Exogenous Thiols Research Articles

Two-electron reduced mercuric reductase binds Hg(II) to the active site dithiol but does not catalyze Hg(II) reduction.

Mercuric reductase contains FAD and a redox-active disulfide which is reduced to a thiol/thiolate pair in two-electron reduced enzyme (EH2) (Fox, B. and Walsh, C.T. (1982) J. Biol. Chem. 257, 2498-2503). A charge transfer interaction between the thiolate and oxidized FAD gives EH2 a characteristic absorption spectrum, very similar to that found with other flavoprotein disulfide oxidoreductases. We have examined the reaction of EH2 with HgCl2 (+/- mercaptoethanol) in stopped-flow kinetic and static titration experiments. In the absence of mercaptoethanol, reaction of EH2 with HgCl2 yields a final spectrum which is indistinguishable from that of oxidized enzyme. The nature of the final species was examined by titration of enzyme thiols with 5,5'-dithiobis-2,2'-nitrobenzoic acid under denaturing conditions in the presence of NaI to displace any Hg(II) bound to enzyme thiols. These studies demonstrate that EH2 tightly complexes Hg(II) with its active site thiols, but is incapable of reducing Hg(II) to Hg0. For the latter reaction to occur, additional reducing equivalents are required. In catalysis, the enzyme must first be reduced to EH2 after which it cycles between EH2 and EH2 X NADPH forms. This is in contrast to other flavoprotein disulfide oxidoreductases which cycle between Eox and EH2 forms in catalysis (Williams, C. H., Jr. (1976) in The Enzymes (Boyer, P. D., ed) 3rd Ed., Vol. 13, pp. 89-173, Academic Press, New York). With mercuric reductase, exogenous thiols are required for catalytic reduction of Hg(II) to Hg0. We have shown that this is due to prevention or reversal of formation of an abortive complex of Hg(II) with the thiol/thiolate pair of EH2.

Changes in intestinal mucosal permeability caused by nonprotein thiol loss in rats.

The barrier selectivity of the intestinal mucosal membrane permeability may be impaired in certain disease conditions. Membrane permeability was previously shown to be correlated with changes in nonprotein thiol in rat intestinal tissue by the everted sac method. In the present study, the mucosal effects of alloxan-induced diabetes and chronic alcohol administration to intact rats, as well as pre-treatment with diethyl maleate, ethanol, and salicylate, were investigated. In each case, a drop of mucosal nonprotein thiol was associated with an increased absorption of cefoxitin, cefmetazole, and phenol red, hydrophilic compounds that are poorly absorbed through intact membrane, and with a decreased absorption of L-phenylalanine. The effect of nonprotein thiol loss on rectal absorption of cefoxitin, cefmetazole, and phenol red was greater than that on the small intestinal absorption. The increase in phenol red absorption by diethyl maleate in the in vitro everted sac method correlated with Ca(2+) release from the intestinal mucosa, which was induced by nonprotein thiol loss. Resistance to the effect of nonprotein thiol loss on Ca(2+) homeostasis was greater in rat ileum than in rat colon (including rectum). The administration of cysteamine as an exogenous nonprotein thiol restored non-protein thiol levels in the mucosa along with the barrier function of the intestinal mucosa to the absorption of cefoxitin, cefmetazole, and phenol red. In contrast, the transport of L-phenylalanine in the small intestinal mucosa was not restored by cysteamine treatment.

Some protease inhibitors are also inhibitors of poly(ADP-ribose) polymerase.

The low-molecular-weight peptide protease inhibitors, tosyl-lysine-chloromethyl ketone, antipain and leupeptin, inhibited poly(ADP-ribose) [poly(ADP-Rib)] polymerase in permeable cells. The concentrations required for 50% inhibition were 3.6, 5 and 29 mM, respectively. Two peptides without protease inhibitor activity, fibrinopeptide A and phenylalanine-leucine-(glutamine)2-leucine, also inhibited poly (ADP-Rib) synthesis; doses required for 50% inhibition were 0.37 and 11.2 mM, respectively. These concentrations lie within a range bracketed by the 50% inhibition concentrations of the strong and weak poly(ADP-Rib) synthesis inhibitors, 3-amino-benzamide (0.15 mM) and caffeine (greater than 100 mM), respectively. N-Ethylmaleimide also inhibited poly(ADP-Rib) synthesis, at a 50% inhibitory dose of 0.3 mM, in the absence of exogenous thiol reagents. High-molecular-weight protease inhibitors, such as soybean (including Bowman-Birk reagent) and lima bean trypsin inhibitors and human alpha 1-protease inhibitor, had no effect on poly(ADP-Rib) synthesis up to 2 mg/ml. Interference with transformation and other cellular effects that have been reported in carcinogen-damaged cells treated with low-molecular-weight peptide protease inhibitors may therefore involve common mechanisms with poly(ADP-Rib) inhibitors. Similar effects of high-molecular-weight protease inhibitors presumably involve different mechanisms.

The Role of Endogenous Thiols in Intrinsic Radioprotection

Observations are reviewed from experiments performed to study the role of endogenous thiols in the radiation response of cells using a glutathione-deficient and a related glutathione-proficient cell strain. The effect of glutathione in the initial radical reactions was considered and the yield of single-strand DNA breaks was the end-point of the response. The rejoining of breaks and clonogenic survival were chosen as end-points when, in addition, the role of glutathione in the subsequent biochemical processes was studied. The results were interpreted to indicate that glutathione plays a role in both the radical and the biochemical reactions which follow irradiation. In the former case, it functions as a damage-restituting reactant, in general agreement with the 'competition model'. Some biochemical repair processes, in particular those concerned with the rejoining of breaks induced by radiation in the presence of oxygen or misonidazole, appear also to be critically dependent on glutathione. Due, probably, to its particular spatial distribution, endogenous glutathione is specific in the radical processes, and exogenous thiols cannot be substituted for it. No such specificity was indicated in the biochemical processes related to strand break rejoining.

Participation of cellular thiol/disulphide groups in the uptake, degradation and bioactivity of insulin in primary cultures of rat hepatocytes.

The effects on the uptake (cell-associated 125I) and degradation (125I-labelled products released into the medium) of 125I-insulin and bioactivity (protein, glycogen and lipid synthesis) of insulin caused by altering the cellular thiol/disulphide status in primary cultures of rat hepatocytes were studied. Incubation of hepatocyte cultures with various exogenous thiol compounds (reduced glutathione, 2-mercaptoethanol, cysteamine, dithiothreitol) resulted in increased insulin binding, but markedly decreased degradation and bioactivity. These effects could be reversed by washing or by the addition of oxidized glutathione, which alone had no effect. When cultures were exposed to certain thiol-modifying reagents (N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuribenzenesulphonate, iodoacetamide, iodoacetate), some decreases in bioactivity were evident, but the pronounced decrease in insulin degradation observed with the thiol-containing compounds was not observed with this class of compounds. None of the thiol-containing or -modifying agents tested had any significant effect on cellular ATP concentrations, indicating that the effects observed were due to perturbation of the thiol/disulphide status. Depletion of intracellular glutathione by DL-buthionine SR-sulphoximine (a specific inhibitor of glutathionine biosynthesis) decreased the syntheses of glycogen and lipid by about one-half, while having essentially no effect on protein synthesis, ATP concentrations or on the binding and degradation of insulin. The data presented here indicate that although intracellular thiol (glutathione) concentrations may be important for the maintenance of full expression of certain biological activities (glycogen and lipid synthesis), the thiol/disulphide groups on the cell surface and those immediately inside the cell membrane may be more critical in the mediation of insulin action, including the degradation and bioactivity of insulin in primary cultures of rat hepatocytes.

Radioprotective effect of cysteamine in glutathione synthetase-deficient cells.

The radioprotective role of endogenous and exogenous thiols was investigated, with survival as the end-point, after radiation exposure of cells under oxic and hypoxic conditions. Human cell strains originating from a 5-oxoprolinuria patient and from a related control were used. Due to a genetic deficiency in glutathione synthetase, the level of free SH groups, and in particular that of glutathione, is decreased in 5-oxoprolinuria cells. The glutathione synthetase deficient cells have a reduced oxygen enhancement ratio (1.5) compared to control cells (2.7). The radiosensitivity was assessed for both cell strains in the presence of different concentrations of an exogenous radioprotector:cysteamine. At concentrations varying between 0.1 and 20 mM, cysteamine protected the two cell strains to the same extent when irradiated under oxic and hypoxic conditions. The protective effect of cysteamine was lower under hypoxia than under oxic conditions for both cell strains. Consequently, the oxygen enhancement ratio decreased for both cell strains when cysteamine concentration increased. These results suggest that cysteamine cannot replace endogenous thiols as far as they are implicated in the radiobiological oxygen effect.

The generation of a hyperporphyrin spectrum upon thiol binding to ferric chloroperoxidase. Further evidence of endogenous thiolate ligation to the ferric enzyme.

In spite of numerous spectroscopic similarities between chloroperoxidase and cytochrome P-450 (P-450) which suggest endogenous cysteinate axial ligation in chloroperoxidase as has been established for P-450, assignment of the endogenous axial ligand of chloroperoxidase has remained controversial since no available free sulfhydryl groups have been detected in chemical studies of chloroperoxidase. To help clarify this problem, we have carried out extensive studies of thiol-binding properties of native ferric chloroperoxidase and have compared our new results with those previously obtained in our laboratory on thiol adducts of P-450. We have found that the ligation of exogenous thiols to the heme iron of chloroperoxidase generates hyperporphyrin (split Soret) spectra (lambda max = approximately 372 and approximately 455 nm), consistent with the formation of bisthiolate low-spin ferric heme adducts as has been established for P-450 and its heme models. However, in contrast to the results with P-450, thiols not only coordinate to the ferric heme iron of chloroperoxidase in the thiolate form in competition with cyanide but also bind, presumably in the thiol form, to a site in the heme vicinity other than the heme iron without competition with cyanide. The thiol acidity (pK alpha greater than 7) or medium pH (pH 3-7) have little effect on the spectral and equilibrium properties of the adducts. Thiol binding to the latter site, which is presumably the organic substrate-binding site, causes a red shift of the Soret peak (339 vector approximately 420 nm) of the high-spin ferric enzyme; the resulting thiol adducts are still predominantly high spin. Similar spectral changes are also observed upon binding of other neutral sulfur (sulfides and disulfide) and oxygen (alcohols and ketones) donor ligands to native ferric chloroperoxidase. In conclusion, the generation of hyperporphyrin spectra upon exogenous thiol binding to native ferric chloroperoxidase provides considerable support for the presence of an endogenous thiolate ligand to the heme of the enzyme in its ferric state.

Endogenous ligands of rat lung beta-galactoside-binding protein (galaptin) isolated by affinity chromatography on carboxyamidomethylated-galaptin-Sepharose.

Rat lung beta-galactoside-binding protein (galaptin) is developmentally regulated during postnatal lung development. In common with other vertebrate galaptins, it is very labile when purified and dependent on the presence of exogenous thiol reagents. Reaction of rat lung galaptin with iodoacetamide resulted in a stable active carboxyamidomethylated galaptin that could be coupled to Sepharose. The resultant affinity matrix bound asialoglycoproteins, and these could be quantitatively eluted with disaccharide haptens. The carboxyamidomethylated-galaptin-Sepharose affinity matrix was used to search for endogenous ligands in 13-day-rat lung. Cytosolic fractions of developing rat lung contained no moieties that could be specifically eluted with disaccharide hapten. Only when membranous fractions were extracted with 1% Triton were glycoproteins solubilized that bound to the affinity matrix and could be specifically eluted with disaccharide hapten. The eluted glycoproteins were potent inhibitors of galaptin binding to asialo-orosomucoid. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis identified these glycoproteins as being of high Mr, with three components of Mr 160000-200000 and a smaller component of Mr 75000. This is the first evidence for specific membrane-associated glycoproteins being the ligands of rat lung galaptin.

Glutathione depletion and cell radiosensitization in three cell lines irradiated in air: Importance of exogenous thiols

Glutathione depletion and cell radiosensitization in three cell lines irradiated in air: Importance of exogenous thiols

Role of glutathione in gastric mucosal cytoprotection.

Exogenous thiol compounds have been reported to protect the stomach from ethanol-induced necrotic lesions. The gastric mucosa contains high levels of an endogenous thiol, glutathion (GSH). Because of the known role of glutathione in protecting against hepatic injury, its role in gastric mucosal cytoprotection was of interest. By use of an animal model for acute gastric injury from ethanol, a close parallel relation between depletion of endogenous mucosal GSH and induction of mucosal protection was demonstrated. Surprisingly, mucosal protection varied inversely with the level of mucosal GSH obtained after treatment with specific GSH-depleting agents (diethyl maleate and cyclohexene-1-one). Depletion of gastric mucosal GSH was associated with an increase in the mucosal content of prostaglandins 6-keto F1 alpha and F2 alpha but not E2. The protective effect induced by GSH-depleting agents was partially reversed by indomethacin in some but not all studies. Although GSH depletors increased gastric juice volume, protection with these agents persisted after the volume and mucosal GSH had returned to control levels and also was not reversed by increasing the dose of ethanol threefold to overcome a possible dilutional effect. We conclude that, contrary to apparent predictions, depletion of endogenous gastric GSH protects the stomach from acute ethanol-induced injury. Although the mechanism of this protection is unknown, a mediation by endogenous release of prostaglandins seems to play a minor role since diethyl maleate was protective even in indomethacin-treated animals.

Depletion of intracellular GSH and NPSH by buthionine sulfoximine and diethyl maleate: Factors that influence enhancement of aerobic radiation response

Many investigators have observed aerobic sensitization of V79, CHO and A549 (human lung carcinoma) cells upon depletion of GSH using buthionine sulfoximine (BSO). Recently we discovered that this aerobic sensitization can be reversed if WR-2721 or N-acetylcysteine is added to the cells just prior to irradiation. Reversal requires that the exogenous thiols be present during the time of irradiation. One possible explanation was that these thiols entered the cells and either increased the pool of cellular nonprotein thiols or reversed the thiol-depleted state by stimulation of GSH synthesis. Cells treated with BSO do not readily regenerate intracellular GSH because this agent irreversibly inhibits y-glutamyl synthetase. For A549 monolayer cultures, there is approximately 50% regeneration 6 hr after removal of 0.0I mM BSO, 20% 6 hr after 0.I mM BSO, and only 5% 6 hr after 0.5 mM BSO. We found that addition of WR-2721 or N-acetylcysteine to BSO-treated cells did not affect the rate of regeneration of intracellular GSH. Thus, reversal of the aerobic sensitization of A549 cells by BSO cannot be explained on the basis of intracellular thiol levels alone, or by rapid reversal of BSO inhibition. In addition, diethylmaleate (DEM)-treated cells are considerably different from BSO-treated cells with respect to the ability to regenerate GSH. After removal of DEM, A549 cells immediately begin GSH resynthesis, and return to control levels occurs within 2 hr. Exogenous 5 mM GSH increases the rate of resynthesis of GSH in DEM-treated cells, but not in BSO-treated cells.

Influence of thiol substances on in vitro and in vivoL-thyroxine deiodination in rainbow trout, Salmo gairdneri

The effects of the thiols DTT (dithiothreitol) and GSH (reduced glutathione) on hepatic in vitro and in vivo T4 (L-thyroxine) deiodination by rainbow trout held at 11 °C were studied. Hepatic deiodination increased progressively over the DTT range of 0.02–20 mM. GSH was less potent than DTT at low concentrations and strongly inhibited deiodination at high concentrations (> 1 mM). Hepatic deiodination was not increased by 1 mM NADPH or anaerobic conditions and was enhanced and not inhibited by the GSH inhibitor, diamide (2.5 mM), indicating that the low T4 deiodination in the absence of DTT is not due to endogenous GSH deficiency. Intraperitoneally injected GSH consistently increased plasma levels of 125I and [125I]-3,5,3′-triiodo-L-thyronine (T3) in fed or starved [125I]T4-injected trout, suggesting a GSH stimulation of extrahepatic T4 deiodination. However, injected GSH did not elevate plasma T3 concentrations. This was probably due to a demonstrated GSH stimulation of plasma T4 and T3 clearance. Force-fed GSH did not increase [125I]T4 deiodination. It is concluded that exogenous thiols can enhance T4 deiodination both in vitro and in vivo. However, availability of neither endogenous nor dietary GSH appears to regulate T4 deiodination under physiological conditions, including altered nutritional state.

Metabolism of vitamin K and vitamin K 2,3-epoxide via interaction with a common disulfide.

The effects of thiols and sulfhydryl blocking reagents on the reduction of vitamin K to vitamin K hydroquinone and vitamin K 2,3-epoxide to vitamin K and vitamin K hydroquinone catalyzed by rat hepatic microsomes were investigated to determine the mechanism(s) for these reactions. Both vitamin K and vitamin K 2,3-epoxide reductions were catalyzed more effectively with dithiols than with monothiols as the reductant. The sulfhydryl reagent N-ethylmaleimide (NEM) inhibited vitamin K and vitamin K 2,3-epoxide reduction much more effectively when microsomes were initially treated with dithiothreitol (prereduced). In prereduced microsomes iodoacetamide was approximately half as effective an inhibitor of vitamin K and vitamin K 2,3-epoxide reduction as NEM, but in microsomes not prereduced it was more effective. Iodoacetic acid was ineffective as an inhibitor. Vitamin K or vitamin K 2,3-epoxide added to prereduced microsomes blocked subsequent inhibition by NEM of vitamin K and vitamin K 2,3-epoxide metabolism, respectively. Vitamin K added to prereduced microsomes also blocked inhibition by NEM of vitamin K 2,3-epoxide metabolism, and vitamin K 2,3-epoxide addition blocked inhibition by NEM of vitamin K metabolism. Vitamin K did not diminish the rate of vitamin K 2,3-epoxide metabolism, however, nor did vitamin K 2,3-epoxide diminish the rate of vitamin K metabolism. These data establish that exogenous thiol compounds promote the reduction of at least one protein disulfide which participates in the metabolism of vitamin K and vitamin K 2,3-epoxide. Presumably, the resultant sulfhydryl groups are reoxidized to the disulfide form during the metabolism of either vitamin which protects them from reaction with NEM.

Thiols, Thiol Depletion, and Thermosensitivity

Hyperthermia sensitization or tolerance is subject to cellular events that may occur at membrane, nuclear, and cytoplasmic sites. We have studied the effects of elevated temperatures on the oxidative-reductive state of the cell by measuring and altering glutathione (GSH) concentrations. GSH plays a pivotal role in maintaining the overall cellular redox state and detoxification of peroxides. Continuous heating at 42.5 degrees C or acute exposure at 43 degrees C or 45.5 degrees C resulted in rapid elevations of cellular GSH to 120-200% of control values. Qualitatively, the more severe the heat exposure, the quicker the maximal GSH levels are attained. Ethanol, a compound that also induces thermal tolerance, likewise increases intracellular GSH concentrations. GSH depletion by two different modalities, diethylmaleate (DEM) and buthionine sulfoximine (BSO), results in thermal sensitization. It was demonstrated that once thermotolerance has been induced, depletion of GSH has minimal effects on subsequent heating and thermotolerance. Heat shock protein (HSP) synthesis is lessened by treatment with buthionine sulfoximine; the extent of the decrease in HSP production correlates with the decrease in thermotolerance. The exogenous thiol cysteine combined with heat treatment results in thermosensitization. Exogenous cysteine is found to oxidize to cysteine and to enhance oxygen consumption. The use of N-acetylcysteine resulted in less oxygen consumption and less thermosensitization. A proposed mechanism of peroxide-induced cell damage is suggested by exogenous thiols, as well as an involvement of GSH in the initial aspects of thermotolerance induction.

Flow Cytometry Techniques for Studying Cellular Thiols

Cellular thiols, and especially glutathione, act as scavenger nucleophiles and can protect against toxicity, mutagenicity, or transformation by ionizing radiation and many carcinogens. Development of a rapid assay to quantitate the cellular content of thiols could thus be useful in assessing or predicting cellular risk to damage. Several fluorescent thiol-reactive drugs, usually maleimide or bromobimane derivatives, have been described for use in histopathology. Most of these agents do not distinguish between protein and nonprotein thiols, and virtually all of these fluorescent stains have normally been used after fixation of the cells or tissues. We have found that some of the probes will, however, rapidly penetrate and bind within viable cells with little associated cytotoxicity; the amount bound can be easily quantified using flow cytometry. We have used several of these agents, in conjunction with fluorescence-activated cell sorting in V79 spheroids, to examine the thiol content of cells as a function of their depth or position in the spheroid. Additionally, the radiation response of cells from different depths has been assessed following addition of exogenous thiols including glutathione and WR-2721, or after treatment with thiol-depleting agents, including DL-buthionine-S,R-sulfoximine (BSO), diethylmaleate (DEM), and dimethylfumarate (DMF). Our studies indicate that examination of the thiol content and radiation response of the sorted cells provides an improved understanding of the modes of action of these compounds.

Modulation of coupling factor ATPase activity in intact chloroplasts. Reversal of thiol modulation in the dark

Illumination of intact pea chloroplasts results in both the pH activation and the thiol modulation of the reversible protonmotive ATPase, or CF 0-CF 1 (Mills, J.D., Mitchell, P. and Schürmann, P. (1980) FEBS Lett. 112, 173–177). We have studied the reversal of the activation and thiol-modulation processes that occurs when illuminated chloroplasts are darkened. (1) Methyl viologen is shown to prevent light-dependent thiol modulation of CF 0-CF 1 in intact chloroplasts. Studies using methyl viologen indicate that the decline in ATPase activity of intact chloroplasts in the dark is due both to pH deactivation and thiol demodulation of CF 0-CF 1. Reillumination in the presence of methyl viologen reactivates CF 0-CF 1 complexes that have undergone pH deactivation, but does not allow thiol modulation to occur. (2) At 20°C, both pH deactivation and thiol demodulation of CF 0-CF 1 are complete within 10 min darkness. At 3.5°C, both processes are retarded, and require more than 30 min for completion. (3) Lysis of intact chloroplasts greatly retards the thiol demodulation of CF 0-CF 1. In the dark, addition of oxidants to lysed chloroplasts promotes thiol demodulation. Under these conditions, thioredoxin behaves as a weak oxidant, but glutathione has very little effect. Illumination of intact chloroplasts followed by rapid lysis and storage on ice stabilises the pH-activated, thiol-modulated state of CF 0-CF 1 for extended periods in the dark and increases the time required for thiol demodulation to well over 2 h. This method may prove to be useful for preparing thiol-modulated chloroplasts without the need to add or remove exogenous thiols. The results suggest that an oxidative system exists in the stroma which actively reverses thiol modulation of CF 0-CF 1 in the dark. Such a system would be expected if thiol modulation of CF 0-CF 1 were an important physiological process regulating reversible ATPase activity in vivo.

Specific modification of gastric K +-stimulated ATPase activity by thimerosal

Treatment of hog gastric microsomes with the sulfhydryl reagent, thimerosal (ethylmercurithiosalicylate), produced differential effects on the K +-ATPase and the K +-stimulated p-nitrophenylphosphatase activities. For example, exposure to 2 mM thimerosal for 3 min severely reduced the activity of K +-stimulated ATPase, while K +- p-nitrophenylphosphatase activity was enhanced 2- to 3-fold. Higher concentration of thimerosal, or longer incubation times, also led to inhibition of K +- p-nitrophenylphosphatase. The activated state of p-nitrophenylphosphatase could be sustained by a 20-fold, or greater, dilution of treated membranes, and could be reversed by reduction of membrane SH groups by exogenous thiols. Significant activation of K +- p-nitrophenylphosphatase was not produced by p-chloromercuribenzene sulfonate, p-chloromercuribenzoate or mersalyl; however, ethyl mercuric chloride had qualitatively similar activity effects as thimerosal. Kinetics of K +- p-nitrophenylphosphatase for thimerosal-treated membranes were altered as follows: V increased; K m for p-nitrophenylphosphate unchanged for K a for K + increased. ATP, which is a potent inhibitor of K +- p-nitrophenylphosphatase activity in native membranes ( K I ≈ 200 μM). These data suggest that there are multiple SH groups which differentially influence the gastric K +-stimulated ATPase activity. Defined treatments with thimerosal are interpreted as an uncoupling of the K +-stimulated phosphatase component of the enzyme (for which p-nitrophenylphosphatase is a presumed model reaction). Such differential modifications can be usefully applied to the study of partial reactions of the enzyme and their specific role in the related H +-transport reaction.

The effect of thiols on the sulfhydryl content and nucleotide binding of thymidylate synthetase

Summary Variability in the endpoints for catalytic sulfhydryl group modification, nucleotide binding, and ternary complex formation has been observed in thymidylate synthetase from amethopterinresistant Lactobacillus casei . Electrophoretic analysis of ternary complexes of various enzyme preparations revealed the presence of as many as three enzyme forms containing one, two, or no nucleotide binding sites or catalytic sulfhydryl groups per enzyme dimer. The relative quantities of multiple forms were dependent on exogenous thiols in the purification scheme, thus resulting in the variability in active site content obtained for thymidylate synthetase.

69] Purification of an enzyme containing a NADP-protected cysteine on organomercuri-agarose 17β-estradiol dehydrogenase

Publisher Summary 17 β-Estradiol dehydrogenase, among other enzymes, possesses essential cysteinyl residues which are protected by the coenzyme against thiol reagents, such as p-hydroxymercuribenzoate (PMB), N-ethylmaleimide (NEM), or 5, 5'-dithiobisnitrobenzoate (DTNB). The incubation of an impure enzyme solution with thiol reagents in the presence of NADP results in the reaction of all cysteinyl residues that are not protected by this coenzyme. After removal of both NADP and exogenous thiol reagents, only the unmodified residues are able to react with an organomercuri-Sepharose derivative. These proteins are readily eluted by β-mercaptoethanol or dithiothreitol. The method provides the basis of a successful purification procedure if activity is retained.

Cr 51-tagged red cell equilibration in dogs

ARTICLESCr 51-tagged red cell equilibration in dogsRA Huggins, EL Smith, and S DeaversRA Huggins, EL Smith, and S DeaversPublished Online:01 Aug 1966https://doi.org/10.1152/ajplegacy.1966.211.2.283MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByInduced Response to Hypercapnia in the Two-Compartment Total Cerebral Blood Volume: Influence on Brain Vascular Reserve and Flow Efficiency28 June 2016 | Journal of Cerebral Blood Flow & Metabolism, Vol. 15, No. 6Evaluation Of An Additive Solution For Preservation Of Canine Red Blood CellsJournal of Veterinary Internal Medicine, Vol. 8, No. 4Effects of endogenous and exogenous thiols on the distribution of mercurial compounds in mouse tissuesArchives of Environmental Contamination and Toxicology, Vol. 15, No. 6Blood volume following diuresis induced by furosemideThe American Journal of Medicine, Vol. 76, No. 4Regional Brain Blood Flow, Blood Volume, and Haematocrit Values in the Adult Rat29 June 2016 | Journal of Cerebral Blood Flow & Metabolism, Vol. 3, No. 2Effect of exercise on erythrocyte count and blood activity concentration after technetium-99m in vivo red blood cell labeling.Circulation, Vol. 66, No. 3The relationship between the pharmacokinetics of intravenous cismethrin and bioresmethrin and their mammalian toxicityPesticide Biochemistry and Physiology, Vol. 13, No. 3Splenic storage volume in the unanesthetized resting beagleEuropean Journal of Applied Physiology and Occupational Physiology, Vol. 38, No. 3Accuracy of blood volume estimations in critically I11 children using 125I-labelled albumin and 51Cr-labelled red cellsEuropean Journal of Pediatrics, Vol. 125, No. 2Effects of a single exposure to carbon disulphide on the rate of urea production and on plasma free fatty acid and glucose concentrations in the rat.Occupational and Environmental Medicine, Vol. 32, No. 2Pulmonary oedema in rats given monocrotaline pyrroleThe Journal of Pathology, Vol. 106, No. 4Das Blutvolumen More from this issue > Volume 211Issue 2August 1966Pages 283-287 Copyright & PermissionsCopyright © 1966 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1966.211.2.283PubMed5921091History Published online 1 August 1966 Published in print 1 August 1966 Metrics