Protective effect of salicylates against hydrogen peroxide stress in yeast

Aims:  To investigate the effects of salicylates in Saccharomyces cerevisiae exposed to oxidative stress induced by hydrogen peroxide (H₂O₂).
Methods and Results:  Saccharomyces cerevisiae was cultured through to the postlogarithmic phase of growth. Stress was induced by the addition of 1·5 mmol l⁻¹ H₂O₂ for 1 h, while N-acetyl-l-cysteine (NAC) and glutathione (GSSG) were used as control agents that affect the redox balance. Sodium salicylate, at 0·01–10 mmol l⁻¹or acetylsalicylic acid, at 0·02–2·5 mmol l⁻¹ was administered at various times before hydrogen peroxide stress. Both agents conferred resistance to a subsequent hydrogen peroxide stress, similarly to the induction of the adaptive response observed upon pretreatment with NAC and GSSG. Sodium salicylate was more potent as a short-term, but not as a long-term pretreatment agent, compared to acetylsalicylic acid.
Conclusions:  Pharmacological pretreatment with salicylates resulted in dose related increases in cell survival, indicating the induction of the protective response in yeast.
Significance and Impact of the study:  The possible role of salicylates in the modulation of the hydrogen peroxide stress response in eukaryotic cells address questions on the effects of these commonly used therapeutic agents in a number of disorders exhibiting an oxidative stress component.     

Purification and some properties of (1R,2S)-1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase from Comamonas testosteroni T-2

Abstract Inducible (1 R ,2 S )-1,2-dihydroxy-3,5-cyclohexadiene-l,4-dicarboxylate (diene-diol) dehydrogenase was found in extracts of Comamonas testosteroni T-2 grown in p -toluate-or terephthalate-salts medium and it was purified using anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is a homodimer with subunit M r 39000. It had a specific activity of 500 mkat/kg of protein and was activated by the addition of Fe²⁺. The dehydrogenase converted 1 mol diene-diol and 1 mol NAD⁺ to 1 mol protocatechuic acid, 1 mol NADH and 1 mol CO₂. Apparent K _m-values of 43 μM (NAD⁺) and about 90 μM (diene-diol) were determined. The hydride ion was transferred to the si face of NAD⁺.     

Purification and characterization of 5-aminolevulinic acid dehydratase from Methanosarcina barken

Abstract 5-Aminolevulinic acid dehydratase from the archaebacterium Methanosarcina barken resembles the mammalian and yeast enzymes in its activation by Zn²⁺, whereas its activation by K⁺ resembles the characteristic of bacterial enzymes. This enzyme is activated with Ni²⁺ which is a component of F₄₃₀, a cofactor present mainly in methanogens. The M _r of 280000 for the native enzyme and 30 000 ± 2000 for the individual subunit suggest that the enzyme is composed of eight apparently indentical subunits similar to mammalian and yeast enzymes. The enzyme has two pH optima, at 8.5 and 9.4. Higher levels of 5-aminolevulinic acid dehydratase in acetate-grown cells suggest the possibility that regulation and control of this enzyme could be different on various growth substrates.     

Note: Purification of amylase secreted from Bifidobacterium adolescentis

Bifidobacterium adolescentis Int-57 isolated from human faeces produced extracellular amylase. The enzyme was purified from the culture supernatant fluids by ammonium sulphate precipitation, gel-filtration chromatography (Sephadex-G-75), ion-exchange chromatography (CM-cellulose) and FPLC. SDS-PAGE of the purified enzyme revealed a major band with an apparent molecular weight of 66 kDa. The pI was 5·2. Enzyme activity was optimal at 50°C, and at pH 5·5. The enzyme was stable at 20–40°C, and at pH 5–6 with a K _m value of 2·4 g l⁻¹ soluble starch. The activation energy was 42·3 kJ mol⁻¹. The enzyme was significantly inhibited by maltose (10%), glucose (10%), Cu²+ (5 mmol l⁻¹), Zn²+ (5 mmol l⁻¹), N- bromosuccinimide (5 mmol l⁻¹), EDTA (5 mmol l⁻¹), I₂ (1 mmol l⁻¹) and activated by β-mercaptoethanol (10 mmol l⁻¹).     

Properties of glutamate dehydrogenase from developing maize endosperm

Glutamate dehydrogenase (EC 1.4.1.3) activity was assayed in homogenates of maize ( Zea mays L. inbred lines Oh43 and Oh43o₂) endosperm during development. During the period 20–35 days after pollination anabolic (aminative) activities were higher than catabolic (deaminating) ones. In order to study the regulation of GDH activity, glutamine or glutamate were injected into the ear peduncle before sample harvesting. The amination and deamination reactions showed similar behaviour with different nitrogen sources: glutamine increased, whereas glutamate decreased, both aminative and deaminative reactions. Partially purified enzyme was active with NADH and NADPH in a ratio 9:1. In Tris-HCl buffer a broad optimum at pH 7.6–8.9 and pH 6.8–8.9 was observed with NADH and NADPH, respectively, NADH activity was activated by Ca²⁺. Saturation curves for (NH₄)₂SO₄ and NADH showed normal Michaelis-Menten kinetics in the presence of 1 m M Ca²⁺, but substrate inhibition occurred without Ca²⁺. The enzyme was inactivated by EDTA. The effect of EDTA was reversed by Ca²⁺ and Mn²⁺, but not by Cu₂₊ and Mg²⁺.     

Characteristics of the reverse reaction of NADP+-isocitrate dehydrogenase from castor bean seeds

The reductive carboxylation of α-ketoglutarate by purified NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) from maturing castor bean seeds ( Ricinus communis L. ) has been characterized. The optimum pH for the reaction was 6.5, whereas pH 8.5 was optimum for oxidation of isocitrate (forward reaction). The enzyme utilized NADH as well as NADPH as the reducing agent in the reverse reaction, but only NADP⁺ in the forward reaction. The Km values for NADPH and NADH were 0.044 and 2.8 m M respectively, and for α-ketoglutarate and HCO₃ 4.1 and 3.7 m M. The enzyme was activated by various cations including Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Ni²⁺ and Co²⁺. Km values for Mg²⁺ Mn²⁺, Co²⁺ and Zn²⁺ were 12, 34, 37 and 49μ M respectively.     

α-Galactosidase from the yeast Torulaspora delbrueckii IFO 1255

The yeast Torulaspora delbrueckii IFO 1255 was selected as the strain fermenting melibiose from 35 strains of Torulaspora species. The strain IFO 1255 produced extracellular and cell-associated forms of α-galactosidase when grown on either melibiose or galactose as the sole carbon source. Most of the enzyme was located outside of the cell membrane: the periplasmic space, or cell walls, or both. α-Galactosidase was purified to homogeneity from the cell-free extract of the strain IFO 1255 by acid treatment and column chromatography on DEAE-Toyopearl 650M and Butyl-Toyopearl 650M. The molecular weight of the purified enzyme was estimated to be 88 000 by SDS-polyacrylamide gel electrophoresis and 530 000 by gel filtration. The enzyme contained 50% of its molecular weight as carbohydrate. Optimum pH and temperature were 4.5–5.5 and 55°C, respectively. The enzyme was inhibited strongly by Ag²⁺, Hg²⁺ and Cu²⁺ each at 1 mmol 1^-1. The K _m (μmol 1^-1) for p -, o -, m -nitrophenyl α-D-galactopyranoside, melibiose, raffinose and stachyose were 2.8, 1.3, 2.8, 4.2, 170 and 230, respectively, and V _max (μmol min^-1 mg protein^-1) for those substrates were 310, 140, 21, 22, 30 and 44, respectively. The properties of α-galactosidase from T. delbrueckii IFO 1255 were similar to those from the related species, Saccharomyces cerevisiae.     

Probable participation of phospholipase A2 reaction in the process of fertilization-induced activation of sea urchin eggs

In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a phospholipase A₂ inhibitor) blocked fertilization envelope formation and transient CN⁻-insensitive respiration in a concentration-dependent manner. BPB had virtually no effect on the increase in [Ca²⁺]_i, (cytosolic Ca²⁺ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN⁻-sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN⁻-insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A₂, induced the envelope formation, acid production, augmentation of CN⁻-insensitive and sensitive respiration, but did not cause any increase in [Ca²⁺]_i, the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A₂ induced by Ca²⁺ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN⁻-insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca²⁺-triggered sequential reactions other than Ca²⁺-caused activation of phospholipase A₂.     

Purification and characterization of the major β-1,4-endoglucanase from Thermomonospora curvata

The major β-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata , contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose. The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg⁻¹ when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70°C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60°C, pH 6.0 and had a half-life of 30 min at 80°C; temperature and pH optima were 70–73°C and 6.0–6.5, respectively. The mol. wt was 100000 and the pI was 4.0. The K _m and V _max values were 7.33 mg ml⁻¹ and 833 μmol min⁻¹, respectively. EG activity was inhibited by Fe^{2 +}, Hg^{2 +}, Ag⁺ and Pb^{2 +}, and enhanced by dithiothreitol and Zn^{2 +}. The first 12 amino acid residues at the N -terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found.     

Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Characterization of cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Cyclic nucleotide phosphodiesterase (3',5'-cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, chromatography on 3',5'-cAMP-agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH₄)₂SO₄ pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca²⁺, Mg²⁺ and Mn²⁺, whereas NaF, PP_i and Fe³⁺ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An M_I of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3',5'-cAMP-agarose a phosphodiesterase was resolved that produced 5'-AMP as sole reaction product.     

Purification and kinetic properties of a castor bean seed acid phosphatase containing sulfhydryl groups

An acid phosphatase (EC 3.1.3.2) has been identified and purified from castor bean ( Ricinus communis L., IAC-80 ) seed through sulphopropyl (SP)-Sephadex, diethylaminoethyl (DEAE)-Sephadex, Sephacryl S-200, and Concanavalin A-Sepharose chromatography. The enzyme was purified 2 000-fold to homogeneity, with a final specific activity of 3.8 μkat mg⁻¹ protein. The purified enzyme revealed a single diffuse band with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis, at pH 8.3. The relative molecular mass, determined by high-performance liquid chromatography (HPLC), was found to be 60 kDa. The acid phosphatase had a pH optimum of 5.5 and an akpparent K_m value for p -nitrophenylphosphate of 0.52 m M . The enzyme-catalyzed reaction was inhibited by inorganic phosphate, fluoride, vanadate, molybdate, p -chloromercuribenzoate ( p CMB), Cu²⁺ and Zn²⁺. The strong inhibition by p CMB, Cu²⁺ and vanadate suggests the presence of sulfhydryl groups essential for catalysis. The castor bean enzyme also recognized tyrosine-phosphate and inorganic pyrophosphate (KPP_i) as substrate. The highest specificity constant (V_max/K_m) was observed with KPP_i, making it a potential physiological substrate.     

Isolation and characterization of cytoplasmic NADP+-malic enzyme from Lupinus luteus leaves

NADP⁺-dependent malic enzyme (L-malate : NADP⁺ oxidoreductase, decarboxylating, EC 1.1.1.40) was extracted from the leaves of yellow lupine. The purification procedure included fractionation with (NH₄)₂SO₄ and Sephadex G-25 chromatography, followed by purification on DEAE-cellulose and Sephadex G-200 columns. The enzyme was purified 122-fold. The enzyme affinity towards L-malate was found to be significantly higher with Mn²⁺ than with Mg²⁺. The Hill coefficient for Mg²⁺ depended on concentration and was 1.6 for the lower and 3.9 for the higher concentrations. The dependence of the enzyme activity on NADP⁺ followed a hyperbolic curve. K_m values and Hill coefficients for NADP⁺ were similar with both Mn²⁺ and Mg²⁺. The enzyme activity was strictly dependent on divalent cations and followed a sigmoidal curve at least for Mg²⁺. The enzyme had 4-fold higher affinity towards Mn²⁺ than towards Mg²⁺, the K_m values being 0.3 and 1.15 m M respectively. Of several tested organic acids, oxalate was the most effective inhibitor followed by oxaloacetate while succinate was the strongest activator.     

Stimulation of plant plasma membrane Ca2+-ATPase activity by acidic phospholipids

The effect of phospholipids on the activity of the plasma membrane (PM) Ca²⁺-ATPase was evaluated in PM isolated from germinating radish ( Raphanus sativus L. cv. Tondo Rosso Quarantino) seeds after removal of endogenous calmodulin (CaM) by washing the PM vesicles with EDTA. Acidic phospholipids stimulated the basal Ca²⁺-ATPase activity in the following order of efficiency: phosphatidylinositol 4,5-diphosphate (PIP2)≈phosphatidylinositol 4-monophosphate>phosphatidylinositol≈phosphatidylserine≈phosphatidic acid. Neutral phospholipids as phosphatidylcholine and phosphatidylethanolamine were essentially ineffective. When the assays were performed in the presence of optimal free Ca²⁺ concentrations (10 μ M ) acidic phospholipids did not affect the Ca²⁺-ATPase activated by CaM or by a controlled trypsin treatment of the PM, which cleaved the CaM-binding domain of the enzyme. Analysis of the dependence of Ca²⁺-ATPase activity on free Ca²⁺ concentration showed that acidic phospholipids increased V_max and lowered the apparent K_m for free Ca²⁺ below the value measured upon tryptic cleavage of the CaM-binding domain; in particular, PIP2 was shown to lower the apparent K_m for free Ca²⁺ of the Ca²⁺-ATPase also in trypsin-treated PM. These results indicate that acidic phospholipids activate the plant PM Ca²⁺-ATPase through a mechanism only partially overlapping that of CaM, and thus involving a phospholipid-binding site in the Ca²⁺-ATPase distinct from the CaM-binding domain. The physiological implications of these results are discussed.     

Shikimate dehydrogenase from pepper (Capsicum annuum) seedlings. Purification and properties

Shikimate dehydrogenase (SKDH, EC 1.1.1.25) was extracted from seedlings of pepper ( Capsicum annuum L.) and purified 347-fold. The purification procedure included precipitation with ammonium sulphate and chromatography in columns of Reactive Red-agarose, Q-Sepharose and Sephadex G-100. Pepper SKDH isozymes are separable only using PAGE. The purified enzyme has a relative molecular mass of 67 000 as estimated by gel filtration. The optimum pH of enzyme activity is 10.5 and the optimum temperature is 50°C, but the enzyme is quickly inactivated at temperatures higher than 40°C. The purified enzyme exhibited typical Michaelis-Menten kinetics and K_m values are 0.087 m M for shikimic acid and 0.017 m M for NADP. The mechanism of reaction is sequential considering NADP as a cosubstrate. Ions such as Ca²⁺, Mg²⁺ and Mn²⁺ activate the enzyme, but Zn²⁺ and Cu²⁺ are strong inhibitors. Some phenolic compounds such as guaiacol, protocatechuic acid and 2,4-D are competitive inhibitors of pepper SKDH, showing K_i values of 0.38 m M , 0.27 m M and 0.16 m M , respectively.     

Systemin triggers an increase of cytoplasmic calcium in tomato mesophyll cells: Ca2+ mobilization from intra- and extracellular compartments

We show here that, within 1–2 min of application, systemin triggers a transient increase of cytoplasmic free calcium concentration ([Ca²⁺]_c) in cells from Lycopersicon esculentum mesophyll. The systemin-induced Ca²⁺ increase was slightly but not significantly reduced by L-type Ca²⁺ channel blockers (nifedipine, verapamil and diltiazem) and the Ca²⁺ chelator [ethylene glycol tetraacetic acid (EGTA)], whereas inorganic Ca²⁺ channel blockers (LaCl₃, CdCl₂ and GdCl₃) and compounds affecting the release of intracellular Ca²⁺ from the vacuole (ruthenium red, LiCl, neomycin) strongly reduced the systemin-induced [Ca²⁺]_c increase. By contrast, no inhibitory effect was seen with the potassium and chloride channel blockers tested. Unlike systemin, other inducers of proteinase inhibitor (PI) and of wound-induced protein synthesis, such as jasmonic acid (JA) and bestatin, did not trigger an increase of cytoplasmic Ca²⁺. The systemin-induced elevation of cytoplasmic Ca²⁺ which might be an early step in the systemin signalling pathway, appears to involve an influx of extracellular Ca²⁺ simultaneously through several types of Ca²⁺ permeable channels, and a release of Ca²⁺ from intracellular stores sensitive to blockers of inositol 1,4,5-triphosphate (IP₃)- and cyclic adenasine 5'-diphosphoribose (cADPR)-mediated Ca²⁺ release.     

Apparent phytotoxicity of mononuclear hydroxy-aluminum to four dicotyledonous species

It has long been assumed that Al³⁺ is an important rhizotoxic ion in acid soils around the world, but the toxicity of Al³⁺ relative to mononuclear hydroxy-Al [AlOH²⁺ and Al(OH)⁺₂] has been examined in detail only for an Al-sensitive wheat variety ( Triticum aestivum L. cv. Tyler). That plant appears to be sensitive to Al³⁺ but not to AlOH²⁺ and Al(OH)⁺₂. New experiments, and reanalyses of previously published experiments, provide evidence that dicotyledonous species may be sensitive to mononuclear hydroxy-Al and that Al³⁺ may be nontoxic, or less toxic, to those plants. Despite these consistently measured differences between wheat and the dicotyledons, the determination of relative toxicities (Al³⁺ vs mononuclear hydroxy-Al) may be an intractable problem. Because of hydrolysis equilibria, (AlOH²⁺) and (Al(OH)⁺₂) are equivalent to (Al³⁺)k₁(H⁺)⁻¹ and (l³⁺)k₂(H⁺)⁻², respectively, in which k₁ and k₂ are the first and second hydrolysis constants (braces denote activities). Thus, any expression of root elongation as a function of mononuclear hydroxy-Al can be alternatively expressed as a function of (Al³⁺) and (H⁺). Toxicity attributed to mononuclear hydroxy-Al may actually be Al³⁺ toxicity that increases as pH rises (i.e. Al³⁺ toxicity ameliorated by H⁺).     

Inhibition of Bradykinin-Induced Calcium Increase by Phosphatase Inhibitors in Neuroblastoma × Glioma Hybrid NG108-15 Cells

Abstract: Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) induced by bradykinin by ∼63%. This inhibition was dependent on the concentration of okadaic acid with an IC₅₀ of 0.15 n M . Okadaic acid treatment only lowered the maximal response of [Ca²⁺]_i increase and had no effect on the EC₅₀ value for bradykinin regardless of the presence of extracellular Ca²⁺. Neither the capacity of ⁴⁵Ca²⁺ accumulation within intracellular nonmitochondrial Ca²⁺ stores nor the magnitude of [Ca²⁺]_i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP₃) generation, the Mn²⁺ influx, and the capacity of mitochondrial Ca²⁺ accumulation. Furthermore, the sensitivity of IP₃ in the Ca²⁺ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca²⁺]_i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP₃, and the slowed rate of Ca²⁺ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca²⁺ signaling cascade in NG108-15 cells.     

Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance?

Elevated levels of salicylic acid (SA) are required for the induction of systemic acquired resistance (SAR) in plants. Recently, a salicylic acid-binding protein (SABP) isolated from tobacco was shown to have catalase activity. Based on this finding elevated levels of hydrogen peroxide (H₂O₂) were postulated to act as a second messenger of SA in the SAR signal transduction pathway. A series of experiments have been carried out to clarify the role of H₂O₂ in SAR-signaling. No increase of H₂O₂ was found during the onset of SAR. Induction of the SAR gene, PR-1, by H₂O₂ and H₂O₂-inducing chemicals is strongly suppressed in transgenic tobacco plants that express the bacterial salicylate hydroxylase gene, indicating that H₂O₂ induction of SAR genes is dependent on SA accumulation. Following treatment of plants with increasing concentrations of H₂O₂, a dose-dependent accumulation of total SA species was found, suggesting that H₂O₂ may induce PR-1 gene expression through SA accumulation. While the results do not support a role for H₂O₂ in SAR signaling, it is suggested that SA inhibition of catalase activity may be important in tissues undergoing a hypersensitive response.     

In vitro evidence for the ionoregulatory role of rainbow trout mucus in acid, acid/aluminium and zinc toxicity

Rainbow trout body mucus dialysed with acidified distilled water at pH 7,5 and 3 experienced ion depletion which was greatest at pH 3 and minimal between pH 7 and 5. Mucus Na⁺ loss is exacerbated in the presence of 1 mg I⁻¹ aluminium as A1₂(SO₄), at pH 5 and 7. Al₂(SO₄), causes greater depletion of Na⁺ from mucus than A1C1₃. A lethal level of zinc (2 mg 1⁻¹) does not deplete mucus Na⁻ or K⁺, unlike a lethal level of aluminium (1 mg 1⁻¹) at pH 7. The results are discussed in terms of the ionoregulatory role of mucus in heavy metal and acid toxicity.