Changes in Inhibitory Effect of Vanadate on the Plasma Membrane H+-ATPase Under Trypsin Treatment

The changes in inhibitory effect of vanadate on the plasma membrane H + ATPase were studied with mild trypsin treatment using plasma membrane vesicles purified from soybean （Glycine max L.）hypocotyles by sucrose gradient centrifugation. Results showed that under mild trypsin treatment the ATPase ATP hydrolysis activity was increased significantly. It was also found that the inhibitory effect of vandate was reduced after proteolysis. In the presence of 2 mmol/L vanadate, the ATP hydrolysis activity of the cleaved ATPase was inhibited by only 53.49%,while that of the un-cleaved ATPase was inhibited by 64.13%. Kinetic studies indicated that both the Km values and the inhibition type of vanadate were not affected by trypsin treatment. Upon proteolysis, Km remained as 0.34 mmol/L,while vanadate was still an uncompetitive inhibitor. Taking together, the structure and activity of the ATPase phosphatase domain were affected by trypsin treatment, implying that this domain might be regulated by the C-terminal end of the plasma membrane H+ ATPase.     

Changes in Photosystem Ⅱ Activity and Leaf Reflectance Features of Several Subtropical Woody Plants Under Simulated SO2 Treatment

The effects of simulated SO2 treatment on the photosynthetic apparatus were investigated in five subtropical forest plants, namely Plnus massonlana Lamb., Schlma superba Gardn. et Champ., Castanopsls flssa （Champ. ex Benth.） Rehd. et Wils., Acmena acuminatissima （BI.） Merr et Perry, and Cryptocarya concinna Hance. After leaf sections had been immersed in 0, 20, 50, and 100 mmol/L NaHSO3 for 20 h, total chlorophyll （Chl） content, Chl a/b, maximal photochemical efficiency, and the photochemical quantum yields of photosystem Ⅱ of all five woody plants were reduced to different degrees, whereas lutein content （Chl base） was increased. Two protective mechanisms, namely the xanthophyll cycle （de-epoxidation） and an anti-oxidant system （1,1-diphenyl-2-picrylhydrazyl radical-scavenging capacity）, showed differences in the degree of modulation under simulated SO2 treatment. Compared with control （distilled water treatment）, the revised normalized difference vegetation index, a leaf reflectance index, was lowered with Increasing concentrations of NaHSO3. Cryptocarya conclnna, a dominant species in the late succession stage of subtropical forests in South China, exhibited less sensitivity to NaHSO3. Conversely, Pinus massonlana, the pioneer hellophyte species, was most susceptible to NaHSO3 treatment. It Is suggested that SO2 pollution may accelerate the succession of subtropical forest.     

Comparison of Plasma Membrane H^＋-ATPase Activity in Two Ecotypes of Reed （Phragmites communis） Leaves from Different Habitats

Plasma membrane (PM) vesicles of the leaves of two ecotypes of reed (Phragrnites communis Trin.), swamp reed (SR) and heavy salt meadow reed (HSMR) growing in the desert region of Northwest China, were purified by two-phase partitioning and the properties of their PM H^ -ATPases (EC 3.6.1.35) were compared. The specific activity of this enzyme was greater in HSMR than in SR and the Km lower (1.27mmol/L in SR and 0.30mmol/L in HSMR), and the Vmax of ATP hydrolysis activity showed no significant difference between the two ecotypes. The PM H^ -ATPase was more sensitive to denaturing temperatures in HSMR than in SR, and the pH profile also showed a slight difference, suggesting that the catalytic mechanism of this enzyme was different in HSMR compared with that in SR. The p-nitrophenyl phosphate (PNPP) hydrolysis activity of H^ -ATPase was higher in HSMR than in SR at low concentrations of PNPP, but showed no difference at high PNPP concentration. The Km for PNPP hydrolysis was 3.61mmol/L and 1.92mmol/L in SR and HSMR, respectively. And the Vmax of PNPP hydrolysis showed no significant difference in the two reed ecotypes. An experiment with the inhibitor vanadate showed that the catalytic mechanisms of the phosphatase domain of the ATPase were different in the two ecotypes. The data obtained following trypsin treatment showed a difference in the enzyme activity pattern, suggesting that there existed a possible change in the C-terminus of the ATPase, either in the structure or in the property or both of them. In addition, compared with SR, the ATP-dependent H^ pumping activity of ATPase and the coupling between proton transport and ATP hydrolysis in HSMR were increased. These results indicated that the properties of PM H^ -ATPase were changed in HSMR with compared to those in SR, which might include enzyme modifications and different isoforms expressed. The alterations of the properties of this enzyme might be an adaptive response to the habitat.     

Estimation of Membrane Potential Δψ in Reconstituted Plasma Membrane Vesicles Using a Numerical Model of Oxonol VI Distribution

A model of membrane potential-dependent distribution of oxonol VI to estimate the electrical potential difference across Schizosaccharomyces pombe plasma membrane vesicles (PMV) has been developed. was generated by the H⁺-ATPase reconstituted in the PMV. The model treatment was necessary since the usual calibration of the dye fluorescence changes by diffusion potentials (K⁺ + valinomycin) failed. The model allows for fitting of fluorescence changes at different vesicle and dye concentrations, yielding in ATP-energized PMV of 80 mV. The described model treatment to estimate may be applicable for other reconstituted membrane systems.     

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase (III. Is There a Direct Interaction between the Fusicoccin Receptor and the Plasma Membrane H+-ATPase?)

A radioimmunoassay using antibodies raised against bovine serum albumin-conjugated fusicoccin (FC) was applied to measure FC bound to the plasma membrane (PM) isolated from seedlings of radish (Raphanus sativus L.) and of Arabidopsis thaliana treated in vivo plus or minus the toxin. FC bound to the PM from seedlings treated with 5 [mu]M FC was 2-fold (radish) to 7-fold (A. thaliana) higher than the binding capacity of control PM. FC binding depended on the duration of the in vivo treatment but was unaffected by cycloheximide. When FC binding and the PM H+-ATPase activity were compared under different conditions (in vivo or in vitro treatment of different lengths or with different concentrations of FC), a strict linear relation between FC binding and the activation of the PM H+-ATPase was observed in both plant materials under all the conditions tested. Comparison between the maximum binding capacity and the amount of H+-ATPase observed in PM from the two plant materials suggest a one-to-one stoichiometry between the FC receptor and the PM H+-ATPase.     

Activity of β-glucuronidase in Root Tips of Different Types of Transgenic Sugar Beet Plants

Expression of the -glucuronidase (GUS) reporter gene driven by the CaMV 35S, rolC, nos and mas promoters was assessed in the tips of 12 independent clones of transgenic sugar beet (Beta vulgaris) roots. Three questions were addressed: 1) expression pattern specific for a given promoter, 2) expression pattern variability, and 3) relationship between gene expression and cell differentiation. Characteristic patterns of tissue-specific expression were distinguished for each promoter. Striking differences, however, were found between some clones, bearing the same construct. Statistical analysis of the pattern variability proved that the variability is significantly lower within the construct than between constructs. rolC-GUS clones exhibited the lowest and CaMV 35S clones the highest pattern variability. Comparisons between the four promoters showed consistent GUS activity in areas playing a key role in tissue determination (the elongation zone) where cells switch from frequent mitosis and mostly isodiametrical growth, typical for the promeristem, to rapid elongation and differentiation. All of the clones were highly GUS-positive in the elongation zone of stele. Activity was commonly localised in the stele of the maturation zone for CaMV 35S, rolC and mas-GUS clones. CaMV 35S-GUS clones were highly active in the promeristem.     

Aporphine Alkaloids from Ocotea puberula with Anti-Trypanosoma Cruzi Potential – Activity of Dicentrine-β-N-Oxide in the Plasma Membrane Electric Potentials

One new aporphine, dicentrine-β-N-oxide ( 1 ), together with five related known alkaloids dehydrodicentrine ( 2 ), predicentrine ( 3 ), N-methyllaurotetanine ( 4 ), cassythicine ( 5 ), and dicentrine ( 6 ) were isolated from the leaves of Ocotea puberula (Lauraceae). Antiprotozoal activity of the isolated compounds was evaluated in vitro against trypomastigote forms of Trypanosoma cruzi. Among the tested compounds, alkaloid 1 exhibited higher potential with EC₅₀ value of 18.2 μM and reduced toxicity against NCTC cells (CC₅₀>200 μM – SI>11.0), similar to positive control benznidazole (EC₅₀ of 17.7 μM and SI=10.7). Considering the promising results of dicentrine-β-N-oxide ( 1 ) against trypomastigotes, the mechanism of parasite death caused by this alkaloid was investigated. As observed, this compound reached the plasma membrane electric potential directly after 2 h of incubation and triggered mitochondrial depolarization, which probably leads to trypomastigote death. Therefore, dicentrine-β-N-oxide ( 1 ), reported for the first time in this work, can contribute to future works for the development of new trypanocidal agents.     

Combined Effect of Diazepam and GABAA-ergic Ligands on the Activity of Cl–-ATPase in Plasma Membrane of Bream Brain (Abramis brama L.)

We studied the combined effect of diazepam and GABA_A-ergic ligands on the activity of Cl^–-ATPase in plasma membrane of bream brain. The membrane fraction were preincubated and incubated with diazepam as well as with other GABA_A-ergic ligands at physiological pH (7.4), i.e. under the conditions when Cl^–-ATPase activity is undetectable. GABA (0.1–100 M) induced Cl^–-ATPase activity with the maximum effect at 10 M. Diazepam (0.1 M) enhanced the effect of low GABA concentrations (0.1–1 M) on Cl^–-ATPase activity but had no effect on the enzyme in the presence of high GABA concentrations (10–100 M). At the same time, GABA (1 M) enhanced the effect of low diazepam concentrations (0.1–1 M) on the enzyme activity but had no effect on it in the presence of high concentrations of the ligand. Blockers of GABA_A-ergic receptors, picrotoxin (50 M) and bicuculline (5 M), canceled the combined effect of diazepam and GABA on the enzyme activity. The obtained data demonstrate that the combined effect of diazepam and GABA_A-ergic ligands on Cl^–-ATPase activity at physiological pH is similar to the effect of these ligands on GABA_A/benzodiazepine/Cl^– channel.     

Influence of Macroelements’ Uneven Distribution on the Content of Hormones and Extension of the Roots in Wheat Plants

Russian Journal of Plant Physiology - Increase in the level of mineral nutrition reduces the relative growth rate of the roots, which adversely affects the drought resistance of plants. This was...     

Differential Response to the Herbicidal Activity of λ-Aminolevulinic Acid in Plants with High and Low Sod Activity

6-aminolevulinic acid (ALA) is the obligatory precursor for tetrapyrroles and for chlorophylls in plants. 1 Under illumination, these photosensitizers generate singlet oxygen, thus causing bleaching and death of treated tissues. We have examined whether superoxide is involved in the mode of action of ALA and whether SOD provides protection. Bean genotypes with similar carotenoid content but differing in SOD activity and cucumber seedlings were used throughout. Cucumber plants treated with 10 mM ethanolamine (EA) prior to ALA, had higher levels of chlorophyll fluorescence and lower values of electrolyte leakage than control. Bean cultivars with high SOD activity were considerably more tolerant to membrane damage caused by ALA than those with low SOD activity. SOD activity was greatly reduced in cucumber leaves treated with diethyldithiocarbamate (DDTC). Electrolyte leakage was markedly increased and chlorophyll fluorescence values were significantly lower in DDTC and ALA treated tissues as compared with those treated with ALA only. The results indicate that superoxide is involved in the toxicity caused by ALA and that, by breeding for high SOD activity. resistance to ALA can be achieved. thus allowing the use of ALA as a selective herbicide in the field.     

Kinetics of Δψ-Dependent Sucrose Transport in Plasma Membrane Vesicles from Higher Plant Cells

The inside-out vesicles of plasma membranes were isolated from pumpkin stem cells, and the kinetics of sucrose efflux induced by the K⁺-diffusion potential (_D) was studied by measuring light transmission. Two phases differing in their rates and duration were identified in _D-dependent changes of light transmission. The increase in _Delevated the rate and magnitude of the fast phase in light transmission changes but did not markedly affect the rate of the slow phase. These two phases also differed in their sensitivity to inhibitors and to changes in sucrose concentration in the external medium. Measurements of _Dduring sucrose transport by means of the fluorescence probe dis-C₃-(5) revealed differences in the magnitude of _Dand its stability in vesicles loaded with sucrose and mannitol, as well as under the action of inhibitors. The two-phase dependence of sucrose efflux from vesicles on the applied diffusion potential is discussed in the context of modern concepts on the functioning of sucrose carriers in the membranes.     

Transport of Cl– across the Plasma Membrane during Pollen Grain Germination in Tobacco

The role of Cl- transport across the plasma membrane was studied in an early step of pollen grain germination in tobacco Nicotiana tabacum L. The Cl- channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid, completely suppress the germination with IC(50) approximately 8 micro M. At this concentration NPPB reduces the rate of Cl- efflux out of pollen grain by 1.8-fold in the interval 5-12 min, and niflumic acid reduces the rate 1.2-fold. 4,4;-Diisothiocyanatostilbene-2,2;-disulfonic acid, a known inhibitor of Cl- channels and antiporters, completely suppresses germination as well (IC(50) = 240 micro M), but has no effect on the rate of Cl- efflux. Inhibitors of chloride co-transporters, such as furosemide, bumetanide, and bis(1,3-dibutylbarbituric acid)pentamethine oxonol, suppress the germination by less than 50%. This set of data suggests that NPPB-sensitive anion channels are involved in the activation of pollen grains in the early stage of germination.     

High-cholesterol Diets Induce Changes in Lipid Composition of Rat Erythrocyte Membrane Including Decrease in Cholesterol,Increase in α-Tocopherol and Changes in Fatty Acids of Phospholipids

Effects of high dietary cholesterol on erythrocyte membrane lipids were studied. Feeding rats with a diet containing 0.5% cholesterol and 0.15% sodium cholate for two weeks induced changes in erythrocyte membrane lipids including a decrease in cholesterol, an increase in α-tocopherol (α-Toc) and changes in the fatty acid composition of phospholipids. Oleic acid and linoleic acid increased, while arachidonic acid decreased in phosphatidylcholine. Saturated fatty acids decreased and unsaturated fatty acids increased in phosphatidylethanolamine. Almost the same changes in membrane lipids were also noted after six weeks of feeding rats with the diet. A diet containing 0.5% cholesterol but without sodium cholate caused a decrease in erythrocyte cholesterol and an increase in erythrocyte α-Toc after two weeks of feeding, as compared to the basal diet, indicating that high dietary cholesterol, but not sodium cholate, was responsible for these changes in the erythrocyte membrane.     

How Does the Plant Plasma Membrane H+-ATPase Pump Protons?

The plasma membrane H⁺-ATPase couples ATP hydrolysis to protontransport thereby establishing the driving force for solutetransport into and out of plant cells. As such, this enzymeparticipates in a number of cellular processes important tothe overall physiology of plants. From biochemical studies andthe recent application of molecular approaches, the enzyme reactionmechanism and structure of this protein have been characterized.However, our basic understanding of how this enzyme links theendergonic reaction of ATP hydrolysis to proton translocationis far from complete. In this review, several significant questionsregarding the energy coupling mechanism will be addressed interms of information available on the plant plasma membraneH⁺-ATPase and from studies on other P-type transport ATPases.These questions focus on the chemical nature of proton translocation,how this is linked or driven by the ATP hydrolysis reactionand what role, if any, K⁺ has in the transport process. Key words: Energy coupling, membranes, bioenergetics, ion transport, P-type transport ATPase     

Changes of α-Glycerophosphate Dehydrogenase Activity in Fatty Liver of Rats by Amino Acid Imbalance

An aminopeptidase was purified from an aqueous extract of mullet roe in the presence of 2-mercaptoethanol by fractionation with ammonium sulfate and column chromatography on DEAE-cellulose and Sephadex G-200. The molecular weight of the enzyme was 184,000 by gel filtration, and the enzyme appeared to consist of two homogenous subunits. The optimal pH and optimal temperature for activity were 7.4 and 45°C, respectively. Puromycin, p-chloromercuribenzoic acid, and o-phenanthroline inhibited the enzyme n on-competitively (their Ki = 1.34 μm, 0.113mm and 0.145 mm, respectively), while 2-mercaptoethylamine was competitive (Ki = 0.056 mm). The enzyme was also inhibited by l-amino acids, in particular glutamic acid. The enzyme could hydrolyze a variety of α-aminoacyl β-naphthylamides and was most active on l-alanyl-β-naphthylamide. Judging from these properties, the mullet roe aminopeptidase resembles soluble alanyl amino-peptidase [EC 3.4.11.14].     

Changes in Unsaturated Levels of Fatty Acids in Thylakoid PSⅡ Membrane Lipids During Chilling-induced Resistance in Rice

Temperature is one of the abiotic factors limiting growth and productivity of plants. In the present work, the effect of low non-freezing temperature, as an inducer of "chilling resistance", was studied in three cultivars of rice (Oryza sativa L.), japonica cv. 9516 (j-9516), the two parental lines of superhigh-yield hybrid rice between subspecies,Peiai/E32 (ji-PE), and the traditional indica hybrid rice Shanyou 63 (i-SY63). Leaves of chill-treated rice showed chilling-induced resistance, as an increase of their low-temperature tolerance was measured using chlorophyll fluorescence measurements, revealing a change in photosystem Ⅱ (PSⅡ) efficiency. After 5 d of exposure to 11℃ under low light (100 μ mol·m-2·s-1), levels of unsaturated fatty acids in PSⅡ thylakoid membrane lipids decreased during the initial 1-2 d, then increased slowly and reached 99.2％, 95.3％ and 90.1％ of the initial value (0 d) in j-9516,ji-PE and i-SY63, respectively, on the fifth day. However, under medium light (600 μmol·m-2·s-1), all cultivars experienced similar substantial photoinhibition, which approached steady state levels after a decline in levels of unsaturated fatty acids in PSII thylakoid membrane lipids to about 57.1％, 53.8％ and 44.5％ of the initial values (0 d) in j-9516,ji-PE and i-SY63 on the fifth day. Under either chilling-induced resistance (the former) or low temperature photoinhibition (the latter) conditions, the changes of other physiological parameters such as D1 protein contents,electron transport activities of PSII (ETA), Fv/Fm, xanthophyl cycle activities expressed by DES (deepoxide state)were consistent with that of levels of unsaturated fatty acids in PSⅡ thylakoid membrane lipids. So there were negative correlations between saturated levels of fatty acids (16:1(3t), 16:0, 18:0), especially the 16:1(3t) fatty acid on thylakoid membrane and other physiological parameters, such as D1 protein contents, ETA and (A+Z)/(A+V+Z). A specific role of desaturation of fatty acids and the photoprotective pigments of the xanthophyl cycle, leading to an acclimation response in thylakoid membrane lipids may be involved. We conclude that chilling-induced resistance is accelerated by the unsaturation of thylakoid membranes, and the ability of rice plants to cold-harden can be enhanced by genetic engineering.