Evidence for an inhibitor in the control of globin synthesis by hemin in a reticulocyte lysate

Maxwell, Charles R.; Rabinovitz, M.

doi:10.1016/0006-291x(69)90485-9

Cited by 100 publications

(18 citation statements)

References 13 publications

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“…(A) the incorporation at 0-10 min; (B), 0-90 min. (11). To distinguish between hemin-sensitive inhibitory activity being formed and an essential factor being reversibly A /~i nactivated, the supernate was warmed and then added to a complete incorporating preparation without hemin.…”

Section: Resultsmentioning

confidence: 99%

“…This hemin-sensitive inhibitory activity appears to be distinct from the irreversible inhibitor (11)(12)(13). The formation of the irreversible inhibitor is prevented by hemin, but, once formed, it is neither inactivated by hemin nor is its action blocked by hemin.…”

Section: Resultsmentioning

confidence: 99%

“…This inhibitor can be inactivated by hemin. When the supernatant fraction is incubated in the absence of hemin for longer periods, an inhibitor, which cannot be reversed by hemin, is formed (11)(12)(13).…”

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confidence: 99%

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Control of Globin Synthesis in Cell-Free Preparations of Reticulocytes by Formation of a Translational Repressor That is Inactivated by Hemin

Gross

Rabinovitz

1972

Proc. Natl. Acad. Sci. U.S.A.

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109

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The control of globin synthesis by hemin in cell-free preparations of rabbit reticulocytes is mediated by an inhibitor (translational repressor) of globin chain initiation that is inactivated by hemin. When the ribosome-free supernatant fraction is warmed at 34°with-out hemin, it quickly acquires the ability to inhibit a fresh cell-free preparation. If the warmed supernatant fraction is further incubated with hemin, its inhibitory activity is lost. This reversible inhibitor, which is observable only during the early period of incubation without hemin, is distinct from an irreversible inhibitor that becomes prevalent during longer incubations. The reversible inhibitor may be an intermediate in the formation of the irreversible inhibitor. The sensitivity of the reversible inhibitor to inactivation by hemin, which permits resumption of globin synthesis in both intact cells and lysates, indicates that the inhibitor is a physiological regulator.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Control of Globin Synthesis in Cell-Free Preparations of Reticulocytes by Formation of a Translational Repressor That is Inactivated by Hemin

Gross

Rabinovitz

1972

Proc. Natl. Acad. Sci. U.S.A.

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109

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“…[14C ]hemoglobin, 4 A&Ci/mg, was prepared by incubating intact rabbit reticulocytes for 24 hr at 30'C in medium (3) containing [14C ]leucine. Preincubated, dialyzed S-30 extracts (1), containing 30-35 mg of ribosomes per ml, were centrifuged at 150,000 X g for 2 hr and the top half of the supernatant solution (S-150) removed.…”

Section: Methodsmentioning

confidence: 99%

Aurintricarboxylic Acid: Inhibitor of Initiation of Protein Synthesis

Stewart

Grollman

Huang

1971

Proc. Natl. Acad. Sci. U.S.A.

112

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Aurintricarboxylic acid prevents the attachment of bacteriophage messenger RNA to ribosomes. As a consequence, initiation of protein synthesis in cellfree extracts prepared from Escherichia coli or rabbit reticulocytes is inhibited at concentrations of dye that do not prevent chain extension. Its properties can be distinguished from other agents that inhibit protein synthesis, including sodium fluoride, cycloheximide, and pactamycin.Aurintricarboxylic acid (ATA) is a triphenylmethane dye that prevents attachment of bacteriophage mRNA to Escherichia coli ribosomes but does not dissociate complexes composed of ribosomes, mRNA, formymethionyl-tRNA, and initiation factors (1). The results reported in the present paper indicate that the primary inhibitory effect of ATA in cell-free extracts prepared from E. coli or rabbit reticulocytes is on the initiation of protein synthesis. Protein synthesis is inhibited at concentrations of dye that show little or no effect on the rate or extent of chain extension; the inhibition is accompanied by sequential release of ribosomes and completed peptides from the polyribosomes. MATERIALS AND METHODSThe preparation and sources of ATA, bacteriophage f2 RNA, and the other materials used in these experiments are described elsewhere (1-3). As prepared, ATA is impure and contains significant quantities of isomeric and inactive materials.[14C ]hemoglobin, 4 A&Ci/mg, was prepared by incubating intact rabbit reticulocytes for 24 hr at 30'C in medium (3) containing [14C ]leucine. Preincubated, dialyzed S-30 extracts (1), containing 30-35 mg of ribosomes per ml, were centrifuged at 150,000 X g for 2 hr and the top half of the supernatant solution (S-150) removed. Reticulocyte lysates were prepared and the synthesis of globin was measured, as described by Maxwell and Rabinovitz (4). Density gradient centrifugation (5), acrylamide gel electrophoresis (6), and the determination of radioactivity in precipitates collected on Millipore membrane filters (3) or on crushed acrylamide gels (6) were performed according to published procedures. RESULTSEffects of ATA on initiation of protein synthesis in extracts of E. coli:The inhibitory eftects of ATA on f2 RNA-directed peptide synthesis in extracts of E. coli are shown in Fig. 1A. If the dye is added prior to the addition of f2 RNA, complete inhibition of peptide synthesis is observed. On the other hand, if the same concentration of ATA is added 10 or 15 min after initiating the reaction, the rate of protein synthesis remains unaffected for several minutes, then decreases rapidly. In contrast to this delayed inhibitory effect of ATA, chloramphenicol, an antibiotic that inhibits chain elongation (7), stops peptide synthesis immediately after addition to the reaction mixture (Fig. 1B).Effects of ATA on the synthesis of phage coat and other proteins The effects of ATA on the synthesis of coat and non-coat proteins are compared in the experiments shown in Fig. 2. Synthesis of coat protein is measured by the incorporation of valine (8); synthesis of...

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“…Sucrose gradient analysis of polysome, ribosome, and subunit aggregation has been extensively used in other studies of reticulocyte lysates (11,19,22,23,55,(65)(66)(67)69,71). In studies on lysates that have been impaired Applied Biochemistrg and Biotechnology Vol.…”

Section: Normal Lysatementioning

confidence: 99%

Protein synthesis in cell-free reticulocyte lysates on multi-hour incubation

Findeis

Whitesides

1987

Appl Biochem Biotechnol

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Cell-free protein synthesis in rabbit reticulocyte lysate translation mixtures has been studied during multi-hour incubations. In an impaired lysate obtained from cells stored at 0'C before lysis, and showing a low initial rate of synthesis, translation could be stimulated by a factor of 4by including RNase inhibitor and additional ATP and GTP. In translation mixtures prepared from normal lysates, protein svnthesis could be improved by -50oh by the addition of excess GTP. The observed increases in protein synthesis were obtained by improved maintenance of the initial rate of synthesis.Index Entries: Cell-free protein synthesis; cell-free translation; reticulocyte lysate, and behavior on multi-hour incubation. INTRODqCTIONThe in vitro, cell-free synthesis of proteins is a much more difficult enterprise than in vivo synthesis using recombinant DNA technology, but it is a technique of real potential utility for the synthesis of proteins containing labeled or unnatural aminoacids, and for synthesis of proteins containing certain types of unnatural amino acid sequences. Chemical methods work well for the synthesis of relatively small peptides ( < 40 amino acid residues in length) but lose efficiency as synthesis of larger products is pursued (1,2). For larger proteins (MW>20,000), chemical synthesis may never be able to rival the combination of rDNA methods coupled with fermentation.

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Evidence for an inhibitor in the control of globin synthesis by hemin in a reticulocyte lysate

Cited by 100 publications

References 13 publications

Control of Globin Synthesis in Cell-Free Preparations of Reticulocytes by Formation of a Translational Repressor That is Inactivated by Hemin

Control of Globin Synthesis in Cell-Free Preparations of Reticulocytes by Formation of a Translational Repressor That is Inactivated by Hemin

Aurintricarboxylic Acid: Inhibitor of Initiation of Protein Synthesis

Protein synthesis in cell-free reticulocyte lysates on multi-hour incubation

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