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PubMed1988

Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu.

Elongation factor Tu from Thermus thermophilus was treated successively with periodate-oxidized GDP or GTP and cyanoborohydride. Covalently modified cyanogen bromide or trypsin fragments of the protein were isolated, and the position of their modification was determined. Lysine residues 52 and 137 were heavily labeled, lysine-137 being considerably more reactive in the GTP form as compared to the GDP form of the protein. These residues are in the proximity of the GDP/GTP binding site. Lys-325 was also labeled, but to a lower extent. The part of the EF-Tu containing residue 52 is missing in crystallized EF-Tu.GDP from Escherichia coli [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36]. These results place the part of T. thermophilus EF-Tu corresponding to the missing fragment in E. coli EF-Tu in the vicinity of the nucleotide binding site and allow its role in the interaction with aminoacyl-tRNA and elongation factor Ts to be evaluated. Cross-linking of EF-Tu.GDP by irradiation at 257 nm showed that a sequence of 10 amino acids residues which is found in the Thermus thermophilus elongation factor Tu but not in other homologous bacterial proteins is located in the vicinity of the GDP/GTP binding site.

Affinity Labels↗
PubMed1990

Peptide and protein carboxyl-terminal labeling through carboxypeptidase Y-catalyzed transpeptidation.

A survey of carboxypeptidase Y-catalyzed carboxyl-terminal modification of short peptides in the presence of various amino acids revealed that transpeptidation occurred in significant yield only with peptides containing a proline at the penultimate or antepenultimate position. For these peptides, transpeptidation was shown to occur specifically at the carboxyl side of the proline, thus suggesting a determining role of this residue for transpeptidation. Two model peptides, YPFP-GPI and YPFVEPI, were studied in detail. Initial yields of transpeptidation in the presence of various nucleophiles were compared. Among natural amino acids, the highest yield was obtained with methionine, followed by other amino acids bearing hydrophobic side chains. In order to transpose the method of transpeptidation to a protein, a variant of Escherichia coli methionyl-tRNA synthetase bearing the carboxyl-terminal Glu-Pro-Met sequence was genetically created. Under the conditions optimized for the transpeptidation of YPF-VEPI with methionine, this protein could be labeled specifically at its carboxyl-terminal end. Moreover, the parameters of the labeling reaction were in agreement with those observed in the transpeptidation of the model peptide.

Amino Acid Sequence↗
PubMed1978

Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase: inhibition of interaction of Met-tRNAfMet binding factor with another initiation factor in formation of Met-tRNAfMet.40S ribosomal subunit complexes.

Protein synthesis in reticulocytes and their lysates is regulated by heme. In heme deficiency a heme-regulated translational inhibitor (HRI) that blocks initiation of polypeptide chains is activated. HRI is a protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNA(f) (Met) binding factor (IF), which forms a ternary complex with Met-tRNA(f) (Met) and GTP, a finding that suggests that the inhibition by HRI involves the phosphorylation of IF. We have investigated the effect of HRI in the partial reactions of protein chain initiation in which the IF-promoted binding of Met-tRNA(f) (Met) to 40S ribosomal subunits is enhanced by another initiation factor [ternary complex dissociation factor (TDF)] and AUG. The results show that HRI at very low concentrations markedly inhibits the binding of Met-tRNA(f) (Met) to 40S subunits. The inhibitory effect of HRI requires ATP. Under these conditions HRI phosphorylates only the 38,000-dalton subunit of IF. The TDF preparations not only promote the binding of the ternary complex to 40S subunits but also promote the dissociation of the ternary complex in the presence of 5 mM Mg(2+) at 0 degrees . The preincubation of purified IF alone with low concentrations of HRI and ATP does not significantly affect its capacity to form the ternary complex; however, the TDF-promoted dissociation of the ternary complex is inhibited. The nonhydrolyzable analog adenosine 5'-[beta,gamma-imido]triphosphate does not substitute for ATP. These findings suggest that phosphorylation causes a conformational modification in IF, which results in inhibition of the interaction between the ternary complex and TDF that is required for the binding of the ternary complex to 40S subunits.

Adenosine Triphosphate↗
PubMed1987

Characterization by tandem mass spectrometry of structural modifications in proteins.

Tandem mass spectrometry can be used to solve a number of protein structural problems that are not amenable to conventional methods for amino acid sequencing. Typical problems that use this approach involve characterization of peptides with blocked amino termini or peptides that have been otherwise posttranslationally processed, such as, by phosphorylation or sulfation. The structure and homogeneity of synthetic peptides can also be evaluated. Since peptides can be selectively characterized in the presence of other peptides or contaminants, the need for extensive purification is reduced or eliminated.

Amino Acid Sequence↗
PubMed1980

Folding up a transfer RNA molecule is not simple.

For much of its history, molecular biology has concerned itself with the implications and consequences of the "Central Dogma" (Crick, 1958): DNA leads to RNA leads to protein. This "pathway" is of course atypical in that it describes, not chemical interconversions, but rather the flow of information from gene to product. It has been only recently appreciated, however, that in those cases where the final gene product is an RNA molecule, this pathway is not simply truncated. To the contrary, in virtually every instance for which sufficient data are now available, it is found that the RNA transcript is subject to a series of reactions in which the primary gene product undergoes nucleotide additions, deletions, and modifications. The set of biosynthetic reactions that intervene between transcription of the gene and production of the mature functional product is collectively referred to as RNA processing. It now appears that the existence of these complex biosynthetic events cannot always be adequately explained by the necessity to overcome otherwise insurmountable topological or energetic constraints: the final product can, at least in some cases, "self-assemble." The genesis of transfer RNA by this indirect route appears to insure the delivery of a functional product, at the right rate, at the right time. We suggest, moreover, that this process is primarily determined by recognition of some of the same structural elements in the precursor that are required for the function of the mature tRNA molecule.

Base Sequence↗
PubMed1981

Chemical structure and thermal properties of initiator tRNA from Euphausia sperba in comparison with those of other eucaryotic initiator tRNAs.

The nucleotide sequence of initiator tRNA (tRNAiMet) from Euphausia sperba, which was harvested in the Antarctic Sea, was determined to be pA-G-C-A-G-A-G-U-m1G-m2G-C-G-C-A-G-U-G-G-A-A-G-C-G-U-m2G-C-U-G-G-G-C-C-C-A-U-t6 A-A-C-C-C-A-G-A-G-m7G-U-C-G-G-U-A-G-A-psi-C-G-m1A-A-A-C-U-A-C-U-C-U-C-U-G-C-U-A -C-C-AOH by using post-labeling methods recently developed. The nucleotide sequence was very similar to that of mammalian tRNAiMet except for changes in six bases and three modifications: C16, U55, D47 and m5C48 are replaced by U16, psi 55 and unmodified U47 and C48, respectively. A50-U64 and G52-C62 base pairs of mammalian tRNAiMet are reversed in Euphausia tRNAiMet. In addition, the G49-C65 pair of the former is replaced by a less stable G49-U65 pair in Euphausia tRNAiMet. The sequence homology was compared between Euphausia tRNAiMet and over ten different species of eucaryotic tRNAiMet so far sequenced. The melting temperature of Euphausia tRNAiMet was 72.5 degrees C, which is 4.2 degrees C and 8.3 degrees C lower than those of rat liver and yeast tRNAiMet's, respectively. The origin of the thermal instability of Euphausia tRNAiMet is discussed in comparison of its secondary structure compared with those of other eucaryotic tRNAiMet's.

Animals↗
PubMedDate not supplied

Biosynthesis and biological properties of compounds containing highly reactive, reduced sulfane sulfur.

The covalent modifications of sulfhydryl groups (-SH) may occur through oxidation to mixed disulfides (S-thiolation), S-nitrosylation, as well as persulfide and trisulfide formation. The latter possibilities of -SH group modification connected with compounds containing sulfur called sulfane sulfur are described in this paper. Sulfane sulfur compounds contain a labile, highly reactive sulfur atom at a reduced oxidation state with a valence of 0 or -1, covalently bound to another sulfur atom. These compounds include persulfides, polysulfides, polythionates, thiosulfate, elemental sulfur and disulfides, which enable tautomerization to thiosulfoxides. Sulfane sulfur compounds are formed in the anaerobic cysteine sulfur metabolism with the participation of such enzymes as cystathionase (CST), 3-mercaptopyruvate sulfurtransferase (MpST) and rhodanese (thiosulfate: cyanide sulfurtransferase). Compounds containing sulfane sulfur participate in cell regulation processes through activation or inactivation of some enzymes. Other important roles of sulfane sulfur compounds are their antioxidative properties, significance in the processes of carcinogenesis, participation in the tRNA sulfuration as well as an influence on the activity of immune cells. To recognize completely the biological role of compounds with sulfane sulfur it is necessary to have sensitive methods of quantitative determination, so a review of these methods is presented in this paper. Moreover, biosynthetic pathways and biological properties of these compounds have been discussed.

Animals↗
PubMed2005

Effects of nucleotide substitution and modification on the stability and structure of helix 69 from 28S rRNA.

The helix 69 (H69) region of the large subunit (28S) rRNA of Homo sapiens contains five pseudouridine (Psi) residues out of 19 total nucleotides (26%), three of which are universally or highly conserved. In this study, the effects of this abundant modified nucleotide on the structure and stability of H69 were compared with those of uridine. The role of a loop nucleotide substitution from A in bacteria (position 1918 in Escherichia coli 23S rRNA) to G in eukaryotes (position in 3734 in H. sapiens) was also examined. The thermodynamic parameters were obtained through UV melting studies, and differences in the modified and unmodified RNA structures were examined by 1H NMR and circular dichroism spectroscopy. In addition, a [1,3-15N]Psi phosphoramidite was used to generate H69 analogs with site-specific 15N labels. By using this approach, different Psi residues can be clearly distinguished from one another in 1H NMR experiments. The effects of pseudouridine on H. sapiens H69 are consistent with previous studies on tRNA, rRNA, and snRNA models in which the nucleotide offers stabilization of duplex regions through PsiN1H-mediated hydrogen bonds. The overall secondary structure and base-pairing patterns of human H69 are similar to the bacterial RNA, consistent with the idea that ribosome structure and function are highly conserved. Nonetheless, pseudouridine-containing RNAs have subtle differences in their structures and stabilities compared to the corresponding uridine-containing analogs, suggesting possible roles for Psi such as maintaining translation fidelity.

Circular Dichroism↗
PubMed2003

Proteomic consequences of a human mitochondrial tRNA mutation beyond the frame of mitochondrial translation.

Numerous severe neurodegenerative and neuromuscular disorders, characterized biochemically by strong perturbations in energy metabolism, are correlated with single point mutations in mitochondrial genes coding for transfer RNAs. Initial comparative proteomics performed on wild-type and Myoclonic Epilepsy and Ragged Red Fibers (MERRF) mitochondria from sibling human cybrid cell lines revealed the potential of this approach. Here a quantitative analysis of several hundred silver-stained spots separated by two-dimensional gel electrophoresis was performed in the specific case of a couple of mitochondria, containing or not mutation A8344G in the gene for mitochondrial tRNALys, correlated with MERRF syndrome. Computer-assisted analysis allowed us to detect 38 spots with significant quantitative variations, of which 20 could be assigned by mass spectrometry. These include nuclear encoded proteins located in mitochondria such as respiratory chain subunits, metabolic enzymes, a protein of the mitochondrial translation machinery, and cytosolic contaminants. Furthermore, Western blotting combined with mass spectrometry revealed the occurrence of numerous isoforms of pyruvate dehydrogenase subunits, with subtle changes in post-translational modifications. This comparative proteomic approach gives the first insight for nuclear encoded proteins that undergo the largest quantitative changes, and pinpoints new potential molecular partners involved in the cascade of events that connect genotype to phenotype.

Humans↗
PubMed1986

Amino-acid-transfer reactions in isolated nuclei of Ehrlich ascites tumor cells.

Nuclear enzymatic activities incorporating amino acids into acid-insoluble material were investigated with respect to their differentiation from protein biosynthesis, reaction optima, requisites and localization. The product of the reaction was analyzed with respect to its localization and nature. The nuclear activities are not inhibited by a number of inhibitors for protein biosynthesis. The reaction optima found are similar to those of other residual nuclear syntheses including the stringent dependence on ATP. All naturally occurring amino acids are utilized with different efficiencies. Their incorporation is neither cooperative nor competitive which points to individual incorporation mechanisms. Aminoacylation of tRNA may be involved because the incorporation is RNase-sensitive and aminoacylation of tRNA can be shown under the reaction conditions. The enzymatic activities are exclusively nuclear. Significant activity with unchanged characteristics is released by sonication. 70% of the radiolabel incorporated is exported across the nuclear envelope during the incubation. The residual 30% of the radiolabel is distributed without enrichment in any nuclear subfraction. The products are exclusively of polypeptide nature. Since distinct nuclear proteins (e.g. histones) which are definitely preformed in the cytoplasm by protein biosynthesis become radiolabelled by the incorporation of radiolabelled amino acids, it is evident that the incorporation takes place at preformed polypeptides. This is unequivocally proven by the incorporation of radiolabelled amino acids into exogenous proteins by means of the solubilized nuclear activities. The results indicate that the nuclear activity under investigation reflects a nuclear modification system for polypeptides which may be of similar importance as other post-translational modification systems.

Amino Acids↗
PubMedDate not supplied

Chemical, biochemical and genetic endeavours characterizing the interaction of sparsomycin with the ribosome.

Sparsomycin interaction with the ribosome and characteristics of the drug binding site in the particle were studied using chemical modification of the drug, affinity labeling methods and isolation of drug resistant mutants. The structure-function relationship studies, performed with a large number of drug derivatives, indicate that the drug interacts with the ribosome by its western and eastern moieties. The uracil ring, in the western end of the drug molecule, probably forms hydrogen bonds with the rRNA, while the apolar CH3-S-CH3 group in the eastern end interacts with a hydrophobic ribosomal domain that affinity labeling results seem to indicate is formed by protein. An increase in lipophilicity in this part of the antibiotic results in a dramatic increase in the inhibitory activity of the drug. The sparsomycin binding site is not accessible in free ribosomes, but the presence of an N-blocked amino acyl-tRNA at the P-site turns the particles capable of reversible interaction with the drug. After failure using Escherichia coli, a sparsomycin-resistant mutant was obtained by direct mutagenesis on Halobacterium halobium, a species with a unique copy of rRNA genes, stressing the role of rRNA on the drug interaction site.

Binding Sites↗
PubMed1988

Specific recognition of the 3'-terminal adenosine of tRNAPhe in the exit site of Escherichia coli ribosomes.

Ribosomes from Escherichia coli possess, in addition to A and P sites, a third tRNA binding site, which according to its presumed function in tRNA release during translocation has been termed the exit site. The exit site exhibits a remarkable specificity for deacylated tRNA; charged tRNA, e.g. N-AcPhe-tRNAPhe, is not bound significantly. To determine the molecular basis of this discrimination, we have measured the exit site binding affinities of a number of derivatives of tRNAPhe from E. coli, modified at the 3' end. Binding to the exit site of the tRNAPhe derivatives was measured fluorimetrically by competition with a fluorescent tRNAPhe derivative. We show here that removal of the 2' and 3' hydroxyl groups of the 3'-terminal adenosine decreases the affinity of tRNAPhe for the exit site 15 and 40-fold, respectively. Substitutions at the 3' hydroxyl group (aminoacylation, phosphorylation, cytidylation) as well as removal of the 3'-terminal adenosine (or adenylate) of tRNAPhe lower the affinity below the detection limit of 2 x 10(5) M-1, i.e. more than 100-fold. Modification of the adenine moiety (1,N6-etheno adenine) or replacement of it with other bases (cytosine, guanine) has the same dramatic effect. In contrast, the binding to both P and A sites is virtually unaffected by all of the modifications tested. These results suggest that a major fraction (at least -12 kJ/mol, probably about -17 kJ/mol) of the free energy of exit site binding of tRNAPhe (-42 kJ/mol at 20 mM-Mg2+) is contributed by the binding of the 3'-terminal adenine to the ribosome. The binding most likely entails the formation of hydrogen bonds.

Adenosine↗
PubMed1996

Primer selection by HIV-1 reverse transcriptase on RNA-tRNA(3Lys) and DNA-tRNA(3Lys) hybrids.

During reverse transcription of the genomic RNA of human immunodeficiency virus type 1 (HIV-1) into double-stranded DNA, reverse transcriptase (RT) must accommodate RNA-RNA, DNA-RNA, RNA-DNA and DNA-DNA hybrids as primer-template. In this study, we examined extension of RNA-tRNA3Lys, and DNA-tRNA3Lys complexes by HIV-1 RT. When the 3' end of tRNA3Lys is annealed to oligoribonucleotides, tRNA3Lys, but not the complementary RNAs, is extended by HIV-1 RT, indicating that tRNA3Lys is efficiently used as primer and RNA as template. An opposite primer usage is observed when tRNA3Lys is annealed to complementary oligodeoxyribonucleotides. In this case, the oligodeoxyribonucleotides are efficiently used as primer and tRNA3Lys as template. This result indicates that the nature of nucleic acid bound to tRNA3Lys determines which strand of the RNA-tRNA3Lys and DNA-tRNA3Lys hybrids is extended by HIV-1 RT. When an oligoribonucleotide is annealed to an unmodified transcript of tRNA3Lys, both nucleic acids are extended by HIV-1 RT, indicating that specific selection of tRNA3Lys as primer requires the post-transcriptional modifications of tRNA3Lys.

Base Sequence↗
PubMed2000

Mechanistic studies of RNA modifying enzymes. RNA pseudouridine synthase and m5cytosine methyl transferase.

RNA has numerous post-transcriptional modifications, but relatively little is known about the enzymes that catalyze such modifications or about the functions of the modified residues. Our laboratory has been engaged in studies of the structure and function of enzymes that catalyze the conversion of Urd residues in RNA to pseudouridine (psi Synthase), and Cyd to 5-methylcytidine methyl transferase (RNA m5C Mtase). The presentation will summarize recent results from our laboratory.

Amino Acid Motifs↗
PubMed1987

Regulation of the ubiquitin-mediated proteolytic pathway: role of the substrate alpha-NH2 group and of transfer RNA.

Degradation of intracellular proteins via the ubiquitin pathway involves several steps. In the initial event, ubiquitin becomes covalently linked to the protein substrate in an ATP-requiring reaction. Following ubiquitin conjugation, the protein moiety of the adduct is selectively degraded with the release of free and reusable ubiquitin. Ubiquitin modification of a variety of protein targets in the cell plays a role in basic cellular functions. Modification of core nucleosomal histones is probably involved in regulation of gene expression at the level of chromatin structure. Ubiquitin attachment to cell surface proteins may play roles in processes of cell-cell interaction and adhesion, and conjugation of ubiquitin to other yet to be identified protein(s) could be involved in the progression of cells through the cell cycle. Despite the considerable progress that has been made in the elucidation of the mode of action and cellular roles of the ubiquitin pathway, many major problems remain unsolved. A problem of central importance is the specificity in the ubiquitin ligation system. Why are certain proteins conjugated and committed for degradation, whereas other proteins are not? A free alpha-NH2 group is an important feature of the protein structure recognized by the ubiquitin conjugation system, and tRNA is required for the conjugation of ubiquitin to selective proteolytic substrates and for their subsequent degradation. These findings can shed light on some of the features of a substrate that render it susceptible to ubiquitin-mediated degradation.

Amines↗
PubMed1989

A covalent angiogenin/ribonuclease hybrid with a fourth disulfide bond generated by regional mutagenesis.

Human angiogenin is a blood vessel inducing protein whose primary structure displays 33% identity to that of bovine pancreatic ribonuclease A (RNase A). Angiogenin catalyzes limited cleavage of 18S and 28S ribosomal RNA and is several orders of magnitude less potent than RNase A toward conventional substrates. A striking structural difference between angiogenin and RNase is the virtual absence of sequence similarity within the region of RNase that contains the Cys-65--Cys-72 disulfide bond. Indeed, angiogenin lacks this disulfide linkage. The present report describes the use of regional mutagenesis to generate a covalent angiogenin/RNase hybrid protein, ARH-I, where residues 58-70 of angiogenin have been replaced by the corresponding segment of RNase A (residues 59-73). The protein expressed in Escherichia coli readily folds at pH 8.5 to form the four expected disulfide bonds. The in vivo angiogenic potency of ARH-I is markedly diminished compared with that of angiogenin when examined using the chick chorioallantoic membrane assay. In contrast, its enzymatic activity is dramatically increased. With high molecular weight wheat germ RNA and tRNA, ARH-I is 660- and 300-fold more active than angiogenin, respectively, while with poly(uridylic acid), poly(cytidylic acid), cytidylyl(3'----5')adenosine (CpA), and uridylyl(3'----5')adenosine (UpA) activity is enhanced by about 200-fold. In addition, the specificity of ARH-I toward dinucleoside 3',5'-phosphates is qualitatively similar to RNase A; while angiogenin prefers cytidylyl(3'----5')guanosine (CpG) to UpA, both RNase and the hybrid prefer UpA to CpG. ARH-I also displays greater than 10-fold enhanced activity toward rRNA in intact ribosomes, while abolishing the capacity of the ribosome to support cell-free protein synthesis. The enhanced enzymatic properties of ARH-I parallel a 2-fold increase in chemical reactivity of active-site lysine and histidine residues based on rates of chemical modification. The data indicate that introduction of a region of RNase A containing the Cys-65--Cys-72 disulfide bond into angiogenin dramatically increases RNase-like enzymatic activity while reducing its angiogenicity.

Amino Acid Sequence↗
PubMed1998

Novel temperature-sensitive mutants of Escherichia coli that are unable to grow in the absence of wild-type tRNA6Leu.

Escherichia coli has only a single copy of a gene for tRNA6Leu (Y. Komine et al., J. Mol. Biol. 212:579-598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O2-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA4Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O2-methyluridine) as its anticodon, tRNA6Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA6Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su+6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA6Leu. These tRNA6Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaA gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA4Leu restored the growth of group I mutants at 42 degrees C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA4Leu poorly recognizes the UUG codon at 42 degreesC and that the wild-type tRNA6Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA6Leu remains to be investigated.

Alkyl and Aryl Transferases↗
PubMed1984

Posttranslational protein modification by amino acid addition in intact and regenerating axons of the rat sciatic nerve.

Experiments were performed to determine whether posttranslational addition of amino acids to axonal proteins occurs in axons of the rat sciatic nerve. Two ligatures were placed 1 cm apart on sciatic nerves. Six days later, segments proximal to each ligature were removed, homogenized, centrifuged at 150,000 X g, and analyzed for the ability to incorporate 3H-amino acids into proteins. No incorporation of amino acids into proteins was found in the high-speed supernatant, but when the supernatant was passed through a Sephacryl S-200 chromatography column (removing molecules less than 20 kD), [3H]arginine, lysine, leucine and aspartic acid were incorporated into proteins in both proximal and distal nerve segments. Small but consistently greater amounts of radioactivity were incorporated into proteins in proximal segments compared with distal segments, indicating that the components necessary for the reaction are transported axonally. This reaction represents the posttranslational incorporation of a variety of amino acids into proteins of rat sciatic nerve axons. Other experiments showed that the incorporation of amino acids into proteins is by covalent bonding, that the amino acid donor is likely to be tRNA, and that the reaction is inhibited in vivo by a substance whose molecular mass is less than 20 kD. This inhibition is not affected by incubation with physiological concentrations of unlabeled amino acids, by boiling, or by treatment with Proteinase K. When the axonally transported component of the reaction was determined in regenerating nerves, the amount of incorporation of amino acids into protein was 15-150 times that in intact nerves.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗