Cross-linked iron dextran is an efficient oral phosphate binder in the rat.

BACKGROUND: There is a need for alternative oral phosphate binders. In-vitro studies showed that iron(III)oxide-hydroxide-modified cross-linked dextran is a promising, insoluble phosphate-binding agent. The present study was designed to assess its in-vivo efficacy and safety in the rat. STUDY, DESIGN AND METHODS: Iron(III)oxide-hydroxide modified dextran beads were mixed with normal rat feed in a proportion of 8% by weight. With this formula rats were fed for 4 weeks. A control group received the same diet without added phosphate binder. Samples of blood, urine, and faeces were taken from each animal before the phosphate binder was administered, 2 weeks later, and at the end of the examination period (day 29). Phosphate, calcium, iron were analysed in the blood samples. Calcium and phosphate concentrations were determined in the urine, phosphate, calcium, and iron concentrations in the excrements. Stability of the material in the duodenum was also simulated. RESULTS AND CONCLUSIONS: The results demonstrate an excellent phosphate-binding capacity of the material and a good tolerance during the intestinal passage. No significant chemical or enzymatic degradation, histological alterations, or other treatment-related macroscopic findings were recorded. The present efficacy and toxicity study has shown effective phosphate binding with no toxicity and no iron release after ingestion of this novel phosphate binding agent. We propose clinical evaluation studies to assess whether similar efficacy and safety can be shown in humans.

Fidelity in the aminoacylation of tRNA(Val) with hydroxy analogues of valine, leucine, and isoleucine by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli.

Several analogues of valine, leucine, and isoleucine carrying hydroxyl groups in the gamma- or delta-position have been tested in the aminoacylation of tRNA by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli. Results of the ATP/PPi exchange and of the aminoacylation reactions indicate that the amino acid analogues not only can form the aminoacyl adenylate intermediate but are also transferred to tRNA. However, the fact that the reaction consumes an excess of ATP indicates that the misactivated amino acid analogue is hydrolytically removed. Thus, valyl-tRNA synthetase from S. cerevisiae shows a high fidelity in forming valyl-tRNA. Although the much bulkier amino acid analogues allo- and iso-gamma-hydroxyvaline and allo- and iso-gamma-hydroxyisoleucine are initially charged to tRNA, the misaminoacylated tRNA(Val) is enzymatically deacylated. This cleavage reaction is mediated by the hydroxyl groups of the amino acid analogues which are converted into the corresponding lactones.

The proofreading of hydroxy analogues of leucine and isoleucine by leucyl-tRNA synthetases from E. coli and yeast.

Three analogues each of leucine and isoleucine carrying hydroxy groups in gamma- or delta- or gamma- and delta-position have been synthesized, and tested in the aminoacylation by leucyl-tRNA synthetases from E. coli and yeast. Hydrolytic proofreading, as proposed in the chemical proofreading model, of these analogues and of homocysteine should result in a lactonisation of these compounds and therefore provide information regarding the proofreading mechanism of the two leucyl-tRNA synthetases. Leucyl-tRNA synthetase from E. coli shows a high initial substrate discrimination. Only two analogues, gamma-hydroxyleucine and homocysteine are activated and transferred to tRNALeu where a post-transfer proofreading occurs. Lactonisation of gamma-hydroxyleucine and homocysteine could be detected. Leucyl-tRNA synthetase from yeast has a relatively poor initial discrimination of these substrates, which is compensated by a very effective pre-transfer proofreading on the aminoacyl-adenylate level. No lactonisation nor mischarged tRNALeu is detectable.

Biochemical comparison of the Neurospora crassa wild type and the temperature-sensitive and leucine-auxotroph mutant leu-5. Purification of the cytoplasmic and mitochondrial leucyl-tRNA synthetases and comparison of the enzymatic activities and the degradation patterns.

The cytoplasmic leucyl-tRNA synthetases of Neurospora crassa wild type (grown at 37 degrees C) and mutant (grown at 28 degrees C) were purified approximately 1770-fold and 1440-fold respectively. Additional enzyme preparations were carried out with mutant cells grown for 24 h at 28 degrees C and transferred then to 37 degrees C for 10-70 h of growth. The mitochondrial leucyl-tRNA synthetase of the wild type was purified approximately 722-fold. The mitochondrial mutant enzyme was found only in traces. The cytoplasmic leucyl-tRNA synthetase from the mutant (grown at 37 degrees C) in vivo is subject of a proteolytic degradation. This leads to an increased pyrophosphate exchange, without altering aminoacylation. Proteolysis in vitro by trypsin or subtilisin of isolated cytoplasmic wild-type and mutant leucyl-tRNA synthetases, however, did not establish and difference in the degradation products and in their catalytic properties. Comparing the cytoplasmic wild-type and mutant enzymes (grown at 28 degrees C) via steady-state kinetics did not show significant differences between these synthetases either. The rate-determining step appears to be after the transfer of the aminoacyl group to the tRNA, e.g. a conformational change or the release of the product. Besides leucine only isoleucine is activated by the enzymes with a discrimination of approximately 1:600; however, no Ile-tRNALeu is released. Similarly these enzymes, when tested with eight ATP analogs, cannot be distinguished. For both enzymes six ATP analogs are neither substrates nor inhibitors. Two analogs are substrates with identical kinetic parameters. The mitochondrial wild-type leucyl-tRNA synthetase is different from the cytoplasmic enzyme, as particularly exhibited by aminoacylating Escherichia coli tRNALeu but not N. crassa cytoplasmic tRNALeu. The presence of traces of the analogous mitochondrial mutant enzyme could be demonstrated. Therefore, the difference between wild-type and mutant leu-5 does not rest in the catalytic properties of the cytoplasmic leucyl-tRNA synthetases. Differences in other properties of these enzymes are not excluded. In contrast the activity of the mitochondrial leucyl-tRNA synthetase of the mutant is approximately 1% of that of the wild-type enzyme.

Formycin 3' end modified tRNATrp. Recognition by avian myeloblastosis virus reverse transcriptase and primer function.

Primer tRNATrp has been modified at the 3' end by adenosine analogues: 2'deoxyadenosine, 3'deoxyadenosine, 3' amino-3' deoxyadenosine and formycin. Aminoacylation of modified tRNATrp with cognate aminoacyl-tRNA synthetase and primer function for DNA synthesis catalyzed by AMV reverse transcriptase have been studied. The tRNATrp was able to accept tryptophan but did not initiate the DNA synthesis directed by 35S AMV RNA. Recognition of modified tRNATrp by AMV reverse transcriptase was not affected as followed by enzyme-tRNA complex formation. The functional consequences of these effects are discussed.

Evolutionary aspects of accuracy of phenylalanyl-tRNA synthetase. A comparative study with enzymes from Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, and turkey liver using phenylalanine analogues.

The phenylalanyl-tRNA synthetases from Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, and turkey liver activate a number of phenylalanine analogues (tyrosine, leucine, methionine, p-fluorophenylalanine, beta-phenylserine, beta-thien-2-ylalanine, 2-amino-4-methylhex-4-enoic acid, mimosine, N-benzyl-L- or N-benzyl-D-phenylalanine, and ochratoxin A), as demonstrated by Km and kcat of the ATP/PPi pyrophosphate exchange. Upon complexation with tRNA, the enzyme-tRNAPhe complexes show a significantly increased initial discrimination of these amino acid analogues expressed in higher Km and lower kcat values, as determined by amino-acylation of tRNAPhe-C-C-A(3'NH2). The overall accuracy is further enhanced by a second discrimination, a proofreading step. The strategies employed by the enzymes with respect to accuracy differ. Better initial discrimination in the aminoacylation and less elaborated proofreading for the E. coli enzyme can be compared to a more efficient proofreading by other synthetases. In this way the comparatively poor initial amino acid recognition in the case of the S. cerevisiae and N. crassa enzymes is balanced. The extent of initial discrimination is therefore inversely coupled to the hydrolytic capacity of the proofreading. A striking difference can be noted for the proofreading mechanisms. Whereas the enzymes from E. coli, S. cerevisiae, and N. crassa follow the pathway of posttransfer proofreading, namely, enzymatic hydrolysis of the misaminoacylated tRNA, the turkey liver enzyme uses tRNA-dependent pretransfer proofreading in the case of natural amino acids. In spite of the same subunit structure and similar molecular weight, the phenylalanyl-tRNA synthetases from a prokaryotic and lower and higher eukaryotic organisms show obvious mechanistic differences in their strategy to achieve the necessary fidelity.

Involvement of tRNA in retrovirus expression: biological implications of reverse transcriptase-primer tRNA interactions.

We have previously studied the topographical and functional implications of the recognition of primer tRNATrp by avian retrovirus reverse transcriptase. Here we have presented evidence that the enzyme is able to deacylate beef liver Trp-tRNATrp, provided that 35-S viral RNA is present in the incubation mixture. No effect of dNTPs on this activity was observed. The extensive modification of tRNATrp with acrylonitrile led to a marked loss of priming activity by tRNATrp if the annealing between primer and template was performed at 37 degrees C, while the annealing of cyanoethylated tRNA with the viral genome at 75 degrees C gave almost normal levels of cDNA synthesis. We have also studied the priming behaviour of tRNATrp, modified by incorporation of various analogs of adenosine. Only tRNATrp-2'dA was active in cDNA initiation; 3'dA, 3'NH2-3'dA, and primer tRNA with formycin in the 3' end showed low or nonexistent priming activity.

Avian myeloblastosis reverse transcriptase deacylates tryptophanyl-tRNA.

Tryptophanyl-tRNA can be used as a primer for RNA-dependent DNA synthesis by avian reverse transcriptase. We have determined that whereas the retroviral polymerase is not by itself capable of deacylating Trp-tRNATrp, the ester linkage between the 3' OH of the ribose moiety and the aminoacid can be very efficiently hydrolyzed when both the polymerase and the viral 35 S RNA are present.

Isoleucyl-tRNA synthetase from Baker's yeast. Action of ATP analogs in pyrophosphate exchange and aminoacylation, two pathways of the aminoacylation depending on concentration of pyrophosphate.

The order of substrate addition to isoleucyl-tRNA synthetase from baker's yeast has been investigated by steady-state kinetics with inhibition by four different inhibiting ATP analogs acting competitively, uncompetitively and noncompetitively with respect to ATP, namely purineriboside (= nebularin), 3'-deoxy-adenosine (= cordycepin), 8-amino-adenosine and 8-azido-adenosine 5'-triphosphates. The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase. Additionally, bisubstrate kinetics and product inhibition studies were carried out. The inhibition patterns indicate a multisite system with a minimum number of two sites for each of the substrates. The results of the pyrophosphate exchange studies are consistent with formation of E . Ile-AMP . ATP . Ile complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, subsequent release of pyrophosphate and random addition of a second molecule of ATP and isoleucine. For the aminoacylation in the absence of pyrophosphatase an ordered ter-ter mechanism is postulated; in the presence of pyrophosphatase the mechanism is random bi-uni uni-bi ping-pong. Both the pyrophosphate and the analogs of this compound such as imidodiphosphate or methylenediphosphonate can induce the enzyme to act in the ter-ter mechanism.

Fluorimetric study of yeast tRNAPheCCF in the complex with phenylalanyl-tRNA synthetase. Evidence for a correlation between the structural adaptation of both macromolecules and the appearance of the acylation activity.

The fluorescence properties of yeast tRNAPheCCF (tRNAPhe in which the 3'-terminal adenosine has been replaced by formycin) and tRNAPheCCFoxi-red (tRNAPheCCF after periodate oxidation followed by borohydride reduction) were studied in the complex with the cognate aminoacyl-tRNA synthetase. In both cases a conformational change affecting the 3' end was observed in a magnesium concentration range close to 1 mM. The modification of formycin fluorescence could be ascribed simultaneously to the existence of a tautomeric equilibrium of the fluorescent probe and to a pH effect raising from a prototropic effect at the active site of phenylalanyl-tRNA synthetase, and to a partial destacking of the 3'-formycin from the adjacent C residue. The observed transconformation, which can be related to the structure modification of the anticodon loop previously reported [Ehrlich, Lefèvre, and Remy (1980) Eur. J. Biochem. 103, 145-153], takes place in the magnesium concentration range allowing the transfer of the activated amino acid from the adenylate to the tRNA. The interconnection between the anticodon loop and the accepting end was further supported by the observation that wybutine excision hinders the specific structure modification of 3'-formycin upon binding to the synthetase. The tRNAPhe transconformations occurring in the complex with the cognate synthetase probably reflect a reciprocal adaptation of both macromolecules which might lead to the optimal aminoacylation velocity and thus contribute to the specificity of aminoacylation, since it was previously established that this specificity relies more strongly on the kinetics of the reaction than on a discrimination of tRNAs according to different affinities.

A novel enzymatic activity of phenylalanyl transfer ribonucleic acid synthetase from baker's yeast: zinc ion induced transfer ribonucleic acid independent hydrolysis of adenosine triphosphate.

Phenylalanyl-tRNA synthetase from baker's yeast in the presence of phenylalanine or other amino acids misactivated by the enzyme, ATP, and low concentrations of Zn2+ is able to hydrolyze ATP to AMP and PPi very efficiently. After dialysis of the enzyme against ethylenediaminetetraacetic acid (EDTA), this amino acid dependent but tRNAPhe-independent hydrolysis is suppressed to negligible levels. The ATP hydrolysis can be restored by the addition of Zn2+ to the EDTA-dialyzed enzyme. During aminoacylation of tRNAPhe the Zn2+-induced ATP hydrolysis parallels the aminoacylation reaction, leading to nonstoichiometric production of AMP. Mechanistically, we conclude that Zn2+ can be bound to phenylalanyl-tRNA synthetase and can influence the stability of ATP if an activatable amino acid is present. The influence of Zn2+, if any, on the aminoacylation of tRNAPhe is not known. In practice, this side reaction is of the utmost importance in all cases in which the fate of ATP during aminoacylation is followed, especially if the stoichiometry of ATP consumption in relation to Phe-tRNAPhe formation has to be determined.

Conformation transitions of a tRNA--aminoacyl-tRNA synthetase complex induced by tRNAs bearing different modifications in the 3' terminus.

The influence of modifications of the 3'-terminal adenosine of tRNAPhe (yeast) on the complex formation between this tRNA and phenylalanyl-tRNA synthetase (yeast) has been investigated by using fluorescence titrations and fast kinetic techniques. Subtle changes in the 3' terminus are reflected by distinct alterations in the two-step recognition process which had been demonstrated earlier for the native substrate tRNAPheCCA [Krauss, G., Riesner, D., & Maass, G. (1977) Nucleic Acids Res. 4, 2253--2262]. Binding experiments with tRNAPheCC, tRNAPheCCA-ox-red, tRNAPheCC2'dA, tRNAPheCC3'dA, tRNAPheCC-formycin, and tRNAPheCC-formycin-ox-red confirm that the 3'-terminal adenosine participates in a conformational change of the tRNA--synthetase complex. This is valid in both the absence and presence of phenylalaninyl-5'-AMP, the alkyl analogue of the aminoacyladenylate. As compared to tRNAPheCCA, a slower conformational change is observed with the competitive inhibitor tRNAPheCC-formycin-ox-red. The reaction enthalpy and/or the quench of the Y-base fluorescence that accompany the conformational change are altered upon binding of tRNAPheC2'dA, tRNAPheCC3'dA, and tRNAPheCC-formycin. It is evident that the final adaptation between tRNA and its synthetase in the complex is determined by the chemical nature of the 3'-terminal nucleotide. This is of vital importance for the specificity of the aminoacylation process.