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F Cramer

Publications and source records attributed to F Cramer.

At least 73 records · Page 4Linked to original sources
PubMed1982

Phenylalanyl-tRNA synthetases from hen liver cytoplasm and mitochondria, yeast cytoplasm and mitochondria, and from Escherichia coli: substrate specificity relationship with regard to ATP analogs.

Twelve structural analogs of ATP have been tested in the aminoacylation reaction of phenylalanyl-tRNA synthetases from hen liver cytoplasm and mitochondria, yeast cytoplasm and mitochondria and E. coli. Three compounds are substrates for all five phenylalanyl-tRNA synthetase, three are completely inactive, while the other ATP analogs show differing properties with the different enzymes. Their Km, Ki and V values have been determined. The importance of the amino group in Position 6, the nitrogen in Position 7 and an unsubstituted Position 8 of the purine moiety as well as the supposed anti-conformation of the glycosidic bond and coordination of the magnesium cation to N-7 appear to be conserved through evolution. Bulky substituents on the 2' and 3' of the ribose moiety are generally not tolerated. Graduation of substrate properties of some analogs are similar for the intracellular heterotopic isoenzymes from yeast and hen liver.

Adenosine Triphosphate↗
PubMed1982

Phenylalanyl-tRNA synthetases from cytoplasm and mitochondria of yeast and hen liver: comparison of their structural and catalytic properties.

Amino acid compositions and tryptic maps of the cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases from yeast and hen liver, respectively, demonstrate the similarity of these enzymes, although they are clearly not identical. Moreover, similarity is noted for catalytic properties like stoichiometries of complex formation with tRNAPhe and negative cooperativity of tRNAPhe binding, triggered by substrates. Analysis of the kinetics at saturating and subsaturating substrate concentrations indicates the contribution of the transfer of phenylalanine from the adenylate to tRNAPhe to the rate-determining step in aminoacylation and subunit interactions in the tetrameric enzymes. Furthermore, the locations of substrate-binding sites appear rather constant within species and in interspecies comparison. Subtle differences at certain sites, although homology exists, are exemplified by a special regulatory effect on activity by other amino acids only in the case of the cytoplasmic enzyme from hen liver. The results support the idea of a common ancestry by gene duplication for the cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases in fungi and animals, respectively.

Amino Acids↗
PubMed1981

Minor conformational changes of yeast tRNAPhe anticodon loop occur upon aminoacylation as indicated by Y base fluorescence.

The Y base fluorescence of highly purified yeast tRNAPhe was measured in order to detect possible conformational changes of the anticodon loop, which were induced as a consequence of aminoacylation. A small enhancement of Y base fluorescence intensity in the order of 5% was observed in situ during aminoacylation. The rotational mobility of the Y base of Phe-tRNAPhe and tRNAPhe was determined by measuring the fluorescence polarization at various temperatures between 5 degrees C and 35 degrees C. Differences in the fluorescence polarization of the Y base between these tRNAs were however not observed. These results confirm that minor changes in the microenvironment of the Y base occur upon aminoacylation, whereas significant conformational changes of the anticodon loop can be excluded.

Anticodon↗
PubMed1981

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.

Adenosine Triphosphate↗
PubMed1981

Arginyl-tRNA synthetase from Baker's yeast. Order of substrate addition and action of ATP analogs in the aminoacylation reaction; influence of pyrophosphate on the catalytic mechanism.

The order of substrate addition to arginyl-tRNA synthetase from baker's yeast has been investigated by bisubstrate kinetics, product inhibition and inhibition by three different inhibiting ATP analogs, the 6-N-benzyl, 8-bromo and 3'-deoxy derivatives of ATP, each acting competitively with respect to one of the substrates. The kinetic patterns are consistent with a random ter-ter mechanism, an addition of the three substrates and release of the products in random order. The different inhibitors are bound to different enzyme . substrate complexes of the reaction sequence. Addition of inorganic pyrophosphatase changes the inhibition patterns and addition of methylenediphosphonate as pyrophosphate analog abolishes the effect of pyrophosphatase, showing that the concentration of pyrophosphate is determinant for the mechanism of catalysis.

Acylation↗
PubMed1981

Survey on substrate specificity with regard to ATP analogs of aminoacyl-tRNA synthetases from E. coli and from Baker's yeast. Correlation to synthetase families.

The substrate specificity of twenty aminoacyl-tRNA synthetases from E. coli and thirteen enzymes from baker's yeast with regard to eight ATP analogs is investigated for a comparison of the active-site topography. The enzymes are arranged in a scheme of possible "enzyme families" and compared to earlier schemes.

Adenosine Triphosphate↗
PubMed1980

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.

Adenosine Triphosphate↗
PubMed1980

Phenylalanyl-tRNA, lysyl-tRNA, isoleucyl-tRNA and arginyl-tRNA synthetases. Substrate specificity in the ATP/PPi exchange with regard to ATP analogs.

The analogs of ATP have been tested in the ATP/PPi exchange reaction of phenylalanyl-tRNA, lysyl-tRNA, isoleucyl-tRNA and arginyl-tRNA synthetases from baker's yeast. Three compounds are substrates for phenylalanyl-tRNA, seven for lysyl-tRNA, two for isoleucyl-tRNA and five for arginyl-tRNA synthetase. Their Km and V values have been determined. No analog was an inhibitor. (3'-dATP), 3'-Deoxyadenosine 5'-triphosphate which is an inhibitor of the four enzymes in the aminoacylation reaction, becomes a good substrate in the PPi exchange. Additionally lysyl-tRNA synthetase accepts two analogs with modifications at position 6 of the purine and three analogs modified at the ribose moiety as substrates in the PPi exchange, whereas these compounds are inactive or inhibitors in the aminoacylation reaction. In general the enzymes are less specific in the ATP/PPi exchange and the results indicate a more sophisticated proof of the nucleotide moiety upon aminoacylation. This could occur with the aminoacyladenylate intermediate as well as with any other intermediate.

Adenosine Triphosphate↗
PubMed1979

Proton nuclear magnetic resonance of minor nucleosides in yeast phenylalanine transfer ribonucleic acid. Conformational changes as a consequence of aminoacylation, removal of the Y base, and codon--anticodon interaction.

The assignments of the resonances of the methyl and methylene groups belonging to the residues dihydro-uridine-16 and -17 (C5 and C6), dimethylguanosine-26, N-2-methylguanosine-10, and 7-methylguanosine-46 of yeast tRNAPhe at low temperature are reported. Observing the high-field proton NMR spectral region at different temperatures, the effects of aminoacylation, removal of the Y base, and codon-anticodon interaction on the tertiary structure of yeast tRNAPhe were investigated. The following are the results of this study. (1) The two dihydrouridine residues of tRNAPhe have different environments in aqueous solution: dihydro-uridine-16 is more shielded than dihydrouridine-17. (2) The ribothymidine residue from the fragment (47--76) of yeast tRNAPhe and from a tRNA with a partially disrupted structure exhibits multiple conformations arising from different stacking modes between the ribothymidine-54 and the guanosine-53 residue. (3) Upon aminoacylation the type of guanosine-53 interaction with ribothymidine-54 in the tRNAPhe changes. (4) Removal of the Y base from the anticodon loop of yeast tRNAPhe weakens the thermal stability of the tertiary interactions. (5) The interaction of two complementary anticodons in the absence of proteins and of ribosomes results in stabilization of the tertiary structure. Codon-anticodon interaction dependent rearrangement of the tertiary structure of yeast tRNAPhe was not observed. The spin-lattice relaxation times of the methyl and methylene groups of the minor nucleosides in yeast tRNAPhe demonstrate that the minor nucleosides undergo rotational reorientation (tau c) in the nano-second range. The observed differences in these tau c values indicate a similarity of structure of tRNAPhe in solution and in crystalline form.

Anticodon↗
PubMed1979

Reversible inactivation of tRNA nucleotidyltransferase from baker's yeast by tRNAPhe containing iodoacetamide-alkylated 2-thiocytidine in normal and additional positions.

2-Thiocytidine 5'-triphosphate, s2CTP, is able to replace CTP as a substrate for tRNA nucleotidyltransferase. s2CMP can be incorporated into both cytidine sites of the C-C-A terminus common to all tRNAs, and in the absence of ATP into at least two additional positions. This was shown by alkylation of the 2-thiocytidine residues with iodo[14C]acetamide, total nucleoside analysis, microgel electrophoresis and analysis of RNase T1 fragments of these tRNAs. The incorporation of the 3'-terminal AMP is not influenced by the additional s2CMP residues at pH 9.0. However, at pH 7.6 the additional s2CMP residues are hydrolysed and AMP can be incorporated into the normal position. Two different tRNAs with terminal 2-thiocytidine alkylated by iodoacetamide inhibit tRNA nucleotidyltransferase. This inhibition is significantly slower if an elongated species is used compared to a tRNA with alkylated 2-thiocytidine in the normal position 75. The addition of 2-mercaptoethanol reactivates the enzyme and leads to a cytidine containing tRNA. This reaction identifies the attacking nucleophile of the enzyme as cysteine residue, which is probably identical to a cysteine residue found in a similar experiment reported previously. The mechanism of the enzymatic and chemical reactions is discussed.

Adenosine Monophosphate↗
PubMed1979

Synthesis of carbonic anhydrase in rabbit and chicken reticulocyte lysates.

The synthesis of carbonic anhydrase, the second most abundant soluble protein of red blood cells, is shown to occur in rabbit and chicken reticulocyte lysates. This translation product was identified by chloroform/ethanol extraction, polyacrylamide gel electrophoresis in sodium dodecylsulphate and peptide mapping. In rabbit retic-locyte lysates, predominantly one of the two red cell isozymes, carbonic anhydrase I, is synthesised. The proportion of carbonic anhydrase synthesis (0.2-0.8% of total protein synthesis) in vitro is comparable to that (0.2-1.0%) in vivo for both rabbit and chicken reticulocytes.

Amino Acids↗