Relationship Between Effect of Ethanol on Proton Flux Across Plasma Membrane and Ethanol Tolerance, inPichia stipitis

Pichia stipitisefficiently converts glucose or xylose into ethanol but is inhibited by ethanol concentrations exceeding 30 g/L. InSaccharomyces cerevisiae, ethanol has been shown to alter the movement of protons into and out of the cell. InP. stipitisthe passive entry of protons into either glucose- or xylose-grown cells is unaffected at physiological ethanol concentrations. In contrast, active proton extrusion is affected differentially by ethanol, depending on the carbon source catabolized. In fact, in glucose-grown cells, the H⁺-extrusion rate is reduced by low ethanol concentrations, whereas, in xylose-grown cells, the H⁺-extrusion rate is reduced only at non-physiological ethanol concentrations. Thus, the ethanol inhibitory effect on growth and ethanol production, in glucose-grown cells, is probably caused by a reduction in H⁺-extrusion. Comparison of the rates of H⁺-flux with the relatedin vitroH⁺-ATPase activity suggests a new mechanism for the regulation of the proton pumping plasma membrane ATPase (EC 3.6.1.3) ofP. stipitis, by both glucose and ethanol. Glucose activates both the ATP hydrolysis and the proton-pumping activities of the H⁺-ATPase, whereas ethanol causes an uncoupling between the ATP hydrolysis and the proton-pumping activities. This uncoupling may well be the cause of ethanol induced growth inhibition of glucose grownP. stipitiscells.     

Pyrazole and 4-methylpyrazole inhibit oxidation of ethanol and dimethyl sulfoxide by hydroxyl radicals generated from ascorbate, xanthine oxidase, and rat liver microsomes

Pyrazole and 4-methylpyrazole, which are potent inhibitors of alcohol dehydrogenase, inhibited the oxidation of ethanol and of dimethyl sulfoxide by two model hydroxyl radical-generating systems. The systems used were the iron-catalyzed oxidation of ascorbic acid and the coupled oxidation of xanthine by xanthine oxidase. Pyrazole and 4-methylpyrazole were more effective inhibitors at lower substrate concentrations than at higher substrate concentrations; the oxidation of ethanol was inhibited to a greater extent than the oxidation of dimethyl sulfoxide. These results are consistent with competition between pyrazole or 4-methylpyrazole with the substrates for the generated hydroxyl radicals. Pyrazole and 4-methylpyrazole appear to be equally effective in reacting with hydroxyl radicals. An approximate rate constant of about 8 × 10⁹m^?1 s^?1 was calculated from the inhibition curves, indicating that pyrazole and 4-methylpyrazole are potent scavengers of the hydroxyl radical. Previous studies have implicated a role for hydroxyl radicals in the microsomal pathway of ethanol oxidation. In the presence of azide (to inhibit catalase), pyrazole and 4-methylpyrazole inhibited the NADPH-dependent microsomal oxidation of ethanol, as well as several other hydroxyl radical-scavenging agents. This inhibition by pyrazole and by 4-methylpyrazole may reflect a mechanism involving competition for hydroxyl radicals generated by the microsomes. However, the kinetics of inhibition by pyrazole were mixed, not competitive, and pyrazole and 4-methylpyrazole also inhibited aminopyrine demethylase activity. Pyrazole has been shown by others to interact with cytochrome P-450. It is suggested that pyrazole and 4-methylpyrazole affect microsomal oxidation of ethanol via effects on the mixed-function oxidase system and via competition for the generated hydroxyl radicals. In view of these results, low concentrations of pyrazole and 4-methylpyrazole should be used in studies on pathways of alcohol metabolism, and caution should be made in interpreting the actions of these compounds when used at high concentrations.     

Interaction of anthelmintic benzimidazoles with AscarisSuum embryonic tubulin

The ability of mebendazole and fenbendazole to bind to tubulin in cytosolic fractions from 8-day Ascaris suum embryos was determined by inhibition studies with [³H]colchicine. Colchicine binding in the presence of 1·10^?6 M mebendazole was completely inhibited during a 6 h incubation period at 37°C. Inhibition of colchicine binding to A. suum embryonic tubulin by mebendazole and fenbendazole appeared to be noncompetative. The inhibition constants of mebendazole and fenbendazole for A. suum embryonic tubulin were 1.9·10^?8 M and 6.5·10^?8 M, respectively. Mebendazole and fenbendazole appeared to be competitive inhibitors of colchicine binding to bovine brain tubulin. The inhibition constants of mebendazole and fenbendazole for bovine brain tubulin were 7.3·10^?6 M and 1.7·10^?5 M, respectively. These values are 250–400 times greater than the inhibition constants of fenbendazole and mebendazole for A. suum embryonic tubulin. Differential binding affinities between nematode tubulin and mammalian tubulin for benzimidazoles may explain the selective toxicity. The importance of tubulin as a receptor for anthelmintic benzimidazoles in animal parasitic nematodes is discussed.     

Steady-state Inhibitory Kinetic Studies on Almond β-Glucosidase-catalyzed Reaction——Binding Sites of Monosaccharides

Steady-state inhibitory kinetic studies on almond β-glucosidase-catalyzed reactions were done to elucidate the binding subsite of several monosaccharides on this enzyme.

Glucono-1,5-Iactone (a transition-state analog), glucose, 2-deoxy glucose, fucose, and methyl α-glucoside showed mixed-type inhibition, but galactose, galactosamine, mannose, N-acetyl glucosamine, and glucosamine showed pure competitive inhibition on the hydrolysis of P-nitrophenyl β-glucoside.

These results are reasonably accounted for by assuming that the former monosaccharides (the mixed type inhibitors) bind to subsite 1 (the nonreducing-end side subsite to which the nonreducing-end glucose residue of a substrate binds in a productive binding mode), and that the latter (the competitive inhibitors) bind to subsite 2, the adjacent subsite to subsite 1.

The binding affinity ( — ΔG°) of glucono-1,5-lactone (— ΔG° = 6.7 kcal mol ¹ at pH 5.0, 25°C) was significantly greater than those of the others (0.3 ~ 1.6 kcal mol^-1).     

Amyloid aggregation of lysozyme: The synergy study of red wine polyphenols

The amyloidoses are diseases associated with nonnative folding of proteins and characterized by the presence of protein amyloid aggregates. The ability of quercetin, resveratrol, caffeic acid, and their equimolar mixtures to affect amyloid aggregation of hen egg white lysozyme in vitro was detected by Thioflavin T fluorescence assay. The anti‐amyloid activities of tested polyphenols were evaluated by the median depolymerization concentrations DC₅₀ and median inhibition concentrations IC₅₀. Single substances are more efficient (by at least one order) in the depolymerization of amyloid aggregates assay than in the inhibition of the amyloid formation with IC₅₀ in 10^?4 to 10^?5M range. Analyzed mixture samples showed synergic or antagonistic effects in both assays. DC₅₀ values ranged from 10^?5 to 10^?8M and IC₅₀ from 10^?5 to 10^?9M, respectively. We observed that certain mixtures of studied polyphenols can synergistically inhibit production of amyloids aggregates and are also effective in depolymerization of the aggregates. Synergic or antagonistic effects of studied mixtures were correlated with protein–small ligand docking studies and AFM results. Differences in these activities could be explained by binding of each polyphenol to a different amino acid sequence within the protein. Our results indicate that synergic/antagonistic anti‐amyloid effects of studied mixtures depend on the selective binding of polyphenols to the known amyloidogenic sequences in the lysozyme chain. Our findings of the effective reduction of amyloid aggregation of lysozyme by polyphenol mixtures in vitro are of the utter physiological relevance considering the bioavailability and low toxicity of tested phenols. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

Optimal Conditions for [3H]Apomorphine Binding and Anomalous Equilibrium Binding of [3H]Apomorphine and [3H]Spiperone to Rat Striatal Membranes: Involvement of Surface Phenomena Versus Multiple Binding Sites

I. Binding of [³H]apomorphine to dopaminergic receptors in rat striatum was most reproducible and clearly detectable when incubations were run at 25°C in Tris-HCl buffer, pH 7.5, containing 1 mM-EDTA and 0.01% ascorbic acid, using a washed total-membrane fraction. The receptor binding was stereospecifically inhibited by (+)-butaclamol, and dopamine agonists and antagonists showed high binding affinity for these sites. Unlabelled apomorphine inhibited an additional nonstereospecific binding site, which was unrelated to dopamine receptors. EDTA in the incubation mixture considerably lowered nonstereospecific [³H]apomorphine binding, apparently by preventing the complexation of the catechol moiety with metal ions which were demonstrated in membrane preparations. Stereospecific [³H]apomorphine binding was not detectable in the frontal cortex, whereas in the absence of EDTA much saturable nonstereospecific binding occurred. II. Kinetic patterns of stereospecific [³H]spiperone and [³H] apomorphine binding to rat striatal membranes and the inhibition patterns of a dopamine antagonist and an agonist were evaluated at different temperatures in high-ionic-strength Tris buffer with salts added and low-ionic-strength Tris buffer with EDTA. Apparent K_D, values of spiperone decreased with decreasing tissue concentrations. K_D, values of both spiperone and apomorphine were little influenced by temperature changes. Scatchard plots of the stereospecific binding changed from linear to curved; the amount of nonstereospecific binding of the ³H ligands varied considerably, but in opposite directions for spiperone and apomorphine in the different buffers. In various assay conditions, interactions between agonists, and between antagonists, appeared fully competitive, but agonist-antagonist interactions were of mixed type. The anomalous binding patterns are interpreted in terms of surface phenomena occurring upon reactions of a ligand with complex physicochemical properties and nonsolubilized sites on membranes suspended in a buffered aqueous solution. It is concluded that anomalous binding patterns are not necessarily an indication of binding to multiple sites or involvement of distinct receptors for high-affinity agonist and antagonist binding.     

The effect of ethanol on erythrocyte carbonic anhydrase isoenzymes activity: An in vitro and in vivo study

The ethanol is a widely consumed as sedative-hypnotic drug throughout the world. In this study, the effects of ethanol were investigated on carbonic anhydrase (CA) enzyme activities both in vitro in human erythrocyte and in vivo in Sprague-Dawley rat erythrocyte. For in vitro study, the human carbonic anhydrase-I (HCA-I) and -II (HCA-II) are purified by Sepharose 4B–L-tyrosine-sulphanilamide affinity chromatography. In vivo CA enzyme activity was determined colorimetrically by using CO₂-hydration method of Wilbur and Anderson. Rat blood samples were taken from each rat before and after the ethanol administration at different times (1 h, 3 h, and 5 h). Rat erythrocyte CA activity was significantly inhibited by pharmacological dosage of the ethanol (2 mL.kg^{? 1}) for up to 3 h (p < 0.001) following intraperitoneally administration. The ethanol showed in vitro inhibitory effects on HCA-I and HCA-II hydratase activity, determined by colorimetrically using the CO₂-hydratase method. The inhibitor concentrations causing up to 50% inhibition (IC₅₀) were 2.09 M for HCA-I (r²:0.9273) and 1.83 M for HCA-II (r²:9749). In conclusion, it was demonstrated that carbonic anhydrase enzyme in erythrocytes was significantly inhibited by the ethanol both in in vitro and in vivo.     

Regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide

The regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide (NEM) was investigated using the agonist ligand, [³H] cis methyldioxolane ([³H] CD). Characterization studies on rat forebrain homogenates showed that [³H] CD binding was linear with tissue concentration and was unaffected by a change in pH from 5.5 to 8.0. The regional variation in [³H] CD binding in the rat brain correlated generally with [³H] (?)3-quinuclidinyl benzilate ([³H] (?)QNB) binding, although the absolute variation in binding was somewhat less. At a concentration of 100 μM, the GTP analogue, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], caused a 43–77% inhibition of [³H] CD binding in the corpus striatum, ileum, and heart. The results of binding studies using several Gpp(NH)p concentrations demonstrated that the potency of this guanine nucleotide for inhibition of [³H] CD binding was greater in the heart than in the ileum. In contrast to its effects on [³H] CD binding, Gpp(NH)p caused an increase in [³H] (?)QNB binding in the heart heart and ileum and no change in [³H] (?)QNB binding in the corpus striatum. When measured by competitive inhibition of [³H] (?)QNB binding to the longitudinal muscle of the ileum, Gpp(NH)p (100 μM) caused an increase in the IC₅₀ values of a series of agonists in a manner that was correlated with the efficacy of these compounds. The results of binding studies on NEM treated forebrain homogenates revealed an enhancement of [³H] CD binding by NEM.     

A comparison of calcium binding inCallinectes sapidus premolt and postmolt cuticle homogenates: Implications for regulation of biomineralization

Cuticle tissue homogenates (CTHs) fromCallinectes sapidus premolt cuticle bound approximately 367% more Ca²⁺ ions than did those from the postmolt cuticle. ThepH-stat assay which was used to comparein vitro CaCO₃ nucleation times confirmed that the premolt CTHs had greater inhibitory activity than did the postmolt CTHs. This inhibitory activity was indicated by CaCO₃ nucleation times in excess of control values. Premolt nucleation times exceeded those of postmolt samples by approximately 340%. A positive correlation was observed between Ca²⁺ binding and calcification inhibitory activity for both premolt and postmolt CTHs. Heat pretreatment of CTHs at 70°C for a 24-hr period had no significant effect on their Ca²⁺ binding. However, this heat pretreatment decreased their calcification inhibitory activity. Pretreatment of CTHs with Ca²⁺ diminished their calcification inhibitory activity. These results are consistent with a mechanism for inhibition of biocalcification by these proteins which involves their initial reversible binding to nascent calcite nuclei growth steps and kinks, rather than theirin vivo interaction with free Ca²⁺ ions in solution.     

Alcohol interactions with brain opiate receptors

Ethanol, added $\text{in vitro}$ to mouse caudate membranes, inhibited high-affinity binding of 0.2 nM ³H-dihydromorphine (³H-DHM) over an ethanol concentration range of 250–1,000 mM. At lower, physiologically-attainable ethanol concentrations (e.g.: 50 mM), ³H-DHM binding was significantly increased. Over the concentration range of 50–1,000 mM, ethanol inhibited 0.5 nM ³H-[D-Ala², D-Leu⁵] enkepha l in (³H-ENK) binding to mouse caudate tissue, and no stimulation of Ethanol inhibits opiate binding in a pseudo-competitive manner and, therefore, the concentration of ligand used to assess the effects of ethanol is of major importance. Results obtained with other alcohols which differ in their membrane: water partition coefficients suggest that alcohol effects on opiate binding are not solely dependent on the membrane-disordering properties of the alcohols.     

Muscarinic receptor binding in rat brain using the agonist, [3H]cis methyldioxolane

Muscarinic receptor binding was measured in rat forebrain preparations using the muscarinic agonist, [³H]cis methyldioxolane ([³H]CD). The results of equilibrium binding studies using [³H]CD concentrations between 0.5–64 nM showed that [³H]CD binding did not saturate in this concentration range, although the binding isotherm was concave downward. Nonlinear regression analysis of the binding data revealed the presence of two populations of muscarinic receptors having dissociation constants of 1.83 and 123 nM and binding capacities of 85 and 1320 fmol/mg protein, respectively. Competitive inhibition experiments showed that [³H]CD binding was readily displaced by several muscarinic agonists and antagonists. The stereospecificity of [³H]CD binding was demonstrated in competitive inhibition experiments using the stereoisomers of benzetimide and acetyl-β-methylcholine. Dexetimide was 10,000 times more potent than levetimide and l-acetyl-β-methylcholine was 520 times more potent than d-acetyl-β-methylcholine. A variety of nonmuscarinic cholinergic drugs were not effective at inhibiting [³H]CD binding at a concentration of 10 μM.     

Chronic ethanol alters the receptor binding characteristics of the delta opioid receptor ligand,DAla2DLeu5 enkephalin in mouse brain

The binding characteristics of the delta opioid receptor ligand, ³HDAla²DLeu⁵ enkephalin, were markedly altered in brains obtained from mice fed an ethanol-containing diet for five days. Control mice exhibited both a high and low affinity site for ³HDAla²DLeu⁵ enkephalin, whereas those consuming the ethanol diet were found to possess only one binding site. This singular site has an intermediate K_D value with an increase in receptor number when compared to the high and low affinity sites observed in control mice. The $\text{in}$$\text{vitro}$ addition of ethanol to a brain membrane preparation obtained from untreated mice, at a concentration equivalent to that found in the blood of the ethanol-treated mice, did not markedly affect DAla²DLeu⁵ enkephalin binding characteristics. No alteration in the binding characteristics of ³H-naloxone, a mu receptor ligand, was noted following five days of ethanol consumption. Mice maintained on the ethanol-containing diet were tolerant to the activity-stimulating effects of acute ethanol administration. These results suggest that mice consuming an ethanol diet in sufficient quantities to render them tolerant exhibit a specific loss of a ³HDAla²DLeu⁵ enkephalin binding site, while the binding of ³H-naloxone was unchanged.     

Characterization of Insulin-Like Growth Factor Binding to Human Granulosa Cells Obtained During in Vitro Fertilizationd

Abstract

Insulin and IGF-I affect in vitro ovarian stromal and follicular cell function in several species. We previously characterized insulin receptors on human granulosa cells obtained from in vitro fertilization procedures but were unable to demonstrate specific binding of IGF-I.

Following modification of the assay conditions, we now report specific, high affinity IGF-1 binding sites on human granulosa cells. Substitution of equimolar concentrations of sucrose for sodium chloride in the buffer solution increased binding of IGF but not insulin in equilibrium assays. Maximal specific IGF-I binding was 2.69 ± 0.30%/10⁵ cells (SEM, n=9) with half-maximal inhibition of binding at 2 ng/ml IGF-I. Unlabeled insulin recognized the type I IGF receptor with low affinity. An IGF-I receptor monoclonal antibody (αIR-3) inhibited ¹²⁵I-IGF-I but not ¹²⁵I-insulin binding. Affinity crosslinking followed by SDS/PAGE under reducing conditions revealed IGF-I binding at a molecular weight compatible with the αsubunit of the type I IGF receptor and with a pattern of inhibition by various ligands that paralleled the equilibrium binding assays.

IGF-I receptors are present on freshly isolated human ovarian granulosa cells obtained following pharmacologic stimulation with gonadotrophin according to the protocols of in vitro fertilization. The biologic function of these receptors currently is being investigated.     

Inhibition of 3H-clozapine binding in rat brain after oral administration of neuroleptics

Clozapine, thioridazine, perlapine, clothiapine, chlorpromazine, NT 104–252, loxapine or haloperidol were administered orally, and atropine subcutaneously, to rats. The animals were decapitated, brain tissue was removed and homogenized in tris buffer and incubated with ³H-clozapine. Total and nonspecifically bound ³H-clozapine were measured in each preparation. A dose-dependent inhibition of specific ³H-clozapine binding of more than 50% was observed only after the administration of clozapine or thioridazine. There was no correlation between inhibition of ³H-clozapine binding and that of ³H-haloperidol, a specific ligand for DA-receptors. ³H-Clozapine receptor density was much greater than ³H-haloperidol receptor density, suggesting that the majority of clozapine binding sites are not DA-receptors.     

36种中药材体外抗菌活性筛选研究

该文研究36种常用中药材80%乙醇提取物在体外抗临床常见致病菌的抗菌活性。采用药敏纸片法测耐药菌的耐药谱,中药粗粉用80%乙醇浸泡提取,提取液减压浓缩得浸膏,通过琼脂打孔法测定提取物抑菌圈,再通过微量倍比稀释法测定最低抑菌浓度(MIC)和最低杀菌浓度(MBC)。结果表明:36种中药材醇提物中,有15种具有广谱抗菌活性,对实验中各标准菌表现出不同程度的抑制作用,对MRSA抗菌活性也较强。其中,岩陀、卷柏、首乌藤、苏木、乌药、夏枯草6种药材的抗菌活性比较突出,抑菌圈均大于11 mm,细菌对其表现为中高度敏感;它们对7株标准菌的MIC/MBC值除个别为12.5 mg·m L~(-1)以外,均小于1.563 mg·m L~(-1),对16株MRSA的MIC/MBC值均小于1.563 mg·m L~(-1),它们的萃取层活性均小于1 mg·m L~(-1)。所筛选出的15种抗菌活性较强的中药材,可为后续研究其活性单体化合物和作用机制,研发有效的抗多重耐药菌的中药制剂以及解决细菌耐药性问题提供一定的参考。     

Metal complexes of naphthoquinone based ligand: synthesis,characterization, protein binding,DNA binding/cleavage and cytotoxicity studies

Protein binding, DNA binding/cleavage and in vitro cytotoxicity studies of 2-((3-(dimethylamino)propyl)amino)naphthalene-1,4-dione (L) and its four coordinated M(II) complexes [M(II) = Co(II), Cu(II), Ni(II) and Zn(II)] have been investigated using various spectral techniques. The structure of the ligand was confirmed by spectral and single crystal XRD studies. The geometry of the complexes has been established using analytical and spectral investigations. These complexes show good binding tendency to bovine serum albumin (BSA) exhibiting high binding constant values (10⁵ M^?1) when compared to free ligand. Fluorescence titration studies reveal that these compounds bind strongly with CT-DNA through intercalative mode (K_app 10⁵ M^?1) and follow the order: Cu(II) > Zn(II) > Ni(II) > Co(II) > L. Molecular docking study substantiate the strength and mode of binding of these compounds with DNA. All the complexes efficiently cleaved pUC18-DNA via hydroxyl radical mechanism and the Cu(II) complex degraded the DNA completely by converting supercoiled form to linear form. The complexes demonstrate a comparable in vitro cytotoxic activity against two human cancer cell lines (MCF-7 and A-549), which is comparable with that of cisplatin. AO/EB and DAPI staining studies suggest apoptotic mode of cell death, in these cancer cells, with the compounds under investigation.     

STEREOSPECIFIC IN VlTRO BINDING OF [3H]DEXETIMIDE TO BRAIN MUSCARINIC RECEPTORS

A binding assay for muscarinic cholinergic receptors has been developed using labelled dexetimide as ligand and a filtration technique. The main features of this assay are its stereospecific nature, the very high affinity of the ligand for the specific receptors sitcs and its very low affinity for non-specific binding sites. The latter point was further investigated using labelled levitimide, the inactive enantiomer. The binding was found to be neither stereospecific nor saturable and displacement by both enantiomers revealed a particular curve with a very flattened course. Kinetic experiments with [³H]dexetimide suggest the occurrence of a heterogenous population of muscarinic receptors in the rat striatum. A study of the regional distribution of muscarinic receptors in rat brain showed a high concentration in the dopaminergic areas, the cortex and the hippocampus, but practically none in the cerebellum. The subcellular distribution pattern revealed a marked enrichment of [³H]dexetimide stereospecific binding sites in the microsomal fraction of rat striatum and hippocampus. Such a distribution was not found with [³H]levitimide. All the characteristics of this binding assay make dexetimide a very appropriate ligand for labelling muscarinic receptors in vitro as well as in vivo.     

Differential effect of ethanol on muscarinic cholinergic binding to rat and locust neural membranes

We have compared the effect of ethanol, a membrane perturbant, on the muscarinic binding sites in neural membranes from a vertebrate (rat) and an insect (locust). The binding of the muscarinic antagonist [³H]quinuclidinyl benzilate ([³H]QNB) to both rat and locust neural membranes was inhibited by ethanol at 10–500 mM concentrations; but this inhibition was greater in the locust. Ethanol (500 mM) increased the apparent dissociation constant (K d) of [³H]QNB binding to rat membranes from 0.13±0.01 nM in control to 0.20±0.02 nM; there was also an small but significant reduction in the number of binding sitesB _max. In locust, 500 mM ethanol reduced theB _max of [³H]QNB binding from 590±30 in control to 320±40 pmol/g protein; no significant alteration in theK _D was detected. The dissociation rate constant (k _off) of [³H]QNB increased from 0.020±0.003 in controls to 0.031±0.004 (min^–1) in the presence of 500mM ethanol, the association rate constant (k _on) did not change significantly. In locust, 500 mM ethanol did not affect eitherk _on ork _off. Competition experiments revealed that the binding affinities of both the agonist carbamylcholine and the antagonist atropine to the rat membranes were reduced in the presence of ethanol. In contrast, ethanol caused no alteration in the binding affinities of these ligands to the locust membranes. This differential effect of ethanol on rat and locust muscarinic binding suggests a difference in the hydrophobic domains and/or the membrane interactions of the muscarinic receptors in the two species.     

Mechanism for Cd2+ inhibition of (K++ Mg2+)ATPase activity and K+ (86Rb+) uptake join roots of sugar beet (Beta vulgaris)

Steady state kinetics were used to examine the influence of Cd²⁺ both on K⁺ stimulation of a membrane-bound ATPase from sugar beet roots (Beta vulgaris L. cv. Monohill) and on K⁺(⁸⁶Rb⁺) uptake in intact or excised beet roots. The in vitro effect of Cd²⁺ was studied both on a 12000–25000 g root fraction of the (Na⁺+K⁺+Mg²⁺)ATPase and on the ATPase when further purified by an aqueous polymer two-phase system. The observed data can be summarized as follows: 1) Cd²⁺ at high concentrations (>100 μM) inhibits the MgATPase activity in a competitive way, probably by forming a complex with ATP. 2) Cd²⁺ at concentrations <100 μM inhibits the specific K⁺ activation at both high and low affinity sites for K⁺. The inhibition pattern appears to be the same in the two ATPase preparations of different purity. In the presence of the substrate MgATP, and at K⁺ <5 mM, the inhibition by Cd²⁺ with respect to K⁺ is uncompetitive. In the presence of MgATP and K⁺ >10 μM, the inhibition by Cd²⁺ is competitive. 3) At the low concentrations of K⁺, Cd²⁺ also inhibits the 2,4-dinitrophenol(DNP)-sensitive (metabolic) K⁺(⁸⁶Rb⁺) uptake uncompetitively both in excised roots and in roots of intact plants. 4) The DNP-insensitive (non metabolic) K⁺(⁸⁶Rb⁺) uptake is little influenced by Cd²⁺. As Cd²⁺ inhibits the metabolic uptake of K⁺(⁸⁶Rb⁺) and the K⁺ activation of the ATPase in the same way at low concentrations of K⁺, the same binding site is probably involved. Therefore, under field conditions, when the concentration of K⁺ is low, the presence of Cd²⁺ could be disadvantageous.     

Anoxia tolerance and ethanol sensitivity of rice and oat coleoptiles

Anoxia tolerance and ethanol sensitivity of rice (Oryza sativa L.) and oat (Avena sativa L.) seedlings were evaluated to clarify their growth habit in anoxia. Anoxic stress inhibited elongation and dry weight gain of coleoptiles of the oat and rice seedlings; however, the inhibition of the oat coleoptiles was much greater than that of the rice coleoptiles. Anoxic stress increased endogenous ethanol concentration and alcohol dehydrogenase activity in oat and rice coleoptiles and their increases in the rice coleoptiles were much greater than those in the oat coleoptiles. At concentrations greater than 30 mM and 300 mM, exogenously applied ethanol inhibited the elongation and weight gain for the oat and the rice coleoptiles, respectively, and the inhibition was increased with increasing ethanol concentrations with marked inhibition being achieved on the oat coleoptiles. These results suggest that anoxia tolerance and induction of ethanolic fermentation in anoxia may be greater in rice than oat, and ethanol sensitivity of rice may be lower than that of oat.