Marco Rabinovitz

Control of reticulocyte polyribosome content and hemoglobin synthesis by heme

Abstract Hemin was found to enhance polyribosome stabilization and formation as well as globin synthesis, thus duplicating the previously demonstrated effects of iron salts. Similar enhancing effects on polyribosome and globin production were seen with cobaltous ion, deuterohemin and zinc protoporphyrin. Nickel deuteroporphyrin was partially effective. Little or no effect was noted with other metal cations, metalloporphyrins or protoporphyrin IX. Lead ion, an inhibitor of both heme and globin synthesis in the reticulocyte, caused disaggregation of polyribosomes and marked suppression of protein synthesis. Hemin was more effective than iron in preventing the effect of lead on polyribosomes, but was less able to prevent the inhibition of hemoglobin synthesis. Hemin, but not iron, was shown to protect polyribosomes against subsequent disruption by a high concentration of 2,2′-bipyridine and to reverse an established inhibition by lead. These results indicate that hemin is the intracellular mediator of polyribosome assembly; the metalloporphyrin in some manner induces the aggregation of monomeric ribosomes and, presumably, messenger RNA into functional polyribosomes.

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

Abstract 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.

Actinomycin D: Inhibition of Protein Synthesis Unrelated to Effect on Template RNA Synthesis

Incubation of sarcoma-37 ascites cells in vitro with actinomycin D resulted in inhibition of synthesis of nuclear and cytoplasmic proteins. The overall inhibition could be prevented or relieved by glucose; it is thus unrelated to breakdown of template RNA.

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

Abstract The synthesis of hemoglobin by a reticulocyte lysate is stimulated several fold by the addition of hemin. The ability to respond to hemin is completely lost if the lysate is held for a short time at 34°C, but only slightly reduced if held at 0°C. Hemoglobin synthesis by reconstituted lysates prepared by combinations of ribosomal and supernatant fractions from hemin-responsive and -unresponsive lysates indicated that the factor mediating hemin stimulation is in the supernatant fluid. Addition of supernatant fraction from an unresponsive lysate to an incorporating system containing a responsive lysate markedly decreased the stimulation of globin synthesis by hemin. These results suggest that hemin promotes globin synthesis by preventing the formation of an inhibitor of chain initiation in the cell sap.

Characteristics of the inhibition of hemoglobin synthesis in rabbit reticulocytes by threo-α-amino-β-chlorobutyric acid

Abstract 1. 1. Addition of the valine antagonist, α-amino-β-chlorobutyric acid, to rabbit reticulocytes produces a rapid inhibition of hemoglobin synthesis with a subsequent increase in labelling of protein of the ribosomal fraction. 2. 2. At appropriate antagonist: valine ratios, the incorporation of valine itself occurs at the ribosomal level, but is inhibited in hemoglobin. This indicates that the block in hemoglobin synthesis is caused by substitution of the analogue for valine at specific valine sites, and it is suggested that the loci of these substitutions may be the Val-Val residues on the precursor protein of the β-chain. 3. 3. The antagonist also inhibits [ 14 C]isoleucine incorporation into hemoglobin, but unlike other amino acids, isoleucine does not accumulate in protein of the ribosomal fraction. This observation is discussed in relation to the characteristics of isoleucine incorporation into reticulocyte proteins and the location of residues of this amino acid near the NH 2 -terminal end of the α-chain of rabbit hemoglobin. 4. 4. Synthesis of both the α- and β-chains of hemoglobin is blocked by the antagonist and there is no evidence for the preferential synthesis and release of either chain. 5. 5. Abortive protein formed in the presence of the antagonist is rapidly degraded into trichloroaceeric acid-soluble products.

Amino acid-conferred protection against melphalan--characterization of melphalan transport and correlation of uptake with cytotoxicity in cultured L1210 murine leukemia cells.

Abstract The uptake of melphalan by L1210 cells was reduced to one-sixth of controls by physiological concentrations of the l -isomers of leucine and glutamine, and this decrease was accompanied by a corresponding decrease in cytotoxicity. Cytotoxicity was estimated by treatment of cells with melphalan for 35 min in phosphate-buffered saline containing bovine serum albumin and glucose followed by clonal growth of the surviving cells in soft agar. It was prominent within a critical region of net melphalan uptake of 2–5 pmoles/10 5 cells. Inhibition analysis revealed that at cytotoxic concentrations melphalan is transported by a high-affinity amino acid transport system of the leucine ( l ) type. The values of the Michaelis constants ( K m ) for l -leucine, a protective amino acid, l -valine, a minimally protective amino acid, and melphalan were 6, 58 and 19 μM respectively. These results suggest that the ability of amino acids to protect L1210 cells from melphalan cytotoxicity is related to their affinities for the leucine carrier sites.

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

Abstract 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.

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

Abstract 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.

Inhibition of an initiation codon function by hemin deficiency and the hemin-controlled translational repressor in the reticulocyte cell-free system.

The trinucleotide codon, AUG, promotes the combination of reticulocyte 40S and 60S native ribosomal subunits as well as recombination of those derived by dissociation factor. This interaction is inhibited by hemin deficiency or the hemin-controlled repressor and results in the loss of methionine from ribosomal bound met-tRNAf. The locus of inhibition among the partial reactions of peptide chain initiation is between met-tRNAf and initiation codon binding to the 40S ribosomal subunit and peptide bond formation.

2,9-Dimethyl-1,10-phenanthroline (neocuproine): a potent, copper-dependent cytotoxin with anti-tumor activity

2,9-Dimethyl-1,10-phenanthroline (2,9-DMP), a copper-specific chelator, was a potent cytotoxin against L1210 cells in vitro; its activity was dependent upon available Cu2+ in the medium. Other divalent ions, Fe2+ and Zn2+, were ineffective as promoters of growth inhibition. As the copper chelate, a 4 microM solution produced a 4 log kill after a 1-hr incubation. This was in marked contrast to 1,10-phenanthroline, whose inhibition of cell growth was overcome by added Cu2+, Fe2+ and Zn2+. Cellular uptake of labeled 2,9-dimethyl-1,10-phenanthroline also required added Cu2+ in the medium. This transport was energy dependent, and the drug was concentrated over 200-fold by the cells. In preliminary evaluations, copper-2,9-DMP showed significant chemotherapeutic activity against the P388 murine lymphoma in vivo.