Search

The presence of two different aminoacyl-RNA synthetases for phenylalanine and aspartic acid has been demonstrated by Imamoto et al. (1965) and by Barnett and Epler (1966) in Neurospora Crassa; Yu and Rappaport (1966) have separated two leucyl-RNA synthetase activities in E. Coli by acrylamide-gel electrophoresis. Very recently (1967) Strehler et al. have shown chromatographycally different leucyl-synthetase activities in rabbit heart. In the present communication the separation of two leucyl-RNA synthetase activities from rat liver is reported; moreover, kinetic experiments and a different behaviour to heat lead to the conclusion that the two activities are bound to two different enzymes.

An enzyme which catalyzes the hydrolytic cleavage of various ribonucleosides to free base and ribose components has been obtained from cells of Pseudomonas fluorescens and purified about 300-fold by fractionation with protamine sulfate, ammonium sulfate, alumina Cγ, and diethylaminoethyl cellulose. The preparation thus purified catalyzes the hydrolysis of about 50 µmoles of uridine per min per mg of protein at 37°, and is stable when stored at -15°. The enzyme is highly specific for nucleosides containing the β-d-ribofuranosyl carbon-nitrogen linkage. A broad specificity is shown with respect to the base components: both pyrimidine and purine ribonucleosides are hydrolyzed. Because the maximum rate of hydrolysis of a pyrimidine nucleoside is generally much greater than that of a purine nucleoside, the systematic name N-ribosylpyrimidine ribohydrolase is suggested for this enzyme.

Phenylalanine and 2-amino-4-hydroxy-5,6-dimethyltetrahydropteridine, but not oxygen, protect phenylalanine hydroxylase ( l -phenylalanine tetrahydropteridine: oxygen oxidoreductase (4-hydroxylating), EC 1.14.3.1) from thermal inactivation. Several nucleosides also protect the enzyme from inactivation by heat. Most, but not all, of the nucleosides and related compounds which protect the enzyme also inhibit the activity of the enzyme. Adenosine is a linear competitive inhibitor of the enzyme with respect to the tetrahydropteridine cofactor. The inhibition constant for adenosine is 12.2 mM; the dissociation constant for the adenosine-enzyme complex is 2.7 mM as measured by the ability of adenosine at several concentrations to protect the hydroxylase from slow inactivation under the conditions of the assay. Dithiothreitol has a dual effect on the stability of the enzyme. It labilizes the enzyme to thermal inactivation at low concentrations of adenosine or phenylalanine. However, at high concentrations of adenosine or phenylalanine dithiothreitol stabilizes the enzyme. Several other sulfhydryl compounds affect the stability of the enzyme like dithiothreitol. On the other hand, most chelators without a sulfhydryl group do not alter the stability of the enzyme. Some possible implications of these findings are discussed.

As shown by viscosity and optical rotation dispersion measurements, subtilisin Carlsberg is not denatured in the presence of 10 M urea or 6 M guanidinium chloride. This unusual structural stability made it possible to investigate the effects of these hydrophobic-bond breaking solutes on various aspects of the enzymic interaction with substrates and inhibitors. The binding of the competitive inhibitor N- benzoylarginine was decreased by urea or guanidinium chloride. The nature of this effect was such as to implicate hydrophobic interaction as making a major contribution to the binding. By contrast, K s for the substrates N- acetyltyrosine ethyl ester, N- benzoylarginine ethyl ester and N- trans-cinnamoylimidazole was apparently unchanged by the presence of urea or guanidinium chloride. The influence of these solutes on k cat for the substrates was rather involved. Tentative hypotheses are put forward to account for the effects seen.

1. 1. An unusually stable esterase which catalyzes the hydrolysis of malathion to its monoacid was extreacted from irradiation-sterilized soil and non-irradiated soil with 0.2 M NaOH and partially purified by MnCl2 treatment, (NH4)2SO4 precipitation, dialysis and ion exchange chromatography. A 240-g sample of Chehalis clay loam yielded 28 mg of final preparation. It contained 9.8 mg of protein and hydrolyzed 31 μmoles of malathion in 4 h at pH 7.0 and 37°. Lineweaver-Burk plots obtained for two concentrations of the enzyme gave identical Km values of 2.12·10−4 M. The enzyme was optimally active around pH 7.0. The enzyme was very stable, being denatured only at temperatures above 70° and by 24-h exposures at pH 10.0. Lyophilization partially destroyed the activity but no loss of activity occurred during extended storage of enzyme solutions at 4° or frozen at − 10°. 2. 2. When partially purified enzyme was applied to soil, activity was detected for the duration of the experimental period (8 weeks). Its existence as a stable, cell-free soil enzyme is postulated from experimental evidence based on the persistence and adsorptive characteristics of the partially purified enzyme in soil. This esterase should be an excellent tool for investigating enzymatic biological transformations in soil.

Three temperature-sensitive, adenine-requiring mutants of Neurospora crassa were found to be osmotic-remedial when non-penetrating solutes were used to increase the osmolarity of the growth medium. The affected enzyme (adenylosuccinase) from one of the mutants (ad-4, 44206t) was found to have higher levels of activity when the organism was grown at non-permissive temperatures under osmotic-remedial conditions than when it was grown with adenine as a nutritional supplement. The enzyme synthesized at 30 degrees C in the presence of adenine exhibited increased sensitivity to inhibition by high salt concentrations and a lowered stability toward heat denaturation, indicating that the remedial effect may be the result of changes in the physical properties of the enzyme molecule. Temperature shift experiments indicate that the enzyme which is synthesized at permissive temperatures or under osmotic-remedial conditions is also stable in vivo under non-permissive conditions. This suggests that the critical period for temperature sensitivity, and conversely osmotic remediability, may be during protein synthesis or during the conformational folding of the protein.

Diploid human fibroblasts accumulate heat labile enzymes during the final stages of their life-span in culture. The RNA base analogue 5-FU induces premature senescence, which is preceded by the appearance of altered enzyme. These observations support Orgel's "error catastrophe" theory of ageing.

Results of the chemical and X-ray analyses are combined to locate the active site and the calcium binding sites.

Phospholipase was isolated (in two fractions) from Vipera palestinae venom and it was shown to possess phospholipase A activity (hydrolyzing diacyl-sn-glycerophosphorylcholines, e.g., lecithin, in the 2-position) as well as lysophospholipase (phospholipase B) activity (hydrolyzing 1-monoacyl-sn-glycerophosphorylcholines, e.g., lysolecithin, yielding free fatty acid and glycerophosphorylcholine). Each of the two purified enzyme fractions was homogeneous as judged by electrophoresis on acrylamide gel and by immunodiffusion and immunoelectrophoresis, and both had essentially equal activities. The ratio of the specific activity, at various purification stages, to the specific activity of the whole venom was the same for A activity (substrate lecithin) as for B activity (substrate lysolecithin). The enzyme has a molecular weight of 16,000, six S-S bridges, and no free thiol groups. At pH 7, dimerization was observed in the ultracentrifuge. A dissociation constant of about 10(-5) m was estimated. The amino acid composition for both fractions (140 amino acid residues) was found to be essentially the same. The A activity had a pH optimum at 9; B activity was low at this pH but increased steadily beyond pH 10.5. For the hydrolysis of lysolecithin the Lineweaver-Burk plot was found to be linear, giving K(m) = 1.1 mm and k(cat) = 0.55 sec(-1) at 37 degrees C and pH 10. 2-Deoxylysolecithin was also hydrolyzed by the enzyme at pH 10, with k(cat) = 0.01 sec(-1) (zero-order kinetics in the range 0.5-2.5 mm). For lecithin these constants could not be determined, but at 0.25 mm substrate the hydrolysis rate (at pH 9) of lecithin was about 1000 times the hydrolysis rate of lysolecithin (at pH 10).