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Distinct FMN reductases specific for NADH and NADPH were identified in extracts of Beneckea harveyi. These enzymes differ in their physical (molecular weight, thermostability) as well as in their chemical properties (binding constants for NADH and NADPH). The NADH-specific enzyme is more efficient than the NADPH-specific one with respect to the bioluminescent reaction.Some years ago Strehler and Cormier [l] and McElroy et al. [2] identified the components required for maximum luminescence in a cell-free extract of Photobacterium fischeri, i.e. FMNH2, a long-chain aldehyde, oxygen and luciferase. FMNH, which readily oxidises in the presence of O2 is generated by the oxidation of NADH or NADPH through an enzyme which has been given the names of FMN reductase or NAD(P)H dehydrogenase [3]. In wildtype Photobacterium fischeri, it is difficult to separate the dehydrogenase activity from luciferase, suggesting a possible association in vivo between the two activities [4]. Using a purification procedure adapted from the method of Gunsalus et al. [5], we were able to separate and characterize NADH-specific and NADPH-specific FMN reductase activities in Beneckea harveyi. MATERIALS AND METHODS Growth and Harvesting of CellsBeneckea harveyi cells (previously described as Photobacterium fischeri strain MAV [6] and recently identified by Reichelt and Bauinann [7]), kindly provided by Dr J. W. Hastings, were subcultured in the solid agar medium previously described [5]. The cells were grown at 25 "C with vigorous aeration; anti-foam was added to the medium. Cells were cooled at the time of maximum luminescence, at a density of 1 x lo9 cells/ml, and harvested by continuous Enzymes. Luciferase; NADH-specific and NADPH-specific F M N reductase (EC 1.6.99.-); alcohol dehydrogenase (EC 1.1.1.1).Eur. J. Biochem. 57 (1975) centrifugation in a Sorvall centrifuge (rotor SS 34) at a speed of 16000 rev./min and a flow rate of 250 ml/ min. The yield of packed wet cells was about 4 g/1 medium. Purification ProcedureWe followed the method of Gunsalus-Miguel et al. [5], with minor modifications for the purification of luciferase. Frozen cells were thawed and lysed in cold water adjusted to pH 7.0 with NaOH and containing 0.1 mM dithioerythritol, 5 mM MgSO, and a trace of DNase, in a ratio of 6 ml/g wet pellet. After overnight stirring at 4 " C , EDTA was added to a final concentration of 15 mM. The crude extract was centrifuged for 30 min at 8000 rev./min. Dry DEAE-cellulose (Whatman DE-32) was added to the supernatant (0.22 g/g wet pellet); the pH was maintained at 7.0 by thc addition of 0.5 N acetic acid. Protein was extracted batchwise by increasing phosphate concentrations (0.1, 0.15, 0.5 M potassium phosphate pH 7.0). The fraction extracted by 0.5 M phosphate was then fractionated with ammonium sulfate (special enzyme grade, Mann Research Laboratories). Material precipitating between 40 and 75% saturation was dissolved in 0.25 M phosphate buffer pH 7.0 (0.1 mM dithioerythritol) and dialysed for 16 h against a large volume of the s...

Arginyl-tRNA synthetase from Escherichia coli K12 has been purified more than 1000-fold with a recovery of 17%. The enzyme consists of a single polypeptide chain of about 60 000 molecular weight and has only one cysteine residue which is essential for enzymatic activity. Transfer ribonucleic acid completely protects the enzyme against inactivation by p-hydroxymercuriben zoate. The enzyme catalyzes the esterification of 5000 nmol of arginine to transfer ribonucleic acid in 1 min/mg of protein at 37 degrees C and pH 7.4. One mole of ATP is consumed for each mole of arginyl-tRNA formed. The sequence of substrate binding has been investigated by using initial velocity experiments and dead-end and product inhibition studies. The kinetic patterns are consistent with a random addition of substrates with all steps in rapid equilibrium except for the interconversion of the cental quaternary complexes. The dissociation constants of the different enzyme-substrate complexes and of the complexes with the dead-end inhibitors homoarginine and 8-azido-ATP have been calculated on this basis. Binding of ATP to the enzyme is influenced by tRNA and vice versa.

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5')tetraphospho(5')adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

I n this paper we report on the properties of the pursed isoleucyl-tRNA synthetase from Bacillus stearothermophilus. The enzyme which was purified approximately 500-fold, has a molecular weight of about 110000.The effect of temperature on the activation and transfer activities has been studied. The thermostable enzyme shows an optimal activity a t 65 "C for the isoleucine-dependent ATP-PPI exchange reaction and a t 45 "C for the formation of isoleucyl-tRNA. Attempts to heat inactivate the amino-acid-transfer activity selectively, i.e. without loss of the activation reaction, were unsuccessful. The protection of the enzyme against heat inactivation in presence of tRNA makes it possible to study the binding of this molecule to the protein.An association constant of 0.11 nM-l was determined a t 70.5 "C. Formation of this tRNA enzyme complex was also demonstrated by binding onto nitrocellulose filters. By this technique one tRNA site per enzyme molecule was detected and an affinity constant of 0.4 nM-1 measured. The same results were obtained either with tRNA from Escherichia coli or B. steurothermophilus, showing close resemblance of the two nucleic acids. Periodate-oxidation of the 3'-terminal adenosine of the tRNA does not perceptibly affect the binding or the degree of protection of the enzyme against heat inactivation. These effects are still strictly dependent on magnesium ions and a clearcut requirement of these ions for the formation and/or stabilization of the tRNA * enzyme complex has been demonstrated.The intermediate isoleucine * AMP * enzyme complex with a molar ratio of 1 : 1 : 1 was isolated by molecular sieving and by chromatography on DEAE-cellulose. Mg2+ ions are required for the formation of the complex but are not necessary to stabilize the isoleucyl-adenylate once this is bound to the enzyme. The transfer of activated isoleucine to tRNA again requires magnesium ions and must probably be related to the formation of a tRNA -enzyme complex.Part only of the activated isoleucine is transferred to tRNA; this results essentially from the breakdown of a fraction of the isoleucine. AMP-enzyme complex, induced by tRNA and Mg2+ ions, acting together. Periodate-oxidized tRNA of B. stearothermophilus or of E. coli in presence of Mg2+ ions completely releases isoleucine from the complex.The major purpose of our investigation was to study the structural basis for the great specificity of the aminoacyl-tRNA synthetases. These enzymes select a single amino acid among 20 species, activate it by formation of an enzyme-bound aminoacyladenylate and subsequently transfer it to one, or a few, amino-acid-specific transfer RNAs.The specificity of the second step is higher than that of the activation step [1-31. Misrecognition between the enzyme and tRNA has been observed Enzymes. Isoleucyl-tRNA synthetase or L-isoleucine