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PubMed1988

Interactions of bromide, iodide, and fluoride with the pathways of chloride transport and diffusion in human neutrophils.

Isolated human neutrophils possess three distinct pathways by which Cl- crosses the plasma membrane of steady state cells: anion exchange, active transport, and electrodiffusion. The purpose of the present work was to investigate the selectivity of each of these separate processes with respect to other external halide ions. (a) The bulk of total anion movements represents transport through an electrically silent anion-exchange mechanism that is insensitive to disulfonic stilbenes, but which can be competitively inhibited by alpha-cyano-4-hydroxycinnamate (CHC; Ki approximately 0.3 mM). The affinity of the external translocation site of the carrier for each of the different anions was determined (i) from substrate competition between Cl- and either Br-, F-, or I-, (ii) from trans stimulation of 36Cl- efflux as a function of the external concentrations of these anions, (iii) from changes in the apparent Ki for CHC depending on the nature of the replacement anion in the bathing medium, and (iv) from activation of 82Br- and 125I- influxes by their respective ions. Each was bound and transported at roughly similar rates (Vmax values all 1.0-1.4 meq/liter cell water.min); the order of decreasing affinities is Cl- greater than Br- greater than F- greater than I- (true Km values of 5, 9, 23, and 44 mM, respectively). These anions undergo 1:1 countertransport for internal Cl-. (b) There is a minor component of total Cl- influx that constitutes an active inward transport system for the intracellular accumulation of Cl- [( Cl-]i approximately 80 meq/liter cell water), fourfold higher than expected for passive distribution. This uptake is sensitive to intracellular ATP depletion by 2-deoxy-D-glucose and can be inhibited by furosemide, ethacrynic acid, and CHC, which also blocks anion exchange. This active Cl- uptake process binds and transports other members of the halide series in the sequence Cl- greater than Br- greater than I- greater than F- (Km values of 5, 8, 15, and 41 mM, respectively). (c) Electrodiffusive fluxes are small. CHC-resistant 82Br- and 125I- influxes behave as passive leak fluxes through low-conductance ion channels: they are nonsaturable and strongly voltage dependent. These anions permeate the putative Cl- channel in the sequence I- greater than Br- greater than Cl- with relative permeability ratios of 2.2:1.4:1, respectively, where PCl approximately 5 X 10(-9) cm/s.

Anions↗
PubMed1990

Differences among human tumor cell lines in the expression of glutathione transferases and other glutathione-linked enzymes.

A large number of human tumor cell lines of various origins have been investigated with respect to expression of glutathione-linked enzymes in the cytosol fraction. The amounts of the different enzymes were estimated by use of activity measurements and by silver staining or immunoblot analysis after electrophoresis of cytosol fractions purified by affinity chromatography on S-hexylglutathione Sepharose. Class Pi glutathione transferase was the most abundant enzyme in most tumor cells; the cell lines HepG2 and Raji were exceptions in not expressing significant amounts of this enzyme. HepG2 cells derive from hepatocytes, which normally do not express the class Pi enzyme, whereas Raji cells originate from B-lymphocytes, which normally do express a class Pi glutathione transferase. The highest level of the class Pi transferase, in terms of protein reacting with antibodies as well as enzyme activity, was noted in the colon carcinoma cell line LS174T. Hu549Pat cells, EBV-transformed B-lymphocytes, also expressed high levels of a protein reacting with antibodies specific for class Pi glutathione transferases, but did not display any significant activity with ethacrynic acid, a substrate characteristic for this class. Class Alpha and class Mu glutathione transferases, in cell lines expressing these isoenzymes, were present in significantly lower concentrations than the class Pi enzyme. Most of the tumor cells contained a class Alpha transferase composed of 27.5 kd subunits, which has the physicochemical and immunological properties of the most basic glutathione transferase found in human skin. In several cell lines, a protein was detected with an apparent subunit Mr value of 30 kd that was tentatively identified as an additional class Alpha glutathione transferase not previously described. In addition, other glutathione-linked enzyme activities, namely glutathione peroxidase, glutathione reductase and glyoxalase I, were assayed with specific substrates in the cytosolic fraction of the tumor cells; glyoxalase I could also be estimated semiquantitatively by silver staining of SDS-PAGE cells after affinity chromatography. Like the glutathione transferases, these enzymes displayed distinctly different levels of expression in the various cell lines. Thus, virtually every cell line was found to have a unique pattern of glutathione-linked enzymes, suggesting that the resistance phenotypes of the cells differ accordingly.

Cell Line↗
PubMed1994

Protection by transfected glutathione S-transferase isozymes against carcinogen-induced alkylation of cellular macromolecules in human MCF-7 cells.

Increased expression of glutathione S-transferase (GST) isozymes has been correlated with development of resistance both to cytotoxic anticancer agents and to genotoxic carcinogens. While most anticancer agents are poor GST substrates, the model alkylating carcinogen 4-nitroquinoline-1-oxide (NQO) is a good substrate for human pi class GST (hGSTP1-1) and murine GST mu-1 (mGSTM1-1), but not human GST alpha-2 (hGSTA2-2). We investigated whether expression of these GST isozymes in stably transfected clonal cell lines could protect against the genotoxic and cytotoxic effects of NQO. Compared to parental MCF-7 or pSV2neotransfected control cell lines, covalent labeling of total cellular macromolecules by [3H]NQO (0.1-1.0 mM) was reduced by 70% and 92% in hGSTP1-1- and mGSTM1-1-transfected cell lines, respectively, but was not affected in the hGSTA2-2 expressing line. The observed protection was closely correlated with the relative specific activity of each cell line for conjugation of NQO by the transfected GST isozymes and this protection was reversible by pretreatment of cells with the GST inhibitor ethacrynic acid. Similar results were obtained when covalent labeling of total cellular nucleic acid or DNA was measured. However, clonogenic survival assays indicated that the sensitivity of these cell lines to the cytotoxic effects of NQO was similar for the control and GST-transfected MCF-7 cell lines. Thus, while expression of hGSTP1-1 and mGSTM1-1 (but not hGSTA2-2) was highly protective against alkylation of cellular macromolecules by NQO, this protection was not effective against cytotoxicity induced by NQO as measured by clonogenic assay. These results indicate that expression of GST isozymes can protect differentially against the acute genotoxic and potentially mutagenic effects, as compared to the cytotoxic effects, of electrophiles that are detoxified by glutathione conjugation.

4-Nitroquinoline-1-oxide↗
PubMed1995

The high non-enzymatic conjugation rates of some glutathione S-transferase (GST) substrates at high glutathione concentrations.

Enzymatic properties of five human glutathione S-transferases (GSTs) and non-enzymatic conjugation rates of GST substrates were examined as a function of glutathione (GSH) concentration and pH. GSTP1-1 showed a broad substrate specificity with both low and high GSH (10 mM) concentrations at pH 7.0, and the inhibitor insensitivity was then prominent. Among the GST substrates tested, ethacrynic acid, p-nitrophenylacetate, 1-chloro-2,4-dinitrobenzene and trans-4-phenyl-3-buten-2-one conjugated nonenzymatically with half-times of 0.39, 3.1, 9.4 and 10.0 min respectively at 10 mM GSH, pH 7.0 and 35 degrees C. The half-time for acrolein estimated by extrapolation was approximately 0.5 s. While the enzymatic reaction rates were independent of GSH at concentrations larger than Km values according to Michaelis-Menten kinetics and relatively insensitive to a pH change from 6.5 to 7.0, the non-enzymatic ones were linearly proportional to the GSH concentration and sensitive to pH. The enhanced non-enzymatic conjugation of various electrophiles at high GSH concentrations may in part account for the physiological significance of GSH elevation in preneoplastic and neoplastic cells.

Female↗
PubMed1988

Glutathione S-transferase isoenzymes in human lung tumors.

In the present studies we have compared the levels of glutathione (GSH) and GSH-related enzymes in lung tumors and corresponding normal tissues obtained from the same individuals. We have also immunologically quantitated the relative amounts of glutathione S-transferase pi (or GST-P) type antigen in tumors and adjacent normal tissues from five patients. GST activities towards 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid were found to be elevated in tumors from two out of five patients (patients #1 and 4), whereas the activity towards these substrates was markedly suppressed in the tumor tissue from one of the patients (#5). Immunotitration and Western blot studies using antibodies raised against pi-type GST isoenzymes of human lung and placenta indicated induction of GST pi-type isoenzyme in tumors from patients #1 and 4 and suppression of this isoenzyme in tumor from patient #5. The tumors from patients #2 and 3 did not show any increase in GST activity or GST pi-type antigen. Except for the tumor from patient #5, the GSH content was higher in the tumors from other patients. GSH reductase activity was found to be elevated in tumors of all the patients examined in this study. These results indicate that GSH and GSH related enzymes are differentially altered in lung tumors and GSH levels and GST pi- or GST-P-type isoenzyme(s) are not uniformly elevated in all tumors.

Carcinoma↗
PubMed2005

Garlic organosulfur compounds upregulate the expression of the pi class of glutathione S-transferase in rat primary hepatocytes.

The chemopreventive property of garlic is related in part to its induction of phase II detoxification enzymes. In the present study, we investigated the modulatory effect of 3 garlic organosulfur compounds, i.e., diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), which differ in their number of sulfur atoms, on the gene expression of the pi class of glutathione S-transferase (GSTP). Hepatocytes isolated from male Sprague-Dawley rats were cultured with 50-200 micromol/L of DAS, DADS, or DATS for 24 h. DADS and DATS increased GST activity toward ethacrynic acid by 40 and 66%, respectively (P < 0.05). Moreover, both garlic allyl sulfides dose dependently induced GSTP mRNA and protein expression. DATS increased the protein level more than DADS (P < 0.05). In contrast, DAS did not affect the activity or the protein or mRNA levels of this phase II drug-metabolizing enzyme. In Clone 9 liver cells, the pTA-luciferase reporter assay showed that luciferase activity in DADS- and DATS-treated cells was 2.8- and 3.9-fold higher than that in control cells, respectively (P < 0.05). Again, luciferase activity was not affected by treatment with DAS. Deletion of -2.7 to -2.6 kb in the GSTP promoter region, which contains the GSTP enhancer (GPE) I element, abolished the upregulation of GSTP transcription by DADS and DATS. Deletion of GPE II, however, did not affect the induction of reporter activity. In conclusion, the effectiveness of 3 garlic allyl sulfides on GSTP expression was related to the number of sulfur atoms in the molecules, and GPE I was responsible for this upregulation.

Allyl Compounds↗
PubMed1996

Characterization of glutathione S-transferases in needles of Norway spruce trees from a forest decline stand.

Glutathione S-transferases (GST) detoxify many electrophilic xenobiotics, including several volatile organic compounds and pesticides. The GST activity for the conjugation of several xenobiotic substances was isolated from needles of Norway spruce (Picea abies L. Karst.) trees from a forest decline stand in the northern alps. Trees that exhibited different degrees of damage were selected from several stands in an altitude profile. The GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) in crude protein extracts of needles showed a seasonal pattern with highest activity during summer. The GST activity exhibited a strong dependence on the altitude of the stand showing highest activities in trees growing in the valley and lowest activities in trees growing in the summit regions of the mountain. When cytosolic GST from needles of healthy and damaged trees was purified, trees of healthy appearance exhibited three distinct GST isozymes with activities for the conjugation of CDNB and 1,2-dichloro-4-nitrobenzene (DCNB), whereas severely defoliated trees exhibited four GSTs with additional activity for the conjugation of ethacrynic acid. The main GST isozymes catalyzing the conjugation of CDNB differed in molecular weight, isoelectric point and catalytic properties between damaged and healthy trees.

Journal Article↗
PubMed1987

Acute renal failure following traumatic injury or major operation.

Acute oliguria in the critically ill postoperative patient, or in the trauma victim after resuscitation, is a substantial clinical problem. The mortality associated with ARF in these settings remains unacceptably high. Evaluation of the oliguric patient must include thorough monitoring for, and correction of, prerenal and postrenal causes of oliguria. In this sense, diagnosis of ARF is one of exclusion. Differential diagnosis is facilitated by microscopic examination of urine and by biochemical analyses of blood and urine for calculating indices of tubular function (urinary-to-plasma ratios of blood urea nitrogen and creatinine, sodium excretion, and clearances of sodium, creatinine, solute, and water). The early detection of an intrarenal defect, as accomplished by using serial measurements of free water clearance, may allow interruption of the process and prevention of ARF. Preventive measures include optimization of hemodynamic status and the use of osmotic diuretic agents (mannitol) and loop diuretics (furosemide, ethacrynic acid, and bumetanide). Dopamine is useful for increasing both renal blood flow and urine flow and may be useful for preventing ARF, but this is not firmly established. Experimentally, other approaches such as modulating the renin-angiotensin system, prostaglandin system, and cellular calcium fluxes have been attempted, but the clinical applicability of these measures is not established. The best approach to ARF is preventing it by knowing which patients are at high risk, by studiously preventing renal insults, and by aggressively treating early indications of renal malfunction using established therapies.

Acute Kidney Injury↗
PubMed1998

The endogenous antioxidant glutathione as a factor in the survival of physically injured mammalian spinal cord neurons.

Glutathione is part of the system of cellular defenses against lipid peroxidation and other free radical-mediated damage. An established in vitro trauma model was utilized to evaluate whether glutathione is a factor in the survival of mammalian spinal cord neurons following physical injury. Cultured murine spinal neurons were subjected to a standard lesion: transection of a primary dendrite 100 microm from the perikaryon. Prior reduction of glutathione with ethacrynic acid or buthionine sulfoximine caused a dose-dependent decrease in neuronal survival 24 hours after dendrotomy. Prior glutathione augmentation with gamma-glutamylcysteine or L-2-oxo-4-thiazolidine carboxylic acid significantly increased survival, but N-acetyl-cysteine was not protective. Gamma glutamylcysteine effected the most rapid increase in glutathione (peak at 10 min), and survival was 72% +/- 10 when 0.2 mM gamma-glutamylcysteine was added immediately after dendrotomy compared with 38% +/- 4 in the control group (p < 0.0001). These results indicate that the level of glutathione is a factor in spinal cord neuron survival after physical trauma, and that glutathione augmentation may be an effective acute phase spinal cord injury (SCI) intervention strategy.

Animals↗
PubMed1998

University of Miami Division of Clinical Pharmacology Therapeutic Rounds: managing hypertension in patients with kidney disease-implications for preservation of renal function.

Renal parenchymal hypertension is defined as hypertension caused by disease of the kidneys. Impaired renal sodium excretion leading to extracellular fluid volume (ECFV) expansion is the most clinically important mechanism leading to hypertension in patients with kidney disease. Most patients with renal parenchymal hypertension have sodium-sensitive hypertension, and, consequently, sodium restriction and loop diuretics constitute the initial steps in effective antihypertensive therapy in patients with renal disease. The loop diuretics (furosemide, ethacrynic acid, bumetanide, torasemide) are used for the management of ECFV and hypertension in patients with renal disease because thiazide diuretics are generally not effective in patients with serum creatinine values above 2.0 mg/dL or creatinine clearances below 30 mL/min. The Joint National Committee VI recommends the use of angiotensin-converting enzyme (ACE) inhibitors in patients with hypertension and chronic renal disease to control hypertension and to slow progressive renal failure. Antihypertensive treatment with ACE inhibitors may favorably alter renal hemodynamics, thereby slowing the progression of renal dysfunction. ACE inhibitors have been found to be useful agents in preventing the progression of renal disease in the settings of established insulin-dependent diabetes mellitus (IDDM) nephropathy, non-insulin-dependent diabetes mellitus (NIDDM) nephropathy, IDDM patients with normal blood pressures and microalbuminuria, NIDDM patients with microalbuminuria and normal renal function, and a variety of non-diabetic renal diseases, especially in the setting of significant proteinuria. Calcium antagonists are effective for treating hypertensive patients with chronic renal impairment, and the initial results for calcium antagonists and for combination calcium antagonist-ACE inhibitor therapy have been encouraging. Although promising, the calcium antagonists and the new angiotensin II antagonists have not been studied as intensively as ACE inhibitors in regard to their ability to slow the progression of renal insufficiency.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1998

Isolation and characterization of glutathione S-transferase isozymes from sorghum.

Two glutathione S-transferase (GST) isozymes, A1/A1 and B1/B2, were purified from etiolated, O-1,3-dioxolan-2-yl-methyl-2,2,2, -trifluoro-4'-chloroacetophenone-oxime-treated sorghum (Sorghum bicolor L. Moench) shoots. GST A1/A1, a constitutively expressed homodimer, had a subunit molecular mass of 26 kD and an isoelectric point of 4.9. GST A1/A1 exhibited high activity with 1-chloro-2, 4, dinitrobenzene (CDNB) but low activity with the chloroacetanilide herbicide metolachlor. For GST A1/A1, the random, rapid-equilibrium bireactant kinetic model provided a good description of the kinetic data for the substrates CDNB and glutathione (GSH). GST B1/B2 was a heterodimer with subunit molecular masses of 26 kD (designated the B1 subunit) and 28 kD (designated the B2 subunit) and a native isoelectric point of 4.8. GST B1/B2 exhibited low activity with CDNB and high activity with metolachlor as the substrate. The kinetics of GST B1/B2 activity with GSH and metolachlor fit a model describing a multisite enzyme having two binding sites with different affinities for these substrates. Both GST A1/A1 and GST B1/B2 exhibited GSH-conjugating activity with ethacrynic acid and GSH peroxidase activity with cumene hydroperoxide, 9-hydroperoxy-trans-10, cis-12-octadecadienoic acid and 13-hydroperoxy-cis-9, trans-11-octadecadienoic acid. Both GST A1/A1 and GST B1/B2 are glycoproteins, as indicated by their binding of concanavalin A. Polyclonal antibodies raised against GST A1/A1 exhibited cross-reactivity with the B1 subunit of GST B1/B2. Comparisons of the N-terminal amino acid sequences of the GST A1, B1, and B2 subunits with other type I theta-GSTs indicated a high degree of homology with the maize GST I subunit and a sugarcane GST.

Amino Acid Sequence↗
PubMed1987

Properties of Single K and Cl Channels in Asclepias tuberosa Protoplasts.

Potassium and chloride channels were characterized in Asclepias tuberosa suspension cell derived protoplasts by patch voltage-clamp. Whole-cell currents and single channels in excised patches had linear instantaneous current-voltage relations, reversing at the Nernst potentials for K(+) and Cl(-), respectively. Whole cell K(+) currents activated exponentially during step depolarizations, while voltage-dependent Cl(-) channels were activated by hyperpolarizations. Single K(+) channel conductance was 40 +/- 5 pS with a mean open time of 4.5 milliseconds at 100 millivolts. Potassium channels were blocked by Cs(+) and tetraethylammonium, but were insensitive to 4-aminopyridine. Chloride channels had a single-channel conductance of 100 +/- 17 picosiemens, mean open time of 8.8 milliseconds, and were blocked by Zn(2+) and ethacrynic acid. Whole-cell Cl(-) currents were inhibited by abscisic acid, and were unaffected by indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology and development.

Journal Article↗
PubMed1991

Glutathione S-transferases in human and rodent skin: multiple forms and species-specific expression.

The glutathione S-transferases (GST) are a family of widely distributed multifunctional detoxification enzymes that catalyze the reaction between reduced glutathione and a variety of electrophiles. Of interest is the fact that several extracutaneous tissues exhibit a distinct spectrum of isozymes that are expressed in a highly controlled fashion. Despite the fact that the skin is continuously exposed to numerous injurious agents, little is known about the expression of GST isozymes and their role in metabolism of physiologic and xenobiotic substrates in cutaneous tissue. Using specific polyclonal antibodies to the Alpha, Mu, and Pi classes of GST, we identified their expression in rat, mouse, and human skin cytosol. In each species, GST isozymes expressed activities towards 1-chloro-2,4-dinitrobenzene, benzo(a)pyrene 4,5-oxide, styrene 7,8-oxide, leukotriene A4, and ethacrynic acid, but not towards bromosulfophthalein and cumene hydroperoxide. Western blot analysis indicated the predominant expression of Pi isozyme in all three species. Alpha class of isozyme(s) was present only in human skin, whereas Mu class of isozyme(s) was detected only in rat and mouse skin. Similarly, in normal and transformed cultured human keratinocytes Pi was the predominant isozyme. In situ localization studies using immunohistochemical techniques confirmed the observations of Western blotting. In mouse skin, Pi and Mu isozyme(s) were found to be predominantly localized in sebaceous glands, whereas no reactivity was observed with the Alpha class of isozymes. Our data show that multiple forms of GST exist in rodent and human skin and that GST Pi is the predominant isozyme in each species. Furthermore, cutaneous GST can metabolize both endogenous substrates and foreign compounds.

Animals↗
PubMed1996

Lentinan enhances sensitivity of mouse colon 26 tumor to cis-diamminedichloroplatinum (II) and decreases glutathione transferase expression.

We investigated the influence of a combination of lentinan, a biological response modifier, and cis-diamminedichloroplatinum(II) (CDDP) on the growth and glutathione S-transferase (GST) content of colon 26 tumor to examine whether lentinan represses GST expression and enhances the therapeutic effects of CDDP. Female CDF1 mice inoculated subcutaneously with transplantable colon 26 adenocarcinoma cells (1 X 10(6)/mouse) received intraperitoneal administrations of lentinan, CDDP, or the two drugs in combination, on days 10, 14, 17 and 21 after the inoculation. On day 24, tumor weights (estimated from their length and width) were significantly lower in the CDDP+ lentinan group (2.7+/-1.3 g) than in the CDDP alone group (4.3+/-0.7 g, P<0.05), both values being less than in the nontreated control group (7.2+/-1.5 g). The major GST form of colon 26 tumor was identified as GST-II, the Pi class form, and a minor form as GST-III belonging to the Mu class. Both GST-II and GST-III values on day 24 were significantly decreased in the lentinan alone (0.90+/-0.29 and 0.26 +/-0.11 microg/mg protein, respectively) and lentinan + CDDP groups (0.98+/-0.22 and 0.29+/-0.07 microg/mg protein), as compared with the control levels (1.39+/-0.20 and 0.52+/-0.11 microg/mg protein). However, these values were not different between the CDDP alone and lentinan + CDDP groups. Neither tissue interleukin (IL)-6, glutathione nor platinum values were different between the two groups. IL-6 values were elevated in about half of the samples treated with lentinan or CDDP and exhibited a modest inverse correlation with GST-II levels (r= -0.46). A GST inhibitor, ethacrynic acid, enhanced the sensitivity of cultured colon 26 cells to CDDP, suggesting the possible involvement of GST in modulating the cytotoxicity of CDDP to this cell line. These results indicated that lentinan administration decreases tissue GST-II and GST-III contents and enhances the sensitivity of colon 26 tumor to CDDP.

Adenocarcinoma↗
PubMed1974

Effect of bovine parathyroid hormone 1-34 fragment on renal production and excretion of adenosine 3', 5' monophosphate in man.

Biologically active bovine parathyroid hormone (b PTH) 1-34 fragment infused over one hour in normal subjects produced an immediate and sharp increase in the excreted fractions of filtered bicarbonate, sodium and potassium, followed by the return to pre-infusion levels as soon as the administration of b PTH was stopped. There was a gradual but steady increase in the excreted fraction of filtered phosphate but a decrease in the excreted fraction of filtered calcium and magnesium. The excreted fractions of these ions were still abnormal 150 minutes after the completion of PTH infusion. The urinary excretion of 3'5'-cyclic AMP increased immediately about one hundred-fold but returned rapidly to pre-infusion levels. Urinary clearance of cyclic AMP approximated glomerular filtration rate in control periods and was twenty to thirty times greater during b PTH infusion. In subjects overloaded with bicarbonate, b PTH brought about a decrease in bicarbonate T(m) and the same effects on the urinary excretion of other electrolytes. 3'5'-cyclic AMP excretion was clearly higher in control periods and reached higher levels during b PTH infusion when compared to subjects without an alkaline load. 3'5'-cyclic AMP excretion and fractional clearance were also clearly higher in subjects not given b PTH when control periods were compared to periods with bicarbonate infusion or after acetazolamide administration. During distal blockade obtained by simultaneous administration of chlorothiazide and ethacrynic acid, there was a delay in the rise of 3'5'-cyclic AMP excretion after b PTH administration. It can be concluded from these studies that the pattern of excretion of 3'5'-cyclic AMP is similar to that of bicarbonate, sodium and potassium. The increase of 3'5'-cyclic AMP excretion when urinary pH is above 7 suggests a diffusion trapping mechanism for the secretion into the lumen of this nucleotide. Distal diuretics used in distal blockade did not inhibit 3'5'-cyclic AMP production but delayed its secretion into urine.

Adenosine Monophosphate↗
PubMed1991

Class pi glutathione S-transferase from pig lung. Purification, biochemical characterization, primary structure and crystallization.

A cytosolic glutathione S-transferase from pig lung was purified 210-fold to apparent homogeneity. The enzyme was classified as a class pi isoenzyme on the basis of its physical and chemical properties. It is homodimeric with a subunit Mr of 23,500, has a pI of 7.2, and shows a high specific activity towards ethacrynic acid. The glutathione analogues, S-hexylglutathione and glutathione sulfonate, were strong reversible inhibitors. The enzyme's primary structure, established entirely by protein chemical methods, consists of 203 amino acids and is highly similar (82-84% residue identity) to the rat and human class pi isoenzymes. Furthermore, there was no evidence of microheterogeneity or post-translational modifications. Each subunit contains a highly reactive cysteine residue, the modification of which leads to enzyme inactivation. None of the cysteine residues in the pig enzyme appear to form intramolecular disulfide bonds. Singel crystals of the glutathione-S-transferase-glutathione-sulfonate complex were obtained by the hanging-drop method of vapour diffusion from poly(ethylene glycol) 4000 solutions. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions of a = 10.125 nm, b = 8.253 nm and c = 5.428 nm and diffract to better than 0.22 nm.

Amino Acid Sequence↗
PubMed1996

Class-pi glutathione S-transferase is unable to regain its native conformation after oxidative inactivation by hydrogen peroxide.

The oxidation of protein sulphydryls to disulphides and their reduction back to sulphydryls is an early cellular response to oxidative stress. Hydrogen-peroxide-mediated oxidation of class-pi glutathione S-transferase results in the formation of disulphide bonds, which inhibits its catalytic function yet allows it to retain its non-substrate-ligand-binding properties. The overall hydrodynamic volume of the oxidised class-pi glutathione S-transferase type-1 homodimer (GSTP1-1) is decreased, and its tertiary-structural and secondary-structural elements are changed with respect to the native protein. Structural differences appear to be prominent in domain 1 of oxidised GSTP1-1, in that the exposure of both tryptophan residues is increased, while the electric potential about one of them is altered. Treatment of the oxidised protein with dithiothreitol or glutathione restores its enzymatic capabilities, albeit with lower specific activities for 1-chloro-2,4-dinitrobenzene and ethacrynic acid. The hydrophobic binding site (H-site) for electrophilic substrates is negatively affected in that the K(m) and catalytic-efficiency values are diminished significantly with respect to those values obtained for the native protein. The dithiothreitol-treated oxidised GSTP1-1 is able to regain its overall hydrodynamic volume; however, both its secondary-structural and tertiary-structural elements remain modified with respect to the native protein, as do both tryptophanyl environments. Furthermore, the oxidised glutathione S-transferase and dithiothreitol-treated oxidised glutathione S-transferase are less thermostable (tM = 55.5 degrees C and 56.3 degrees C, respectively) than the native enzyme (tM = 59 degrees C). These results indicate that the class-pi glutathione S-transferase is unable to regain its native conformation after oxidative inactivation.

Chromatography, High Pressure Liquid↗
PubMed1989

Changes in hepatic cytosolic glutathione S-transferase enzymes induced by clotrimazole treatment in rats.

1. The influence of the antifungal agent clotrimazole on cytosolic glutathione S-transferase activities was studied in male Wistar rats. 2. Animals received clotrimazole by gastric lavage for 3 days (75 mg/kg per day). Hepatic glutathione S-transferase activity was determined with five different substrates: 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), p-nitro-benzyl chloride (PNBC), ethacrynic acid (EA) and trans-4-phenyl-3-buten-2-one (TPBO). 3. The largest increases in glutathione S-transferase activity were found with CDNB, DCNB and PNBC (+61%, +50% and +50%, respectively, when expressed per mg of cytosolic protein). Enzyme activity toward EA was induced to a lower extent (+33%). Changes in the formation of the conjugate of TPBO were relatively small (+22%). 4. These data indicate a differential induction of glutathione S-transferase isoenzymes and suggest that clotrimazole is a phenobarbital-type inducer of enzyme activity.

Animals↗