Protein synthesis in rabbit reticulocytes: characteristics of a ribosomal factor that reverses inhibition of protein synthesis in heme-deficient lysates.

Ralston, Robert; Das, Abhimanyu; Dasgupta, Asim; Roy, R.; Palmieri, Steven; Gupta, Naba K.

doi:10.1073/pnas.75.10.4858

Cited by 29 publications

(6 citation statements)

References 34 publications

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“…Data presented in this paper show that eIF-2 and RF, which have been reported to reverse protein synthesis inhibition in heme-deficient reticulocyte lysates (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), possibly reverse inhibition by the same mechanism; in both cases the active components include the eIF-2 a subunit and the 67-kDa polypeptide. The active components in eIF-2 preparations include the 67-kDa polypeptide in addition to the usual three subunits contained in eIF-2.…”

Section: Discussionmentioning

confidence: 86%

Roles of a 67-kDa polypeptide in reversal of protein synthesis inhibition in heme-deficient reticulocyte lysate.

Datta

Chakrabarti²,

Roy³

et al. 1988

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

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During heme deficiency in reticulocyte lysates, the heme-regulated protein synthesis inhibitor, HRI, phosphorylates the a subunit of eukaryotic initiation factor 2 (eIF-2) and thus inhibits protein synthesis. Two factors, eIF-2 and a reticulocyte-lysate supernatant factor that we term RF, reverse this inhibition. We now report the following. (i) An active eIF-2 preparation contained, in addition to the three subunits (a, (3, and y), a 67-kDa polypeptide. Pretreatment of eIF-2 with polyclonal antibodies against either isolated a subunit or 67-kDa polypeptide almost completely inhibited the reversal activity. Upon further fractionation, three-subunit eIF-2 and the 67-kDa polypeptide were resolved. Neither the three-subunit eIF-2 nor the 67-kDa polypeptide alone was active in protein synthesis inhibition reversal. The activity was, however, restored by combining both the three-subunit eIF-2 and the 67-kDa polypeptide. (ii) Active RF preparations contained eIF-2 a (unphosphorylated) and (3 subunits and the 67-kDa polypeptide. As with eIF-2, prior treatment of the RF preparation with antibodies to either the a subunit or the 67-kDa polypeptide almost completely inhibited the reversal activity. The RF preparation devoid of eIF-2 y subunit did not form ternary complex (Met-tRNA;MeteIF-2 GTP). The eIF-2 y subunit in the free form was isolated, and addition of this isolated y subunit to RF promoted significant ternary-complex formation. (iii) Purified HRI efficiently phosphorylated the a subunit in the three subunit eIF-2. However, the extent of such phosphorylation was significantly reduced when eIF-2 containing the 67-kDa polypeptide was used. The 67-kDa polypeptide apparently protected eIF-2 a subunit from HRI-catalyzed phosphorylation but did not inhibit HRI activity. Based on these results, we suggest that the protein synthesis inhibition reversal activity in both eIF-2 and RF is due to the same components-namely, eIF-2 a subunit and the 67-kDa polypeptide. The 67-kDa polypeptide protects eIF-2 a subunit from HRI-catalyzed phosphorylation and may also be a necessary component of the functioning eIF-2 molecule.

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Section: Discussionmentioning

confidence: 86%

Roles of a 67-kDa polypeptide in reversal of protein synthesis inhibition in heme-deficient reticulocyte lysate.

Datta

Chakrabarti²,

Roy³

et al. 1988

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

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“…I shall use the last of these terms, which is the functional definition, in this review. In addition, GEF activity is probably the basis of action of stimulatory activities described in earlier reports as anti-inhibitor [34], ESP [35,36], sRF [37] and Co-eIF2C [38,39].…”

Section: Vol 235mentioning

confidence: 77%

Initiation of protein synthesis in mammalian cells

Pain¹

1986

Biochemical Journal

361

157

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“…Furthermore only the 81 000-M,, 60000-M, and 41 000-M, polypeptides are present both in the 17-S complex and in the anti-inhibitor. It is likely therefore, that the anti-inhibitor can be isolated both free and complexed to eIF-2, which explains the results of other groups [20,23]. However, since the anti-inhibitor could not be prepared completely free of other proteins and since the 17-S complex contained at least two other proteins in addition to the ones mentioned, no unequivocal conclusion about the number and nature of polypeptides of the anti-inhibitor could be reached.…”

Section: Fraction Numbermentioning

confidence: 70%

“…However, Ralston et al [23] have isolated a similar factor (RF) from the ribosomal wash containing polypeptides of approximately the same molecular weight. The anti-inhibitor presumably plays a role in the control of globin synthesis, whereby the rate of protein synthesis is the result of the combined (antagonistic) action of both inhibitor and anti-inhibitor.…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of a Protein Factor that Reverses the Inhibition of Protein Synthesis by the Heme‐Regulated Translational Inhibitor in Rabbit Reticulocyte Lysates

Amesz

Goumans

Haubrich‐Morree

et al. 1979

European Journal of Biochemistry

107

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We have purified and partially characterized a supernatant factor which reverses the effect of the heme‐regulated translational inhibitor on protein synthesis in rabbit reticulocyte lysates. The anti‐inhibitor restores protein synthesis activity in heme‐deficient lysates (and in lysates to which the inhibitor has been added) to the level observed in the presence of heme. The factor has no effect on the phosphorylation of eIF‐2 by the inhibitor nor on any reaction carried out with purified initiation factors. The anti‐inhibitor probably consists of three subunits with molecular weights of 81000, 60000 and 41000. The factor is isolated from the postribosomal supernatant of rabbit reticulocytes both free and complexed to eIF‐2. A possible mechanism of action is discussed.

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Protein synthesis in rabbit reticulocytes: characteristics of a ribosomal factor that reverses inhibition of protein synthesis in heme-deficient lysates.

Abstract: A ribosomal salt (0.5 M KCI) wash factor (RF) that reverses inhibition of protein synthesis in heme-deficient reticulocyte lysates has been resolved from the bulk of Met-

Cited by 29 publications

References 34 publications

Roles of a 67-kDa polypeptide in reversal of protein synthesis inhibition in heme-deficient reticulocyte lysate.

Roles of a 67-kDa polypeptide in reversal of protein synthesis inhibition in heme-deficient reticulocyte lysate.

Initiation of protein synthesis in mammalian cells

Purification and Characterization of a Protein Factor that Reverses the Inhibition of Protein Synthesis by the Heme‐Regulated Translational Inhibitor in Rabbit Reticulocyte Lysates

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