Binding of periodate-oxidized guanine nucleotides to eukaryotic elongation factor 2.

Periodate-oxidized guanine nucleotides (GTPox and GDPox) were shown to bind stoichiometrically to rat liver elongation factor 2 (EF-2). This binding was quantitatively inhibited in the presence of GTP. After binding, oxidized nucleotides remained on EF-2 despite extensive dialysis. They exchanged, however, with free quanine nucleotides in the course of prolonged (greater than 1 h) incubations. The prior reduction EF-2.GTPox with NaBH4 abolished, to a large extent, this slow exchange. Thus, a Schiff's base was implicated to be formed between EF-2 and oxidized guanine nucleotides. Mg2+ increased the GTPox concentration necessary for a stoichiometric binding to EF-2. EF-2-oxidized nucleotide conjugates bound in the presence of ribosomes a second molecule of GTP (or GTPox). GTPox bound to EF-2 in the presence of ribosomes appeared to exchange readily with free GTP. Moreover, GTPox proved to be active as substrate in EF-2 and ribosome-dependent GTPase reaction: Km values found for GTPox and GTP were 7.7 and 3.4 microM, respectively. The binding of GTPox to EF-2 inhibited only partially the subsequent ribosome-dependent GTP binding, and GTPase reaction or polyphenylalanine (polyPhe) synthesis. On the other hand, the binding of GuoPP[CH2]Pox to EF-2 inhibited all of these reactions strongly. The nature of the binding site involved in the direct interactions of EF-2 with guanine nucleotides is discussed in the light of these results.

Characterization of N-ethylmaleimide-reactive proteins from human tonsillar ribosomes by two-dimensional polyacrylamide gel electrophoresis.

Human tonsillar 80-S ribosomes were 17% and 43% inactivated by 1 mM N-ethylmaleimide after 12 min at 30 or 37 degrees C, respectively. The ribosomes were unaffected by the reagent during the same period of time at 0 or 20 degrees C. 4, 12, 27 and 59 sulfhydryl groups per 80-S ribosomes were found labeled by 1 mM N-ethyl[14C] maleimide after 12 min at 0, 20, 30 or 37 degrees C, respectively. The analysis of radioactively labeled proteins by two-dimensional gel electrophoresis revealed the following: after 3 min at 37 degrees C only two 40-S proteins, S3 and S7, displayed a significant amount of label. After 12 min at 37 degrees C, there was a several-fold increase in the extent of radioactivity found in each of these proteins and, additionally, S1, S2, S4, S5, S15, S22 and S31 were also found among labeled 40-S proteins. S3 appeared to be the most N-ethylmaleimide-reactive 40S protein. After 3 min at 37 degrees C, L10, L17, L20 (and/or S20), L26, L32 and L33, and after 12 min at 37 degrees C, additionally L1, L2, L7, L9, L11, L15, L16, L18, and L25 were labeled among 60-S proteins. l17 and 32 were the most N-ethylmaleimide-reactive proteins under these conditions. After 12 min at 37 degrees C, approx. 26% and 39% of the radioactivity incorporated into the 80 S or 60 S ribosomal protein, respectively, was found in these two proteins. After 12 min at 0 degrees C, S3, L17, L32 and L33 were the only labeled proteins.

Changes in NAD/ADP-ribose metabolism in rectal cancer.

The extent of ADP-ribosylation in rectal cancer was compared to that of the corresponding normal rectal tissue. Twenty rectal tissue fragments were collected during surgery from patients diagnosed as having rectal cancer on the basis of pathology results. The levels of ADP-ribosylation in rectum cancer tissue samples (95.9 +/- 22.1 nmol/ml) was significantly higher than in normal tissues (11.4 +/- 4 nmol/ml). The level of NAD+ glycohydrolase and ADP-ribosyl cyclase activities in rectal cancer and normal tissue samples were measured. Cancer tissues had significantly higher NAD+ glycohydrolase and ADP-ribosyl cyclase activities than the control tissues (43.3 +/- 9.1 vs 29.2 +/- 5.2 and 6.2 +/- 1.6 vs 1.6 +/- 0.4 nmol mg(-1) min(-1)). Approximately 75% of the NAD+ concentration was consumed as substrate in rectal cancer, with changes in NAD+/ADP-ribose metabolism being observed. When [14C]-ADP-ribosylated tissue samples were subjected to SDS-PAGE, autoradiographic analysis revealed that several proteins were ADP-ribosylated in rectum tissue. Notably, the radiolabeling of a 113-kDa protein was remarkably greater than that in control tissues. Poly(ADP)-ribosylation of the 113-kDa protein in rectum cancer tissues might be enhanced with its proliferative activity, and poly(ADP)-ribosylation of the same protein in rectum cancer patients might be an indicator of tumor diagnosis.

Functional groups of elongation factor 2 involved in interactions with guanosine nucleotides and ribosomes.

Treatment of rat liver EF-2 with N-ethylmaleimide (MalNEt) did not affect the direct interactions of the factor with guanine nucleotides or with ribosomes, but inhibited the binding of guanosine 5'-(beta, gamma-methylene)triphosphate (GuoPP(CH2)P) to the EF-2-ribosome complex. The amino group reactive reagent 2,4,6-trinitrobenzenesulfonate (TNBS), however, inhibited specifically the direct interactions of EF-2 with guanine nucleotides, but not the binding of GuoPP(CH2)P to the EF-2-ribosome complex. The different sensitivities of EF-2 to MalNEt and to TNBS suggested that the binding sites involved in the binary vs. ternary complex might correspond to different conformational states or might even be distinct physical entities.

Interactions of eukaryotic elongation factor 2 with actin: a possible link between protein synthetic machinery and cytoskeleton.

Eukaryotic elongation factor 2 (EF-2) was shown to bind to F-actin as assayed by co-sedimentation. In the presence of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) binding was increased fourfold. At saturation level a molar ratio of about 0.12 EF-2 per F-actin (subunit) was observed. Our results suggest a single type of binding site with an apparent dissociation constant of 0.85 microM. The stoichiometry was independent of the filament length, and ADP-ribosylation had no effect on the binding. Experimental data indicated the involvement of SH-groups of both EF-2 and actin in the binding. The interaction EF-2 with F-actin appeared to be inhibited competitively by EF-1 alpha and non-competitively by G-actin.

A translational inhibitor activated in rabbit reticulocyte lysates under high pO2.

An inhibitor of protein synthesis was activated under high oxygen partial pressure (pO2) in hemin-supplemented and glutathione disulfide-free lysates from rabbit reticulocytes. This inhibitor shared some common features with other translational inhibitors from rabbit reticulocytes; that is, hemin-controlled repressor, glutathione disulfide-activated inhibitor and high pressure-activated inhibitor. It caused biphasic kinetics of inhibition which could be potentiated by ATP. Its activation was prevented by cAMP or glucose 6-phosphate. The high pO2-inhibitor could be partially purified from post-ribosomal supernatant containing ribosomal salt wash by precipitation between 0-50% (NH4)2SO4-saturation, Sephadex G-100, and DEAE-cellulose chromatography.

Cu(2+)-mediated complex formation between polyacrylic acid (PAA) and bovine serum albumin.

Cu(2+)-mediated complex formation between polyacrylic acid (PAA) and negatively charged bovine serum albumin (BSA) was studied in neutral water in the presence of Cu2+. Depending on the concentration of Cu2+, the reaction between PAA-Cu2+ complexes and BSA appeared to follow one of two possible paths. At low Cu2+ concentrations (nCu/nAA < 0.15), a further increase in BSA concentration led to the breakdown of the complex as in mechanism I: [formula: see text] At higher Cu2+ concentrations (nCu/nAA > 0.15), a further increase in BSA concentration led to the formation of non-stoichiometric polycomplexes (mechanism II): [formula: see text] The immunogenic properties of ternary mixtures of BSA-Cu(2+)-PAA were investigated and the relationship between immunogenicity and complex formation in solution was analyzed. The addition of Cu2+ to solutions of PAA with BSA gave rise to a considerable increase in BSA-specific immunogenicity. Data obtained from the analysis of the immunogenicity of BSA-Cu(2+)-PAA mixtures formed using different ratios of the components suggested that (1) the highest immunogenic activity is exhibited by stable ternary complexs, and (2) immunoactive polyelectrolyte complexes have a non-stoichiometric composition. We thus propose a novel method, based on Cu2+ mediated complex formation, to enhance protein-specific antibody responses.

Enhancement of the immune response to hepatitis B virus vaccine by antigen specific IgM.

In the present study, modulation of antibody response induced by Hepatitis B virus vaccine-IgM complex was investigated. Purified IgM-type anti HBv monoclonal antibody (1B11) was complexed to commercially available HBv vaccine (GenHevac B Pasteur, France) at varying concentrations of HBsAg (0.5, 1, 1.5 microg of HBsAg) and used to immunize BALB/c mice. An enhanced humoral immune response was obtained with the HBv vaccine-IgM complex at all the doses compared with those immunized by vaccine alone and increased antibody levels were observed with increased concentrations of HBsAg in vaccine formulation. Immunization with HBv vaccine-IgM complex mostly generated IgG-type antibodies in the sera of mice, and also gave rise to the development of hybrid cells which predominantly produced IgG-type monoclonal antibodies. Hence, results from this study indicate that 1B11 can be effectively used to obtain a better immune response to HBv vaccine.

Immune response to progesterone involved in Cu(2+)-mediated polyanion-protein complex--antigen specificity and affinity of hybridoma clones.

The immunogenic properties of water soluble (PAA-Cu(2+)-BSA) and colloidal (PAA-Cu(2+)-BSA.P) polycomplexes were investigated, and the specificity of antibodies produced was analyzed. Polycomplexes containing progesterone appeared to possess a high steroid-specific immunogenic activity. A comparative study of immunogenic properties of polycomplexes versus BSA.P + incomplete Freund's adjuvant (IFA) mixtures revealed differences in regards to the specificity of antibody production. In contrast to the IFA system, polycomplexes were able to generate P- as well as BSA-specific antibodies. Such a response is determined, possibly, by increases in the immunogenicity of weak antigenic determinants on the surface of protein globules and or by the representation of dormant determinants existing in the miner site upon complex formation with polyelectrolytes. Finally, using a short immunization procedure based on use of PAA-Cu(2+)-BSA polycomplexes, we produced seven monoclonal antibodies against progesterone included in polyelectrolyte complexes with affinities Kd ranging between 1.3 x 10 (-5) and 9 x 10(-8) M.

ADP-ribosylation of serum proteins: evaluation as a potential tumor marker.

Serum samples from cancer patients revealed elevated levels of in vitro ADP-ribosylation through non-enzymic binding of ADP-ribose to free acceptor sites on serum proteins. Low concentrations of serum ADP-ribose caused by high NAD glycohydrolase activity together with elevated rates of ADP-ribose transport into erythrocytes appeared to account for under-saturation of the acceptor sites on serum proteins. ADP-ribosylation of serum proteins was assessed as an indicator of cancer disease, and an attempt was made to determine the correlation of ADP-ribosylation levels with carcinoembryonic antigen (CEA) values. Based on positive test results for all tumor patients and negative test results for all healthy controls, sensitivity and specificity of ADP-ribosylation as a tumor indicator were estimated as 67% and 95%, respectively. A close correlation appeared to exist with CEA (r = 0.67; P < 0.001). Similarly, the changes in the levels of ADP-ribosylation correlated with the changes in the levels of CEA during the clinical course (r = 0.58; P < 0.05).

Serum proteins with NAD+ glycohydrolase activity and anti-CD38 reactivity--elevated levels in serum of tumour patients.

NAD+ glycohydrolase activities in serum samples from cancer patients were two- to three-fold higher than the activities in samples from healthy controls. SDS-PAGE analysis of serum samples followed by Western blotting revealed the presence of two proteins of approximately 45 and approximately 21 kDa that were immunoreactive with human CD38-specific monoclonal antibodies T16, HIT2 and OKT 10. These proteins appeared to be more abundant in serum from cancer patients. NAD+ glycohydrolase activity in serum could be enriched by immunoaffinity chromatography by using T16-Sepharose 4B. The results suggest that the relative abundance of proteins immunologically related to CD38 may account for the elevated levels of glycohydrolase activities in serum of tumour patients.

Regional distributions of beta-thalassemia mutations in Turkey.

The distributions of twelve beta-thalassemic mutations in samples (n = 139 chromosomal samples) from four regions of Turkey were determined. The frequencies of these mutations did not reveal a notable region specific heterogeneity. In particular, the four mutations, IVS.1/nt.110(G/A), IVS.1/nt.6(T/C), IVS.1/nt.1(G/A) and nonsense codon.39(C/T), with country-scale frequencies of 35.9%, 21.6%, 13.0% and 7.2%, respectively, were found to be distributed with rather similar frequencies also on a regional scale.

Effects of mitogenic agents upon glucocorticoid action in human tonsillar T-lymphocytes.

The treatment of human tonsillar T-lymphocytes with 4-phorbol 12-myristate 13-acetate (PMA), resulted in about two fold increase in glucocorticoid receptor (GR) number, without any significant change in the receptor affinity. This increase disappeared in the presence of cycloheximide. Alone, PMA and calcium ionophore A23187 did not affect, but together stimulated, like phytohaemagglutinin (PHA), leucine and, in particular, thymidine incorporation. PMA enhanced slightly the stimulatory effect of PHA. Alone, these agents failed to alter the suppressive effect of dexamethasone on thymidine and leucine incorporation; however, PMA-A23187 and PMA-PHA combinations appeared to antagonize the suppression by dexamethasone.

Immune response to 17beta-estradiol involved in polymer gels: antigen specificity and affinity of hybridoma clones.

The immunogenic properties of 17beta-estradiol, immobilized in negatively charged polymer gels, were investigated, and the specificity of antibodies produced was analyzed. The polymer gels developed were composed of a hydrophobic estradiol core surrounded by hydrophilic polyanions as corona. As an immunogen, it was conceived to function via a dual mode, that is as a hapten-delivery system (prolongation effect) and as a polyelectrolyte adjuvant. Polymer gels containing estradiol appeared to possess a high estradiol-specific immunogenicity even without the addition of traditional adjuvants. A comparative study of estradiol trapped in polymer gels versus estradiol conjugated to bovine serum albumin (BSA.E) + Incomplete Freund's Adjuvant (IFA) mixtures revealed similar immunogenic properties in terms of induction of specific antibodies. Following a short immunization procedure based on the use of 17beta-estradiol immobilized in polymer gels, we developed 10 specific monoclonal antibodies with Kd values ranging between 1.2 X 10(-7) and 8 X 10(-8) M.

Immune response to 17 beta-estradiol in polyelectrolyte complex: antigen specificity and affinity of hybridoma clones.

Complex formation between synthetic polyelectrolytes (PE) [poly-4-vinyl-N-ethyl (cetyl), pyridine bromides-PVP(R2, R16)], bovine serum albumin (BSA), or 17 beta-estradiol-BSA conjugate (BSA.E) was studied in neutral water. Weakly water-soluble (colloidal) complex was formed upon addition of BSA.E to PVP (R2, R16) solution at pH 7. A nonrandom distribution of the protein molecules between the coils of polycations and self-assembly in the nonstochiometric polycomplex particles took place. The immunogenic properties of PVP (R2, R16)-BSA.E polycomplex were investigated and the specificities of produced antibodies analyzed. 17 beta-Estradiol introduced in polyelectrolyte complexes (PE-BSA) was found to invoke considerable increases in the steroid-specific immunoresponse. However, a comparative study of immunogenic activity of polycomplexes versus BSA.E+incomplete Freund's adjuvant (IFA) mixtures revealed some differences in regards to the specificity of antibody production. In contrast to IFA+BSA.E systems, polycomplexes were able to generate estradiol-as well as BSA-specific antibodies. Such a carrier-directed response may be determined by increase in immunogenicity of weak antigenic determinants and/or by the exposure of internally located determinants upon complex formation with polyelectrolytes. Fusions following the two different immunization procedures resulted in the growth of comparable numbers of estradiol-specific monoclonal antibodies with apparently similar antigen affinities. Thus, immunizations using antigens in PEC appear to provide an efficient alternative to IFA.

Interactions of adenosine diphosphate-ribosylated elongation factor 2 with ribosomes.

The binding of adenosine diphosphate-ribosylated elongation factor 2 (ADPRib-EF-2) to ribosomes was inhibited both in the presence and absence of GTP in proportion to the amounts of unmodified EF-2 added. Concomitant with this inhibition, an increase in the activity of ribosome-bound EF-2 in polyphenylalanine synthesis was observed. On the other hand, the addition of ADPRib-EF-2 reduced the rate of poly(Phe) synthesis observed in the presence of a saturating amount of EF-2 and increased the amount of EF-2 required for the half-maximal rate of poly(Phe) synthesis. Phe-tRNA, nonenzymatically bound to the ribosome in the presence of poly(U), inhibited the subsequent binding of ADPHRib-EF-2. The same ribosomal population appeared to preferentially bind either aminoacyl-tRNA or ADPRib-EF-2. The Scatchard plot of the binding of ADPRib-EF-2 to the ribosome in the presence of GTP revealed the presence of two ribosomal binding sites (or ribosomal populations) with apparent different affinities for the modified factor (K371 degrees d,1 = 6.6 nM and K37 degrees d,2 = 126 nM). At saturating concentrations of ADPRib-EF-2, a maximum of about 1 molecule of the factor was bound per ribosome. The binding of ADPRib-EF-2 to the ribosome was stimulated by GTP. The binding of radioactive GTP to the ribosome was observed concomitantly with the binding of ADPRib-EF-2. One mole of GTP was bound per mole of ADPRib-EF-2. No significant difference could be found in the binding of GTP to ribosome required in the presence of either EF-2 or ADPRib-EF-2. The binding of ADPRib-EF-2 to the ribosome required the presence of Mg2+ and reached a maximum at 5 mM. The binding was greatest at K+ concentrations below 20 mM. ADPRib-EF-2 was bound primarily to the large ribosomal subunit. A slight, but reproducible binding to the 40 S subunit was also observed. The addition of 40 S to 60 S subunits stimulated the binding of ADPRib-EF-2. GTP displayed a stimulatory effect on the binding only in the presence of recombined subunits. Human ADPRib-EF-2 was bound to rat liver ribosomes as efficiently as to human tonsil ribosomes, while the binding to Escherichia coli ribosomes was insignificant.

Interactions of elongation factor 2 (EF-2) with guanine nucleotides and ribosomes. Binding of periodate-oxidized guanine nucleotides to EF-2.

Interactions of rat liver elongation factor 2 (EF-2) with guanine nucleotides and ribosomes were studied by equilibrium dialysis and sedimentation methods. GDP (Kd = 0.5 microM) or GDP-Mg2+ (Kd = 1.57 microM) displayed a higher affinity in the formation of a binary complex with EF-2 than GTP (Kd = 2.68 microM), GTP-Mg2+ (Kd = 2.77 microM), or guanosine 5'-[beta, gamma-methylene]triphosphate (GuoPP[CH2]P) (Kd = 24.0 microM). NaIO4-oxidized guanine nucleotides (oGDP) (Kd = 38 microM) and oxidized/reduced guanine nucleotides (orGDP) (Kd = 27 microM) had lower affinites to the binding site on EF-2 than those of GDP or GTP. However, the binding of oGDP, oGTP or oGuoPP[CH2]P to EF-2 resulted in the formation of a stable product which could be recovered by the nitrocellulose filter technique or by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In the presence of ribosomes and EF-2 the formation of a new binding site (or a different conformation of the binding site) with a higher affinity for GuoPP[CH2]P-Mg2+ (Kd = 0.26 microM) than fof GDP-Mg2+ (kd = 9.3 microM) became apparent. The presence of ribosomes thus appeared to favor the formation of a complex involving guanosine triphosphates. Adenosine diphosphate ribosylated EF-2 (ADP-Rib-EF-2) in its turn could bind to the ribosome with high affinity even without guanosine nucleotides (Kd = 0.18 microM). GuoPP[CH2]P increased to some extent the affinity of ADP-Rib-EF-2 for its ribosomal binding site (Kd = 0.05 microM).