Seung-Hee Cha

Change in glutathione S-transferase and glyceraldehyde-3-phosphate dehydrogenase activities in the organs of mice treated with 2-chloroethyl ethyl sulfide or its oxidation products

Various organs or skin from male ICR mice treated intraperitoneally with 2-chloroethyl ethyl sulfide (CEES) or its oxidation derivatives 2-chloroethyl ethyl sulfoxide (CESSO) and 2-chloroethyl ethyl sulfone were analysed for changes in two thiol-containing enzymes, namely glutathione S-transferase (GST) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). CEES was more potent than its oxidation derivatives with respect to the decrease in organ weight and the loss in GAPDH activity, although the reverse was found in GST induction. Whereas the induction of GST was highest in the lung after multiple intraperitoneal intoxication with CEESO (8 and 32 mg/kg), the decrease in GAPDH activity after exposure to CEES (8 mg/kg body weight) was most remarkable in the spleen, the most susceptible organ to toxicity of CEES. GST and GAPDH activities in the skin of male hairless mice exposed subcutaneously to CEES (2 mg/kg body weight) were not altered significantly at 2-hr exposure, but decreased up to 60% of that of controls at 8 hr, when oedema formation was greatest. Taken together, it appears that GAPDH activity is a more sensitive biochemical parameter than GST activity in organs of mice treated with CEES or its oxidation products.

Glyceraldehyde-3-phosphate dehydrogenase as a biochemical marker of cytotoxicity by vinyl sulfones in cultured murine spleen lymphocytes.

Recently, vinyl sulfones have been observed to selectively inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is an important ATP-generating enzyme in glycolysis. The possibility of using GAPDH as a biochemical parameter of cytotoxicity by vinyl sulfones was investigated using mouse lymphocytes. Incubation of lymphocyte GAPDH with ethylvinyl sulfone resulted in a pseudo-first-order loss of enzyme activity. The exposure of lymphocytes to ethylvinyl sulfone resulted in the decrease of GAPDH activity followed by ATP depletion and cell death, which were both dependent on the concentration of ethylvinyl sulfone. A further study on the time-dependent change indicated that cell death was preceded by ATP loss. Compared to ethylvinyl sulfone, divinyl sulfone was more than 8 times more potent in causing either ATP depletion or cell death.

The release of lysosomal arylsulfatase from liver lysosomes exposed to 2-chloroethylethyl sulfide

Treatment of a lysosome-rich fraction from liver with 2-chloroethylethyl sulfide resulted in a dose-dependent release of arylsulfatase. The inclusion of Ca2+ enhanced the enzyme release by approximately 2.3-fold. The enhancing effect of Ca2+, showing an EC50 value of 30 mM, was mimicked by neither Mg2+ nor Mn2+. Studies on a structural requirement and a time-dependent release suggest that the Ca(2+)-dependent release proceeds via a specific process involving the alkylation of lysosomal membranes by 2-chloroethylethyl sulfide. Furthermore, the Ca(2+)-dependent process was prevented partially by either leupeptin or gentamycin, but neither pepstatin nor PMSF, implying that the enzyme release may be partially mediated by lysosomal cysteine-protease or phospholipase. Meanwhile, the Ca(2+)-independent release seems to be expressed non-specifically by various compounds.

Inactivation of microsomal Ca2+-ATPase by 2-chloroethylethyl sulfide

Exposure of liver microsomes to 2-chloroethylethyl sulfide (CEES) led to a dose-dependent decrease of Ca(2+)-ATPase activity. Studies on a structural requirement and a time dependence suggest that the enzyme inhibition may proceed via an instantaneous process involving an alkylation by an unstable intermediate, presumably a sulfonium form. It is noteworthy that the microsomal Ca(2+)-ATPase was more sensitive to CEES than the Na+/K(+)-ATPase from erythrocyte membranes. The Ca(2+)-ATPase was inhibited non-competitively by CEES, and its inhibitory action was independent of Ca2+ concentrations. The involvement of membrane phospholipid in the enzyme inhibition is excluded, since the temperature dependence of microsomal Ca(2+)-ATPase was not affected by CEES. Moreover, Triton X-100-solubilized Ca(2+)-ATPase was inactivated by the compound to the same extent as the membrane-bound enzyme was. Thus, it is suggested that CEES inactivates Ca(2+)-ATPase by alkylating the enzyme molecule at a region other than the active site.

Differential inhibition of soluble and membrane-bound acetylcholinesterase forms from mouse brain by choline esters with an acyl moiety of an intermediate size

Differential inhibitions of soluble and membrane-bound acetylcholinesterase forms purified from mouse brain were examined by the comparison of kinetic constants such as a Km value, a Kss value (substrate inhibition constant), and IC50 values of active site-selective ligands including choline esters. Membrane-bound acetylcholinesterase form (solubilized only in the presence of detergent) showed lower Km and Kss values than soluble acetylcholinesterase form (easily solubilized without detergent). Edrophonium expressed a slightly but significantly (p<0.01) higher inhibition of detergent-soluble acetylcholinesterase form than aqueous-soluble acetylcholinesterase form, while physostigmine inhibited both forms with a similar potency. A remarkable difference in inhibition was observed using choline esters; although choline esters with acyl chain of a short size (acetyl-to butyrylcholine) or a long size (heptanoyl- to decanoylcholine) showed a similar inhibitory potency for two forms of acetylcholinesterase, pentanoylcholine and hexanoylcholine inhibited more strongly aqueous-soluble acetylcholinesterase than detergent-soluble acetylcholinesterase. Thus, it is suggested that the two forms of AChE may be distinguished kinetically by pentanoyl- or hexanoylcholine.

Properties of acetylcholinesterase reconstituted in liposomes of a different charge.

Acetylcholinesterase (AChE) purified from mouse brain was reconstituted in liposomes of a different charge, and the properties of liposome-associated AChE were investigated. Relative to the Km value (38.5 μM) of AChE bound to a neutral liposome, the value of AChE reconstituted in a negatively-charged liposome decreased to 23.3 μM, whereas that of AChE in a positively-charged liposome increased to 90.9 μM. Additionally, AChE bound to a positively-charged liposome expressed a wider range of optimum pH than the enzyme in a negatively-charged liposome. In a stability study, it was found that soluble AChE was unstable at pH 5.5 and 7.4, while it was relatively stable at pH 10. Noteworthy, the immobilization of AChE to liposome enhanced the stability of soluble enzyme at acidic and neutral pH. Moreover, in the stabilization of the enzyme, a neutral liposome was more effective than charged liposomes, of which a positively-charged liposome was more effective than a negatively-charged liposome at acidic pH. Based on these results, it is proposed that while the Km value and the pH dependence of AChE activity are affected by the charge of liposome, the stability of AChE is determined mainly by a hydrophobic binding to a phospholipid membrane.

Protection by lysosomal hydrolase inhibitors against cytotoxicity of 2-chloroethylethyl sulfide

A possible participation of lysosomal hydrolases in the cytotoxicity of 2-chloroethylethyl sulfide in spleen lymphocytes was investigated using inhibitors of lysosomal phospholipases and proteases. Pepstatin (6 microM) and leupeptin (60 microM), inhibitors of lysosomal proteases, raised the viability of lymphocytes exposed to 2-chloroethylethyl sulfide from 63 to 87 and 88% of control, respectively. Serine protease inhibitors showed no significant effect on viability. Aminoglycoside inhibitors of lysosomal phospholipases were also found to prevent the decrease in viability of spleen lymphocytes exposed to 2-chloroethylethyl sulfide, and the effectiveness of these aminoglycosides (30 microM) was as follows: gentamicin > kanamycin > streptomycin, with viability increased to 89, 79 and 67%, respectively. In contrast to a co-operative action between leupeptin and gentamicin, the protection by pepstatin was reduced in the presence of gentamicin. Moreover, the order of the aminoglycosides in terms of the extent to which they antagonized the protective action of pepstatin was the same as their order of efficacy in preventing the cytotoxicity of CEES. It is suggested that inhibitors of lysosomal hydrolases reduce the cytotoxicity of 2-chloroethylethyl sulfide, presumably through lysosomal stabilization in spleen lymphocytes.

Effect of choline esters on the decarbamylation of dimethylcarbamyl-acetylcholinesterase

Acetylcholine and butyrylcholine exhibited the dose-dependent decarbamylation up to 0.2 mM, although at higher concentrations the decarbamylation degree declined. In combination with choline, butyrylcholine potentiated the choline-catalyzed decarbamylation by 30-100%, and was found to be more effective than acetylcholine in enhancing the decarbamylation. In kinetic analysis, it was observed that Ka value of choline was not remarkably altered by butyrylcholine whereas the maximum rate for decarbamylation was enhanced significantly in the presence of butyrylcholine, suggesting that butyrylcholine may affect the decarbamylation by interacting with the peripheral sites, different from the central active site which choline is known to interact with. In support of the suggestion, butyrylcholine was observed to compete with gallamine, a well known peripheral activator, and the effect of butyrylcholine was enhanced by three times at low ionic strength. In addition, acetylcholinesterase from mouse brain or bovine erythrocyte seemed to differ from electric eel enzyme in the interaction with butyrylcholine.