Addition Of N-ethylmaleimide Research Articles

3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2): A simple assay adapted to human blood cells

A product of cysteine catabolism, 3-mercaptopyruvate, is enzymatically degraded to sulfur and pyruvate by a sulfurtransferase (EC 2.8.1.2.) present in human red and white blood cells. A simple sulfurtransferase assay is reported which takes advantage of the fact that pyruvate is conveniently measured enzymatically by lactate dehydrogenase (LDH) after the elimination of 3-mercaptopyruvate, also a substrate of LDH, by addition of N-ethylmaleimide. Sulfite is employed as sulfur acceptor, and conditions for a reproducible assay including data for preparation and storage of reactants, their assay, and their optimal concentrations are given. The apparent K m for sulfite is 6.5 · 10 −3 M and for 3-mercaptopyruvate is 1.9 · 10 −3 M. A reducing agent, in this assay dithiothreitol (Cleland's reagent), is essential for active transsulfuration. A normal metabolite, 3-mercaptopyruvate is reported elsewhere to have the capacity of producing polyploidy and chromosomal endoreduplication, a feature rendering it of interest in tumor metabolism.

DNA repair and degradation in x-irradiated E. coli B-r cells after treatment with ultra-violet light and N-ethylameimide.

A study was made of the effect of 5 x 10/sup -4/M N-ethylmaleimide (NEM) on the survival of Escherichia coli cells that had or had not been previously exposed to ultraviolet radiation at 600 or 1200 ergs/mm/sup 2/ before irradiation with x rays in suspensions saturated with air or nitrogen. The dose range of the x rays was 0 to 70 Krads. The enhancement by ultraviolet of the response to x rays in air was slightly more pronounced in Ni. NEM sensitized control cells to a greater extent in anoxia, but in air had practically no effect on cells previously exposed to 600 ergs/mm/sup 2/, and actively protected cells previously exposed to 1200 ergs/mm/sup 2/. In N/sub 2/, the results concerning cells previously exposed to uv light were qualitatively the same as in air, but significantly less evident. ln air the protective effect of NEM was reduced by ~40% in cells photoreactivated after exposure to 1200 ergs/mm/sup 2/ of uv light, and before the addition of NEM and x irradiation. In N/sub 2/, the sensitizing activity of the drug was partially restored by photoreactivation. In the presence of NEM, it was shown that a greater number of single-strand DNA breaksmore » was produced immediately after x irradiation. These were repaired out at a slower rate than under control conditions (30% versus 65% of breaks repaired in 80 min). In cells exposed only to uv light, there was no evidence of single- strand breaks immediately after exposure, but they appeared after incubation and were repaired to about 60% after 40 min. In cells exposed to uv and then to x rays, the number of breaks immediately after irradiation appeared to be the same as in those treated only with x rays, but after 40 and 80 min there was evidence of extensive DNA degradation with no repair. In the combined treatment with uv, NEM, and x rays, the number of breaks immediately after irradiation corresponded to that of samples treated with NEM and x rays, with a slow but significant repair taking place. (UK)« less

On the Formation of Yuba-like Film from Wheat Gluten

To elucidate the mechanism of the formation of Yuba-film, wheat gluten was applied instead of soybean protein, and the conditions of film formation, strength, strain and other properties of the film were studied. The Yuba-like films from gluten solution were formed under conditions as follows; at pH 10.2-12.5 and pH 4.9-2.2, or in the solution containing 3.3-4.0M urea or 0.25-0.5% sodium dodecylbenzenesulfonate. It seems to be necessary that protein is completely solubilized and unfolded to a certain extent and that coagulation of protein molecules on the surface of the solution is not prevented. These conditions of solvents varied the properties of the film. Addition of 2-mercaptoethanol decreased the strength of the film from alkaline solution, while it increased the strength and decreased the strain of the film from urea solution. Addition of N-ethylmaleimide, the modifying reagent of sulfhydryl group, decreased the strength and increased the strain of the film. Remarkable differences were observed between the fractions of gliadin and glutenin on strength, strain and transparency of film.

N-ethyl maleimide's effect on the recovery latency of the ERG a-wave after light adaptation.

The delay in the onset of the ERG a-wave in the albino rat's eye after light adaptation can be decreased by addition of N-ethyl maleimide into the vitreous as soon as the adapting light is turned off. It is suggested that the sulfhydryl groups of the opsin liberated in the rhodopsin — light energy reaction serves as an inhibitor of the reactions leading to the generation of the a-wave.

Studies on the thionucleotides in transfer ribonucleic acid. Addition of N-ethylmaleimide and formation of mixed disulfides with thiol compounds.

Studies on the thionucleotides in transfer ribonucleic acid. Addition of N-ethylmaleimide and formation of mixed disulfides with thiol compounds.

Rapid mixing in radiobiology.

THE mechanism by which N-ethylmaleimide (NEM)1,2 sensitizes bacteria to the lethal effects of radiation has not yet been elucidated. (For a recent review see ref. 3.) It has often been suggested that removal of naturally occurring radioprotective SH groups may explain this effect of NEM and related sulphydryl-reacting agents. This hypothesis could be eliminated if it were possible to demonstrate the sensitization when the interval of time between addition of NEM and irradiation of the mixture is too short for the thermal reaction between SH and the sensitizer to take place. Here we describe experiments using a rapid-mixing apparatus in which this condition is fulfilled.

Inhibition of dopamine β-hydroxylase by sulfhydryl compounds and the nature of the natural inhibitors

1. 1. Dopamine β-hydroxylase (3,4-dihydroxyphenylethylamine, ascorbate:O 2 oxidoreductase (hydroxylating), EC 1.14.2.1) was found to be inhibited by various sulfhydryl compounds such as cysteine, glutathione, coenzyme A and mercaptoethanol. d-Cysteine was equally as effective an inhibitor as l-cysteine. Cystine did not inhibit the enzyme. N-Ethylmaleimide, which reacts with sulfhydryl groups, completely reversed the inhibition by cysteine. Preincubation of the enzyme with cysteine had no effect on inhibition. The inhibition produced by cysteine was reversed by dialysis. The extent of inhibition was not related to the concentration of substrate, ascorbate or fumarate. The inhibition of dopamine β-hydroxylase by cysteine seems to be the result of a chelation of the copper atom at the active site of the enzyme. 2. 2. The inhibition of the enzyme by natural inhibitors of brain and adrenal medulla was completely protected by the addition of N-ethylmaleimide. Decrease in sulfhydryl contents of the tissue preparation caused a parallel decrease in the inhibitory activity for the enzyme. The results indicate that the natural inhibitors of dopamine β-hydroxylase are sulfhydryl compounds.

Cholesterol esterification in rat-liver cell sap

The existence of a cholesterol-est erifying enzyme was investigated with sap from liver cells. After subfractionation of liver homogenates, cholesterol esterification was checked with particulate and cell sap fractions and compared with the activity in plasma. Cholesterol was esterified on incubation with cell sap. The enzyme of cell sap esterified cholesterol in the sam e manner as that of plasma ; it did not require ATP, CoA or Mg 2+. The fatty acids esterified were derived from the added phospholipids, lecithin and cephalin. Pretreatment with phospholipase A or the addition of N-ethylmaleimide caused a marked lowering of the esterification. The optimal pH (6.5) was lower than that (7.3) of plasma. Free [1- 14C]linoleic acid added to reaction mixtures was not incorporated into cholesterol ester whether or not ATP, CoA and Mg 2+ were present. On the other hand, the particulate fraction esterified cholesterol only when ATP, CoA and Mg 2+ were present. It is concluded that, in cell sap, cholesterol ester was synthesized by a transfer of the fatty acids from lecithin, and in the particulates by a transfer of the fatty acids from acyl-CoA.