[3H] Conduritol C cis-epoxide (1,2-anhydro-epi-inositol, I) was synthesized as an active-site-directed inhibitor for lacZ P-galactosidase from Escherichia coli. A considerable kinetic isotope effect was noted in the reduction by [3H]NaBH, of the p-benzoquinone-derived precursor for I.Complete loss of fl-galactosidase activity occurred on incorporation of 4 mol I/mol fl-galactosidase tetramer. The inhibitor was very labile in the denatured enzyme at pH > 8, implying the formation of an ester bond between I and a carboxylate at the active site. The radioactive material released from the labeled enzyme was identified as allo-inositol. The stereochemistry of the expoxide reaction (trans-diaxial ring opening) is thus the same as for P-glucosidases with the corresponding epoxides.The binding site for I was identified as Glu-461 by the isolation and partial sequence analysis of a radioactive octapeptide from the cyanogen bromide and pepsin fragments of the labeled enzyme. A failure to determine the Nterminal amino acid of the labeled peptide is ascribed to the great reactivity of the esterified 11-carboxyl group of its N-terminal Glu-461 which causes rapid cyclisation of this residue to pyroglutamate, even under weakly basic conditions.The participation of the carboxylate of Glu-461 in catalysis is discussed.At least two functional groups are thought to be essential for catalysis by P-galactosidase from Escherichia coli: an acidic group donating a proton to the galactosyl oxygen and a carboxylate that aids bond breaking by partially neutralizing a positive charge on C-1 in the transition state and formation of an a-galactosyl ester intermediate [I]. Evidence for these groups is mainly indirect. The acid is inferred from pHdependence of k,,, [2] and from the influence of Mg2+ on the hydrolysis of 0-and N-galactosides [3] and on the tight binding of basic N-galactosyl derivatives [4]. A possible candidate for this acid is T~r -5 0 3~ which is adjacent to a Met residue that can be labeled by the active-site-directed inhibitors N-bromoacetyl-P-galactosylamine [7] and P-galactosylmethyl-p-nitrophenyltriazene [8]. The participation of a carboxylate is founded on kinetic considerations, on the ability of conduritol C cis-exposide (I) to act as an active-site-directed inhibitor [9] and on the formation of an alkali-labile 2-deoxygalactosyl-enzyme intermediate on reaction with D-We now report the identification of the binding site for I as Glu-461 and give evidence that this site represents the carboxylate group taking part in catalyis.The numbering of the amino acid residues has been changed from that given by Fowler and Zabm [5] to accomodate two additional amino acids as required by the base sequence of the lacZ gene [6].Part of this paper is taken from the doctoral thesis of M. Herrchen (University of Cologne, 1983).Ahhreaiations. TLC, thin-layer chromatography. Enzyme. P-Galactosidase, P-D-galactoside galactohydrolase (EC 3.2.1.23) MATERIALS A N D METHODS Specific radioactivity o j f H] NaBH4[3H]NaBH,, 0.5 -2 pmol in 0.1 ...
BackgroundCationic polyacrylamide copolymers (PAMs) are used for sludge dewatering in municipal waste water treatment and might enter the environment by spreading of the sludge on agricultural land. Concern has been expressed since little is known about the degradation of PAMs in soils. To obtain detailed information on the polymer’s fate in the soil compartment, the degradation of 14C-radiolabelled PAM in an outdoor lysimeter was studied.ResultsNo plant uptake and leaching of radioactivity was observed. There was practically no vertical movement of polymer and no transformation products found at the end of the study. For the top 10 cm soil layer, a mass balance was established throughout the study. About 10% of applied radioactivity was not extractable from soil even with a matrix destructive method, and this was concluded to be bound residue. Characterization of extractable radioactivity by means of GPC-analysis showed a significant decrease of the molecular weight of the PAM with time. The decrease in molecular weight indicates a breakdown of the polymer backbone (the C–C-chain), and is assumed to be primary degradation. The total radioactivity content in the 10 cm top soil layer was quantified every 6 months over a period of 3 years. The results show a significant decrease of the total radioactivity over time and this is defined as ultimate degradation following the definition of OECD and EPA. Based on the data, a half-life time of 2.0 × 103 days and a rate constant of 0.00035/day were calculated. With a χ2 of 12.0 the results of the calculation are thus valid and reliable. The rate constant indicates a mineralization of 22.5% within a period of 2 years based on the total recovered radioactivity. This half-life time is solely based on mineralization and does not take into account the degradation of the polymer backbone, hydrolysis of the side chains, incorporation into the soil matrix, and thus is a conservative approach.Conclusions14C-PAM degrades very slowly in soil after land-spreading as a component of sewage sludge. Even in a very conservative evaluation which only considered the loss of radioactivity, a half-life time of 5.4 years was determined.Electronic supplementary materialThe online version of this article (10.1186/s12302-018-0143-3) contains supplementary material, which is available to authorized users.
BackgroundEngineered nanomaterials (ENMs) are marketed as a substance or mixtures and are additionally used due to their active agent properties in products such as pesticides or biocides, for which specific regulations apply. Currently, there are no specific testing strategies for environmental fate and effects of ENMs within the different regulations. An environmental test and risk assessment strategy for ENMs have been developed considering the general principles of chemical assessment.ResultsThe test strategy has been developed based on the knowledge of national and international discussions. It also takes into account the conclusions made by the OECD WPMN which held an expert meeting in January 2013. For the test strategy development, both conventional and alternative endpoints were discussed and environmental fate and effects were addressed separately.ConclusionA tiered scheme as commonly used in the context of precautionary environmental risk assessment was suggested including the use of mathematical models and trigger values to either stop the procedure or proceed to the next tier. There are still several gaps which have to be filled, especially with respect to fate, to develop the test strategy further. The test strategy features a general approach. It is not specified to fulfil the information requirements of certain legislation (e.g. plant protection act, biocide regulation, REACH). However, the adaption of single elements of the strategy to the specific needs of certain legislation will provide a valuable contribution in relation to the testing of nanomaterials.
Grouping can replace animal testing to demonstrate the safe use of nanomaterials, but previously case studies were missing.
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