Transacylation rates of (aminoacyl)adenosine moiety at the 3'-terminus of aminoacyl transfer ribonucleic acid

The rates of migration of the aminoacyl group (transacylation) between 2'-O-(aminoacyl)-tRNA and 3'-O-(aminoacyl)-tRNA were studied by the nuclear magnetic resonance (NMR) analyses of 3'-terminal fragment models, with regard to the significance of transacylation in the process of protein biosynthesis. 2'(3')-O-L-Alanyladenosine, -valyladenosine, -isoleucyladenosine, -phenylalanyladenosine, and -methionyladenosine, and 2'(3')-O-L-phenylalanyladenosine 5'-phosphate and methionyladenosine 5'-phosphate were chemically synthesized, and the rates of transacylation in deuterated buffer were directly measured by the NMR saturation transfer method. The dependences of transacylation rates on p2H and temperature were analyzed. The results indicate that the transacylation rates are significantly affected by the ionization states of the alpha-amino group of the amino acid moiety but not by the presence of the 5'-phosphate group of the adenylate moiety. The second-order rate constants for the base-catalyzed transacylation reactions were also determined for the ionized form (with alpha-N2H3+ group) of (aminoacyl)adenosines. The transacylation rates of (aminoacyl)adenosines in 1H2O solution at p1H 7.3 and 37 degrees C (intracellular environment) were evaluated as 3-11 s-1 for the 2' leads to 3' transacylation and 1-4 s-1 for the 3' leads to 2' transacylation, indicating that the transacylation rate of free aminoacyl-tRNA is slower than the overall rate of polypeptide chain elongation per ribosome. This suggests the presence of some enzymatic factor for enhancing the transacylation rates of aminoacyl-tRNAs in the polypeptide chain elongation process in vivo.     

N-(3-(p-azido-m-[125I]iodophenyl)propionyl)-succinimide--a heterobifunctional reagent for the synthesis of radioactive photoaffinity ligands: synthesis of a carrier-free 125I-labeled cardiac glycoside photoaffinity label

A new heterobifunctional reagent, N-(3-(p-azido-m-iodophenyl)propionyl)-succinimide (AIPPS), was synthesized and chemically characterized. The radiochemical form of the reagent, [125I]AIPPS, should be of general use as a photoactive reagent for the derivatization of free amino groups on a large variety of biologically active compounds, including many hormones. Amino-containing ligands can be derivatized with [125I]AIPPS in a method which is similar to that used for the 125I-labeled Bolton-Hunter reagent (N-(3-(p-hydroxyphenyl)propionyl)-succinimide). The added advantage with [125I]AIPPS, however, is that the ligand derivative is made both photoactive and radioactive in a single step. As an example of how this reagent can be used, we have prepared carrier-free [125I]AIPPS and reacted it with the amino-containing cardiac glycoside, 4-amino-4,6-dideoxyglucosyl digitoxigenin (GluD). The radioiodinated cardiac glycoside, [125I]AIPP-GluD, was purified by thin-layer chromatography and was carrier-free with a specific radioactivity of 2175 Ci/mmol. [125I]AIPP-GluD was an effective photoaffinity label for Na,K-ATPase as shown by specific photoaffinity labeling of purified canine kidney enzyme and human erythrocyte enzyme.     

Synthesis and characterization of a spin-labeled aminoacyl transfer ribonucleic acid

Unfractionated yeast transfer ribonucleic acid (tRNA) was reacted in aqueous acetone solution with the sulfhydryl spin-labeling reagnent, N-(l-oxyl-2,2,5,5-tetramethyl-3-Pyrrolidinyl)iodoacetamide. Whereas tRNA stripped of amino acids reacted only slowly, there were sites on tRNA charged with cysteine which combined rapidly with the reagent. The latter class of spin-label was quickly cleaved from the tRNA upon incubation in mildly alkaline solution (pH 8.0), suggesting that it was attached to the cysteinyl side chains. The paramagnetic resonance spectrum of column-purified spin-labeled cysteinyl tRNA showed that the spin-label was partially immobilized as a result of its interaction with the tRNA. When the tRNA was slowly heated, an abrupt increase occurred in the rotational mobility of the paramagnetic amino-acid side chain.     

Enzymatic binding of aminoacyl transfer ribonucleic acid to ribosomes: the study of binding sites of 2' and 3' isomers of aminoacyl transfer ribonucleic acid.

The mechanism of enzymatic binding of AAtRNA to the acceptor site Escherichia coli ribosomes has been studied using the following aminoacyl oligonucleotides as models of the 3' terminus of AA-tRNA: C-A-Phe, C-A-(2'-Phe)H, and C-A(2'H)Phe. T-psi-C-Gp was used as a model of loop IV of tRNA. The EF-T dependent binding of Phe-tRNA to ribosomes (in the presence of either GTP or GMPPCP) and the GTPase activity associated with EF-T dependent binding of the Phe-tRNA were inhibited by C-A-Phe,C-A(2'Phe)H, and C-A(2'H)Phe. These aminoacyl oligonucleotides inhibit both the formation of ternary complex EF-Tu-GTP-AA-tRNA and the interaction of this complex with the ribosomal A site. The uncoupled EF-Tu dependent GTPase (in the absence of AA-tRNA) was also inhibited by C-A-Phe, C-A(2'Phe)H, and C-A(2'H)Phe, while nonenzymatic binding of Phe-tRNA to the ribosomal A site was inhibited by C-A-Phe and C-A(2'-Phe)H, but not by C-A(2'H)Phe. The tetranucleotide T-psi-C-Gp inhibited both enzyme binding of Phe-tRNA and EF-T dependent GTP hydrolysis. However, inhibition of the latter reaction occured at a lower concentration of T-psi-C-Gp suggesting a specific role of T-psi-C-Gp loop of AA-tRNA in the GTPase reaction. The role of the 2' and 3' isomers of AA-tRNA during enzymatic binding to ribosomes is discussed and it is suggested that 2' leads to 3' transacylation in AA-tRNA is a step which follows GTP hydrolysis but precedes peptide bond formation.     

Three photo-cross-linked complexes of yeast phenylalanine specific transfer ribonucleic acid with aminoacyl transfer ribonucleic acid synthetases.

Yeast tRNA-Phe has been cross-linked photochemically to three aminoacyl-tRNA synthetases, yeast phenylalanyl-tRNA synthetase, Escherichia coli isoleucyl-tRNA synthetase, and E. coli valyl-tRNA synthetase. The two non-cognate enzymes are known to interact with tRNA-Phe. In each complex, three regions on the tRNA are found to cross-link. Two of these are common to all of the complexes, while the third is unique to each. Thus, the cognate and non-cognate complexes bear considerable similarity to each other in the way in which the respective enzyme orients on tRNA-Phe, a result which was also established for the complexes of E. coli tRNA-Ile (BUDZIK, G.P., LAM, S.M., SCHOEMAKER, H.J.P., and SCHIMMEL, P.R. (1975) J. Biol. Chem. 250, 4433-4439). The common regions include a piece extending from the 5'-side of the acceptor stem to the beginning of the dihydrouridine helix, and a segment running from the 3' side of the extra loop into the TpsiC helix. These two regions overlap with and include some of the homologous bases found in eight tRNAs aminoacylated by yeast phenylalanyl-tRNA synthetase (ROE, B., SIROVER, M., and DUDOCK, B. (1973) Biochemistry 12, 4146-4153). Although well separated in the primary and secondary structure, these two segments are in close proximity in the crystallographic tertiary structure. In two of the complexes, the third cross-linked fragment is near to the two common ones. The picture which emerges is that the enzymes all interact with the general area in which the two helical branches of the L-shaped tertiary structure fuse together, with additional interactions on other parts of the tRNAas well.     

3-([3-125I]iodo-4-hydroxyphenyl)propionyl carbohydrazide,a new radioiodination reagent for ultrasensitive detection and determination of periodate-oxidized nucleoside derivatives and other carbonyl compounds

A new radioiodination reagent for the identification and quantitation of periodate-oxidized ribonucleosides was developed. The reagent, 3-([3-¹²⁵I]iodo-4-hydroxyphenyl)propionyl carbohydrazide, was prepared by radioiodination of 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester in the presence of chloramine T, followed by reduction of the latter with sodium arsenite and treatment of the radioiodinated ester with an excess of carbohydrazide. The reagent reacted quantitatively with periodate-oxidized nucleosides to form ¹²⁵I-labeled morpholine derivatives which were separated by thin-layer chromatography and quantitated by liquid scintillation counting. The reagent was found to react also with other carbonyl compounds and thus may find more general application in the qualitative and quantitative ultramicroanalysis of aldehydes and ketones.     

Ligand design,synthesis and biological anti-HCV evaluations for genotypes 1b and 4a of certain 4-(3- & 4-[3-(3,5-dibromo-4-hydroxyphenyl)-propylamino]phenyl) butyric acids and 3-(3,5-dibromo-4-hydroxyphenyl)-propylamino-acetamidobenzoic acid esters

4-(4-[N-1-carboxy-3-(3,5-dibromo-4-hydroxyphenyl)-3-oxo-propylamino]phenyl)-4-oxo-butyric acid (V), 4-(3- & 4-[N-1-carboxy-3-(3,5-dibromo-4-hydroxyphenyl)-3-oxo-propylaminophenyl]-2-aryl-4-oxo-butyric acids (Xa–e) and 4-(2-alkyl-2-[N-3-(3,5-dibromo-4-hydroxyphenyl)-1-carboxy-3-oxo-propylamino]acetamido) benzoate esters (XVa–e) were designed, synthesized and biologically evaluated as anti-HCV for genotypes 1b and 4a. The design was based on their docking scores with HCV NS3/4A protease-binding site of the genotype 1b (1W3C), which is conserved in the genotype 4a structure. The docking scores predicted that most of these molecules have higher affinity to the HCV NS3/4A enzyme more than Indoline lead. These compounds were synthesized and evaluated for their cytopathic inhibitory activity against RAW HCV cell cultures of genotype 4a and also examined against Huh 5–2 HCV cell culture of genotype 1b, utilizing Luciferase and MTS assays. Compounds Xa and Xb have 95 and 80% of the activity of Ribavirin against genotype 4a and compounds XVa, XVb and XVd exerted high percentage inhibitory activity against genotype 1b equal 87.7, 84.3 and 82.8%, respectively, with low EC₅₀ doses.     

Liquid chromatography method for quantifying N-(4-hydroxyphenyl)retinamide and N-(4-methoxyphenyl)retinamide in tissues

A simple and accurate high-performance liquid chromatography (HPLC) method was developed to measure levels of N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and its main metabolite N-(4-methoxyphenyl)retinamide (4-MPR) in tissue. Following ultrasonic extraction of fresh tissue in acetonitrile (ACN), 4-HPR and 4-MPR were measured by HPLC with UV absorbance detection at 340 nm, using isocratic elution with ACN, H(2)O, and acetic acid. N-(4-ethoxyphenyl)retinamide (4-EPR) was employed as an internal standard. The 4-HPR and 4-MPR recovery in bovine liver or bovine brain tissue samples spiked with known amounts of 4-HPR and 4-MPR ranged from 93 to 110%. The detection limit of the method was 50 ng/ml. The method was tested on actual samples from an athymic (nu/nu) mouse carrying a subcutaneous tumor xenograft originating from SMS-KCNR neuroblastoma cells. The tissues were harvested and analyzed following a 3 day long treatment with intraperitoneal injections of 4-HPR/Diluent-12. 4-HPR and the metabolite 4-MPR were detected and quantitated in the tested tissues including tumor, liver, and brain. This method can be used to quantify 4-HPR and 4-MPR in different tissues to determine the bioavailability of 4-HPR.