Higher eukaryotes contain tRNA transglycosylases that incorporate the guanine derivative queuine from the nutritional environment into specific tRNAs by exchange with guanine at position 34. Alterations in the queuosine content of specific tRNAs are suggested to be involved in regulatory mechanisms of major routes of metabolism during differentiation.Dictyostelium discoideum has been applied as a model to investigate the function of queuine or queuine-containing tRNAs. Axenic strains are supplied with queuine by peptone, but they grow equally well in a defined queuine-free medium. Queuine-lacking amoebae, starved in suspension culture for 24 h, lose their ability to differentiate into stalk cells and spores, whereas amoebae sufficiently supplied with queuine will overcome this metabolic stress and undergo further development when plated on agar.The results presented here show that D(-)-lactate occurs in the slime mould in millimolar amounts and that its level is remarkably decreased in queuine-lacking cells after 24 h of starvation in suspension culture. The modified nucleoside queuosine, 7-{[(4,5-cis-dihydroxy-2-cyclopenten-1 -yl)-amino]-methyl} -7-deazaguanosine, and its glycosylated derivatives occur at position 34 of the anticodon of tRNATy', tRNAHiS, tRNAAS" and tRNAAsp of eubacteria and all studied eukaryotes except yeast [l, 21. Eubacteria synthesize queuine de novo, the biosynthesis sharing common pathways with that of pteridines. Mammals are supplied with queuine by nutrition or by the intestinal flora. Queuine is inserted into the corresponding tRNAs by exchange with guanine 34 by specific tRNA transglycosylases, which can be inhibited by pteridines. The extent of this modification in tRNAs from eubacteria, protists, and higher eukaryotes depends on the metabolic state of the cells and is remarkably changed in organisms undergoing differentiation and ageing. In tRNAs from neoplastically transformed cells queuosine (Q) is partly or totally replaced by G. The extent of Q deficiency seems to be correlated to the malignancy of the tumors (for summarizing references see [3 -61).Dedicated to Friedrich Cramer on the occasion of his 60th birthday.Abbreviations. Queuosine (Q), { 7-[(4,5-cis-dihydroxy-2-cyclopenten-l-yl)-amino]-methyl}-7-deazaguanosine; nLDH, NADdependent lactate dehydrogenase; iLDH, NAD-independent lactate deh ydr ogenase .Enzymes. NAD-dependent D(-)-lactate dehydrogenase (EC 1.1.1.28); NAD-dependent L(+)-lactate dehydrogenase (EC 1.1.1.27); NAD-independent D(-)-lactate dehydrogenase (EC 1.1.2.4) ; NADindependent L(+)-lactate dehydrogenase (EC 1.1.2.3).Interestingly the extent of Q modification in tRNAs fluctuates in rat tissues in an ordered manner during ageing, being essentially complete at 9 months [5]. Precisely at this time, the activity of lactate dehydrogenase isoenzymes reaches a maximum [7]. An attempt was therefore made to establish whether the observed metabolic changes in response to Q modification in tRNA might be somehow linked to the metabolism of lactate.NAD-dependent lactate d...

A 7-methylguanine (m7G) specific tRNA methyltransferase from E. coli MRE 600 was purified about 1000 fold by affinity chromatography on Sepharose bound with normal E. coli tRNA. The purified enzyme catalyzes exclusively the formation of m7G in submethylated bulk tRNA of E. coli K12 met- rel-. The purified enzyme transfers the methyl group from S-adenosyl-methionine to initiator tRNA of B. subtilis and 0.8 moles m7G residues are formed per mole tRNA. It is suggested that the enzyme specifically recognizes the extra arm unpaired guanylate residue.

To elucidate subtle functions of transfer ribonucleic acid (tRNA) modifications in protein synthesis, pairs of tRNA's that differ in modifications at specific positions were prepared from Bacillus subtilis. The tRNA's differ in modifications in the anticodon loop, the extra arm, and the TWC loop. The functional properties of these species were compared in aminoacylation, as well as in initiation and peptide bond formation, at programmed ribosomes. These experiments demonstrated the following. (i) In tRNAMet the methylation of guanosine 46 in the extra arm to 7-methylguanosine by the 7-methylguanosine-forming enzyme from Escherichia coli changes the aminoacylation kinetics for the B. subtilis methionyl-tRNA synthetase. In repeated experiments the Vmax value is decreased by onehalf. (ii) tRNAfmet species with ribothymidine at position 54 (rT54) or uridine at position 54 (U54) were obtained from untreated or trimethoprim-treated B. subtilis. The formylated fMet-tRNAPet species with U54 and rT54, respectively, function equally well in an in vitro initiation system containing AUG, initiation factors, and 70s ribosomes. The unformylated MetAtRNAmet species, however, differ from each other: "Met-tRNAm't rT" is inactive, whereas the U54 counterupart effectively forms the initiation complex. (iii) Two isoacceptors,tRNAlhe and tRNA he, were obtained from B. subtilis. tRNAIhe accumulates only under special growth conditions and is an incompletely modified precursor oftRNA2he: in the first position of the anticodon, guanosine replaces Gm, and next to the 3' end of the anticodon (isopentenyl)adenosine replaces 2-thiomethyl-N6-(isopentenyl)adenosine. Both tRNA's behave identically in aminoacylation kinetics. In the factor-dependent AUGU3-directed fornation of fMet-Phe, the undermodified tRNAphe is always less efficient at Mg2+ concentrations between 5 and 15 mM than its mature counterpart.

Zusammenfassung:Hydroxynitril-Lyase (EC 4.1.2.10) setzt Cyanwasserstoff mit einer großen Anzahl aliphatischer, aromatischer und heterocyclischer Aldehyde zu optisch aktiven oc-Hydroxynitrilen um. Die Disk-Elektrophorese aus Mandeln gewonnener D-Hydroxynitril-Lyase ergibt eine sehr starke und eine schwache Bande. Präparativ kann das Enzym an Cellulose-Ionenaustauscher Whatman DE 32 in drei Komponenten aufgespalten werden, die deut-Summary: Occurrence and characterisation of the isoenzymes of -hydroxynitrile lyase from almonds. D-Hydroxynitrile lyase (EC 4.1.2.10) catalyses the reactions of hydrogencyanide with a great number of aliphatic, aromatic and heterocyclic aldehydes to optically active a-hydroxynitriles. Disc-electrophoresis of mandelo-D-hydroxynitrile lyase gives one very large and one small band. Ion exchange chromatography on Whatman DE 32 leads to three components of different activity. FAD is the coenzyme for each of the three com- Nach vorbereitenden ArbeitenHydroxynitril-Lyase aus Mandeln rein und kristallin darstellen.lieh unterschiedliche Aktivitäten besitzen und als Coenzym alle FAD enthalten. Diese Isoenzyme besitzen eine unterschiedliche elektrophoretische Beweglichkeit in Medien wie Agar-oder Acrylamidgel und haben differente isoelektrische Punkte. Diffusionsmessungen an Sephadex G-150 ergeben geringe Unterschiede in den Molekulargewichten. Die drei Isoenzyme besitzen gleiche antigene Eigenschaften.ponents, which are isoenzymes of the mandelo-Dhydroxynitrile lyase. These isoenzymes show differences in the elect rophoretic mobility when running in agar-or polyacrylamidgel and have different isoelectric points. According to the diffusion behaviour on Sephadex G-150, there are slight differences in the molecular weight. The three isoenzymes have identical antigenic properties.Hydroxynitril-Lyase gehört zur Gruppe der Flavoproteine. Das Coenzym, Flavin-adenin-dinucleotid, kann mit Säuren in Ammoniumsulfatlösung bei gleichzeitiger Anwesenheit von Bromid reversibel

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