William H. Habig

[51] Assays for differentiation of glutathione S-Transferases

Publisher Summary This chapter provides the spectrophotometric, titrimetric, nitrite, and cyanide assay for the differentiation of glutathione S-transferases. Spectrophotometric assays depend upon a direct change in the absorbance of the substrate when it is conjugated with glutathione (GSH). Because each of the reactions is catalyzed at a finite rate in the absence of enzyme, care is needed to reduce nonenzymatic catalysis by minimizing substrate concentrations and by decreasing pH wherever necessary. Titrimetric assay is based on the principle that the conjugation of alkyl halides with GSH can be measured titrimetrically. Although acid production accompanies many of the transferase catalyzed reactions in which thioethers are formed, titrimetry is only used when more convenient assays are not available. Nitrite assay is based on the principle that nitrite is released when GSH reacts with nitroalkanes or with organic nitrate esters. The nitrite can be assayed as the limiting factor in a diazotization reaction with sulfanilamide that produces a readily quantitatable pink dye. Cyanide assay is based on the fact that when glutathione transferases catalyze the attack of the glutathione thiolate ion on the electrophilic sulfur atom of several organic thiocyanates, it results in the formation of an asymmetric glutathionyl disulfide and cyanide. Cyanide can be readily quantitated by a calorimetric method.

[27] Glutathione S-transferases (rat and human)

Publisher Summary This chapter presents a procedure for the preparation of glutathione transferases of the rat and the human. The glutathione S-transferases are the enzymes catalyzing conjugation reactions with glutathione as the first step in mercapturic acid synthesis. Although these enzymes may be distinguished from each other by their characteristic substrate-activity spectrum; all of the transferases are active with 1-chloro-2,4-dinitrobenzene as substrate. The reaction of this substrate and glutathione occurs spontaneously and the assay, therefore, requires the use of a control from which enzyme is absent. Not all of the data presented in the chapter summarizing the purification procedure were obtained with 1-chloro-2,4-dinitrobenzene. Methyl iodide, 1,2-dichloro-4-nitrobenzene, and 1,2-epoxy-3-(p-nitrophenoxy)propane have also been used. The homogeneous transferases prepared from human and rat liver represent a group that, aside from overlapping substrate specificity, resemble each other in size and subunit number. Although transferases A and C are similar to a greater degree, by reason of cross-reactivity to antibody raised against either protein, the other transferases from rat liver seem to be quite separate species.

Glutathione transferase from human erythrocytes. Nonidentity with the enzymes from liver.

Glutathione transferase ϱ has been purified to homogeneity from human erythrocytes. The enzyme has a molecular weight of 47,500 and is composed of two subunits of the same apparent molecular weight. The enzyme is active in catalyzing the reaction of glutathione, as a nucleophile, with a variety of compounds bearing an electrophilic center. Thioether formation with 1-chloro-2,4-dinitrobenzene and with ethacrynic acid are among the most rapid reactions under standard assay conditions. The erythrocyte enzyme has a different amino acid composition from that of glutathione transferases from human liver, whereas all of the liver enzymes appear to have the same composition. The erythrocyte transferase also differs from the liver transferases in its low isoelectric point of 4.5, its lack of reactivity with antibody produced against a liver glutathione transferase, and its more limited spectrum of substrates. The enzyme is present at a concentration of about 1.2 mg/100 ml of packed human erythrocytes.

Developmental and neurochemical specificity of neuronal deficits produced by electrical impulse blockade in dissociated spinal cord cultures

Blockade of spontaneous electrical activity in dissociated fetal spinal cord cultures produced neuronal deficits as measured by biochemical and morphological techniques. Spinal cord cultures exhibited an age-dependent vulnerability to impulse blockade with tetrodotoxin (TTX) or xylocaine. Neuronal cell counts, [125I]tetanus toxin fixation and [125I]scorpion toxin binding indicated that TTX application produced neuronal deficits during the second or third week in culture. Application of TTX during the first or fourth week did not produce a difference in tetanus toxin fixation from controls. Radioautography of [125I]tetanus toxin revealed no obvious change in the label distribution after TTX treatment. Suppression of electrical activity during the first 6 days in culture had no effect on choline acetyltransferase (CAT) activity and no apparent effect on the appearance of the cultures. Application of TTX during the seventh day in culture decreased CAT activity to 68% of control. Chronic electrical blockade produced a progressively greater loss of CAT activity through 21 days in culture. GABAergic neurons, as indicated by high-affinity GABA uptake, glutamic acid decarboxylase activity and [3H]GABA radioautography, were not affected by electrical blockade. These data indicate that there is developmental and neurochemical specificity in the neuronal death produced by blocking spontaneous electrical activity in dissociated spinal cord cultures.

Tetanus toxin in dissociated spinal cord cultures: long-term characterization of form and action.

Abstract: The clinical course of tetanus is notable, in addition to its often dramatic clinical presentation, by the long duration of the neuromuscular symptoms. Survivors may have tetanic manifestations for several weeks after the onset of the disease. In this article we correlate the duration of specific electrophysiologic effects produced by tetanus toxin with the degradation of cell‐associated toxin in primary cultures of mouse spinal cord neurons. From these studies we can conclude that the toxin has a half‐life of 5–6 days. Both the heavy and the light chains of tetanus toxin degrade at similar rates. Labeled toxin, visualized by radioautography, is associated with neuronal cell bodies and neurites, and its distribution is not altered during a 1‐week period following toxin exposure. Blockade of synaptic activity persists for weeks at the concentration of radiolabeled toxin used in these studies. This blockade of transmission is reversed as the toxin is degraded, suggesting that degradation of toxin may be a sufficient mechanism for recovery from tetanus.

Isolated light chain of tetanus toxin inhibits exocytosis: Studies in digitonin-permeabilized cells

Previous work indicates that the heavy chain of tetanus toxin is responsible for the binding of the toxin to the neuronal membrane and its subsequent internalization. In the present study, the light chain of tetanus toxin mimicked the holotoxin in inhibiting Ca2+‐dependent secretion of [3H]norepinephrine from digitonin‐permeabilized adrenal chromaffin cells. Preincubation of tetanus toxin with monoclonal antibodies to the light chain prevented the inhibition by tetanus toxin. Preincubation of tetanus toxin with nonimmune ascites fluid or with monoclonal antibodies directed against the C fragment (the C‐terminal of the heavy chains or the heavy‐chain portion of the B fragment did not prevent inhibition by tetanus toxin. The data indicate that the light chain is responsible for the intracellular blockade of exostosis.

Reevaluation of the role of gangliosides as receptors for tetanus toxin.

Binding of tetanus toxin to rat brain membranes was of lower affinity and capacity when binding was determined in 150 mM NaCl, 50 mM Tris‐HCl (pH 7.4) than in 25 mM Tris‐acetate (pH 6.0). Binding under both conditions was reduced by treating the membranes with neuraminidase. Pronase treatment, however, reduced toxin binding only in the Tris‐saline buffer (pH 7.4). In addition, the concentration of gangliosides required to inhibit toxin binding was 100‐fold higher in Trissaline compared to Tris‐acetate buffer. The toxin receptors in the membranes were analyzed by ligand blotting techniques. Membrane components were dissolved in sodium dodecyl sulfate, separated by polyacrylamide gel electrophoresis, and transferred to nitrocellulose sheets, which were overlaid with 125I‐labeled toxin. Tetanus toxin bound only to material that migrated in the region of the dye front and was extracted with lipid solvents. Gangliosides isolated from the lipid extracts or other sources were separated by TLC on silica gel and the chromatograms were overlaid with labeled tetanus toxin. The toxin bound to areas where the major rat brain gangliosides migrated. When equimolar amounts of different purified gangliosides were applied to the chromatogram, binding of the toxin was in the order GDlb≅ GTlb≅ GQ1b > GD2 > GD3≫ GD1a≅ GM1. Thus, the toxin appears to have the highest affinity for gangliosides with a disialyl group linked to the inner galactosyl residue. When binding of tetanus toxin to transfers and chromatograms was determined in the Tris‐saline buffer (pH 7.4), the toxin bound to the same components but the extent of binding was markedly reduced compared with the low‐salt and ‐pH conditions. Our results indicate that the interaction of tetanus toxin with rat brain membranes and gangliosides is greatly reduced under more physiological conditions of salt and pH and raise the possibility that other membrane components such as sialoglycoproteins may be receptors for the toxin under these conditions.

Cholesterol-dependent tetanolysin damage to liposomes

Tetanolysin caused membrane damage, resulting in release of trapped glucose from liposomes containing cholesterol. Maximum glucose release occurred from liposomes that contained 50 mol% cholesterol. At higher or lower levels of cholesterol, glucose release was reduced and glucose release did not occur at all below 40 mol% cholesterol. The apparent activity of tetanolysin was not influenced by temperature (24 degrees C compared to 32 degrees C) or by liposomal phospholipid fatty acyl chain length. We conclude that tetanolysin caused cholesterol-dependent lysin-mediated damage to liposomes, possibly by means of a pore consisting of a complex of toxin and cholesterol.

Binding of Tetanus Toxin to Somatic Neural Hybrid Cells with Varying Ganglioside Composition

Abstract: 125I‐labelled tetanus toxin interaction with several somatic hybrid cell lines was investigated. Binding of toxin is most effective in NCB‐20, followed by NBr‐10A, NG108‐C15, and SB21‐B1 cells. Specific binding of toxin to NCB‐20 and SB21‐B1 cells is 7– and 60‐fold lower, respectively, in comparison to enriched rat cerebral neuron cultures. The NCB‐20, NBr‐10A, and NG108‐C15 clones display a complex ganglioside pattern, including the presence of [N‐acetyl‐neuraminyl]‐galac‐tosyl ‐ N ‐ acetylgalactosaminyl[N‐ acetylneuraminyl] ‐ galactosylglucosyl ‐ ceramide (GDla) and two unidentified [14C]galactose‐labelled lipid‐soluble compounds, while the SB21‐B1 is most abundant in [N‐acetyl‐neuraminyl]‐galactosylglucosyl ‐ ceramide (GM3) and N ‐ acetyl ‐ galactosaminyl‐[N‐acetyl‐neuraminyl]‐galactosylglucosyl‐ceramide (GM2) gangliosides. None of the cells tested contain measurable levels of [l4C]galactose‐labelled or re‐sorcinol‐positive bands of galactosyl‐N‐ acetyl ‐galacto‐saminyl ‐ [N ‐ acetylneuraminyl ‐ N ‐ acetylneuraminyl]‐galactosylglucosyl‐ceramide (GDlb) and [N‐acetylneuraminyl] ‐ galactosyl ‐ N ‐ acetylgalactosaminyl ‐ [N ‐ acetylneuraminyl ‐ N ‐ acetylneuraminyl] ‐ galactosylglucosyl ‐ceramide (GTlb) gangliosides. After 2 h at 37°C a near plateau of toxin association with NCB‐20 cells is seen. Binding in low‐ionic‐strength medium is 1.35‐fold higher at 37°C than at 4°C, but is reduced by 21 and 51% at 4°C and 37°C, respectively, in physiologic medium. Treatment of NCB‐20 cells with neuraminidase causes a partial loss (29%) of toxin‐binding sites. Binding to the hybrid cells is significantly different from that of cerebral cultures with respect to temperature, salt effect, and sensitivity to neuraminidase, suggesting perhaps a different class of receptors for the toxin. Unlike dibutyryl cyclic AMP or prostaglandin, long‐term serum removal causes a significant increase in toxin binding to NCB‐20 and NG108‐C15 cells. Binding of tQxin to ganglioside‐supplemented SB21‐B1 cells is sevenfold higher compared to untreated cells; it is impaired by neuraminidase added before but not after cell interaction with the toxin at 37°.

Structure: Function studies of receptors for thyrotropin and tetanus toxin: Lipid modulation of effector binding to the glycoprotein receptor component

Abstract 125 I-labeled tetanus toxin interacts with the glycoprotein component of the thyroid thyrotropin receptor when this component is in solution or when it is incorporated into a liposome. Binding can be inhibited by both unlabeled thyrotropin and tetanus toxin but not by unlabeled prolactin, glucagon, insulin, ACTH, or growth hormone; binding can also be inhibited by a purified fragment of the glycoprotein component of the receptor. Changing the phospholipid of the liposome matrix from dipalmitoyl phosphatidylcholine to dioleoyl phosphatidylcholine significantly increases the binding of 125 I-TSH to the glycoprotein component of the receptor but does not affect 125 I-tetanus toxin binding.