Dictyostelium, a microbial model for brain disease.

BACKGROUND: Most neurodegenerative diseases are associated with mitochondrial dysfunction. In humans, mutations in mitochondrial genes result in a range of phenotypic outcomes which do not correlate well with the underlying genetic cause. Other neurodegenerative diseases are caused by mutations that affect the function and trafficking of lysosomes, endosomes and autophagosomes. Many of the complexities of these human diseases can be avoided by studying them in the simple eukaryotic model Dictyostelium discoideum. SCOPE OF REVIEW: This review describes research using Dictyostelium to study cytopathological pathways underlying a variety of neurodegenerative diseases including mitochondrial, lysosomal and vesicle trafficking disorders. MAJOR CONCLUSIONS: Generalised mitochondrial respiratory deficiencies in Dictyostelium produce a consistent pattern of defective phenotypes that are caused by chronic activation of a cellular energy sensor AMPK (AMP-activated protein kinase) and not ATP deficiency per se. Surprisingly, when individual subunits of Complex I are knocked out, both AMPK-dependent and AMPK-independent, subunit-specific phenotypes are observed. Many nonmitochondrial proteins associated with neurological disorders have homologues in Dictyostelium and are associated with the function and trafficking of lysosomes and endosomes. Conversely, some genes associated with neurodegenerative disorders do not have homologues in Dictyostelium and this provides a unique avenue for studying these mutated proteins in the absence of endogeneous protein. GENERAL SIGNIFICANCE: Using the Dictyostelium model we have gained insights into the sublethal cytopathological pathways whose dysregulation contributes to phenotypic outcomes in neurodegenerative disease. This work is beginning to distinguish correlation, cause and effect in the complex network of cross talk between the various organelles involved. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.

The novel silatecan 7-tert-butyldimethylsilyl-10-hydroxycamptothecin displays high lipophilicity, improved human blood stability, and potent anticancer activity.

We describe the rational design and synthesis of B- and A, B-ring-modified camptothecins. The key alpha-hydroxy-delta-lactone pharmacophore in 7-tert-butyldimethylsilyl-10-hydroxycamptothecin (DB-67, 14) displays superior stability in human blood when compared with clinically relevant camptothecin analogues. In human blood 14 displayed a t(1/2) of 130 min and a percent lactone at equilibrium value of 30%. The tert-butyldimethylsilyl group renders the new agent 25-times more lipophilic than camptothecin, and 14 is readily incorporated, as its active lactone form, into cellular and liposomal bilayers. In addition, the dual 7-alkylsilyl and 10-hydroxy substitution in 14 enhances drug stability in the presence of human serum albumin. Thus, the net lipophilicity and the altered human serum albumin interactions together function to promote the enhanced blood stability. In vitro cytotoxicity assays using multiple different cell lines derived from eight distinct tumor types indicate that 14 is of comparable potency to camptothecin and 10-hydroxycamptothecin, as well as the FDA-approved camptothecin analogues topotecan and CPT-11. In addition, cell-free cleavage assays reveal that 14 is highly active and forms more stable top1 cleavage complexes than camptothecin or SN-38. The impressive blood stability and cytotoxicity profiles for 14 strongly suggest that it is an excellent candidate for additional in vivo pharmacological and efficacy studies.

Identification of adenine binding domain peptides of the NADP+ active site within porcine heart NADP(+)-dependent isocitrate dehydrogenase.

Photoaffinity labeling with [2'-32P]2N3NADP+ and [32P]2N3NAD+ was used to identify two overlapping tryptic and chymotryptic generated peptides within the adenine binding domain of NADP(+)-dependent isocitrate dehydrogenase (IDH). Photolysis was required for insertion of radiolabel, and prior photolysis of photoprobes before addition of IDH prevented insertion. Photoincorportion of 2N3NAD+ inhibited the enzymatic activity of IDH. Photolabeling of IDH with both [32P]2N3NAD+ and [2'-32P]2N3-NADP+ showed saturation effects with apparent Kds of 20 and 14 microM (+/-12%), respectively. The efficiency of photoincorporation at saturation of binding sites was determined to be about 50%. Also, photolabeling was observed with [32P]8N3ATP and [32P]2N3ATP but with saturation effects observed at lower affinity. With all radiolabeled probes reduction of photoinsertion was effected best by the addition of NADP+ followed by NAD+ and then ATP, indicating that photoinsertion with all the probes was within the NADP+ binding site. Isolation of [32P]2N3NAD+ and [2'-32P]2N3NADP+ photolabeled peptides by use of immobilized boronate and immobilized Al3+ chromatography, respectively, followed by HPLC purification resulted in the identification of overlapping peptides corresponding to Ile244-Arg249 and Leu121-Arg133 (tryptic fragments) and Lys243-His248 and Leu121-His135 (chymotryptic fragments). Trp125 and Trp245 were identified as the sites of photoinsertion based on these residues not being detectable on sequencing, the lack of chymotryptic cleavage at these residues, and the decreased rate of trypsin digestion at nearby Lys243 and Lys127. Sequence analysis of [32P]8N3ATP and [32P]2N3ATP photolabeled peptides gave essentially the same peptide regions being photolabeled but at much lower efficiency, indicating that the effects of ATP on IDH activity are dependent on competition for the same site.

Identification of N-terminus peptide of human granulocyte/macrophage colony stimulating factor as the site of nucleotide interaction.

The interaction of nucleotides with recombinant human granulocyte/macrophage colony stimulating factor (rhGM-CSF) has been investigated. Utilizing nucleotide photoaffinity probes [gamma 32P]-8N3ATP and [beta 32P]-8N3Ap4A, an analog of alarmone, the specificity of interaction was demonstrated by saturation of photoinsertion by these analogs and protection of photoinsertion by these analogs in the presence of natural nucleotide. The site of photoinsertion was tentatively determined to be Ser9. The photolabeled cytokine has lost most of its biological activity in a cellular proliferation assay, indicating a possible physiological role for this interaction.

Identification of the NADP(H) binding site of rat liver microsomal 5 alpha-reductase (isozyme-1): purification of a photolabeled peptide corresponding to the adenine binding domain.

We have previously shown that [2'-32P]-2-azido-NADP+ is an effective probe of the NADP-(H) binding site of rat liver microsomal 5 alpha-reductase (5 alpha R-1) [Bhattacharyya et al. (1994) Steroids 59, 634-641]. PEG-fractionated (6.5%) detergent-solubilized preparations (40 mg) containing 5 alpha R-1 activity were UV-photolyzed with [32P]-2-azido-NADP+ and subjected to preparative gel electrophoresis on 8% SDS-PAGE. Fractions corresponding to the second major [32P]-labeled peak following the dye-front were analyzed by 10% SDS-PAGE and showed a single [32P]-labeled species with an apparent molecular mass of approximately 26 kDa (5 alpha R-1). TCA precipitation (13.6%) of the labeled fractions resulted in recovery of > 70% of the total radioactivity in the protein pellet. Trypsin digestion of the resuspended pellet followed by immobilized-Al3+ affinity chromatography indicated that > 90% of the radioactivity remained bound to the affinity column. The [32P]-2N3-NADP(+)-labeled peptide was eluted with potassium phosphate, concentrated, and further purified by reverse-phase (C8) HPLC. Sequence analysis of the purified peptide indicated that it consisted of 11 amino acids with the sequence N-L-R-K-P-G-E-T-G-Y-K, corresponding to residues 170-180 of the rat 5 alpha R-1 sequence [Andersson et al. (1989) J. Biol. Chem. 264, 16249-16255].

Photoaffinity labeling of the ATP binding domain of Rubisco activase and a separate domain involved in the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Photoaffinity labeling of Rubisco activase with 2- and 8-N3ATP was used to identify the adenine binding domain for ATP. Rubisco activase hydrolyzed both of these analogs of ATP and used their hydrolysis to support a low rate of Rubisco activation. When irradiated with ultraviolet light, these and other azido-substituted adenine nucleotides covalently modified Rubisco activase at two distinct binding sites. Competition binding experiments with ATP and ADP showed that one of the sites was the ATP binding domain. The other site was not a nucleotide binding domain per se but would bind adenine nucleotides if an azido moiety was present on the base. Tryptophan and other indoles prevented azidoadenine nucleotides from labeling this domain but afforded little protection to the ATP binding domain. The ability to selectively protect each of the two binding sites made it possible to localize the adenine binding domain for ATP to the region of Rubisco activase from N68-D74 and the other binding domain to a region near the N-terminus from Q10 to D14. Modification of the region from Q10 to D14 by photoaffinity labeling prevented Rubisco activase from promoting activation of Rubisco without affecting ATP hydrolysis. These data suggest that a specific region of Rubisco activase near the N-terminus may be a site of interaction with Rubisco. Binding of azidoadenine nucleotides in this region appears to be fortuitous and may involve base-stacking with the species-invariant Trp at position 16 and hydrogen bonding of the azido moiety.

Photoaffinity labeling of rat liver microsomal steroid 5 alpha-reductase by 2-azido-NADP+.

Preincubation of female rat liver microsomal preparations with [2'-32P]2N3-NADP+ followed by photolysis with UV light (254 nm) and analysis by SDS-PAGE/autoradiography showed incorporation of 32P into at least 3 major protein bands in the molecular weight range of 14-97 Kd. Labeling of a 26 kD band, the apparent molecular weight of 5 alpha-reductase in liver microsomes, was accompanied by a loss of enzyme activity, consistent with its covalent modification. The inclusion of 20-fold excess NADP+ (100 microM) completely inhibited the incorporation of [2'-32P]2N3-NADP+ and preserved the enzyme activity, whereas excess NAD+ (100 microM) failed to protect 5 alpha-reductase (5 alpha R) activity. Similar results were obtained with the detergent-solubilized form of 5 alpha R. Polyethylene glycol (PEG) fractionation of detergent-solubilized preparations of 5 alpha R showed that all the 5 alpha R activity could be recovered in the 6.5% pellet with a 3-4-fold increase in the specific activity. photolysis of this fraction with [2'-32P]2N3-NADP+ resulted in approximately 2-fold increase in 32P labeling of the 5 alpha R band. Increasing photolysis time and concentration of the [2'-32P]2N3-NADP+ indicated that the half-life for photoincorporation and the apparent Kd were 1.0 min and 2 microM, respectively. These results suggest that 2N3-NADP+ is an effective probe of the NADP(H) binding site of 5 alpha R, and is a useful marker during purification of the enzyme.

Interaction of nucleotides with acidic fibroblast growth factor (FGF-1).

A wide variety of nucleotides are shown to bind to acidic fibroblast growth factor (aFGF) as demonstrated by their ability to (1) inhibit the heat-induced aggregation of the protein, (2) enhance the thermal stability of aFGF as monitored by both intrinsic fluorescence and CD, (3) interact with fluorescent nucleotides and displace a bound polysulfated naphthylurea compound, suramin, (4) reduce the size of heparin-aFGF complexes, and (5) protect a reactive aFGF thiol group. The binding of mononucleotides, diadenosine compounds (ApnA), and inorganic polyphosphates to aFGF is enhanced as the degree of phosphorylation of these anions is increased with the presence of the base reducing the apparent binding affinity. The nature of the base appears to have much less effect. Photoactivatable nucleotides (8N3-ATP, 2N3-ATP, 8N3-GTP, and 8N3-Ap4A) were employed to covalently label the aFGF nucleotide binding site. In general, Kd's in the low micromolar range are observed. Protection against 90% displacement is observed at several hundred micromolar nucleotide concentration. Using 8N3-ATP as a prototypic reagent, photolabeled aFGF was proteolyzed with trypsin and chymotrypsin and labeled peptides were isolated and sequenced resulting in the identification of 10 possible labeled amino acids (Y8, G20, H21, T61, K112, K113, S116, R119, R122, H124). On the basis of the crystal structure of bovine aFGF, eight of the prospective labeled sites appear to be dispersed around the perimeter of the growth factor's presumptive polyanion binding site. On residue (T61) is more distally located but still proximate to several positively charged residues, and another (Y8) is not locatable in crystal structures. Using heparin affinity chromatography, at least three distinct photolabeled aFGF species were resolved. These labeled complexes display diminished affinity for heparin and a reduced ability to stimulate mitogenesis even in the presence of polyanions such as heparin. In conclusion, nucleotides bind apparently nonspecifically to the polyanion binding site of aFGF but nevertheless are capable of modulating the protein's activity. Evidence for the presence of a second or more extended polyanion binding site and the potential biological significance of these results in terms of potential natural ligands of aFGF are also discussed but not resolved.

Photoaffinity labeling of human placental NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase with [alpha-32P]2N3NAD+. Identification of a peptide in the adenine ring binding domain.

Oxidation of many prostaglandins at C-15 results in the formation of 15-keto metabolites, which have reduced biological activity. This reaction is catalyzed by NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase. Using the photoaffinity analog of NAD+, [alpha-32P]nicotinamide-2-azidoadenine dinucleotide, we have identified a peptide in the adenine ring binding domain of the NAD+ binding site of 15-hydroxyprostaglandin dehydrogenase. The specificity of photolabeling was demonstrated by saturation and protection experiments. Saturation of photolabeling was observed at approximately 45-50 microM with an apparent Kd of 8-10 microM. Approximately 90% of photolabeling could be protected by 200 microM NAD+ when the protein was photolyzed in the presence of 10 microM probe. The photolabeled protein was digested with Staphylococcus aureus V8 or chymotrypsin, and the photolabeled peptides were purified by either boronate affinity chromatography or Fe+3 chelate chromatography followed by reverse phase HPLC. The photolabeled peptide region was identified to be Val32-Glu40.

Identification and characterization of a nucleotide binding site of ovine prolactin with 2-azido-NAD.

Photoaffinity labeling of ovine prolactin with the NAD+ photoaffinity analog [alpha-32P]nicotinamide-2-azidoadenine dinucleotide has been used to identify an NADH/NADPH binding site. Specificity of nucleotide interaction was demonstrated by saturation and protection of labeling at physiologically relevant concentrations. Saturation of photoinsertion was observed at approximately 100 microM probe with an apparent Kd of approximately 25 microM. Protection of photoinsertion was observed with NAD+ and NADH. The photoinsertion was decreased by 75% and greater than 95%, respectively, upon addition of 200 microM of the above-mentioned compounds. The protection obtained with NADP+ and NADPH was of the same order, respectively. The adenine ring binding domain of NADH/NADPH binding site was identified by trypsin and chymotrypsin digestion of the photolabeled prolactin and purification of the photolabeled peptide by boronate affinity chromatography and immobilized Fe3+ affinity chromatography. The peptide was identified to be Ala22-Tyr28. These studies demonstrate that prolactin contains an NADH/NADPH binding site which may be significant in the mechanism of action of this hormone.

Synthesis and application of bidentate photoaffinity cross-linking reagents. Nucleotide photoaffinity probes with two photoactive groups.

Two "targeted bidentate" photoaffinity cross-linking reagents, the monoanhydride of 8-N3ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([gamma-32P]8-N3ATP gamma BP) and the monoanhydride of 8-N3GDP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([gamma-32P]8-N3GTP gamma BP), were developed for studying the inter- and intramolecular interactions of nucleotide-binding proteins. Experiments using these bidentate reagents with two photoactive groups led to specific cross-linking: [gamma-32P]8-N3GTP gamma BP and [gamma-32P]8-N3ATP gamma BP showed intersubunit cross-linking of glutamate dehydrogenase and [gamma-32P]8-N3GTP gamma BP appeared to cross-link the alpha- and beta-subunits of tubulin. The non-azido "monodentate" versions of these reagents, the monoanhydride of ADP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([gamma-32P]ATP gamma BP) and the monoanhydride of GDP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([gamma-32P]GTP gamma BP), were also synthesized and characterized. The ability of these monodentate reagents with one photoactive group to serve as photoaffinity probes was established by photolabeling specifically the exchangeable GTP-binding domain of tubulin with [gamma-32P]GTP gamma BP and the ATP-binding domain of purified adenylate kinase and several nucleotide-binding proteins in human brain homogenate with [gamma-32P]ATP gamma BP.

Identification and characterization of a nucleotide binding site on recombinant murine granulocyte/macrophage-colony stimulating factor.

Granulocyte/macrophage-colony stimulating factor (GM-CSF) is a regulatory cytokine important in the proliferative and functional activation of hematopoietic cells. It belongs to a family of 20 kDa or less acidic glycoprotein molecules found in a broad range of cellular sources. On the basis of the previously reported nucleotide-binding properties of interleukin-2 (IL-2), atrial natriuretic factor (ANF), and glucagon, the interaction of GM-CSF with nucleotides was investigated. Using radiolabeled 8-azidoadenosine-containing photoprobes of ATP ([gamma-32P]-8N3ATP) and Ap4A, the putative biological alarmone ([beta'-32P]-8N3Ap4A), we have identified a nucleotide binding site on recombinant murine GM-CSF (rmGM-CSF). Specificity of binding was demonstrated by saturation and competition experiments. Saturation of photoinsertion by [gamma-32P]-8N3ATP and [beta'-32P]-8N3Ap4A occurs with apparent Kd's of 10 and 0.7 microM, respectively. Using an immobilized Fe3+ affinity chromatography technique, developed specifically for the isolation of photolabeled peptides, a single radiolabeled peptide was isolated. It was identified as amino acids 5-14 near the N-terminus of GM-CSF. This peptide region has been shown in previous studies to be critical for biological activity. Also consistent with this observation is our finding that the photolabeled GM-CSF has lost most, if not all, of its biological activity, as determined by a cellular proliferation assay.

NAD+ binding site of Clostridium botulinum C3 ADP-ribosyltransferase. Identification of peptide in the adenine ring binding domain using 2-azido NAD.

C3 ADP-ribosyltransferase is an exoenzyme produced by certain strains of Clostridium botulinum types C and D, which specifically ADP-ribosylates rho proteins in eukaryotic cells. Using the photoaffinity probe [alpha-32P]nicotinamide-2-azidoadenine dinucleotide, we have identified the adenine ring binding domain of the NAD+ binding site. The specificity of labeling was demonstrated by saturation effects and protection by the natural compound at physiologically relevant concentrations. Saturation of labeling was observed at 50 microM. Protection experiments indicated an 80% protection of labeling by 100 microM NAD+ when protein was photolyzed in the presence of 10 microM probe. Trypsin or Staphylococcus aureus V8 protease digestion of the photolabeled protein, along with boronate affinity chromatography and immobilized metal affinity chromatography, was used to specifically isolate the peptide region photolabeled with the probe. The peptide corresponded to Phe9-Gly19 near the N terminus.

Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes.

The NADPH oxidase of phagocytic cells is important for the efficient killing and digestion of ingested microbes. A very unusual low-potential cytochrome b (b-245) is the only redox molecule to have been identified in this system. The FAD-containing flavoprotein that binds NADPH and transfers electrons to the cytochrome has eluded identification for three decades. We show here that the haem/FAD ratio in the membranes does not change significantly on activation of this oxidase, indicating that the FAD is present in the membranes from the outset and not recruited from the cytosol. The FAD content of membranes from cells of patients with X-linked chronic granulomatous disease (CGD) lacking the cytochrome b was roughly one-quarter of that in normal subjects and in autosomal recessive CGD patients lacking the cytosolic protein p47-phox. Similar low amounts of FAD were present in uninduced promyelocytic (HL60) cells, suggesting that the low amount of FAD in cells from X-CGD patients was probably unrelated to this oxidase system. Cytochrome b-245 appears to bind both the haem and FAD, in a molar ratio of 2:1. The e.p.r. signal of the purified cytochrome was weak and had an asymmetric g(z) peak at g = 3.31. The purified cytochrome could be partially reflavinated (about 20%) in the presence of lipid. Amino acid sequence homology was detected between the beta-subunit of this cytochrome b and the ferredoxin-NADP+ reductase (FNR) family of reductases in the putative NADPH- and FAD-binding sites. 32P-labelled 2-azido-NADP was used as a photoaffinity label for the NADPH-binding site. Labelling that was competed off with NADP was observed in the region of the beta-subunit of the cytochrome. No labelling was seen in this region in X-CGD in three subjects in whom this cytochrome was missing and in a third in whom it was present but bore a Pro-His transposition in the putative NADPH-binding site. These studies indicate that cytochrome b-245 is a flavocytochrome, the first described in higher eukaryotic cells, bearing the complete electron-transporting apparatus of the NADPH oxidase.

Identification of peptides from the adenine binding domains of ATP and AMP in adenylate kinase: isolation of photoaffinity-labeled peptides by metal chelate chromatography.

Photoaffinity labeling with azidoadenine nucleotides was used to identify peptides from the ATP and AMP binding domains on chicken muscle adenylate kinase. Competition binding studies and enzyme assays showed that the 8-azido analogues of Ap4A and ATP modified only the MgATP2- site of adenylate kinase, whereas the 2-azido analogue of ADP modified the enzyme at both the ATP and AMP sites. The positions of the two nucleotide binding sites on the enzyme were deduced by isolating and sequencing the modified peptides. Photolabeled peptides were isolated by a new procedure that used metal chelate chromatography to affinity purify the photolabeled peptides prior to final purification by reverse-phase HPLC. The sequences of the peptides that were photolabeled with the 8-azido analogues corresponded to residues K28-L44, T153-K166, and T125-E135 of the chicken muscle enzyme. The residues that were present in both tryptic- and Staphylococcus aureus V-8 protease-generated versions of these peptides were assigned to the ATP binding domain on the basis of selective photoaffinity labeling with the 8-azidoadenine analogues. These peptides and an additional peptide corresponding to positions I110-K123 were photolabeled with 2-N3ADP. Since I110-K123 was photolabeled by 2-N3ADP but not by 8-N3Ap4A, it was assigned to the AMP binding domain.

A photoactive phosphonamide derivative of GTP for the identification of the GTP-binding domain in beta-tubulin.

A GTP photoaffinity probe (125I-APTG) was developed that incorporated an [125I]-N-(4-azidophenyl)-2-amino-3-(4-hydroxy-3-iodophenyl)propionamide group at the gamma-position of GTP through a phosphonamide linkage. A combination of saturation and GTP protection studies (90% protection at 25 microM GTP with an apparent Kd of 5 microM) validated the use of this new probe as a satisfactory GTP mimic. This probe offered the advantage of possessing an 125I radiolabel external to the GTP moiety, in contrast to the previously reported [gamma 32P]-8-N3GTP that possessed an internal 32P radiolabel. This novel feature accommodated the purification of photolabeled peptides using a combination of ion-exclusion, gel filtration, and HPLC techniques. [125I]APTG was used to identify a peptide (beta:65-79) in the exchangeable GTP-binding domain of the beta-subunit of tubulin.

Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5'-triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site.

Three photoactive derivatives of the 7-methylguanosine-containing cap of eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive and specific labeling of eIF-4E was observed with the previously described probe, [gamma-32P]-gamma-[[(4-benzoylphenyl)methyl]amido]-7-methyl-GTP [Blaas et al. (1982) Virology 116, 339; abbreviated [32P]BPM]. A second probe was synthesized that was an azidophenyltyrosine derivative of m7GTP [( 125I]APTM), the monoanhydride of m7GDP with [125I]-N-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodop hen yl) propionamide. This probe allowed rapid and quantitative introduction of radioactivity in the last rather than the first step of synthesis and placed the radioactive label on the protein-proximal side of the weak P-N bond. A dissociation constant of 6.9 microM was determined for [125I]APTM, which is comparable to the published values for m7GTP. m7GTP and APTM were equally effective as competitive inhibitors of eIF-4E labeling with [125I]APTM. Like [32P]BPM, [125I]APTM labeled both the full-length (25 kDa) polypeptide and a 16-kDa degradation product, designated eIF-4E*, with labeling occurring in proportion to the amounts of each polypeptide present. A third probe, an azidophenylglycine derivative of m7GTP [( 32P]APGM), the monoanhydride of m7GDP with [32P]-N-(4-azidophenyl)-2-(phosphoramido)acetamide, was also synthesized and shown to label eIF-4E specifically. Unlike [32P]BPM and [125I]APTM, however, [32P]APGM labeled eIF-4E* approximately 4-fold more readily than intact eIF-4E. Tryptic and CNBr cleavage suggested that eIF-4E* consists of a protease-resistant core of eIF-4E that retains the cap-binding site and consists of approximately residues 47-182.