Absence Of Hemin Research Articles

Hemin control of globin synthesis: Effect of a translational repressor on Met-tRNA f binding to the small ribosomal subunit and its relation to the activity and availability of an initiation factor

The block in peptide chain initiation brought about by hemin deficiency of the rabbit reticulocyte lysate system is accompanied by a marked decrease in Met-tRNA f bound to the small ribosomal subunit. Under conditions of hemin supplementation, a similar reduction of Met-tRNA f binding is caused by the addition of the translational repressor which is formed by incubation of the ribosome-free supernatant fraction in the absence of hemin. Both types of inhibition can be reversed by an initiation factor obtained from reticulocyte ribosomes. The activity of a suboptimal concentration of initiation factor in promoting Met-tRNA f binding is incompletely inhibited by a large dose of repressor. In a partial system, where translation is blocked by cycloheximide, the repressor has no effect on Met-tRNA f binding promoted by added initiation factor. Cycloheximide increases the Met-tRNA f bound under conditions of hemin supplementation and also permits its slow accumulation under conditions of hemin deficiency. This is attributed to its action in blocking both translation and the resulting turnover of Met-tRNA f bound to the small ribosomal subunit. No change in the distribution of initiation factor between different ribosome particles was found under inhibited conditions. The results indicate that the hemin-controlled repressor has no direct effect on either the activity or the availability of the initiation factor.

Isolation of bacillus HB-1 from human clinical sources.

Seventeen recent isolates from a variety of human clinical sources of organisms designated as HB-1 according to the subgrouping of King and Tatum11,12 were studied with respect to their morphologic, cultural, and biochemical characteristics, as well as their experimental pathogenicity in mice. These nonfermentative, Gram-negative rods produced striking, dry, flat, radially-spreading colonies consisting of a clear, moist center circled by a highly refractile, speckled, pearl-like zone of growth resembling mercury droplets, surrounded in turn by an outer nonrefractile perimeter of spreading growth. Growth of all HB-1 strains was enhanced by the concomitant presence of X factor (hemin) and Co2, but growth was not influenced by V factor (NAD). However, on brain–heart infusion agar alone, in the absence of hemin, growth was obtained under anaerobiosis which was comparable to that observed on blood and chocolate agar. Biochemically, all strains were nonfermentative, and only oxidase and nitrate were positive. All of the isolates were susceptible to most of the commonly employed antibiotics with the exception of lincomycin and oxacillin. Experimental mouse pathogenicity could not be demonstrated, but microorganisms of the HB-1 group may conceivably possess pathogenic potential, as evidenced by their derivation from human pathologic sources.

Control of globin synthesis by hemin Effect of temperature on globin synthesis by the reticulocyte cell-free system and the activity of translational repressors formed in the absence of hemin

The relationship between temperature and the hemin dependence of globin synthesis by the rabbit reticulocyte cell-free system has been investigated. In the absence of hemin, globin synthesis almost ceases after 5 min at 34 °C but continues at a linear rate for 2 h at 25 °C. If, after 10 or 20 min of incubation at 34 °C in the absence of hemin, the reticulocyte cell-free system is cooled to 25 °C, globin synthesis resumes immediately at the rate of samples incubated at 25 °C from time zero. If the ribosome-free supernatant fraction is incubated without hemin at 25 °C, inhibitory activity which can be inactivated by hemin (reversible inhibitor) appears within 1 h and an inhibitory activity which is insensitive to hemin (irreversible inhibitor) is generated after a 2-h delay. These inhibitors are assayed by addition to fresh cell-free systems incubated at 34 °C. The reversible inhibitor, whether formed at 25 °C or 34 °C, has no effect on fresh cell-free systems when incubated at 25 °C without hemin. The irreversible inhibitor is less inhibitory at 25 °C than at 34 °C. The results suggest that globin synthesis is independent of hemin for 2 h at 25 °C because of the delay in formation of the irreversible inhibitor. Globin synthesis resumes when cell-free systems are first briefly incubated without hemin at 34 °C and then cooled to 25 °C, because the reversible inhibitor formed at 34 °C is ineffective at the lower temperature. If the reversible inhibitor, generated at 34 °C, is cooled for 10 min a either 25 °C or 0 °C, it is still just as active when assayed at 34 °C in the absence of hemin, but it is inactivated more rapidly by hemin at 34 °C. These findings provide additional support for the hypothesis that the hemin-reversible inhibitor is the physiological mediator by which hemin controls globin synthesis in intact reticulocytes and their cell-free systems.

Control of globin synthesis by hemin: Factors influencing formation of an inhibitor of globin chain initiation in reticulocyte lysates

The control of globin synthesis by hemin in rabbit reticulocyte lysates is mediated by the formation of an inhibitor of globin chain initiation. This cytoplasmic repressor does not appear to be a product derived from newly synthesized globin. It is formed in the absence of hemin from a protein in the post-ribosomal fraction without participation of free, low molecular weight components. This proinhibitor and the inhibitor are eluted at the same point on a Sephadex G-200 column, which corresponds to a molecular weight of 3.0 · 10 5. Although the complete transition of proinhibitor to inhibitor in the ribosome-free supernatant fraction takes place over a period of 12 to 15 h, partially purified proinhibitor can be converted to inhibitor within 15 min by reaction with N-ethylmaleimide or o-iodosobenzoate. These findings suggest that inhibitor formation may involve a specific conformational change in the proinhibitor molecule.

Hemin control of globin synthesis: Action of an inhibitor formed in the absence of hemin on the reticulocyte cell-free system and its reversal by a ribosomal factor

The inhibitor of globin synthesis formed by incubation of the ribosome-free supernatant fraction of rabbit reticulocytes may be partially purified by precipitation at pH 5. It is inactivated by incubation with trypsin and is therefore presumed to be a protein. Incubation of a cell-free incorporating system with inhibitor results in disaggregation of the polyribosomes, indicating a block at the site of initiation. There is a lag phase of about 2 min before any effect on the polyribosome profile becomes evident. This suggests either that this time is required for interaction between the inhibitor and a component of the hemoglobin-synthesizing system or that the pool of a component past the block must be depleted before the inhibition becomes evident. The inhibitor blocks the synthesis of the α- and β-chains of hemoglobin equally. It also inhibits the formation of non-hemoglobin proteins, although there is a 10-min delay before inhibition of synthesis of these proteins becomes evident. A factor isolated by KCl extraction of reticulocyte ribosomes and eluted with 0.15 M KCl from a DEAE-cellulose column can overcome the action of the inhibitor. This fraction is required for protein synthesis by a reconstituted system containing the KCl-extracted reticulocyte ribosomes.

Acriflavin resistance in the hemoflagellate, Leishmania tarentolae.

The accumulation, metabolism, and distribution of acriflavin (acr) in two culture strains of Leishmania tarentolae were studied. One strain, reported previously, was sensitive to the dye, i.e. became dyskinetoplastic and could not be subcultured in the presence of 470 ng/ml acr, and one was resistant. Accumulation was studied by fluorescence of the dye within cells and by uptake of acr-(3)H by cells. Metabolism was studied by paper chromatography of aqueous extracts from cells grown with acr-(3)H, and distribution was examined by fluorescence and quantitative electron microscope radioautography. Substances affecting the response to acr included hemin and an acr-sensitizing factor initially obtained from red cells but here shown to be distinct from hemoglobin. In the presence of the sensitizing factor or in the absence of hemin, the resistant strain became dyskinetoplastic and could not be subcultured. Acr fluorescence appeared in the nucleus of the resistant strain, and the percentage of radioautography grains appearing in the nucleus increased. Under these conditions the distribution of radioactivity from chromatographed extracts was altered from the normal in a similar fashion. Because sensitization of the resistant strain is associated with increased amounts of acr in the nucleus, that organelle may be implicated in the mode of action of acr. In general, the two strains behaved alike except for (a) the response to acr, (b) the arginine requirement for optimal growth, and (c) the sensitivity to cycloheximide. Thus, one cannot exclude the wider possibility that acr may act on the cytoplasm and the nucleus as well as on the mitochondrion.

The nature of the inhibition of δ-aminolevulinic acid synthetase by hemin

The feedback inhibition of δ-aminolevulinic acid (ALA) synthetase by hemin 2 2 Hemin is ferric heme and hemichrome is ferric haemochrome; heme and hemochrome unless otherwise specified are used as generic names denoting no particular iron valence state. in cell-free extracts of Rhodopseudomonas spheroides was greatly diminished by the prior conversion of the hemin to the bis-imidazole chelate, suggesting that hemin inhibits by forming, through its iron atom, a coordination complex with the enzyme. This suggestion was further confirmed by the finding that the ability of a number of ligands to diminish hemin inhibition was correlated with their affinities for hemin. Increase in imidazole concentration to approximately 0.1 m partly reversed hemin inhibition of ALA synthetase of R. spheroides but above this concentration imidazole inhibited ALA synthetase even in the absence of hemin. The protection afforded ALA synthetase by albumin against inhibition by hemin was found to be due to nonspecific adsorption of the metalloporphyrin to the albumin. The affinities of a number of ligands for ferrous and ferric protoheme were determined and the extinction coefficients for these hemochromes are reported. In addition, the effect of pH on the affinity of imidazole for ferrous and ferric protoheme is described.

Hemin as a Requirement in the Development In vitro of Hymenolepis microstoma (Cestoda: Cyclophyllidea)

Hymenolepis microstoma developed in continuous axenic culture from juvenile to prepatent adult in a diphasic culture system. Strobilization and maturation occurred only when hemin was added to the overlay of Triple Eagle's Medium or NCTC 135, or when blood was present in the agar base. After 20 days in culture, the average length of the organisms varied from 37 to 52 mm. At this time they contained mature segments often containing preoncospheres. Organisms cultured in the absence of hemin or blood attained a maximum length of 1.6 mm after 4 days in culture and did not progress beyond this point. Preliminary investigations using 14C-labeled hemin suggest that small quantities of this tetrapyrrole are incorporated after 3 days in culture. Hemin and other porphyrin derivatives are commonly found in plants and animals. In most instances, they are synthesized de novo from simple precursors. It has long been noted, however, that some microorganisms and protozoa have strict requirements for hemin or related tetrapyrrole compounds for growth and development. The discovery by Thjotta and Avery that Hemophilus influenzae required a substance associated with blood pigment was one of the earliest reports of a defined bacterial nutrient (see Lascelles, 1962). Other microorganisms, including strains of Staphylococcus aureus, Escherichia coli, Physarum polycephalum, and Bacteroides spp. require hemin or a hemeprotein for growth (Lascelles, 1962). Certain Trypanosomatidae also have a hemin requirement (Lwoff, 1951). Hemin and hemeproteins may play an important role in the development of helminths. Hemoglobin and other porphyrin-containing proteins have been demonstrated in trematodes, nematodes, and in a few cestodes. Recent investigations by Hieb and Stokstad (1970) demonstrated a heme requirement for the in vitro reproduction of the nematode Caenorhabditis briggsae. Complete development of the tetrathyridia of Mesocestoides occurs when hemoglobin is substituted for whole blood in the culture media (Voge and Seidel, 1968). Similar results may be obtained when hemin is Received for publication 17 September 1970. * Supported by Research Grant Number AI 07 332-05, U. S. Public Health Service, NIH, Bethesda, Maryland. added to the media (Seidel and Voge, unpublished). The effect of hemin on the in vitro development of Hymenolepis microstoma will be discussed below. MATERIALS AND METHODS Cysticercoids were dissected from the grain beetle, Tribolium confusum, and transferred into 2-inch petri dishes containing 10 ml of sterile Earle's saline, 900 tLg dihydrostreptomycin sulfate, and 900 units of penicillin. They were transferred into fresh solution every 10 min for a total of 3 washes. Sterile procedures were followed throughout. Cysticercoids were then excysted by Rothman's technique (1959), washed once in culture medium, and inoculated into 150by 25-mm screw cap tubes.

Hemin control of globin synthesis: An assay for the inhibitor formed in the absence of hemin and some characteristics of its formation

In the absence of hemin, an inhibitor of globin chain initiation is formed in the ribosome-free supernatant fraction of rabbit reticulocytes. The inhibitor may be assayed in a hemoglobin-synthesizing system containing the complete lysate and hemin. The potency of a unit of inhibitor is not altered over a broad hemin concentration range. A lag phase in inhibitor formation of two and four hours occurs if the supernatant fraction is incubated at 25 °C and 20 °C respectively. Although hemin almost completely suppresses formation of inhibitor if present from the beginning of an incubation, it only partially suppresses formation if added at the end of the lag phase. This is interpreted as indicating the presence of an intermediate in the conversion of a pro-inhibitor to inhibitor. Hemin blocks the formation of the intermediate but not its conversion to inhibitor. The formation of inhibitor continues for eight to twelve hours at 34 °C but stops in one hour at 50 °C, suggesting the possible participation of a heat labile factor. This is supported by the observation that a mixture of two supernatant fractions, one blocked in inhibitor formation with hemin and the other by heating at 50 °C, can form additional inhibitor at 37 °C.

Induction of cytochrome formation and stimulation of oxidative dissimilation by hemin in Streptococcus lactis and Leuconostoc mesenteroides.

Aerobic glucose dissimilation of washed cells ofStreptococcus lactis grown in peptone-glucose-yeast extract medium is characterized by the formation of large amounts of lactic acid, a small amount of acetic acid, and traces of acetoin: a corresponding amount of oxygen is taken up. Aerobic metabolism by washed cells ofS. lactis andLeuconostoc mesenteroides is far more oxidative when the cells have been grown on peptone-glucose-yeast extract agar supplied with 10 ppm of hemin than when they have been grown in the absence of hemin. In the former case respiration is strongly inhibited by KCN and only slightly by bis(tributylgermanium) oxide, (Bu3Ge)2O. Respiration of cells grown without hemin, on the other hand, is strongly inhibited by (Bu3Ge)2O but only moderately by KCN. In cells grown in the presence of hemin, spectra of ana2- andb-type cytochrome were recognized but not in cells grown without hemin. The NADH-oxidase activity of such cells is not affected by KCN.