ADP-ribosyltransferase from beef liver which ADP-ribosylates elongation factor-2

Fragment A of diphtheria toxin and Pseudomonas toxin A intoxicate cells by ADP-ribosylating the diphthamide residue of elongation factor-2 (EF-2) resulting in an inhibition of protein synthesis [1-3]. A cellular enzyme from polyoma virus transformed baby hamster kidney (pyBHK) cells ADP-ribosylates EF-2 in an identical manner [4]. Here we describe a similar cellular enzyme from beef liver which transfers [adenosine-14C]ADP-ribose from NAD to EF-2. The 14C-label can be removed from the EF-2 by snake venom phosphodiesterase as a soluble product which comigrates with AMP on TLC plates, indicating the 14C-label is present on EF-2 as monomeric units of ADP-ribose. Furthermore, the forward transferase reaction catalyzed by the beef liver ADP-ribosyltransferase is reversible by excess diphtheria toxin fragment A, with the formation of 14C-labeled NAD, indicating that both transferases ADP-ribosylate the same site on the diphthamide residue of EF-2. Thus, beef liver and pyBHK mono(ADP-ribosyl)transferases both modify the diphthamide residue of EF-2, in a manner identical to diphtheria toxin fragment A and Pseudomonas toxin A. These results suggest the cellular enzyme is probably ubiquitous among eukaryotic cells.     

Characterization of the endogenous ADP-ribosylation of wild-type and mutant elongation factor 2 in eukaryotic cells.

Anti-[ADP-ribosylated elongation factor 2 (EF-2)] antiserum has been used to immunoprecipitate the modified form of EF-2 from polyoma-virus-transformed baby hamster kidney (pyBHK) cells [Fendrick, J. L. & Iglewski, W. J. (1989) Proc. Natl Acad. Sci. USA 86, 554-557]. This antiserum also immunoprecipitates a 32P-labelled protein of similar size to EF-2 from a variety of primary and continuous cell lines derived from many species of animals. One of these cell lines, chinese hamster ovary CHO-K1 cells was further characterized. The time course of labelling of ADP-ribosylated EF-2 with [32P]orthophosphate was similar in pyBHK cells and in CHO-K1 cells. The kinetics of labelling were more rapid for cells cultured in 2% serum than 10% serum, with incorporation of 32P reaching a maximum at 6 h and 10 h, respectively. EF-2 mutants of pyBHK and CHO-K1 cells resistant to diphtheria-toxin-catalyzed ADP-ribosylation of EF-2 remain sensitive to cellular ADP-ribosylation of EF-2. The 32P-labelled moiety of ADP-ribosylated EF-2 was digested by snake venom phosphodiesterase and the product was identified as AMP. The same 32P-labelled tryptic peptide was modified by toxin in wild-type EF-2 and by the cellular transferase in mutant EF-2. When purified EF-2 from pyBHK cells was incubated with [carbonyl-14C]nicotinamide and diphtheria toxin fragment A, under conditions for reversal of the ADP-ribosylation reaction, [14C]NAD was generated. The results suggest that cellular ADP-ribosylated EF-2 exists in a variety of cell types, and the ribosylated product is identical to that produced by toxin ADP-ribosylation of EF-2, except in diphthamide mutant cells. Studies with the mutant cell lines indicate that the toxin and the cellular transferase, however, recognize different determinants at the ADP-ribose acceptor site in EF-2. The cellular transferase does not require the diphthamide modification of the histidine ring in the amino acid sequence of EF-2 for the transfer of ADP-ribose to the ring. Therefore, we would expect the cellular transferase active site to be similar to, but not identical to, the critical amino acids demonstrated in the active site of diphtheria toxin and Pseudomonas exotoxin A.     

1-N6-Etheno-ADP-ribosylation of elongation factor-2 by diphtheria toxin

Diphtheria toxin fragment A is able to inhibit protein synthesis in the eukaryotic cell by ADP-ribosylating the diphthamide residue of elongation factor-2 (EF-2) [(1980) J. Biol. Chem. 255, 10710-10720]. The reaction requires NAD as ADP-ribose donor. This work reports on the capacity of an NAD analog, the nicotinamide 1-N6-ethenoadenine dinucleotide (epsilon NAD), to be a substrate of diphtheria toxin fragment A in the transferring reaction of the fluorescent moiety, the epsilon ADP-ribose, to the EF-2. As a consequence of the transfer of the epsilon ADP-ribosyl moiety to the EF-2, there is an increase in the emission intensity of the fluorophore and a blue shift in its emission maximum. The epsilon ADP-ribosylated EF-2, like ADP-ribosylated EF-2, retains the capacity to bind GTP and ribosome. The utility of introducing a fluorescent probe in a well defined point of the EF-2 molecule for conformational or binding studies is discussed.     

Identification of the major Mr 100,000 substrate for calmodulin-dependent protein kinase III in mammalian cells as elongation factor-2

The major substrate for Ca2+/calmodulin-dependent protein kinase III in mammalian cells is a species of Mr 100,000 that has a primarily cytoplasmic localization. This substrate has now been identified as elongation factor-2 (EF-2), a protein that catalyzes the translocation of peptidyl-tRNA on the ribosome. The amino acid sequence of 18 residues from the N-terminal of the Mr 100,000 CaM-dependent protein kinase III substrate purified from rat pancreas was found to be identical to the N-terminal sequence of authentic rat EF-2 as previously deduced from nucleic acid sequencing of a cDNA (Kohno, K., Uchida, T., Ohkubo, H., Nakanishi, S., Nakanishi, T., Fukui, T., Ohtsuka, E., Ikehara, M., and Okada, Y. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4978-4982). CaM-dependent protein kinase III phosphorylated EF-2 in vitro with a stoichiometry of approximately 1 mol/mol on a threonine residue. Amino acid sequencing of the purified tryptic phosphopeptide revealed that this threonine residue lies within the sequence: Ala-Gly-Glu-Thr-Arg-Phe-Thr-Asp-Thr-Arg (residues 51-60 of EF-2). The Mr 100,000 protein was stoichiometrically ADP-ribosylated in vitro by the addition of diphtheria toxin and NAD. The Mr 100,000 protein was photoaffinity labeled with a GTP analog and the protein had an endogenous GTPase activity that could be stimulated by the addition of salt-washed ribosomes. These properties are all characteristic of EF-2. Dephospho-EF-2 could support poly(U)-directed polyphenylalanine synthesis in a reconstituted elongation system when combined with EF-1. In the same system, phospho-EF-2 was virtually inactive in supporting polypeptide synthesis; this effect could be reversed by dephosphorylation of phospho-EF-2. These results suggest that intracellular Ca2+ inhibits protein synthesis in mammalian cells via CaM-dependent protein kinase III-catalyzed phosphorylation of EF-2.     

Introduction of Additional Charges as an Aid in Protein Purification: Isolation of Elongation Factor 2 from Sulfolobus acidocaldarius by Preparative Isoelectric Focusing Before and After ADP-Ribosylation

Diphtheria toxin catalyzes the ADP-ribosylation of elongation factor 2 (EF-2) in eukaryotes and archaebacteria. As the reaction is strictly EF-2 specific and introduces two negative charges into the molecule, the resulting shift in the isoelectric point (pI) by 0.2 pH units was used to establish a new purification method for EF-2 from Sulfolobus acidocaldarius. The cells were lysed with dithiothreitol at pH 9 and EF-2 was purified by ammonium sulfate precipitation, gel filtration on Sephadex G-200, and three isoelectric focusing steps. The EF-2-containing fractions from the first isoelectric focusing step at pH 4-9 were refocused in a more narrow pH-gradient (pH 5-7). The EF-2 peak from the second step was eluted, collecting only the fractions above the pH region where ADP-ribosylated EF-2 would focus. The EF-2 was then ADP-ribosylated with diphtheria toxin and NAD and subjected to further isoelectric focusing (pH 5-7). The EF-2 was almost homogeneous since ADP-ribosylation had shifted it into a region of the pH gradient free of contaminating proteins. Diphtheria toxin was immobilized on CNBr-activated Sepharose to prevent a possible contamination by proteins from the diphtheria toxin preparation which might have the same pI as ADP-ribosylated EF-2. Finally, the ADP-ribosyl group was removed by equilibrium dialysis using diphtheria toxin and nicotinamide at pH 6.3. The obtained EF-2 was active in protein synthesis.     

Reduced ribosomal binding of eukaryotic elongation factor 2 following ADP-ribosylation. Difference in binding selectivity between polyribosomes and reconstituted monoribosomes

The biological activity of elongation factor 2 (EF-2) following NAD+ - and diphtheria-toxin-dependent ADP-ribosylation was studied (i) in translation experiments using the reticulocyte lysate system and (ii) in ribosomal binding experiments using either reconstituted empty rat liver ribosomes or programmed reticulocyte polysomes. Treatment of the lysates with toxin and NAD+ at a NAD+/ribosome ratio of 4 resulted in a 90% inhibition of the amino acid incorporation rate. The inhibition was overcome by the addition of native EF-2. At this level of inhibition more than 90% of the EF-2 present in the lysates was ADP-ribosylated and the total ribosome association of EF-2 was reduced by approx. 50%. All of the remaining unmodified factor molecules were associated with the ribosomes, whereas only about 3% of the ribosylated factor was ribosome-associated. The nucleotide requirement for the binding of EF-2 to empty reconstituted rat liver ribosomes and programmed reticulocyte polysomes was studied together with the stability of the resulting EF-2 X ribosome complexes using purified 125I-labelled rat liver EF-2. With both types of ribosomes, the complex formation was strictly nucleotide-dependent. Stable, high-affinity complexes were formed in the presence of the non-hydrolysable GTP analogue guanosine 5'-(beta, gamma-methylene)triphosphate (GuoPP[CH2]P). In contrast to the reconstituted ribosomes, GTP stimulated the formation of high-affinity complexes in the presence of polysomes, albeit at a lower efficiency than GuoPP[CH2]P. The formation of high-affinity complexes was restricted to polysomes in the pretranslocation phase of the elongation cycle. Low-affinity post-translocation complexes, demonstrable after fixation, were formed in the presence of GTP, GuoPP[CH2]P and GDP. In polysomes, these complexes involved a different population of particles than did the high-affinity complexes. In the binding experiments using reconstituted or programmed ribosomes, the pretranslocation binding of EF-2 observed in the presence of GuoPP[CH2]P was reduced by approx. 50% after ADP-ribosylation, whereas the post-translocation binding in the presence of GDP was unaltered. The data indicate that the inhibition of translocation caused by diphtheria toxin and NAD+ is mediated through a reduced affinity of the ADP-ribosylated EF-2 for binding to ribosomes in the pretranslocation state.     

Pseudomonas aeruginosa exotoxin A interaction with eucaryotic elongation factor 2. Role of the His426 residue.

Pseudomonas aeruginosa exotoxin A (ETA) catalyzes the transfer of the ADP-ribose moiety of NAD+ onto eucaryotic elongation factor 2 (EF-2). To study the ETA site of interaction with EF-2, an immobilized EF-2 binding assay was developed. This assay demonstrates that ETA, in the presence of NAD+, binds to immobilized EF-2. Additionally, diphtheria toxin was also found to bind to the immobilized EF-2 in the presence of NAD+. Comparative analysis was performed with a mutated form of ETA (CRM 66) in which a histidine residue at position 426 has been replaced with a tyrosine residue. This immunologically cross-reactive, ADP-ribosyl transferase-deficient toxin does not bind to immobilized EF-2, thus explaining its lack of ADPRT activity. ETA bound to immobilized EF-2 cannot bind the monoclonal antibody TC-1 which specifically recognizes the ETA epitope containing His426. Immunoprecipitation of native ETA by mAb TC-1 is only achieved by incubating ETA in the presence of NAD+. Diethyl pyrocarbonate modification of the His426 residue blocks ETA binding to EF-2 and prevents the binding of the TC-1 antibody. Analogs of NAD+ containing a reduced nicotinamide ring or modified adenine moieties cannot substitute for NAD+ in the immobilized binding assay. Collectively, these data support our proposal that the site of ETA interaction with EF-2 includes His426 and that a molecule of NAD+ is required for stable interaction.     

cAMP-dependent activation of protein synthesis correlates with dephosphorylation of elongation factor 2

The addition of 5 mM cAMP to a cell-free translation system from rabbit reticulocytes increases the rate of protein synthesis 3 5-fold. Lower concentrations of cAMP (0.005, 0.05 and 0.5 mM) have no effect on translation in this system. cAMP at all the concentrations tested stimulates the phosphorylation of the same pattern of polypeptides, while 5 mM cAMP additionally stimulates dephosphorylation of the 95 kDa polypeptide identified as elongation factor 2 (EF-2). Testing of the preparations of EF-2 with a different content of the phosphorylated form in poly(U)-directed poly(Phe) synthesis reveals that the EF-2 activity correlates with the fraction of non-phosphorylated EF-2. Thus cAMP-dependent activation of protein synthesis seems to be due to dephosphorylation of EF-2.     

Protein synthesis and aging: studies with cell-free mammalian systems.

A cell-free system devoid of polysomes, which translates natural mRNA, has been prepared from rat liver. It contains ribosomal subunits, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and protein factors necessary for translation. Protein synthesis required an energy-generating system, mRNA, and 3 mM Mg2+ concentration, and it was inhibited by 7-methylguanylic acid. The total extent and the rate of protein synthesis were approximately 30% greater when the translating system was prepared from livers of 3-month-old rats, as compared to 30-month-old rats. A ribosome-free fraction containing the protein factors required for translation was also prepared from 3-month-old and 30-month-old rat livers and brains, by extraction with 0.5 M KCl. The high-salt extracts were analyzed for elongation factors EF-1 and EF-2 in a poly(U) translating system. Although the activity of EF-2 was similar in preparations from young and old rats, the EF-1 activity in the 3-month-old rat livers and brains was 30 to 40% greater than in 30-month-old animals. The protein synthesizing activity of high salt-washed ribosomes stripped of endogenous peptidyl-tRNA and mRNA, from livers and brains of young and old animals, was the same.     

Identification of an altered elongation factor in temperature-sensitive mutant ts 7'-14 of Saccharomyces cerevisiae

Postpolysomal extracts from wild-type (wt A364A) and temperature-sensitive (ts 7'-14) yeast cells were preincubated for short periods of time at the nonpermissive temperature (37-41 degrees C) prior to incubations for protein synthesis at 20 degrees C. Whereas wt A364A extracts were relatively unaffected by preincubation at the elevated temperature, mutant extracts lost their ability to translate exogenous natural mRNA and poly(U). Phe-tRNA synthetase and ribosomes from ts 7'-14 cells were not inactivated by preincubation at 37-41 degrees C, but a cytosolic component required for chain elongation, as measured by poly(U) translation, was extensively inactivated. The three elongation factors (EF-1, EF-2, and EF-3) required for chain elongation in yeast were resolved chromatographically. Only one factor, EF-3, was able to restore the poly(U)-translational activity of mutant extracts inactivated at the elevated temperature. Heat-inactivated yeast cytosols, which did not support protein synthesis with yeast ribosomes, were perfectly able to translate poly(U) with rat liver ribosomes, which require only EF-1 and EF-2. These and other experiments indicated that the genetically altered component in 7'-14 mutant cells is EF-3.     

Modulation of diphthamide synthesis by 5′-deoxy-5′-methylthioadenosine in murine lymphoma cells

The histidine derivative diphthamide occurs uniquely in eukaryotic elongation factor 2 (EF-2), and is the specific target for the diphtheria toxin mono(ADP-ribosyl)transferase. The first step in diphthamide biosynthesis may involve the transfer of an aminocarboxypropyl moiety from S-adenosylmethionine to the imidazole ring of histidine in EF-2, to yield 2-(3-carboxy-3-aminopropyl)histidine and 5′-deoxy-5′-methylthioadenosine (MeSAdo). As the possible nucleoside product of the initial reaction in the diphthamide biosynthetic pathway, MeSAdo could be an inhibitor of diphthamide formation. In the present experiments, we have analyzed the effects of MeSAdo on diphthamide synthesis in a MeSAdo phosphorylase-deficient mutant murine lymphoma cell line (R1.1, clone H3). As measured by susceptibility to diphtheria toxin-induced ADP-ribosylation, MeSAdo inhibited the formation of diphthamide in EF-2. The inhibition was not due to a nonspecific effect on protein synthesis. Indeed, exogenous MeSAdo substantially protected the lymphoma cells from the lethal effects of diphtheria toxin. These results suggest that MeSAdo can specifically modulate the biosynthesis of diphthamide in EF-2 in murine malignant lymphoma cells.     

Estimating the amounts of ADP-ribosylatable active elongation factor-2 in mammalian cell-free extracts

The content of the elongation factor (EF-2) can be measured by diphtheria toxin-dependent ADP-ribosylation in cell-free extracts of samples prepared from small amounts of tissues and cells containing less than 100 micrograms of total protein. A 20 min in vitro assay, in which a radioactive ADP-ribosyl residue is transferred specifically and 1:1 stoichiometrically to EF-2, is sufficient to estimate the total amounts of ADP-ribosylatable active EF-2. The method is very useful for monitoring changing levels of EF-2 during various pathological and biological processes, including cell cycle, ageing, cancer and other diseases.     

Inhibition of protein synthesis by didemnin B: how EF-1alpha mediates inhibition of translocation

The antineoplastic cyclic depsipeptide didemnin B (DB) inhibits protein synthesis in cells and in vitro. The stage at which DB inhibits protein synthesis in cells is not known, although dehydrodidemnin B arrests translation at the stage of polypeptide elongation. Inhibition of protein synthesis by DB in vitro also occurs at the elongation stage, and it was shown previously that DB prevents EF-2-dependent translocation in partial reaction models of protein synthesis. This inhibition of translocation displays an absolute requirement for EF-1alpha; however, the dependence upon EF-1alpha was previously unexplained. It is shown here that DB binds only weakly to EF-1alpha/GTP in solution, but binds to ribosome. EF-1alpha complexes with a dissociation constant K(d) = 4 microM. Thus, the inhibition of protein synthesis by DB appears to involve an interaction with both EF-1alpha and ribosomes in which all three components are required. Using diphtheria toxin-mediated ADP-ribosylation to assay for EF-2, it is demonstrated that DB blocks EF-2 binding to pre-translocative ribosome.EF-1alpha complexes, thus preventing ribosomal translocation. Based on this model for protein synthesis inhibition by DB, and the proposed mechanism of action of fusidic acid, evidence is presented in support of the Grasmuk model for EF-1alpha function in which this elongation factor does not fully depart the ribosome during polypeptide elongation.     

Modulation of diphthamide synthesis by 5'-deoxy-5'-methylthioadenosine in murine lymphoma cells

The histidine derivative diphthamide occurs uniquely in eukaryotic elongation factor 2 (EF-2), and is the specific target for the diphtheria toxin mono(ADP-ribosyl)transferase. The first step in diphthamide biosynthesis may involve the transfer of aminocarboxypropyl moiety from S-adenosylmethionine to the imidazole ring of histidine in EF-2, to yield 2-(3-carboxy-3-aminopropyl)histidine and 5'-deoxy-5'-methylthioadenosine (MeSAdo). As the possible nucleoside product of the initial reaction in the diphthamide biosynthetic pathway, MeSAdo could be an inhibitor of diphthamide formation. In the present experiments, we have analyzed the effects of MeSAdo on diphthamide synthesis in a MeSAdo phosphorylase-deficient mutant murine lymphoma cell line (R1.1, clone H3). As measured by susceptibility to diphtheria toxin-induced ADP-ribosylation, MeSAdo inhibited the formation of diphthamide in EF-2. The inhibition was not due to a nonspecific effect on protein synthesis. Indeed, exogenous MeSAdo substantially protected the lymphoma cells from the lethal effects of diphtheria toxin. These results suggest that MeSAdo can specifically modulate the biosynthesis of diphthamide in EF-2 in murine malignant lymphoma cells.     

Production of an antiserum specific to the ADP-ribosylated form of elongation factor 2 from archaebacteria and eukaryotes.

An antiserum to ADP-ribosylated elongation factor 2 (ADPR-EF-2) from S. acidocaldarius was raised in rabbits using stained, homogenized, ADPR-EF-2-containing slices from SDS-gels as a source of antigen. Elongation factor 2 (EF-2) from S. acidocaldarius was cloned in E. coli and the expressed gene product was used in order to adsorb all anti-EF-2 antibodies which do not contain the ADP-ribosyl group within their epitopes, as E. coli is unable to synthesize the ADP-ribosyl acceptor diphthamide. The remaining antibodies were specific to ADP-ribosylated EF-2 from Thermoplasma acidophilum, S. acidocaldarius and Desulfurococcus mucosus. ADP-ribosylated EF-2 from eukaryotic sources also reacted with the adsorbed antiserum as shown for EF-2 isolated from the killi-fish Cynolebias whitei, the mouse species BALB/c and Han/Wistar rats. The adsorbed antiserum did not cross-react with ADP-ribosylated actin or rho protein or with FAD-containing D-amino acid oxidase.     

Glucose regulates EF-2 phosphorylation and protein translation by a protein phosphatase-2A-dependent mechanism in INS-1-derived 832/13 cells

The role of elongation factor (EF)-2 phosphorylation in the regulation of pancreatic beta-cell protein synthesis by glucose was investigated in the INS-1-derived cell line 832/13. Incubation of cells in media containing 1 mm glucose resulted in a progressive increase in EF-2 phosphorylation that was maximal by 1-2 h. Readdition of 10 mm glucose promoted a rapid dephosphorylation of EF-2 that was complete in 10 min and maintained over the ensuing 2 h. Similar results were obtained using primary rat islets or Min-6 insulinoma cells. The glucose effect in 832/13 cells was replicated by addition of pyruvate or alpha-ketocaproate, but not 2-deoxyglucose, suggesting that mitochondrial metabolism was required. Accordingly, glucose-mediated dephosphorylation of EF-2 was completely blocked by the mitochondrial respiratory antagonists antimycin A and oligomycin. The hyperglycemic effect was not mimicked by incubation of cells in 100 nm insulin, 30 mm potassium chloride, or 0.25 mm diazoxide, indicating that insulin secretion and/or depolarization of beta cells was not required. The locus of the high glucose effect appeared to be protein phosphatase-2A, the principal phosphatase acting on EF-2. Protein phosphatase-2A activity was stimulated by glucose addition to 832/13 cells, but neither protein phosphatase-1 nor calmodulin kinase III (EF-2 kinase) activity was affected under these conditions. The slower rephosphorylation of EF-2 during the transition from high to low glucose may involve effects on EF-2 kinase activity. Addition of 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside in high glucose led to a marked stimulation of EF-2 phosphorylation, consistent with the possibility that increased AMP kinase activity in low glucose stimulates EF-2 kinase. In parallel with the effects on EF-2 dephosphorylation, addition of high glucose to 832/13 cells markedly increased the incorporation of [(35)S]methionine into total protein. Taken together, these results suggest that modulation of extracellular glucose impacts protein translation rate in beta cells at least in part through regulation of the elongation step, via phosphorylation/dephosphorylation of EF-2.     

Monoclonal antibody specific for yeast elongation factor 3

Hybridomas have been prepared by fusing mouse myeloma (P3 X 63 Ag8) cells with spleen cells of mice immunized with a yeast fraction enriched with respect to non-ribosomal translational components. Cloned hybridoma lines were grown in the form of ascites tumors, and the monoclonal antibodies produced were purified from the ascites fluid by chromatography on DEAE-Affi-Gel Blue. One of the antibodies, from a hybridoma cell line designated as PSH-1, inhibited the translation of natural mRNA and poly(U) and polysomal chain elongation in a cell-free protein-synthesizing system from yeast. Resolution and partial purification of the elongation factors indicated that the monoclonal antibody from PSH-1 did not interact with EF-1 or EF-2 but reacted with and inactivated EF-3, the 125 000 molecular weight additional elongation factor specifically required with yeast ribosomes. The EF-3 purified from the cytosol by immunoaffinity chromatography was comparable to that prepared by ion-exchange chromatography. Evidence was obtained which indicated that EF-3 was essential for the translation of natural mRNA as well as poly(U), was associated with polysomes but not ribosomal subunits, and was required for every cycle in the elongation phase of protein synthesis.     

Inhibition of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, and DNA ligase II by poly(ADP-ribosyl)ation reaction in vitro

Incubation of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, or DNA ligase II in a reconstituted poly(ADP-ribosyl)ating enzyme system markedly suppressed the activity of these enzymes. Components required for poly(ADP-ribose) synthesis including poly(ADP-ribose) polymerase, NAD+, DNA, and Mg2+ were all essential for the observed suppression. Purified poly(ADP-ribose) itself, however, was slightly inhibitory to all of these enzymes. Furthermore, the suppressed activities of DNA polymerase alpha, DNA polymerase beta, and terminal deoxynucleotidyl transferase were largely restored (3 to 4-fold stimulation was observed) by a mild alkaline treatment, a procedure known to hydrolyze alkaline-labile ester linkage between poly(ADP-ribose) and an acceptor protein. All of these results strongly suggest that the four nuclear enzymes were inhibited as a result of poly(ADP-ribosyl)ation of either the enzyme molecule itself or some regulatory proteins of these enzymes.     

Mechanism of elongation factor 2 (EF-2) inactivation upon phosphorylation. Phosphorylated EF-2 is unable to catalyze translocation

Previously we have found that elongation factor 2 (EF-2) from mammalian cells can be phosphorylated by a special Ca2+/calmodulin-dependent protein kinase (EF-2 kinase). Phosphorylation results in complete inactivation of EF-2 in the poly(U)-directed cell-free translation system. However, the partial function of EF-2 affected by phosphorylation remained unknown. Here we show that phosphorylated EF-2, unlike non-phosphorylated EF-2, is unable to switch ribosomes carrying poly(U) and Phe-tRNA in the A site to a puromycin-reactive state. Thus, phosphorylation of EF-2 seems to block its ability to promote a shift of the aminoacyl(peptidyl)-tRNA from the A site to the P site, i.e. translocation itself.