactive proinhibitor is regulated by the amount of heme.

Adenosine Triphosphate↗
PubMed1979

Effect of treatment with hemin on rat liver catalase.

Rats were injected with a single or repeated doses of hemin intraperitoneally, and the effect on liver catalase [EC 1.11.1.6] was studied. A single administration of hemin caused a reduction in the concentration of liver catalase, both in enzymatic activity and in catalase protein determined immunochemically. The reduction occurred a few hours after the hemin injection, and is probably due to stimulated degradation. Disappearance of radioactivity from liver catalase prelabelled with [14C]leucine was enhanced following the administration of hemin. No evidence for a repression in vivo incorporation of [14C]leucine and [3H]sigma-aminolevulinic acid into liver catalase was obtained with hemin-treated rats. When the hemin was given repeatedly at 12-h intervals, the level of liver catalase decreased considerably. However, the impairment in catalase-synthesizing activity of liver cells of rats thus treated was rather slight, when examined in a cell-free system. Some differences were noted between the results in the present study and those in previous investigations with Sedormid-treated rats.

Animals↗
PubMed1979

The role of hemin in the regulation of heme synthesis by fetal mouse liver erythroblasts in culture.

The regulatory role of exogenous hemin on the heme synthetic pathway was studied in fetal mouse liver erythroblasts in culture. Hemin added to culture medium of 13th day embryo liver cells inhibited, dose dependently, the incorporation of the porphyrin precursors, 59Fe, 14C-2-glycine and 14C-5-aminolevulinic acid (ALA) by 85%, 70% and 45%, respectively. This suggests a multiple effect of hemin on the porphyrin biosynthetic enzymes. Exogenous ALA competed with 14 C-2-glycine as a porphyrin precursor, but the rate of heme synthesis, measured by 59Fe incorporation, remained unaltered. Protoporphyrin mimicked the hemin effect on the inhibition of glycine incorporation into heme, but reduced iron incorporation by only 20%. Erythroblasts, with an inhibited porphyrin biosynthesis, utilized exogenous 59Fe-hemin for hemoglobin assembly and maintained an undecreased level of hemoglobin synthesis. The results indicate that hemin inhibits the porphyrin biosynthesis in fetal mouse liver erythroblasts mainly at the iron incorporation stage.

Aminolevulinic Acid↗
PubMed1992

Hemin uptake system of Yersinia enterocolitica: similarities with other TonB-dependent systems in gram-negative bacteria.

The hemin receptor HemR of Yersinia enterocolitica was identified as a 78 kDa iron regulated outer membrane protein. Cells devoid of the HemR receptor as well as cells mutated in the tonB gene were unable to take up hemin as an iron source. The hemin uptake operon from Y. enterocolitica was cloned in Escherichia coli K12 and was shown to encode four proteins: HemP (6.5 kDa), HemR (78 kDa), HemS (42 kDa) and HemT (27 kDa). When expressed in E.coli hemA aroB, a plasmid carrying genes for HemP and HemR allowed growth on hemin as a porphyrin source. Presence of genes for HemP, HemR and HemS was necessary to allow E.coli hemA aroB cells to use hemin as an iron source. The nucleotide sequence of the hemR gene and its promoter region was determined and the amino acid sequence of the HemR receptor deduced. HemR has a signal peptide of 28 amino acids and a typical TonB box at its amino-terminus. Upstream of the first gene in the operon (hemP), a well conserved Fur box was found which is in accordance with the iron-regulated expression of HemR.

Alkaline Phosphatase↗