PubMed · 563842
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S H Kim, J L Sussman. 1977. Transfer RNA: structure-function correlation.. https://pubmed.ncbi.nlm.nih.gov/563842/
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Specific inhibition of mammalian lysyl-tRNA synthetase by polyU is shown. Inhibition of the enzyme is dependent on the length of the oligonucleotide, since oligoU molecules with a length of less than 8 residues do not inhibit the aminoacylation, whilst the effect of oligoU molecules with a length of about 30 residues is the same as that of polyU. Inhibition is a result of recognition by the enzyme of the tRNALys anticodon sequence (UUU) coded by polyU. Aminoacylation of the oligoU molecule with attached CCA sequence (G(U)20-CCA) by yeast and mammalian lysyl-tRNA synthetases is demonstrated.
Cultured porcine aortic endothelial cells were conditioned in normal (5.2 mM) and elevated (15.6 mM) glucose, prelabeled with [14C]arachidonic acid and stimulated with ionophore A23187. Elevated glucose cultures released less radiolabeled products and less [14C]arachidonic acid. Analysis of cellular lipids revealed that elevated glucose reduced net loss of radiolabel from diacylphosphatidylethanolamine, did not affect early phosphatidylinositol hydrolysis, and increased net loss from diacylphosphatidylcholine and alkenylacylphosphatidylethanolamine. Uptake of radiolabel upon stimulation was examined to measure the role of reacylation on the diminished net release of radiolabel in elevated glucose cultures. Enhanced acylation of [3H]arachidonic acid into cellular lipids, especially PI, was observed in stimulated and resting cultures with elevated glucose. Further, pretreatment of the cultures with an acyltransferase inhibitor, thimerosal, prior to A23187 stimulation in radiolabeled cultures, abolished the effects of glucose on eicosanoid and arachidonic acid release. Differences in the ionophore-induced net loss of radiolabel from diacylphosphatidylethanolamine and phosphatidylinositol of the two glucose treatments were diminished by thimerosal exposure, while net loss of radiolabel from diacylphosphatidylcholine and alkenylacylphosphatidylethanolamine were unaffected. The data indicate that elevated glucose alters deacylation and enhances reacylation of arachidonic acid into endothelial cells and particularly into phosphatidylinositol. Enhanced reacylation may explain some of the altered lipid pathways that have been observed in experiments that elevate glucose concentrations or involve diabetes.
The initial rate steady-state kinetics of carboxypeptidase-Y-catalyzed hydrolysis and aminolysis reactions with some alpha-N-benzoyl-L-tyrosinyl compounds has been investigated using L-valinamide as the nucleophile in aminolysis. Hydrolysis of alpha-N-benzoyl-L-tyrosinyl ethyl ester, 4-nitroanilide, and -amide has been studied in the pH range 4-9. The results are interpreted in terms of the classical serine proteinase mechanism, which involves enzyme-substrate complex formation, followed by acylation and deacylation of the enzyme. The three reactions share the same deacylation step. It is rate-determining with the ester substrate, but with the 4-nitroaniline acylation is and this is even more pronounced with the amide. From the pH dependencies, no change of rate-determining step is apparent in the range pH 4-9. For the 4-nitroanilide and the amide substrates, the kinetic parameter, Kc/Km, is influenced by an ionizing group with a pK value of 6. Probably this is the active-site histidine residue, which thus is active in acylation in its deprotonated form. That group affects the deacylation reaction similarly as seen from the kinetics of the ester substrate. Aminolysis occurs in parallel to hydrolysis in the presence of reactive nucleophiles. Here L-valinamide was used as model nucleophile. The analysis of the observed kinetic effects of L-valinamide on the initial rate behaviour of carboxypeptidase-Y-catalyzed hydrolysis reactions suggests a reaction mechanism which involves (a) the binding of the free nucleophile to the free enzyme and (b) reaction of the free nucleophile with the acyl-enzyme complex forming an enzyme-aminolysis product complex, which dissociates into the free enzyme and the aminolysis product. The reactions are characterized by a number of kinetic parameters, the values of which are determined. The results of aminolysis progress reactions indicate that the formation of the product in high yields is strongly dependent on the leaving group of the substrate. The initial production of aminolysis product, however, is the same for the three substrates. But the fact that their Kc/Km values differ by several orders of magnitude leads to significantly different progresses of the aminolysis. The ester substrate is the only one that efficiently competes with and hinders the hydrolysis of the aminolysis product.