Substitution of His-84 (-~ Gin and -~ Ala), a residue of the switch II region of E. coli elongation factor (EF) Tu, hardly affected the binding of GTP or GDP. The activity in poly(Phe) synthesis and GTP hydrolysis of EF-Tu H84Q were both reduced to about 35%, as compared to EF-Tu wt, whereas EF-Tn H84A was inactive in poly(Phe) synthesis but still showed a 10% residual GTPase activity. Phe-tRNA Phe exerted a similar inhibitory effect on the GTPase activity of EF-Tn wt and EF-Tu H84Q while abolishing that of EF-Tu H84A. Ribosomes enhanced the GTPase activity of EF-Tu H84Q, but not that of EF-Tu H84A, on which they even seemed to exert an inhibitory effect. The one-round GTP hydrolysis associated with the EF-TuH84Q-dependent binding of Phe-tRNA phe to poly(U)-programmed ribosomes was less efficient than with EF-Tu wt. Kirromycin stimulated the GTPase activities of both mutants less than EF-Tu wt. The results of this work do not support a catalytic role of His-84 in the intrinsic GTPase of EF-Tu, but they emphasize the importance of its side-chain for polypeptide synthesis and GTP hydrolysis. a residual GTPase activity, making it unlikely that H84 is solely responsible for the catalysis [5].In order to shed further light on its function, His-84 has been substituted by glutamine or alanine in the intact EF-Tu. The former substitution introduces the same residue as found in the homologous position in p21, which shares several properties with histidine including the ability of forming a hydrogen bond; the latter one investigates the effects due to the elimination of interactions but the hydrophobic ones. We did not introduce a glycine, as in the case of the G domain H84G, to avoid possible side-effects associated with the increased backbone flexibility of this residue.The results show that the substitution H84Q is compatible with polypeptide synthesis and GTPase activity, whereas EFTu H84A can only sustain a low intrinsic GTPase activity. This emphasizes the importance of the nature of the side-chain in position 84 for a physiological function of EF-Tu in protein biosynthesis and the associated hydrolysis of GTP.
Hemoglobin, methemoglobin, and blood nitrite concentrations were determined during and after 96-hour exposures of sea bass Dicentrarchus labrax to sublethal concentrations of nitrite in seawater at 26 C. As exposure time or exposure concentration increased, total and functional hemoglobin concentrations decreased in blood, while percent methemoglobin increased. The 96-hour median effective concentrations of nitrite in seawater (EC50, the concentration causing 50% reduction in blood) were 87.2 mg/liter for total hemoglobin (95% confidence limits, 64-118 mg/liter) and 62.4 mg/liter (45-87 mg/liter) for functional hemoglobin. Histochemical analysis of spleen tissues from fish exposed to 50 and 75 mg/liter NO2-N, which reduced total hemoglobin to 17% and 49%, respectively, showed Fe ++ + originating from hemoglobin destruction and indicated hemolytic anemia caused by spleen macrophages. Nitrite concentrations in blood were lower than those in test water. Inhibition of nitrite uptake by chloride in seawater may protect sea bass against nitrite toxicity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.