Plasma Membrane ATPase Activity Research Articles

Phosphorus protects against cadmium-induced phytotoxicity by stimulating plasma membrane H+-ATPase activity

Proper plant nutrition can be an effective strategy to alleviate the phytotoxicity and damaging effects of Cd stress on plants and to avoid its entry into the food chain. This study aimed to investigate effects of phosphorus concentrations, and pH levels in the nutrient solution on Cd uptake, growth, photosynthetic characters, total phytochelatins (PCs), and plasma membrane ATPase activity of sugar beet (Beta vulgaris L.) plants grown in sand culture. Two successive experimental studies were carried out under controlled conditions. In the first study, 10-day-old sugar beet seedlings were irrigated with a half-strength Hoagland and Arnon nutrient solution, adjusted to different pH (3.5, 5.0, 6.0, 7.0, or 8.0), and containing 0, 0.15, or 0.30 mg Cd L−1 as CdCl2. In the second study, 10-day-old sugar beet seedlings were irrigated with the same nutrient solution (pH 6), but combined with different phosphorus concentrations (0, 10, 25 and 50 µg P ml−1 as KH2PO4) and Cd levels (0, 0.15, and 0.30 mg Cd L−1 as CdCl2). The first study revealed that pH had a strong influence on Cd uptake by sugar beet roots. Cd stress significantly decreased sugar beet growth and Mg2+-ATPase activity, whereas it increased total phytochelatins (PCs). Second study indicated an antagonistic effect between P and Cd. Furthermore, phosphorus had the potential to stimulate Mg2+-ATPase activity and synthesis of photosynthetic pigments and to promote growth of sugar beet seedlings. Cadmium induced phytotoxicity in sugar beet seedlings can be alleviated by a proper pH and phosphorus nutrition.

Effect of pressure-assisted thermal sterilization combining with ε-polylysine on Bacillus subtilis spore proteins, nucleic acids and other intraspore substances

Pressure-assisted thermal sterilization (PATS) is a new technology to inactivate bacterial spores and ensure food safety. Little has been known about the effects of PATS combining with ε-PL on the spore’s nucleic acid, enzymes and other key substances. This study aimed to investigate the inactivation effect of PATS combining with ε-PL on the spores of B. subtilis. The spores were treated with pressure 600 MPa at 25 °C, 65 °C and 75 °C, and ε-PL at 0.1% and 0.3%. After treatment, the survival rate of B. subtilis spores, leakage of nucleic acid and protein, the change in the cell membrane ATPase activity, the leakage of dipicolinic acid, and the damage on protein and nucleic acid of the spores were determined. The results showed that PATS combining with ε-PL inactivated more spores, and significantly increased the release of protein and nucleic acid compared to the control. ATPase activity reached the lowest value after the treatment of 600 MPa/75 °C combining with 0.3% ε-PL. The release of dipicolinic acid from the spores was increased by 600 MPa/75 °C combining with 0.3% ε-PL as compared with 600 MPa/75 °C treatment alone. FTIR analysis showed that a combination of PATS with ε-PL changed the spectral features of B. subtilis functional groups of proteins and nucleic acids. The PATS treatments when combined with ε-PL were found to shift the symmetric and antisymmetric stretching vibrational absorption peaks of phosphodiester group in nucleic acid molecules (P=O). This change suggested that the combined treatment denatured nucleic acid. The combined treatment also changed the protein from an ordered state to a disordered state, and decreased protein stability. The results improved our understanding on the principle of spore inactivation by PATS combining with ε-PL.

Conjugated polyamines are involved in conformation stability of plasma membrane from maturing maize grain embryos under drought stress

Polyamines are closely associated with drought stress in plants. However, the function which polyamines play in the maturing maize grain under drought is not still clear. Especially, whether the conjugated polyamines are involved in H + -ATPase activity and conformation of plasma membrane remains to be answered. Therefore, in the study, the contents of conjugated polyamines, H + -ATPase activity and conformation of plasma membrane were detected with maturing grain embryos of two maize ( Zea mays L.) cultivars, which were different in drought stress-tolerance. Under drought stress, the cell plasmolysis of the drought-sensitive Xundan No. 22 cv. was more marked than that of the drought-tolerant Yedan No. 13 cv., while the increases in the contents of conjugated non-covalently spermidine and spermine, conjugated covalently putrescine and spermidine in plasma membrane from Yedan 13 were more obvious than those in Xundan 22. The results suggested that these conjugated polyamines might be involved in stability of plasma membrane. Furthermore, H + -ATPase activity in plasma membrane from Yedan 13 increased more significantly than that in Xundan 22, indicating that these conjugated polyamines were closely correlated with the activity of H + -ATPase. To justify further the inferences, the additional experiments with exogenous spermidine, spermine and polyamine biosynthesis inhibitors were carried out in the study. The results showed that the application of exogenous spermidine or spermine increased the levels of conjugated polyamines in plasma membrane, coupled with alleviating cell plasmolysis and increasing H + -ATPase activity. The application of inhibitors decreased the levels of conjugated polyamines, coupled with aggravating cell plasmolysis and decreasing H + -ATPase activity. From these results together, it could be concluded that non-covalently conjugated spermidine and spermine, conjugated covalently putrescine and spermidine in plasma membrane functioned in increasing H + -ATPase activity, mitigating cell plasmolysis and maintaining the plasma membrane conformation of the maturing maize grain embryos under drought stress. • Drought stress increased the levels of conjugated PAs in plasma membrane. • Conjugated PAs in plasma membrane mitigated cell plasmolysis induced by drought. • Conjugated PAs enhanced the H + -ATPase activity in plasma membrane under drought. • Conjugated PAs in plasma membrane were closely associated with drought tolerance.

ATPase Activity of the Subcellular Fractions of Colorectal Cancer Samples under the Action of Nicotinic Acid Adenine Dinucleotide Phosphate.

In tumor cells with defects in apoptosis, autophagy allows prolonged survival. Autophagy leads to an accumulation of damaged mitochondria by autophagosomes. An acidic environment is maintained in compartments of cells, such as autophagosomes, late endosomes, and lysosomes; these organelles belong to the “acid store” of the cells. Nicotinic acid adenine dinucleotide phosphate (NAADP) may affect the release of Ca2+ from these organelles and affect the activity of Ca2+ ATPases and other ion transport proteins. Recently, a growing amount of evidence has shown that the variations in the expression of calcium channels or pumps are associated with the occurrence, disease-presentation, and the prognosis of colorectal cancer. We hypothesized that activity of ATPases in cancer tissue is higher because of intensive energy metabolism of tumor cells. The aim of our study was to ascertain the effect of NAADP on ATPase activity on tissue samples of colorectal cancer patients’ and healthy individuals. We tested the effect of NAADP on the activity of Na+/K+ ATPase; Ca2+ ATPase of endoplasmic reticulum (EPR) and plasma membrane (PM) and basal ATPase activity. Patients’ colon mucus cancer samples were obtained during endoscopy from cancer and healthy areas (control) of colorectal mucosa of the same patients. Results. The mean activity of Na+/K+ pump in samples of colorectal cancer patients (n = 5) was 4.66 ± 1.20 μmol Pi/mg of protein per hour, while in control samples from healthy tissues of the same patient (n = 5) this value was 3.88 ± 2.03 μmol Pi/mg of protein per hour. The activity of Ca2+ ATPase PM in control samples was 6.42 ± 0.63 μmol Pi/mg of protein per hour and in cancer −8.50 ± 1.40 μmol Pi/mg of protein per hour (n = 5 pts). The mean activity of Ca2+ ATPase of EPR in control samples was 7.59 ± 1.21 μmol Pi/mg versus 7.76 ± 0.24 μmol Pi/mg in cancer (n = 5 pts). Basal ATPase activity was 3.19 ± 0.87 in control samples versus 4.79 ± 1.86 μmol Pi/mg in cancer (n = 5 pts). In cancer samples, NAADP reduced the activity of Na+/K+ ATPase by 9-times (p < 0.01) and the activity of Ca2+ ATPase EPR about 2-times (p < 0.05). NAADP caused a tendency to decrease the activity of Ca2+ ATPase of PM, but increased basal ATPase activity by 2-fold vs. the mean of this index in cancer samples without the addition of NAADP. In control samples NAADP caused only a tendency to decrease the activities of Na+/K+ ATPase and Ca2+ ATPase EPR, but statistically decreased the activity of Ca2+ ATPase of PM (p < 0.05). In addition, NAADP caused a strong increase in basal ATPase activity in control samples (p < 0.01). Conclusions: We found that the activity of Na+/K+ pump, Ca2+ ATPase of PM and basal ATPase activity in cancer tissues had a strong tendency to be higher than in the controls. NAADP caused a decrease in the activities of Na+/K+ ATPase and Ca2+ ATPase EPR in cancer samples and increased basal ATPase activity. In control samples, NAADP decreased Ca2+ ATPase of PM and increased basal ATPase activity. These data confirmed different roles of NAADP-sensitive “acidic store” (autophagosomes, late endosomes, and lysosomes) in control and cancer tissue, which hypothetically may be connected with autophagy role in cancer development. The effect of NAADP on decreasing the activity of Na+/K+ pump in cancer samples was the most pronounced, both numerically and statistically. Our data shows promising possibilities for the modulation of ion-transport through the membrane of cancer cells by influence on the “acidic store” (autophagosomes, late endosomes and lysosomes) as a new approach to the treatment of colorectal cancer.

Polyamines conjugated to plasma membrane functioned in enhancing the tolerance of cucumber seedlings to osmotic stress via elevating H+-ATPase activity

Polyamine (PA), one of the important plant growth regulators, is closely associated with drought stress. However, the function of conjugated PA is not still clear in the roots of cucumber seedlings under polyethylene glycol (PEG) osmotic stress. Therefore, in this study the relationship between the levels of conjugated polyamines and the activity of H+-ATPase in plasma membrane was elucidated with the roots of two cucumber (Cucumis sativus L.) cultivars, which were different in drought tolerance, as experimental materials. Furthermore, the contents of free PAs and the activities of S-adenosylmethionine decarboxylase (SAMDC) and transglutaminase (TGase), which were closely related to the levels of conjugated polyamines, were also determined. Results showed that under osmotic stress, the increases of the levels of non-covalently conjugated (non-CC) spermidine (Spd) and spermine (Spm), covalently conjugated (CC) putrescine (Put) and Spd in plasma membrane of drought-tolerant Tangshan 5 were more obvious than those of drought-sensitive Jinyou 1. Furthermore, the conjugated PAs mentioned above were closely correlated with increase rate of seedling dry weight, plasma membrane permeability, water content and H+-ATPase activity in plasma membrane. Results of the additional tests, in which exogenous Spd, Spm and two inhibitors, MGBG and phenanthrolin were used, were complementary to the results above. From these results, it could be concluded that non-CC Spd and Spm, CC Put and Spd in plasma membrane functioned in enhancing the tolerance of cucumber seedlings to osmotic stress via elevating H+-ATPase activity.

Potassium mitigates salt-stress impacts on photosynthesis by alleviation of the proton diffusion potential in thylakoids

Addition of K+ improves the tolerance of the photosynthetic apparatus to salt stress, but the primary, underlying mechanism is unclear, prompting our study. The main aim of this study was to elucidate the primary mechanism by which supplemental K+ alleviates the effects of salinity stress on photosynthetic parameters in mulberry seedlings. K+ contents in leaves and chloroplasts, plasma-membrane ATPase activity, photosynthetic properties and expression of genes encoding photosynthetic proteins and ion channels/transporters were measured. K+ addition during salt stress improved H+-ATPase activity in plasma-membranes, restored photosynthetic linear and cyclic electron flow, restored the activity of key Photosystem I (PSI) and PSII proteins, and increased dark-adapted PSII photochemical yield Fv/Fm, while reducing non-photochemical quenching. Among the genes encoding ion channels/transporters, that of the Two-Pore-K+ channel in thylakoid membranes (TPK3) was notably responsive to NaCl/K+ treatments: relative to the control (CK) treatment, it decreased slightly in the CK+K treatment, but increased in the NaCl treatment and increased further in the NaCl+K treatment. The above observations are consistent with our hypothesis that K+ supplementation in salt stress restores a high stromal [K+] which, by utilizing TPK3, minimizes a transient, proton diffusion potential that otherwise develops as protons exit the thylakoid lumen through the ATP synthase; thus, rapid charge compensation by K+ movement accelerates both the proton efflux from the lumen and the associated ATP synthesis to enhance carbon assimilation, thereby improving linear as well as cyclic electron flow. The hypothesis pinpoints the primary physicochemical mechanism by which potassium mitigates salt-stress impacts on photosynthesis. It rationalizes both the existence of a K+ channel in thylakoid membranes and the maintenance of a high stromal [K+].

AMELIORATIVE ROLE OF GARLIC (ALLIUM SATIVUM) ON CHROMIUM (VI) - INDUCED MEMBRANE DAMAGE IN MALE ALBINO RATS

Objective: Membrane damage is one of the important consequences of chromium (Cr) induced cytotoxicity. Garlic possesses antioxidant property to scavenge the toxic radicals and cytoprotective activity. The aim of the present study is to evaluate the ameliorative role of aqueous extract of garlic (AEG) on Cr-induced membrane damage of both liver and kidneys in male albino rats.&#x0D; Methods: Male albino rats of Wistar strain (80-100 g) were used for the present study. Rats were divided into three groups of almost equal average body weight. The animals of two groups were injected K2Cr2O7 at a dose of 0.8 mg per 100 g body weight per day for 28 days. The animals of one of the Cr-treated groups served as the supplemented group supplied aqueous extract of garlic (AEG) (250 mg per kg body weight daily at an interval of 6 h after injection of Cr for a period of 28 days). The animals of the remaining group received only the vehicle (0.9% NaCl), served as control. The body weights of the animals were taken in each day of treatment schedule.&#x0D; Results: The results indicated that significant increases in membrane cholesterol level as well as significant decreases in membrane phospholipid level in Cr exposed animals suggest structural alterations in both liver and kidneys plasma membrane. Alkaline phosphatase (ALP), total ATPase, and Na+-K+- ATPase activities of plasma membrane were significantly decreased in both liver and kidneys after Cr treatment. On the other hand, AEG supplementation plays a vital role to restore such alterations induced by Cr in plasma membrane of both liver and kidney.&#x0D; Conclusion: These findings indicate that Cr treatment at the present dose and duration induces structural and functional alterations in the plasma membrane in both liver and kidney. However, AEG supplementation restored those alterations induced by Cr in plasma membrane of both liver and kidneys but was not able to eliminate the deposited Cr from the liver and kidney tissues.

Erythrocyte membrane ATPase activity in Sickle cell crisis

Background: Sickle cell disease is a genetic disease caused by a point mutation in the beta globin gene on chromosome 11 leading to the substitution of valine in place of glutamic acid in beta subunit of globin molecule. The vaso-occlusive crisis, or sickle cell crisis, is a common painful complication of sickle cell disease in adolescents and adults. Many studies have suggested the alteration in intracellular concentration of various ions associated with sickle cell disease.Aims and Objectives: This study aims to evaluate the activity of red blood cell membrane ATPase activity of sickle cell crisis patients in comparison to sickle cell patients in steady state.Material and Methods: Twenty patients of sickle cell crisis were included in the study. Twenty sickle cell patients in steady state were also registered who served as controls. The RBC Membrane ATPase activity were estimated and expressed as μmol of Pi/μg of protein/hour.Results: The activity of membrane ATPase were found to be significantly high in patients of sickle cell crisis in comparison to steady state ones.Conclusion: Membrane ATPases play a crucial role in the pathophysiology of sickle cell crisis. This study can be a based upon for future studies to understand the pathophysiology of sickle cell crisis and development of diagnostic and treatment modalities for the same.Asian Journal of Medical Sciences Vol.8(5) 2017 27-30

The Prenylated Rab GTPase Receptor PRA1.F4 Contributes to Protein Exit from the Golgi Apparatus

Prenylated Rab acceptor1 (PRA1) functions in the recruitment of prenylated Rab proteins to their cognate organelles. Arabidopsis (Arabidopsis thaliana) contains a large number of proteins belonging to the AtPRA1 family. However, their physiological roles remain largely unknown. Here, we investigated the physiological role of AtPRA1.F4, a member of the AtPRA1 family. A T-DNA insertion knockdown mutant of AtPRA1.F4, atpra1.f4, was smaller in stature than parent plants and possessed shorter roots, whereas transgenic plants overexpressing HA:AtPRA1.F4 showed enhanced development of secondary roots and root hairs. However, both overexpression and knockdown plants exhibited increased sensitivity to high-salt stress, lower vacuolar Na+/K+-ATPase and plasma membrane ATPase activities, lower and higher pH in the vacuole and apoplast, respectively, and highly vesiculated Golgi apparatus. HA:AtPRA1.F4 localized to the Golgi apparatus and assembled into high-molecular-weight complexes. atpra1.f4 plants displayed a defect in vacuolar trafficking, which was complemented by low but not high levels of HA:AtPRA1.F4 Overexpression of HA:AtPRA1.F4 also inhibited protein trafficking at the Golgi apparatus, albeit differentially depending on the final destination or type of protein: trafficking of vacuolar proteins, plasma membrane proteins, and trans-Golgi network (TGN)-localized SYP61 was strongly inhibited; trafficking of TGN-localized SYP51 was slightly inhibited; and trafficking of secretory proteins and TGN-localized SYP41 was negligibly or not significantly inhibited. Based on these results, we propose that Golgi-localized AtPRA1.F4 is involved in the exit of many but not all types of post-Golgi proteins from the Golgi apparatus. Additionally, an appropriate level of AtPRA1.F4 is crucial for its function at the Golgi apparatus.

Aluminium-inhibited NO3- uptake is related to Al-increased H2O2 content and Al-decreased plasma membrane ATPase activity in the root tips of Al-sensitive black soybean.

In this study, Al-sensitive black soybean (Glycine max (L.) Merr.) specimens were treated in Hoagland solutions containing 50-400µM Al for 1-4 days. The measurement for NO3- uptake showed that the NO3- uptake decreased gradually as the Al concentration and treatment time increased, suggesting that Al stress significantly reduced the NO3- uptake by soybean. Under 100-µM Al stress for 4 days, the plasma membrane (PM) ATPase activity (inorganic phosphate (Pi) release), H+ pump activity, phosphorylation of PM ATPase and its interaction with 14-3-3 protein in soybean root tips were all smaller than those in the root tips of control plants. The addition of 150µM Mg2+ in Al treatment solutions significantly alleviated the Al inhibition of NO3- uptake in soybean. The presence of Mg2+ in a 100-µM Al solution pronouncedly enhanced PM ATPase activity, H+ pump activity, phosphorylation of PM ATPase and its interaction with 14-3-3 protein in soybean root tips. The application of 2mM ascorbic acid (AsA, an H2O2 scavenger) in Al treatment solutions significantly decreased Al-inhibited NO3- uptake in soybean. The cotreatment of soybeans with 2mM AsA and 100µM Al significantly reduced H2O2 accumulation and increased the PM ATPase activity, H+ pump activity, phosphorylation of PM H+-ATPase and its interaction with 14-3-3 protein in soybean root tips. The evidence suggested that Al-inhibited NO3- uptake is related to Al-increased H2O2 content and Al-decreased phosphorylation of PM ATPase and its interaction with 14-3-3 protein as well as PM ATPase activity in the root tips of soybean.

Mechanistic insights into the mode of action of anticandidal sesamol

Previously we have deciphered the antifungal effect of sesamol (Ses), a phenolic compound obtained from sesame oil, against human fungal pathogen Candida albicans. To gain deeper insights into the possible mechanisms involved, transcription profiling was done in presence of Ses which revealed various targets through which Ses was barricading the growth of C. albicans. We observed that Ses perturbs membrane integrity confirming our previous observations and displayed disrupted plasma membrane ATPase activity. We further investigated that Ses leads to inhibited morphological transition, biofilm formation and epithelial cell adhesion which are significant virulence attributes required for pathogenesis. Interestingly, Ses also causes amendment in iron homeostasis as revealed by hypersensitivity under iron deprivation, ferroxidase assay to estimate iron levels and concomitant upregulation of FTR2, a high affinity iron transporter. Finally we assessed that Ses causes defect in mitochondrial functioning and DNA repair mechanism. Together, being source of consumable natural product, further studies on Ses are warranted so that it can be exploited as effective antifungal agent.

Antifungal Mechanism of Action of Lactoferrin: Identification of H+-ATPase (P3A-Type) as a New Apoptotic-Cell Membrane Receptor.

Human lactoferrin (hLf) is a protein of the innate immune system which induces an apoptotic-like process in yeast. Determination of the susceptibility to lactoferrin of several yeast species under different metabolic conditions, respiratory activity, cytoplasmic ATP levels, and external medium acidification mediated by glucose assays suggested plasma membrane Pma1p (P3A-type ATPase) as the hLf molecular target. The inhibition of plasma membrane ATPase activity by hLf and the identification of Pma1p as the hLf-binding membrane protein confirmed the previous physiological evidence. Consistent with this, cytoplasmic ATP levels progressively increased in hLf-treated Candida albicans cells. However, oligomycin, a specific inhibitor of the mitochondrial F-type ATPase proton pump (mtATPase), abrogated the antifungal activity of hLf, indicating a crucial role for mtATPase in the apoptotic process. We suggest that lactoferrin targeted plasma membrane Pma1p H(+)-ATPase, perturbing the cytoplasmic ion homeostasis (i.e., cytoplasmic H(+) accumulation and subsequent K(+) efflux) and inducing a lethal mitochondrial dysfunction. This initial event involved a normal mitochondrial ATP synthase activity responsible for both the ATP increment and subsequent hypothetical mitochondrial proton flooding process. We conclude that human lactoferrin inhibited Pma1p H(+)-ATPase, inducing an apoptotic-like process in metabolically active yeast. Involvement of mitochondrial H(+)-ATPase (nonreverted) was essential for the progress of this programmed cell death in which the ionic homeostasis perturbation seems to precede classical nonionic apoptotic events.

Insights into the mode of action of anticandidal herbal monoterpenoid geraniol reveal disruption of multiple MDR mechanisms and virulence attributes in Candida albicans.

The anticandidal potential of Geraniol (Ger) against Candida albicans has already been established. The present study reveals deeper insights into the mechanisms of action of Ger. We observed that the repertoire of antifungal activity was not only limited to C. albicans and its clinical isolates but also against non-albicans species of Candida. The membrane tampering effect was visualized through transmission electron micrographs, depleted ergosterol levels and altered plasma membrane ATPase activity. Ger also affects cell wall as revealed by spot assays with cell wall-perturbing agents and scanning electron micrographs. Functional calcineurin pathway seems to be indispensable for the antifungal effect of Ger as calcineurin signaling mutant was hypersensitive to Ger while calcineurin overexpressing strain remained resistant. Ger also causes mitochondrial dysfunction, impaired iron homeostasis and genotoxicity. Furthermore, Ger inhibits both virulence attributes of hyphal morphogenesis and biofilm formation. Taken together, our results suggest that Ger is potential antifungal agent that warrants further investigation in clinical applications so that it could be competently employed in therapeutic strategies to treat Candida infections.

The ATPase activity of the mycobacterial plasma membrane is inhibited by the LL37-analogous peptide LLAP

The emergence of multidrug-resistant Mycobacterium tuberculosis strains has led to the development of new antituberculous agents. In this context, antimicrobial targeting proteins to the cell membrane are interesting due to the avoidance of the plasma membrane permeation. Through this study, the antimicrobial activity, cellular toxicity, as well as the effect on the mycobacterial cell membrane ATPase activity of a cathelicidin-analogous peptide were assessed. By using bioinformatics analyses, a 15 amino acid LL37-analogous peptide called LLAP, which has the amino acid sequence: GRKSAKKIGKRAKRI, was designed to improve its helical structure and antibacterial activity compared to the native sequence. The LLAP peptide was synthesized, purified by RP-HPLC and its structural characteristics were determined by MALDI-TOF MS and circular dichroism. Compared to the native amino acid sequence, the minimum inhibitory concentration and cytotoxic activity of LLAP were 4.0 and 5.6-fold lower, respectively. In addition, the hemolytic activity of LLAP was lower than 1.1% and the cytotoxic activity of peptides was similar for both peptides. Interestingly, the LLAP peptide displayed approximately 50% inhibition of basal ATPase activity of the mycobacterial plasma membrane, which could in turn be associated with the impaired cell viability. The results suggest that LLAP could be considered as potential antimycobacterial compounds against cell membrane targeting ATPases. However, this antimycobacterial activity can be improved. It is expected further applications to be found for other antimicrobial peptides families based on the implemented methodology.

Protective Effects of Ferulic Acid on High Glucose-Induced Protein Glycation, Lipid Peroxidation, and Membrane Ion Pump Activity in Human Erythrocytes.

Ferulic acid (FA) is the ubiquitous phytochemical phenolic derivative of cinnamic acid. Experimental studies in diabetic models demonstrate that FA possesses multiple mechanisms of action associated with anti-hyperglycemic activity. The mechanism by which FA prevents diabetes-associated vascular damages remains unknown. The aim of study was to investigate the protective effects of FA on protein glycation, lipid peroxidation, membrane ion pump activity, and phosphatidylserine exposure in high glucose-exposed human erythrocytes. Our results demonstrated that FA (10-100 μM) significantly reduced the levels of glycated hemoglobin (HbA1c) whereas 0.1-100 μM concentrations inhibited lipid peroxidation in erythrocytes exposed to 45 mM glucose. This was associated with increased glucose consumption. High glucose treatment also caused a significant reduction in Na+/K+-ATPase activity in the erythrocyte plasma membrane which could be reversed by FA. Furthermore, we found that FA (0.1-100 μM) prevented high glucose-induced phosphatidylserine exposure. These findings provide insights into a novel mechanism of FA for the prevention of vascular dysfunction associated with diabetes.

The DosR dormancy regulator of Mycobacterium tuberculosis stimulates the Na(+)/K (+) and Ca (2+) ATPase activities in plasma membrane vesicles of mycobacteria.

The latency global regulator DosR regulon of Mycobacterium tuberculosis, which is stimulated by hypoxia, comprises approximately fifty genes including ctpF (Rv1997), which encodes a putative alkali/alkaline earth ion transporter of the plasma membrane. In this work, the influence of hypoxia and M. tuberculosis DosR on the ATPase activity of mycobacterial plasma membrane was assessed. We performed bioinformatic analyses which indicated that the pma1 gene product is the M. smegmatis ortholog of the M. tuberculosis cation transporter CtpF. In addition, a possible Na(+), K(+) and/or Ca(2+) pumping mediated by Pma1 was also predicted. Enzymatic analyses indicated that the basal ATPase activity of plasma membrane vesicles from M. smegmatiscells cultured under hypoxia and over-expressing DosR, decreased 30 and 40% respectively in comparison to oxygenated cells.In contrast, the specific Na(+)/K(+) and Ca(2+) ATPase activities of the plasma membrane increased 2.8- and 3.5-fold, respectively, under hypoxia, similar to that observed for cells over-expressing the DosR regulator. In agreement, RT-qPCR experiments demonstrated that the transcription level of the pma1 gene increased under hypoxia at levels similar to that of M. smegmatis cells over-expressing the M. tuberculosis DosR regulator. The entire findings suggest that hypoxia stimulates Na(+)/K(+) and Ca(2+) ATPase activities in the mycobacterial plasma membrane, and this is possibly mediated by the dormancy regulator DosR.

Quantitative proteomics of Sesuvium portulacastrum leaves revealed that ion transportation by V-ATPase and sugar accumulation in chloroplast played crucial roles in halophyte salt tolerance

Physiological and proteomic responses of Sesuvium portulacastrum leaves under salinity were investigated. Different from glycophytes, this halophyte had optimal growth at 200-300mM NaCl and accumulated more starch grains in chloroplasts under high salinity. Increased contents of soluble sugars, proline, and Na(+) were observed upon salinity. X-ray microanalysis revealed that Na(+) was mainly compartmentalized into cell vacuole. Quantitative proteomics produced 96 salt responsive proteins, and the majority was chloroplast-located proteins. Gene ontology analysis revealed that proteins involved in ion binding, proton transport, photosynthesis and ATP synthesis were overrepresented. The expressions of a Na(+)/H(+) antiporter and several ATP synthase subunits were activated upon high salinity. ATP hydrolysis assay demonstrated that V-ATPase activity at tonoplast was dramatically increased upon NaCl whereas vacuolar H(+)-pyrophosphatase and plasma membrane P-ATPase activities were not increased, which indicated that sodium compartmentalization was mainly performed by enhancing V-ATPase activity rather than P-ATPase and H(+)-pyrophosphatase. Accumulation of soluble sugars as well as sodium compartmentalization maintained the osmotic balance between vacuole and cytoplasm, which finally established ionic homeostasis in saline cells in true halophytes. Physiological and proteomic analyses of S. portulacastrum leaves under different salinities were investigated. This true halophyte accumulated more soluble sugars, starch, proline and Na(+) under high salinity. Differential proteomics produced 96 salt responsive proteins and the majority was involved in ion binding, proton transport, photosynthesis, and ATP synthesis. A Na(+)/H(+) antiporter and several ATP synthase subunits were induced upon high salinity. ATP hydrolysis assay demonstrated that V-ATPase activity at tonoplast was dramatically increased whereas vacuolar H(+)-pyrophosphatase and plasma membrane ATPase activities were stable upon NaCl. These findings demonstrated that the increased Na(+) was compartmentalized into vacuole by enhancing V-ATPase activity rather than H(+)-ATPase.

Low temperature alters plasma membrane lipid composition and ATPase activity of pineapple fruit during blackheart development

Plasma membrane (PM) plays central role in triggering primary responses to chilling injury and sustaining cellular homeostasis. Characterising response of membrane lipids to low temperature can provide important information for identifying early causal factors contributing to chilling injury. To this end, PM lipid composition and ATPase activity were assessed in pineapple fruit (Ananas comosus) in relation to the effect of low temperature on the development of blackheart, a form of chilling injury. Chilling temperature at 10°C induced blackheart development in concurrence with increase in electrolyte leakage. PM ATPase activity was decreased after 1week at low temperature, followed by a further decrease after 2weeks. The enzyme activity was not changed during 25°C storage. Loss of total PM phospholipids was found during postharvest senescence, but more reduction was shown from storage at 10°C. Phosphatidylcholine and phosphatidylethanolamine were the predominant PM phospholipid species. Low temperature increased the level of phosphatidic acid but decreased the level of phosphatidylinositol. Both phospholipid species were not changed during storage at 25°C. Postharvest storage at both temperatures decreased the levels of C18:3 and C16:1, and increased level of C18:1. Low temperature decreased the level of C18:2 and increased the level of C14:0. Exogenous application of phosphatidic acid was found to inhibit the PM ATPase activity of pineapple fruit in vitro. Modification of membrane lipid composition and its effect on the functional property of plasma membrane at low temperature were discussed in correlation with their roles in blackheart development of pineapple fruit.

Effect of hypoxia on the calcium and magnesium content, lipid peroxidation level, and Ca²⁺-ATPase activity of syncytiotrophoblast plasma membranes from placental explants.

In the current study the possible relationship between the Ca2+/Mg2+ ratio of human syncytiotrophoblast plasma membranes and their lipid peroxidation and Ca2+-ATPase activity was determined. Syncytiotrophoblast plasma membranes of placental explants cultured under hypoxia increased their lipid peroxidation and Ca2+ content, diminished their Ca2+-ATPase activity, and kept their Mg2+ content unchanged. Membranes preincubated with different concentrations of Ca2+ increased their Ca2+ content without changes in their Mg2+ content. There is a direct relationship between Ca2+ content and lipid peroxidation of the membranes, as well as an inverse relationship between their Ca2+ content and Ca2+-ATPase activity. On the contrary, preincubation of membranes with different concentrations of Mg2+ showed a higher Mg2+ content without changing their lipid peroxidation and Ca2+-ATPase activity. Explants cultured under hypoxia in the presence of 4 mM MgSO4 showed similar values of lipid peroxidation and Ca2+-ATPase activity of their membranes compared to those of explants cultured under normoxia. Increased Ca2+ content of the membranes by interacting with negatively charged phospholipids could result in destabilizing effects of the membrane structure, exposing hydrocarbon chains of fatty acids to the action of free radicals. Mg2+ might exert a stabilizing effect of the membranes, avoiding their exposure to free radicals.

C-Terminal Truncations of the Saccharomyces cerevisiae PMA1 H + -ATPase Have Major Impacts on Protein Conformation, Trafficking, Quality Control, and Function

The C-terminal tail of yeast plasma membrane (PM) H(+)-ATPase extends approximately 38 amino acids beyond the final membrane-spanning segment (TM10) of the protein and is known to be required for successful trafficking, stability, and regulation of enzyme activity. To carry out a detailed functional survey of the entire length of the tail, we generated 15 stepwise truncation mutants. Eleven of them, lacking up to 30 amino acids from the extreme terminus, were able to support cell growth, even though there were detectable changes in plasma membrane expression, protein stability, and ATPase activity. Three functionally distinct regions of the C terminus could be defined. (i) Truncations upstream of Lys(889), removing more than 30 amino acid residues, yielded no viable mutants, and conditional expression of such constructs supported the conclusion that the stretch from Ala(881) (at the end of TM10) to Gly(888) is required for stable folding and PM targeting. (ii) The stretch between Lys(889) and Lys(916), a region known to be subject to kinase-mediated posttranslational modification, was shown here to be ubiquitinated in carbon-starved cells as part of cellular quality control and to be essential for normal ATPase folding and stability, as well as for autoinhibition of ATPase activity during glucose starvation. (iii) Finally, removal of even one or two residues (Glu(917) and Thr(918)) from the extreme C terminus led to visibly reduced expression of the ATPase at the plasma membrane. Thus, the C terminus is much more than a simple appendage and profoundly influences the structure, biogenesis, and function of the yeast H(+)-ATPase.