PubMed Health⌕ Search

Biomedical subjects

R Petryshyn

Publications and source records attributed to R Petryshyn.

28 records · Page 2Linked to original sources
PubMed1984

Distribution of reversing factor in reticulocyte lysates during active protein synthesis and on inhibition by heme deprivation or double-stranded RNA.

We have recently shown a direct correlation between protein synthetic activity and the function of reversing factor (RF) as a catalyst of GDP-GTP exchange in whole reticulocyte lysates under normal conditions and on inhibition of protein synthesis by heme deficiency, double-stranded RNA, or oxidized glutathione. In this paper we report that RF is detectable as a nonribosomal complex with eukaryotic initiation factor 2 phosphorylated in its alpha subunit [eIF-2(alpha P)] in whole lysates inhibited by heme deprivation or by double-stranded RNA. The complex contains no unphosphorylated eIF-2 alpha, and the GDP present is freely dissociable. All nonribosomal eIF-2(alpha P) is complexed with RF in fully inhibited lysates; we have not detected free eIF-2(alpha P). RF in this [RF X eIF-2(alpha P)] complex is unavailable to catalyze the release of GDP from eIF-2-GDP. Dephosphorylation of eIF-2(alpha P) present in nonribosomal fractions releases active RF, which is able to carry out its normal guanine nucleotide exchange function.

Animals↗
PubMed1982

Regulation of double-stranded RNA-activated eukaryotic initiation factor 2 alpha kinase by type 2 protein phosphatase in reticulocyte lysates.

Protein synthesis initiation in reticulocyte lysates is inhibited by low concentrations (1-20 ng/ml) of double-stranded RNA (ds RNA) due to the activation of a ds RNA-dependent cAMP-independent protein kinase (ds I) that phosphorylates the alpha subunit of the eukaryotic initiation factor eIF-2. In lysates, ds I is present in the latent inactive form and is associated with the ribosome complement. Latent ds I is solubilized by extraction with high-salt buffers and can be purified in its latent form. Activation of purified latent ds I requires ds RNA and ATP and is accompanied by the ds RNA-dependent autophosphorylation of a polypeptide doublet of 70,000 and 72,000 daltons ("70k/72k"), which represent different phosphorylated states of the same polypeptide. These are phosphorylated in the sequence 70k-->72k; increased phosphorylation of 72k is associated with increased ds I activation. Lysates (or Sepharose 6B ribosomes) treated with ds RNA display a similar ds I phosphoprotein profile, and this is accompanied by the phosphorylation of endogenous eIF-2alpha (38,000 daltons). Delayed (32)P pulses in ds RNA-inhibited lysates indicate that the phosphates on ds I and eIF-2alpha turn over. Under defined conditions, activated ds I in lysates is selectively dephosphorylated by endogenous protein phosphatase(s), and this is accompanied by the dephosphorylation of eIF-2alpha. Similarly, purified activated ds I is rapidly dephosphorylated by unfractionated lysate protein phosphatase(s) and by type 2 protein phosphatase but not by type 1 protein phosphatase. The dephosphorylation of ds I occurs in the sequence 72k-->70k and is correlated with ds I inactivation. The heat-stable protein phosphatase inhibitor-2, which selectively blocks type 1 protein phosphatase, does not significantly affect the dephosphorylation of ds I by type 2 protein phosphatase or by unfractionated lysate phosphatases. The data support the conclusion that a ds I phosphatase activity with type 2 characteristics is involved in the regulation of ds I activity.

Animals↗
PubMed1980

Characterization of double-stranded-RNA-activated kinase that phosphorylates alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) in reticulocyte lysates.

Incubation of reticulocyte lysates with low levels of double-stranded (ds) RNA (1-20 ng/ml) activates a cAMP-independent protein kinase (dsI) that phosphorylates the alpha-subunit (M(r) 38,000) of initiation factor 2 (eIF-2) and produces an inhibition of protein chain initiation similar to that caused by heme deficiency. Activation of dsI from its latent precursor takes place on the ribosomes and requires ATP. dsI can also be activated in ribosomal salt washes and in partially purified preparations of the latent precursor of dsI. In all preparations, activation is accompanied by the ds RNA-dependent phosphorylation of a polypeptide doublet that migrates as bands of 67 and 68.5 kilodaltons (67/68.5) in NaDodSO(4)/acrylamide gels. The rate of phosphorylation of these components in a ribosome salt wash is more rapid than the ds RNA-dependent phosphorylation of eIF-2alpha. Other polypeptides in the salt wash also undergo ds RNA-dependent phosphorylation, but their significance is not clear. All of these phosphorylations are prevented by high concentrations of poly(I).poly(C)(20 mug/ml), but not by an antiserum specific for the heme-regulated eIF-2alpha kinase. Both the latent and activated forms of dsI have been partially purified from a 0.5 M KCl wash of reticulocyte ribosomes. The two species have similar M(r)s ( approximately 120,000) and sedimentation coefficients ( approximately 3.75 S), which suggests that activation of dsI probably does not involve extensive changes. By comparison, the heme-regulated eIF-2alpha kinase has an M(r) of approximately 160,000 and sediments at approximately 6.6 S. However, in vitro, dsI and HRI both phosphorylate the same site(s) of eIF-2alpha. Purified dsI inhibits protein synthesis in hemin-supplemented lysates with the same kinetics induced by the addition of ds RNA; both inhibitions are reversed by eIF-2. dsI that has been activated in the salt wash and then purified does not require ds RNA for expression and no longer displays phosphorylation of the 68.5/67 doublet, which appears to occur only during activation. The data support the view that this component(s) may be the eIF-2alpha kinase activated by ds RNA.

Animals↗
PubMed1979

Regulation of protein synthesis in reticulocyte lysates: immune serum inhibits heme-regulated protein kinase activity and differentiates heme-regulated protein kinase from double-stranded RNA-induced protein kinase.

A specific immune serum to the heme-regulated inhibitor (HRI) has been prepared by immunizing chickens with highly purified reversible HRI prepared from rabbit reticulocyte lysates. Studies with this immune serum demonstrate that the behavior of purified reversible HRI is similar to that of the inhibitor activated in rabbit reticulocyte lysates: the immune serum (i) inhibits the phosphorylation of the small subunit (38,000 daltons) of the eukaryotic initiation factor eIF-2 by both crude and purified inhibitor preparations; (ii) prevents the concomitant inhibition of protein synthesis by both crude and purified inhibitor preparations; and (iii) prevents the autophosphorylation of the 95,000-dalton polypeptide in purified and crude HRI preparations. The protein kinase and inhibitory activities of crude and partially purified preparations of the double-stranded RNA-induced inhibitor of protein synthesis are not affected by the immune serum prepared to reversible HRI. These results indicate that the inhibitor induced by double-stranded RNA is antigenically distinct from the reversible HRI.

Animals↗
PubMed1976

Protein synthesis in dystrophic muscle. Activity of the pH 5 supernatant fraction of muscle in dystrophic mice.

The pH 5 supernatant fractions prepared from homogenates of tissues of normal and dystrophic mice were used to study the incorporation of [14C]phenylalanyl-tRNA into peptide. The incorpoation was markedly reduced using the muscle pH 5 supernatant fraction from dystrophic animals but no reduction was seen with brain, liver or heart preparations from dystrophic mice. The lower incorporation with dystrophic muscle pH 5 supernatant was not due to altered activity of ribonuclease, elongation factors, proteolytic enzymes, GTP or sulfhydryl reagents, but was attributable to the presence of activity that was inhibitory to protein synthesis.

Animals↗