The surgical treatment of anterior knee pain due to infrapatellar fat pad pathology: A systematic review.

BACKGROUND: Anterior knee pain (AKP) encompasses a range of pathologies. As a result, there are a number of therapeutic options used to treat AKP. The non-operative treatments have been analysed in a number of randomised controlled trials and systematic reviews. There is however a scarcity of such publications covering the surgical management of AKP. There are no systematic reviews that have investigated surgical interventions for AKP due to pathology of the infrapatellar fat pad (IFP). The aims of this study were to review the literature systematically, to establish which surgical procedures have been used to treat IFP disease and to determine their efficacy. METHODS: The review was conducted in accordance with the PRISMA reporting guidelines. A search of the literature was performed on 1st January 2014 using multiple databases including CENTRAL, MEDLINE, EMBASE, PubMed, and Google Scholar. The quality of the studies was assessed using Oxford Evidence-Based Medicine Levels of Evidence guidelines and the GRADE approach. RESULTS: Twenty-four eligible studies were found and included. The critical appraisal identified that the current evidence-base has low methodology quality. The clinical findings indicated that there is a positive trend towards the surgical management of IFP disease for AKP symptoms. Excision of IFP tumours and resection of the IFP in Hoffa's disease can lead to improvements in symptoms and function. CONCLUSIONS: Truly robust evidence to support the surgical management of IFP pathology requires randomised controlled trials; however the expenses involved to design such trials means that they are unlikely to be undertaken for this uncommon disorder. Consequently well-designed and well-reported case series need to be undertaken to improve our current understanding that includes recording quantitative measures such as range of knee motion, VAS Pain scores and a validated scoring system.

Purification and some properties of an oxydative inhibitor in rabbit reticulocyte lysates.

Protein synthesis in rabbit reticulocyte lysates in the presence of heme is inhibited by 50% by the addition of 4 mM GSSG (oxidized glutathione). The incubation of the rabbit reticulocyte lysate with 4 mM GSSG at 30 degrees C for 30 min will cause activation of an inhibitor of protein synthesis which could be purified from the lysates through a five-step procedure. The inhibitor results in a 70-80% inhibition after a 1 h incubation. The inhibitor consists of one polypeptide of 23 kDa apparent molecular weight and is 90% pure as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. However, in the presence of cAMP (10 mM) or GEF (guanine nucleotide exchange factor) (0.3 microgram), protein synthesis in the inhibited reticulocyte lysate will be already recovered.

The interaction of rabbit reticulocyte guanine nucleotide exchange factor eIF-2B with chain initiation factor 2: studies with N-ethylmaleimide and trypsin.

Treatment of eIF-2B and eIF-2 with NEM abolishes nucleotide exchange and GTP-binding activities of the proteins. Incubation of eIF-2B with [14C]NEM results in strong labeling of the 82- and 55-kDa subunits and with less labeling of the other subunits. Preincubation of eIF-2B with eIF-2 interferes with [14C]NEM labeling of the 82- and 55-kDa subunits. All three (alpha, beta, and gamma) subunits of eIF-2 are labeled strongly by [14C]NEM. Limited digestion of eIF-2B with trypsin inhibits nucleotide exchange activity but does not interfere with GTP binding. Under these conditions, the 65-kDa subunit is degraded completely while the other subunits remain intact. Treatment of eIF-2 with trypsin results in the generation of eIF-2 lacking the beta-subunit (eIF-2 alpha gamma). eIF-2(alpha gamma) binds [3H]GDP equally well as intact elf-2. In the presence of elf-2B, the exchange of [3H]GDP for GTP from elf-2. [3H]GDP prepared with eIF-2(alpha gamma) is diminished considerably. [3H]GTP binding to eIF-2(alpha gamma) is also four- to five-fold less than to intact eIF-2. In addition, the association of eIF-2B with intact eIF-2, but not with eIF-2(alpha gamma), reduces by two-fold the rate and extent of removal of 32P by alkaline phosphatase from CK-2-phosphorylated 82-kDa subunit.

Modulation of rabbit reticulocyte guanine nucleotide exchange factor activity by casein kinases 1 and 2 and glycogen synthase kinase 3.

The in vitro phosphorylation of the guanine nucleotide exchange factor (eIF-2B) by casein kinase 2 (CK-2) was previously shown to stimulate the binding of GTP to eIF-2B and increase nucleotide exchange [Singh, L. P., Aroor, A. R., & Wahba, A. J. (1994) Biochemistry 33, 9152-9157]. The present study examines the in vitro phosphorylation of the 82-kDa subunit of eIF-2B by CK-1 and glycogen synthase kinase 3 (GSK-3) and the effects of this covalent modification on nucleotide exchange. Phosphorylation with CK-1 adds approximately 0.27 mol of phosphate/mol of eIF-2B and doubles guanine nucleotide exchange activity. Treatment of the phosphorylated eIF-2B with alkaline phosphatase reduces its activity by a factor of 4, and rephosphorylation with CK-1 (0.49 mol of phosphate/mol of eIF-2B) restores its specific activity to that of the phosphorylated protein. GSK-3 phosphorylates the 82-kDa subunit of both isolated and alkaline phosphatase-treated eIF-2B; however, the stoichiometry of phosphorylation is much less (approximately 0. 12 mol/mol of eIF-2B in both preparations) than that obtained with CK-1 or CK-2. Phosphorylation of eIF-2B with GSK-3 neither stimulates nor inhibits GDP/GTP exchange. The results of this study indicate that phosphorylation of eIF-2B with CK-1 and/or CK-2 is required for GTP binding to the protein. Evidence is also presented for a mechanism of regulation of eIF-2B activity whereby phosphorylation by GSK-3 influences the activity of the protein and partially suppresses phosphorylation by CK-1 or CK-2.

Regulation of protein synthesis in eukaryotic cells by the guanine nucleotide exchange factor and chain initiation factor 2.

In eukaryotes, the guanine nucleotide exchange factor (eIF-2B) is a key protein in the control of polypeptide chain initiation. It catalyzes the exchange of chain initiation factor (eIF)-2-bound GDP for GTP and facilitates the formation of a ternary complex (eIF-2.GTP.Met-tRNAf). The activity of eIF-2B is inhibited indirectly by phosphorylation of the smallest subunit of eIF-2 which sequesters eIF-2B into an inactive eIF-2(alpha P).eIF-2B complex. On the other hand, eIF-2B activity may be regulated directly by covalent modification of its largest subunit with different kinases, such as casein kinase (CK)-I, CK-II and glycogen synthase kinase (GSK)-3. After stimulation of mammalian cells by insulin or growth factors, the allosteric activation of eIF-2B activity by sugar phosphates and inositol phosphates may also provide an important parameter in the regulation of protein synthesis.

Allosteric activation of rabbit reticulocyte guanine nucleotide exchange factor activity by sugar phosphates and inositol phosphates.

Sugar phosphates are required to maintain active rates of translation in gel-filtered rabbit reticulocyte lysates. They may stimulate polypeptide chain initiation by acting as NADPH generators or by a direct interaction with initiation factor(s). We now provide evidence for the allosteric activation of the purified guanine nucleotide exchange factor (eIF-2B) by sugar phosphates and inositol phosphates. In the presence of microM fructose 1,6-bisphosphate, the rate of eIF-2B-catalyzed GDP/GTP exchange is increased approximately 2-fold. The half-maximal concentration for stimulation of eIF-2B activity (SC50) is 57 microM. The binding of GTP to isolated eIF-2B is stimulated 1.5-fold, whereas GTP-binding to ALP-treated eIF-2B is not affected by sugar phosphates. Inositol 1,4-bisphosphate, like fructose 1,6-bisphosphate, stimulates 2-3-fold the activity of the isolated eIF-2B (SC50, 140 microM).

Hexamethylene bisacetamide-induced differentiation of Friend virus-transformed murine erythroleukemia cells is associated with parallel changes in casein kinase II and guanine nucleotide exchange factor activities.

In mammalian cells, the guanine nucleotide exchange factor (GEF, eIF-2B) plays a major role in the regulation of initiation of protein synthesis. It catalyzes the exchange of eukaryotic chain initiation factor (eIF)-2-bound GDP for GTP and facilitates the recycling of eIF-2 during polypeptide chain initiation. We used the Friend virus-transformed murine erythroleukemia (MEL) cell system to elucidate the translational regulatory processes that occur during growth and hexamethylene bisacetamide (HMBA)-induced cell differentiation. GEF activity is increased during growth and decreased during MEL cell differentiation, and this parallels the overall changes in protein synthesis during this period. Inhibition of GEF activity in induced cells may occur indirectly by phosphorylation of the alpha-subunit of eIF-2. However, the decrease in GEF activity in induced cells cannot be reversed by increasing the concentration of eIF-2-GDP added as a substrate in the GEF assay. This is diagnostic for the presence of eIF-2 alpha(P)-GDP in cell lysates and suggests that regulation of GEF activity may occur by one or more mechanisms other than eIF-2(alpha) phosphorylation. We have previously shown that the activity of GEF may be influenced directly by phosphorylation with casein kinase II (CK-II) of the 82-kD subunit of the factor. CK-II activity parallels the changes in GEF activity and the rate of protein synthesis during growth and differentiation of MEL cells. Addition of 1mM spermidine, a stimulator of CK-II but not of purified GEF, in induced MEL cell extracts enhances both CK-II and GEF activities approximately 48 and 32%, respectively. The results presented suggest that the inhibition of protein synthesis during MEL cell differentiation may be linked to the decreased CK-II and GEF activities.

Inhibition of rabbit reticulocyte guanine nucleotide exchange factor activity by heparin and its reversal by polyamines.

We have previously demonstrated that phosphorylation of the 82-kDa subunit of the guanine nucleotide exchange factor, eIF-2B, by casein kinases I and II stimulates eIF-2B activity. In the present report, we show that heparin binds to eIF-2B (KD approximately 0.3 microM) and inhibits the GDP/GTP exchange activity of eIF-2B. The effect is dose dependent, with half-maximal inhibition (IC50) achieved at 0.03 microM heparin. Although spermidine alone does not stimulate or inhibit guanine nucleotide exchange, it reverses the inhibitory effect of heparin up to 90 percent (IC50 = 0.22 mM). ATP and NADPH which interact with the 65 and 55-kDa subunits also partially relieve heparin inhibition of nucleotide exchange, and the effects of ATP and NADPH are additive. By using a gel overlay technique, we demonstrate that 125I-heparin binds to the 82, 65 and 55-kDa subunits of eIF-2B.

The translation efficiency of ovalbumin mRNA is determined in part by a 5'-end hairpin structure.

We have investigated the role in translation of the secondary structure in the noncoding leader (NCL) sequence of ovalbumin mRNA. Deletion of a stem-loop structure from the mRNA 5'-end (hairpin-1) produced a 2.5-fold decrease in mRNA translation rate in both rabbit reticulocyte wheat germ cell-free systems. A corresponding 2-fold reduction in mRNA binding affinity for reticulocyte eucaryotic initiation factor 2 (eIF-2) was also observed. These effects were independent of mRNA capping. Both translation rate and eIF-2 binding affinity were restored by addition to the mRNA of a sequence containing a hairpin-1-like structure. The positive correlation between cell free translation rate and mRNA binding to eIF-2 suggests that hairpin-1 is both an initiation signal and part of a specific eIF-2 recognition site. Methylation interference indicated a direct interaction between eIF-2 and hairpin-1 of ovalbumin mRNA.

Utilization of selenocysteyl-tRNA[Ser]Sec and seryl-tRNA[Ser]Sec in protein synthesis.

The UGA selenocysteine (Sec) codon in glutathione peroxidase mRNA and in selenoprotein P and the UGA stop codon in rabbit beta-globin mRNA were employed to study the utilization of Sec-tRNA[Ser]Sec and Ser-tRNA[Ser]Sec in protein synthesis. In vitro Ser-tRNA[Ser]Sec served as a suppressor of the UGA Sec codon as well as the UGA stop codon, while Sec-tRNA[Ser]Sec did not. However, in vivo Sec-tRNA[Ser]Sec did donate Sec to glutathione peroxidase in Xenopus oocytes microinjected with glutathione peroxidase mRNA and Sec-tRNA. A ribosome binding assay was devised to investigate the interaction of aminoacyl-tRNA, rabbit reticulocyte ribosomes, and eukaryotic elongation factor 1 (eEF-1) in response to the appropriate trinucleoside diphosphate template. Ser-tRNA[Ser]Sec bound weakly to ribosomes in the presence of eEF-1 and UGA as compared to Phe-tRNA, Ser-tRNAIGA, and Met-tRNAm which bound more efficiently in the presence of eEF-1 and the appropriate template. No increase in the binding of Sec-tRNA[Ser]Sec was observed under the same conditions as Ser-tRNA[Ser]Sec. The ribosome binding studies substantiated the finding that Ser-tRNA[Ser]Sec serves as a suppressor of UGA codons in protein synthesis, but Sec-tRNA[Ser]Sec does not. In addition, these studies provide strong evidence that a specific elongation factor is required in mammalian cells for insertion of Sec into protein from Sec-tRNA[Ser]Sec.

Phosphorylation of the guanine nucleotide exchange factor and eukaryotic initiation factor 2 by casein kinase II regulates guanine nucleotide binding and GDP/GTP exchange.

In mammalian cells, chain initiation factor (eIF) 2 and guanine nucleotide exchange factor (GEF) play a major role in the regulation of polypeptide chain initiation. Since guanine nucleotide exchange is the rate-limiting step in the recycling of eIF-2, we examined the effects of phosphorylation of GEF and eIF-2 on guanine nucleotide binding and the rate of GDP/GTP exchange. Phosphorylation of the 82-kDa subunit of GEF in vitro by casein kinase (CK) II results in the stimulation of guanine nucleotide exchange [Dholakia, J. N., & Wahba, A. J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 51-54]. CK-II also phosphorylates the beta-subunit of eIF2, but the significance of this phosphorylation has not previously been investigated. In this study we demonstrate that treatment of CK-II-phosphorylated GEF or eIF-2 with alkaline phosphatase specifically removes more than 85% of the phosphate incorporated into the factors and alters guanine nucleotide binding to these proteins. In the presence of 1 mM Mg2+, the amount of GTP bound to dephosphorylated GEF is reduced 3.8-fold as compared to that of the CK-II-phosphorylated GEF. Rephosphorylation with CK-II restores GTP binding and increases 4-5-fold the activity of GEF in the exchange of eIF-2-bound GDP for free GTP. On the other hand, the extent of GDP binding to dephosphorylated eIF-2 is increased 2.3-fold as compared to that to the isolated eIF-2. The rate of GEF-catalyzed exchange of dephosphorylated eIF-2-bound GDP for GTP is approximately 2-fold slower than that with the isolated eIF-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of rabbit reticulocyte guanine nucleotide exchange factor in vivo. Identification of putative casein kinase II phosphorylation sites.

The guanine nucleotide exchange factor (GEF) is a multi-subunit protein which catalyzes the exchange of GDP for GTP in eukaryotic chain initiation factor 2. Phosphorylation of the 82-kDa subunit of GEF in vitro by casein kinase II (CK-II) is associated with a 5-fold increase in nucleotide exchange activity. However, phosphorylation of GEF in vivo has not been studied, and the kinase(s) that phosphorylate GEF have not been identified. The 82-kDa subunit of GEF was partially sequenced, and a synthetic peptide was used to generate polyclonal anti-peptide antibodies that react specifically with this subunit. To examine the phosphorylation of GEF in intact cells, the protein was isolated and purified extensively from metabolically 32P-labeled rabbit reticulocytes. Only the 82-kDa subunit was found to be phosphorylated, and on Western blots the anti-peptide antisera reacted specifically with the labeled subunit. Phosphoamino acid analysis indicated that phosphorylation occurred exclusively on Ser residues. Digestion with cyanogen bromide of in vivo labeled protein and GEF phosphorylated in vitro by CK-II produced comparable phosphopeptide maps. However, additional phosphopeptide bands were also observed with GEF derived from intact cells. Sequence analysis obtained by Edman degradation of the phosphopeptides was compared with the deduced amino acid sequence of a cloned 82-kDa subunit of GEF [Bushman, J. L., Asuru, A. I., Matts, R. L., & Hinnenbusch, A. G. (1993) Mol. Cell. Biol. 13, 1920-1932]. Putative sites of phosphorylation were identified at Ser 703 and/or 704, which contain the sequence S(P)XXD, a CK-II consensus recognition motif.(ABSTRACT TRUNCATED AT 250 WORDS)

Translational control of eukaryotic gene expression. Role of the guanine nucleotide exchange factor and chain initiation factor-2.

In mammalian cells, the guanine nucleotide exchange factor (GEF or eIF-2B) is a key regulator of polypeptide chain initiation. The exchange of GDP bound to chain initiation factor 2 (eIF-2) for GTP by GEF is a rate limiting step in protein synthesis. The multisubunit characteristics of GEF suggest that this protein is composed of several distinct structural and functional domains, and is regulated by allosteric means and by phosphorylation. The activity of GEF may be regulated indirectly by the phosphorylation state of the smallest subunit of eIF-2 (alpha-subunit). On the other hand, phosphorylation of the largest subunit of GEF (82-kD subunit) by casein kinase (CK) I or II stimulates GDP/GTP exchange. GEF contains NADPH which is required for structural integrity of the protein. Upon stimulation of cells by insulin and growth factors, allosteric activation of GEF by sugar phosphates and other effector molecules may also play an important role in the regulation of polypeptide chain initiation. In this article, recent information about structure-function relationship of eIF-2 and GEF in nucleotide exchange and the regulatory mechanisms that influence the rate of polypeptide chain initiation under various physiological and pathological conditions are presented.

Purification and characterization of sea urchin initiation factor 2. The requirement of guanine nucleotide exchange factor for the release of eukaryotic polypeptide chain initiation factor 2-bound GDP.

Protein synthesis in sea urchin eggs is stimulated dramatically upon fertilization. We previously demonstrated that this stimulation is primarily due to an increase in the rate of polypeptide chain initiation which in turn may be regulated at the level of recycling of eukaryotic initiation factor 2 (eIF-2) (Colin, A. M., Brown, B. D., Dholakia, J. N., Woodley, C. L., Wahba, A. J., and Hille, M. B. (1987) Dev. Biol. 123, 354-363). We have now purified eIF-2 from sea urchin Strongylocentrotus purpuratus blastulae to apparent homogeneity by chromatography on DEAE-cellulose, phosphocellulose, Mono Q, Mono P, and Mono S columns. The factor, which differs from mammalian eIF-2, is composed of three non-identical subunits with apparent molecular weights of 40,000-alpha; 47,000-beta, and 58,000-gamma as estimated by sodium dodecyl-polyacrylamide gel electrophoresis. Antibodies raised against rabbit reticulocyte eIF-2 do not cross-react with sea urchin eIF-2. The binding of Met-tRNA(f) to sea urchin eIF-2 is totally dependent on GTP. A 4-fold stimulation in the rate of protein synthesis in unfertilized sea urchin egg extracts is observed by the addition of 1 micrograms of purified eIF-2. The factor also binds GDP to form a binary (eIF-2.GDP) complex which is stable in the presence of Mg2+. GDP binding to sea urchin eIF-2 inhibits ternary (eIF-2-GTP.[35S]Met-tRNA(f) complex formation. The rabbit reticulocyte guanine nucleotide exchange factor (GEF) catalyzes the exchange of GDP bound to sea urchin eIF-2 for GTP and stimulates ternary complex formation. The requirement of GEF for the recycling of eIF-2 suggests that protein synthesis in sea urchins is similar to that in mammalian systems and may also be regulated at the level of GEF activity. The reticulocyte heme-controlled repressor phosphorylates the alpha-subunit of eIF-2 from both sea urchins and rabbit reticulocytes. However, casein kinase II which phosphorylates the beta-subunit of the reticulocyte factor specifically phosphorylates the alpha-subunit of sea urchin eIF-2. In this respect, the sea urchin factor is similar to eIF-2 isolated from other nonmammalian sources. Since both heme controlled repressor and casein kinase II phosphorylate the alpha-subunit of sea urchin eIF-2 caution should be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in sea urchins.

Photoaffinity labeling of the rabbit reticulocyte guanine nucleotide exchange factor and eukaryotic initiation factor 2 with 8-azidopurine nucleotides. Identification of GTP- and ATP-binding domains.

We have covalently modified rabbit reticulocyte polypeptide chain initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) with the 8-azido analogs of GTP (8-N3GTP) and ATP (8-N3ATP). Of the five subunits of GEF, the Mr 40,000 polypeptide binds 8-[gamma-32P]N3GTP, and the Mr 55,000 and 65,000 polypeptides bind 8-[gamma-32P]N3ATP. Both 8-N3GTP and 8-N3ATP specifically label the beta-subunit of eIF-2. Covalent binding of 8-azidopurine analogs to the eukaryotic initiation factors is dependent on UV irradiation. Binding of 8-N3GTP and 8-N3ATP is specific for the guanine- and adenine-binding sites on the protein, respectively. GDP and GTP, but not ATP, inhibit the photoinsertion of 8-N3GTP to the protein. Similarly, ATP, but not GTP, inhibits the photoinsertion of 8-N3ATP. The inclusion of NADP+ in the reaction mixtures also interferes with the binding of 8-N3ATP to GEF. Mg2+ inhibits the binding of the 8-azido analogs of GTP and ATP to both eIF-2 and GEF, whereas EDTA stimulates the photoinsertion of these nucleotides. Identical results are obtained when the binding of GTP and ATP to these proteins, in the presence of Mg2+ or EDTA, is estimated by nitrocellulose membranes. In enzymatic assays, 8-N3GTP supports the activity of eIF-2 and GEF, indicating that the interaction of 8-N3GTP is catalytically relevant.

Mechanism of the nucleotide exchange reaction in eukaryotic polypeptide chain initiation. Characterization of the guanine nucleotide exchange factor as a GTP-binding protein.

Polypeptide chain initiation in mammalian systems is regulated at the level of the guanine nucleotide exchange factor (GEF). This multisubunit protein catalyzes the exchange of GDP bound to eukaryotic initiation factor 2 (eIF-2) for GTP. Although various models have been proposed for its mode of action, the exact sequence of events involved in nucleotide exchange is still uncertain. We have studied this reaction by three different experimental techniques: (a) membrane filtration assays to measure the release of [3H]GDP from the eIF-2.[3H]GDP binary complex, (b) changes in the steady-state polarization of fluorescamine-GDP during the nucleotide exchange reaction, and (c) sucrose gradient analysis of the total reaction. The results obtained do not support the reaction as written: eIF-2.GDP + GEF in equilibrium eIF-2.GEF + GDP. The addition of GEF alone does not result in the displacement of eIF-2-bound GDP. The release of bound GDP is dependent on the presence of both GTP and GEF, and this argues against the possibility of a substituted enzyme (ping-pong) mechanism for the guanine nucleotide exchange reaction. An important finding of the present study is the observation that GTP binds to GEF. The Kd value of 4 microM for GTP was estimated (a) by the extent of quenching of tryptophan fluorescence of GEF in the presence of GTP and (b) by the binding of [3H]GTP to GEF as measured on nitrocellulose membranes. The GEF-dependent release of eIF-2-bound GDP was studied at several constant concentrations of one substrate (GTP or eIF-2.GDP) while varying the second substrate concentration, and the results were then plotted according to the Lineweaver-Burk method. Taken together, the results of GTP and eIF-2.GDP binding to GEF and the pattern of the double-reciprocal plots strongly suggest that the guanine nucleotide exchange reaction follows a sequential mechanism.

Isolation and mapping of a gene for protein synthesis initiation factor 4A and its expression during differentiation of murine erythroleukemia cells.

Eukaryotic protein synthesis initiation factor 4A (eIF-4A), a 46-kDa polypeptide, is involved both in mRNA cap recognition and in the binding of mRNA to 40S ribosomal subunits. A 41-mer oligodeoxynucleotide probe was synthesized complementary to a portion of the published coding sequence of eIF-4A mRNA [Nielsen et al., Nucleic Acids Res. 13 (1985) 6867-6870] and used to screen a mouse genomic library. We have isolated and characterized a full-length clone from that library. The eIF-4A sequence is contained in eleven exons. The eleventh exon also has the 3'-nontranslated sequence and two separate polyadenylation sites. Northern-blot analysis of mouse poly(A)+RNA indicates that there are several distinct mRNA species coding for eIF-4A. Two of these contain the same coding sequence and differ only in the length of the 3'-nontranslated region. Two of the eIF-4A mRNAs are therefore likely to be the result of differential processing at the 3'-end. We have used a fragment of the genomic clone to measure the steady-state levels of eIF-4A mRNA during the induced differentiation of murine erythroleukemia cells. S1 nuclease protection experiments demonstrated that by the fourth day after induction eIF-4A mRNA declined to 25% of its steady-state level in uninduced cells. In contrast, the steady-state level of beta-globin mRNA increased dramatically during differentiation. In vitro transcription assays using nuclei isolated from uninduced and induced cells show that the rate of transcription of eIF-4A mRNA was 40% greater in differentiated cells, indicating a posttranscriptional component is involved in the regulation of the steady-state mRNA level.

Effects of eucaryotic initiation factor 3 on eucaryotic ribosomal subunit equilibrium and kinetics.

In order to understand the possible role of eucaryotic initiator factor 3 (eIF-3) in maintaining a pool of eucaryotic subunits, we have measured the effects of eIF-3 on the equilibria and kinetics of ribosomal subunit association and dissociation. The ribosomal subunit interactions have been studied by laser light scattering, which does not perturb the system. We find that eIF-3 reduces the apparent association rate of reticulocyte, wheat germ, and Artemia ribosomes. The kinetics of the reassociation for a shift in [Mg2+] from 0.5 to 6 mM are best explained by a model where eIF-3 dissociates from the 40S subunits prior to association of the 40S and 60S subunits. Static titrations indicate there is some binding of eIF-3 to 80S ribosomes at lower [Mg2+].

Phosphorylation of the guanine nucleotide exchange factor from rabbit reticulocytes regulates its activity in polypeptide chain initiation.

We have demonstrated that the purified guanine nine nucleotide exchange factor (GEF) may be isolated as a complex with NADPH. Complete inhibition of the GEF-catalyzed exchange of eukaryotic initiation factor 2-bound GDP for GTP was observed in the presence of either 0.5-0.75 mM NAD+ or NADP+. Incubation of GEF with ATP results in the phosphorylation of its Mr 82,000 polypeptide. This phosphorylation is strongly inhibited by heparin but is not affected by heme or H8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride), an inhibitor of cAMP- and cGMP-dependent protein kinases and protein kinase C. The purification of GEF was modified to eliminate any contaminating kinase activity and the isolated protein appears to be homogeneous as judged by NaDodSO4/polyacrylamide gel electrophoresis and silver staining. The Mr 82,000 subunit of GEF is phosphorylated only upon addition of ATP and casein kinase II. The extent of phosphorylation is approximately equal to 0.55 mol of phosphate per mol of GEF, and this results in a 2.3-fold increase in the guanine nucleotide exchange activity. Following treatment of the phosphorylated GEF with alkaline phosphatase, the activity of the protein is reduced by a factor of 5. Rephosphorylation of GEF increases its specific activity to that of the phosphorylated protein. The results of this study suggest that phosphorylation/dephosphorylation of GEF plays a role in regulating polypeptide chain initiation.

Expression of a gene for mouse eucaryotic elongation factor Tu during murine erythroleukemic cell differentiation.

The eucaryotic elongation factor Tu (eEF-Tu) is a single polypeptide with an approximate Mr of 53,000. During protein synthesis eEF-Tu promotes the binding of aminoacyl-tRNA to the ribosome. To study the expression of the gene(s) for this factor, a genomic clone was isolated that contains a mouse eEF-Tu gene. We screened a phage genomic library with a synthetic oligonucleotide probe complementary to a region of the Saccharomyces cerevisiae and Artemia sp. eEF-Tu genes which codes for an area that is highly conserved between both yeast and Artemia sp. eEF-Tu. From approximately 75,000 phage plaques we obtained five isolates with apparently identical inserts. All five clones contained a 3.8-kilobase EcoRI fragment that hybridized to additional oligonucleotide probes corresponding to different conserved regions of eEF-Tu. We sequenced the 5' end of one genomic clone and determined the length of the cloned fragment that was protected by eEF-Tu mRNA in S1 nuclease protection assays. A quantitative S1 nuclease protection assay was used to compare the relative steady-state levels of eEF-Tu mRNA in total mRNA in total RNA isolated from hexamethylene-bisacetamide-induced murine erythroleukemia cells. The results show a dramatic reduction in the steady-state level of eEF-Tu mRNA as differentiation proceeds. A similar reduction in transcription of eEF-Tu mRNA was observed in isolated nuclei. Finally, we examined the in vivo synthesis of eEF-Tu during differentiation and found that it declined in a manner parallel to the decline in the steady-state level of eEF-Tu mRNA. In addition, we have isolated and sequenced a cDNA clone for mouse eEF-Tu. The derived amino acid sequence is compared with sequences from other eucaryotes.