Suppression of Mitochondrial Succinate Dehydrogenase, a Primary Target of β-Amyloid, and Its Derivative Racemized at Ser Residue

Abstract: β-Amyloid cores contain considerable amounts of d -Ser and d -Asp residues in Alzheimer's disease. We investigated the cytotoxic effects of various synthetic β-amyloids, including d -Ser-substituted derivatives, on primary cultured neurons and nonneuronal HeLa cells. β25–35, its d -Ser²⁶-substituted derivative, and β1–40 in 10–100 n M specifically suppressed mitochondrial succinate dehydrogenase activity [MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction] in HeLa cells, which are dependent on ATP production mainly from glycolysis, but did not exert detectable cytotoxicity, assessed by dye exclusion test, NADH levels, and uptake of [³H]Leu and [³H]Tdr. The β-amyloids, on the other hand, did exert neurodegenerative effects on rat hippocampal cultured neurons in which ATP is mostly synthesized by the mitochondrion. The activities of β25–35 and [ d -Ser²⁶]β25–35 are dependent on their having β-structures and not random forms. Although β25–35 was degraded rapidly by proteinase(s) in brain extract or leucine aminopeptidase, [ d -Ser²⁶]β25–35 is fairly resistant. These results indicate that one of the primary targets of β-amyloids is suppression of mitochondrial succinate dehydrogenase, and the vulnerability of the brain to β-amyloids can be explained by its large dependence on mitochondrial energy production. Moreover, racemization of serine residues of β-amyloids may be involved in neurodegeneration and formation of senile plaques through escaping from the degradation process by brain proteinases.     

β-Amyloid-Induced Cell Toxicity: Enhancement of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide-Dependent Cell Death

Abstract: In an attempt to understand the cause of neurodegeneration in Alzheimer's disease, the toxic effects of β-amyloid (Aβ) peptides have been widely studied. At high micromolar concentrations Aβ peptides have been demonstrated to be acutely toxic to various cell types. At submicromolar concentrations, Aβ peptides have been suggested to inhibit cellular metabolic activity, due to their inhibition of the ability of cells to metabolize the oxidoreductase substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Here we show, first, that MTT reduction surprisingly leads to a breakdown in PC12 cell membrane integrity and cell death, presumably through the formation of a crystalline formazan product, and, second, that pretreatment of PC12 cells with nanomolar concentrations of Aβ peptide, rather than inhibiting their metabolic activity, increases the susceptibility of these cells to the secondary toxic effect of formazan crystal formation. These results suggest that low nanomolar concentrations of Aβ render membranes more susceptible to damage by a secondary insult, in this case, MTT reduction. It is plausible that such an effect, when combined with additional risk factors, could contribute to the neurodegeneration that occurs in Alzheimer's disease.     

Actions of Neurotoxic β-Amyloid on Calcium Homeostasis and Viability of PC12 Cells Are Blocked by Antioxidants but Not by Calcium Channel Antagonists

Abstract: The fragment of β-amyloid comprised of amino acids 25–35 induces a rapid, concentration-dependent increase in cytosolic free calcium levels in suspensions of PC12 neuronal cells. This action of β-amyloid 25–35 is not altered by pretreatment with the calcium channel blockers nifedipine or cobalt, with the depleter of intracellular calcium stores cyclopiazonic acid, or with the phospholipase C inhibitor neomycin. However, the effects of β-amyloid 25–35 on cytosolic free calcium are absent in calcium-free buffer and are blocked by the antioxidant lazaroid U-83836E and by vitamin E. β-Amyloid 25–35 is also neurotoxic and produces a concentration-dependent reduction in the viability of PC12 cells in culture. The neurotoxic action of β-amyloid is blocked by U-83836E and vitamin E but not by nifedipine or cobalt. These data indicate that both the disruption of calcium homeostasis and the reduction of cell viability produced by β-amyloid in PC12 cells are mediated by free radical-based processes.     

Steroid Hormones Block Amyloid Fibril-Induced 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Formazan Exocytosis: Relationship to Neurotoxicity

Abstract: Perhaps the most reproducible early event induced by the interaction of amyloid β peptide (Aβ) with the cell is the inhibition of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. We recently demonstrated that cytotoxic amyloid peptides such as Aβ and human amylin inhibit cellular MTT reduction by dramatically enhancing MTT formazan exocytosis. We now show the following: (a) Insulin and glucagon, when converted to fibrils with β-pleated sheet structure, induce MTT formazan exocytosis that is indistinguishable from that induced by Aβ. NAC35, an amyloidogenic fragment of α-synuclein (or NACP), also induces MTT formazan exocytosis. (b) All protein fibrils with the β-pleated sheet structure examined are toxic to rat hippocampal neurons. (c) Many sterol sex hormones (e.g., estradiol and progesterone) block amyloid fibril-enhanced MTT formazan exocytosis as well as MTT formazan exocytosis in control cells by acting at a common late step in the exocytic pathway. Steroids fail, however, to protect hippocampal neurons from acute amyloid fibril toxicity. These findings suggest that the ability to enhance MTT formazan exocytosis and to induce neurotoxicity are common biological activities of protein fibrils with β-pleated sheet structure but that enhanced MTT formazan exocytosis is not sufficient for acute Aβ neurotoxicity.     

Inhibitors of V-type ATPases, bafilomycin A1 and concanamycin A, protect against beta-amyloid-mediated effects on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction

The functional viability of cells can be evaluated using a number of different assay determinants. One common assay involves exposing cells to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which is converted intracellularly to a colored formazan precipitate and often used to assess amyloid peptide-induced cytotoxic effects. The MTT assay was employed to evaluate the role of endosomal uptake and lysosomal acidification in amyloid peptide-treated differentiated PC12 cell cultures using selective vacuolar-type (V-type) ATPase inhibitors. The macrolides bafilomycin A1 (BAF) and concanamycin A (CON) block lysosomal acidification through selective inhibition of the V-type ATPase. Treating nerve growth factor-differentiated PC12 cells with nanomolar concentrations of BAF or CON provides complete protection against the effects of beta-amyloid peptides Abeta(1-42), Abeta(1-40), and Abeta(25-35) and of amylin on MTT dye conversion. These macrolides do not inhibit peptide aggregation, act as antioxidants, or inhibit Abeta uptake by cells. Measurements of lysosomal acidification reveal that the concentrations of BAF and CON effective in reversing Abeta-mediated MTT dye conversion also reverse lysosomal pH. These results suggest that lysosomal acidification is necessary for Abeta effects on MTT dye conversion.     

Cytotoxic Amyloid Peptides Inhibit Cellular 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Reduction by Enhancing MTT Formazan Exocytosis

Abstract: Amyloid β peptide (Aβ) neurotoxicity is believed to play a central role in the pathogenesis of Alzheimer's disease. An early indicator of Aβ toxicity is the inhibition of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction to MTT formazan, a widely used assay for measuring cell viability. In this report we show that Aβ and other cytotoxic amyloid peptides such as human amylin dramatically enhance MTT formazan exocytosis, resulting in the inhibition of cellular MTT reduction. Only the amyloid peptides that are known to be cytotoxic enhanced MTT formazan exocytosis. Basal MTT formazan exocytosis and amyloid peptide-enhanced MTT formazan exocytosis are blocked by several drugs with diverse known effects. These and other data suggest that MTT formazan exocytosis is a multistep process and that cytotoxic amyloid peptides enhance MTT formazan exocytosis through an intracellular signal transduction pathway.     

Salvianolic acid B, an antioxidant from Salvia miltiorrhiza, prevents Abeta(25-35)-induced reduction in BPRP in PC12 cells

Several lines of evidence support that beta-amyloid (Abeta)-induced neurotoxicity is mediated through the generation of reactive oxygen species (ROS) and elevation of intracellular calcium. Salvianolic acid B (Sal B), the major and most active anti-oxidant from Salvia miltiorrhiza, protects diverse kinds of cells from damage caused by a variety of toxic stimuli. In the present study, we investigated the effects of Sal B against beta-amyloid peptide 25-35 (Abeta(25-35))-induced neurotoxicity, focused mainly on the neurotoxic effects of Abeta(25-35) and the neuroprotective effects of Sal B on the expression of brain-pancreas relative protein (BPRP), which is a new protein and mainly expressed in brain and pancreas. Following exposure of PC12 cells to 20 microM Abeta(25-35), a marked reduction in the expression of BPRP was observed, accompanied with decreased cell viability and increased cell apoptosis, as well as increased ROS production and calcium influx. Treatment of the PC12 cells with Sal B significantly reversed the expression of BPRP and cell viability while it decreased ROS production and intracellular calcium. These data indicate that Abeta(25-35) decreases the expression of BPRP via enhanced formation of intracellular ROS and increased intracellular calcium, and that Sal B, as an anti-oxidant, protects against Abeta(25-35)-induced reduction in expression of BPRP through its effects on suppressing the production of ROS, calcium flux, and apoptosis. However, the role(s) of BPRP in AD and the definite mechanisms by which Sal B protects against Abeta(25-35)-induced reduction in the expression of BPRP require further study.     

Amyloid β Toxicity Consists of a Ca2+-Independent Early Phase and a Ca2+-Dependent Late Phase

Abstract: Amyloid β protein (Aβ), which accumulates in the senile plaques in the brain of Alzheimer's patients, is cytotoxic to neurons. A modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in which a yellow redox dye, MTT, is reduced to purple formazan, is very sensitive to the effect of Aβ. In primary hippocampal cultures, inhibition of MTT reduction starts within 2 h after the addition of low concentrations of Aβ and reaches a plateau in 12 h. This effect of Aβ is not blocked by Ca²⁺ channel blockers or in Ca²⁺-free medium. In contrast, lactate dehydrogenase (LDH) release and trypan blue exclusion, which are indices of cell death, start 3 days after exposure to high concentrations of Aβ and are blocked by Ca²⁺ channel blockers such as Co²⁺, nicardipine, and diltiazem. When Aβ was washed out from the medium after 12 h, MTT reduction recovers and LDH release does not occur, suggesting that a long-lasting inhibition of the cellular redox system may be required to induce cell death. These observations demonstrate that Aβ toxicity consists of two phases—a Ca²⁺-independent early phase and a Ca²⁺-dependent late phase—and that the early phase may be required to induce the late phase.     

β-Amyloid1–40 Increases Expression of β-Amyloid Precursor Protein in Neuronal Hybrid Cells

Abstract: Studies of cell injury and death in Alzheimer's disease have suggested a prominent role for β-amyloid peptide (β-AP), a 40–43-amino-acid peptide derived from a larger membrane glycoprotein, β-amyloid precursor protein (β-APP). Previous experiments have demonstrated that β-AP induces cytotoxicity in a neuronal hybrid cell line (MES 23.5) in vitro. Here, we demonstrate that β-APP mRNA content is increased 3.5-fold in 24 h after treatment with β-AP_1–40. Accompanying β-AP_1–40-induced cell injury, levels of cell-associated β-APP and a C-terminal intermediate fragment are increased up to 15-fold, and levels of secreted forms of β-APP and 12- and 4-kDa fragments are also increased. Application of β-APP antisense oligodeoxynucleotide reduces both cytotoxicity and β-APP expression. 6-Hydroxydopamine application or glucose deprivation causes extensive cell damage, but they do not increase β-APP expression. These results suggest a selective positive feedback mechanism whereby β-AP may induce cytotoxicity and increase levels of potentially neurotrophic as well as amyloidogenic fragments of β-APP with the net consequence of further neuronal damage.     

Abeta(31-35) peptide induce apoptosis in PC 12 cells: contrast with Abeta(25-35) peptide and examination of underlying mechanisms

The toxic behaviour of the two shorter sequences of the native Abeta amyloid peptide required for cytotoxicity i.e., Abeta(31-35) and Abeta(25-35) peptides, was studied. We have shown that Abeta(31-35) peptide induces neurotoxicity in undifferentiated PC 12 cell via an apoptotic cell death pathway, including caspase activation and DNA fragmentation. Abeta(25-35) peptide, like the shorter amyloid peptide has the ability to induce neurotoxicity, as evaluated by the MTS reduction assay and by adherent cell count, but the Abeta(25-35) peptide-induced neurotoxicity is not associated with any biochemical features of apoptosis. The differences observed between the neurotoxic properties of Abeta(31-35) and Abeta(25-35) peptides might result on their different ability to be internalised within the neuronal cells. Furthermore, this study reveals that the redox state of methionine residue, C-terminal in Abeta(31-35) and Abeta(25-35) peptides affect in a different way the toxic behaviour of these two short amyloid fragments. Taken together our results suggest that Abeta(31-35) peptide induces cell death by apoptosis, unlike the Abeta(25-35) peptide and that role played by methionine-35 in Abeta induced neurotoxicity might be related to the Abeta aggregation state.     

Role of GSK-3beta activation and alpha7 nAChRs in Abeta(1-42)-induced tau phosphorylation in PC12 cells

β-amyloid peptide 1–42 (Aβ_1–42) and hyperphosphorylated tau are associated with neurodegeneration in Alzheimer's disease. Emerging evidence indicates that Aβ_1–42 can potentiate hyperphosphorylation of tau in cell lines and in transgenic mice, but the underlying mechanism(s) remains unclear. In this study, Aβ_1–42-induced tau phosphorylation was investigated in differentiated PC12 cells. Treatment of cells with Aβ_1–42 increased phosphorylation of tau at serine-202 as detected by AT8 antibody. This Aβ_1–42-induced tau phosphorylation paralleled phosphorylation of glycogen synthase kinase-3β (GSK-3β) at tyrosine-216 (GSK-3β-pY216), which was partially inhibited by the GSK-3β inhibitor, CHIR98023. Aβ_1–42-induced tau phosphorylation and increase in GSK-3β-pY216 phosphorylation were also partially attenuated by α7 nicotinic acetylcholine receptor (α7 nAChR) selective ligands including agonist A-582941 and antagonists methyllycaconitine and α-bungarotoxin. The α7 nAChR agonist and the GSK-3β inhibitor had no additive effect. These observations suggest that α7 nAChR modulation can influence Aβ_1–42-induced tau phosphorylation, possibly involving GSK-3β. This study provides evidence of nAChR mechanisms underlying Aβ_1–42 toxicity and tau phosphorylation, which, if translated in vivo , could provide additional basis for the utility of α7 nAChR ligands in the treatment of Alzheimer's disease.     

异槲皮苷对Aβ25-35导致的PC12细胞损伤的保护作用及机制研究

探讨异槲皮苷对β-淀粉样蛋白(Aβ25-35)导致的PC12细胞氧化损伤的保护作用.首先通过分子对接技术分析异槲皮苷与AMPK的结合情况.采用Aβ25-35(20 μmol/L)损伤PC12细胞建立细胞氧化损伤模型,采用甲基噻唑蓝(MTT)法检测细胞活力,通过试剂盒检测乳酸脱氢酶(LDH)漏出量、活性氧(ROS)含量、...     

Mechanism of Cellular 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Reduction

Abstract: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction is one of the most frequently used methods for measuring cell proliferation and neural cytotoxicity. It is widely assumed that MTT is reduced by active mitochondria in living cells. By using isolated mitochondria from rat brain and B12 cells, we indeed found that malate, glutamate, and succinate support MTT reduction by isolated mitochondria. However, the data presented in this study do not support the exclusive role of mitochondria in MTT reduction by intact cells. Using a variety of approaches, we found that MTT reduction by B12 cells is confined to intracellular vesicles that later give rise to the needle-like MTT formazan at the cell surface. Some of these vesicles were identified as endosomes or lysosomes. In addition, MTT was found to be membrane impermeable. These and other results suggest that MTT is taken up by cells through endocytosis and that reduced MTT formazan accumulates in the endosomal/lysosomal compartment and is then transported to the cell surface through exocytosis.     

Amidation of β-Amyloid Peptide Strongly Reduced the Amyloidogenic Activity Without Alteration of the Neurotoxicity

Abstract: β-Amyloid accumulates in cerebral deposits in Alzheimer's disease, so to test the correlation between the neurotoxic and fibrillogenic capacity of β-amyloid, we synthesized a peptide homologous to fragment 25–35 of β-amyloid (β25–35) and amidated at the C-terminus (β25–35-NH₂). As the amidation strongly reduced the amyloidogenic capacity of β25–35, we compared its neurotoxic activity in the amidated (β25–35-NH₂) and nonamidated forms. The viability of primary cultures from fetal rat hippocampus was reduced in a dose-related manner (10–100 µ M ) similarly by β25–35 and β25–35-NH₂, whereas a scrambled peptide, amidated or nonamidated, did not alter the neuronal viability. The neurotoxic activity of β25–35-NH₂ is mediated by apoptosis as demonstrated by morphological and biochemical investigations. Electron microscopy examination of culture media with β25–35 or β25–35-NH₂ incubated with neuronal cells for 7 days confirmed the high level of fibrillogenic activity of β25–35 and the almost total absence of fibrils in the solution with β25–35-NH₂. Furthermore, staining with thioflavine S was used to identify amyloid fibrils, and only the cultures exposed to β25–35 exhibited intense staining associated with neuronal membranes. These data indicate that the neurotoxic activity of the β-amyloid fragment is independent of the aggregated state of the peptide.     

Genistein inhibits mitochondrial-targeted oxidative damage induced by beta-amyloid peptide 25–35 in PC12 cells

The antioxidative properties of genistein (Gen) have been demonstrated by our previous studies and others, but its potential mechanism was not very clear. Because of the key role of mitochondria in oxidant production, we wondered if mitochondria were one of Gen’s neuroprotective targets. In the present study we investigated whether Gen has protective effects on mitochondria damaged by Aβ25-35. PC12 cells were pre-incubated with or without Gen for 2 h followed by the incubation with 20 μM Aβ25-35 for another 24 h before mitochondrial membrane fluidity (MMF), mitochondrial membrane potential (MMP) , and mitochondrial redox state were measured. The results showed that Gen alleviated the decrease of MMF induced by Aβ25-35, and maintained the MMP. Additionally, Gen promoted the mitochondrial antioxidative capability through increasing the GSH/GSSG ratio, GPx activity and MnSOD protein expression in mitochondria. Moreover, Gen reversed the changes of ChAT mRNA and AChE mRNA expression in cells induced by Aβ25-35. These results suggested that Gen can protect the mitochondrial membrane and maintain redox state in mitochondria damaged by Aβ25-35.     

Amyloid-β(1-42) alters structure and function of retinal pigmented epithelial cells

Age-related macular degeneration (AMD) is characterized by the formation of drusen, extracellular deposits associated with atrophy of the retinal pigmented epithelium (RPE), disturbance of the transepithelial barrier and photoreceptor death. Amyloid-β (Aβ) is present in drusen but its role during AMD remains unknown. This study investigated the in vitro and in vivo effects of the oligomeric form of Aβ(1-42) – OAβ(1-42) – on RPE and found that it reduced mitochondrial redox potential and increased the production of reactive oxygen species, but did not induce apoptosis in RPE cell cultures. It also disorganized the actin cytoskeleton and halved occludin expression, markedly decreasing attachment capacity and abolishing the selectivity of RPE cell transepithelial permeability. Antioxidant pretreatment partially reversed the effects of OAβ(1-42) on mitochondrial redox potential and transepithelial permeability. Subretinally injected OAβ(1-42) induced pigmentation loss and RPE hypertrophy but not RPE cell apoptosis in C57BL/6 J mice. Rapid OAβ(1-42)-induced disorganization of cytoskeletal actin filaments was accompanied by decreased RPE expression of the tight junction proteins occludin and zonula occludens-1 and of the visual cycle proteins cellular retinaldehyde-binding protein and RPE65. The number of photoreceptors decreased by half within a few days. Our study pinpoints the role of Aβ in RPE alterations and dysfunctions leading to retinal degeneration and suggests that targeting Aβ may help develop selective methods for treating diseases involving retinal degeneration, such as AMD.     

Chronic Ethanol Reduces Immunologically Detectable Gqα/11α in NG108–15 Cells

Abstract: The amyloid protein (βA₄) is found in the CNS of patients with Alzheimer's disease; however, the pathogenic role of this protein is not known. In the present study, a peptide fragment of βA₄βA₄ 25–35; Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-NH₂), which contains the conserved C-terminal sequence of substance P (X-Gly-Leu-Met-NH₂), and the neuropeptide substance P (SP) were examined for their ability to modulate nicotine-evoked secretion from cultured bovine adrenal chromaffin cells. Secretion of the released endogenous catecholamines was monitored by electrochemical detection after separation by HPLC. Secretion induced by 10⁻⁵ M nicotine was inhibited by SP and βA₄ 25–35. The IC₅₀ of SP and βA₄ 25–35 was 3 × 10⁻⁶ and 3 × 10⁻⁵ M , respectively. SP and βA₄ 25–35 both protected against nicotinic receptor desensitization. However, βA₄ 25–35 was ∼ 10-fold less effective than SP in its protective effect. The present work shows that βA₄ 25–35 can mimic the modulatory actions of SP on the nicotinic response of cultured bovine chromaffin cells, i.e., inhibition of the nicotinic response and protection against nicotinic desensitization. These modulatory actions may be associated with changes in nicotinic receptor levels reported to occur in Alzheimer's disease.     

β25–35 Alters Calcium Homeostasis and Induces Neurotoxicity in Cerebellar Granule Cells

Abstract: We studied the neurotoxic effects of β25–35 amyloid fragment (β25–35) on cerebellar granule cells and the intracellular mechanisms involved. Treatment for 3 days with peptide greatly reduced the survival of 1 day in vitro (DIV) cultures kept in 5 m M KCl but slightly modified the survival of 25 m M KCl-cultured cerebellar granule cells. We also studied the effect of glutamate on survival of undifferentiated cerebellar granules. We report no neurotoxic effect of glutamate on 3-DIV-treated cultures; whereas in β25–35-pretreated cells, a significant glutamate toxicity was observed. Treatment of 6-DIV cells with β25–35, performed with 25 m M KCl, induced a late but significant neurotoxic effect after 5 days of exposure, and death occurred within 8 days. Differentiated cerebellar granule cells were also sensitive to glutamate-related neurotoxicity, and this effect was enhanced by β25–35 pretreatment. To study the molecular mechanisms underlying the neurotoxic effects of β25–35, changes in calcium homeostasis after glutamate stimulation were evaluated in control and β25–35-treated cells. β25–35 did not affect basal [Ca²⁺]_i but modified glutamate-induced [Ca²⁺]_i increase, causing a sustained plateau phase that persisted even after the removal of the agonist. These results show that β25–35 induces neurotoxicity in cerebellar granule cells and that this effect is related to modifications in the control of calcium homeostasis.     

Inhibition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) endocytosis by ouabain in human endothelial cells

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and reduction is widely used to evaluate cell proliferation and viability. MTT is taken up by the cells through endocytosis. We find that ouabain (1-200 nM) inhibits MTT reduction in human umbilical vein endothelial cells (HUVEC) without affecting cell viability. Ouabain does not inhibit MTT reduction when cell lysates substituted for the intact cells. Disruption of caveolae by cholesterol depletion, completely prevents the effect of ouabain. Treatment of HUVEC with Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine partially abrogates the inhibitory effect of ouabain. The data suggest that ouabain interaction with caveolar Na/K-ATPase inhibits MTT endocytosis through the activation of signaling proteins such as Src kinase.     

A β-(1→4)-xylosyltransferase involved in the synthesis of arabinoxylans in developing barley endosperms

A β-(1→4)-xylosyltransferase (XylTase; EC 2.4.2.24) participating in the synthesis of arabinoxylans was investigated using microsomal membranes prepared from developing barley ( Hordeum vulgare L.) endosperms. The microsomal fraction transferred Xyl from uridine 5'-diphosphoxylose (UDP-Xyl) into exogenous β-(1→4)-xylooligosaccharides derivatized at their reducing ends with 2-aminopyridine. HPLC analysis showed chain elongation of pyridylaminated β-(1→4)-xylotriose (Xyl₃-PA) by repeated attachment of one to five single xylosyl residues depending on the reaction time, leading to the formation of Xyl₄₋₈-PA. Methylation analysis and enzymatic digestions with β-xylosidase (EC 3.2.1.37) and endo -β-(1→4)-xylanase (EC 3.2.1.8) confirmed that the transfer of xylosyl residues into the newly synthesized products occurred through β-(1→4)-linkages. The activity of the XylTase was maximal at pH 6.8 and 20°C and most enhanced in the presence of 0.5% Triton X-100 and 5 m M MnCl₂. The apparent Michaelis constant and maximal velocity of the enzyme for Xyl₃-PA were 2.1 m M and 25 400 pmol min⁻¹ mg protein⁻¹, respectively. The enzyme also transferred [¹⁴C]Xyl from UDP-[¹⁴C]Xyl into higher β-(1→4)-xylooligosaccharides and birchwood xylans through β-(1→4)-linkages. The enzyme activity varied according to the stage of development (7–35 days after flowering) of the endosperms. Maximal activity occurred at 13–16 days; no activity was detectable in mature seeds. A comparison of endosperms from 10 different cultivars of barley harvested 11–22 days after flowering showed no correlation between enzyme activity and the amount of Xyl in the cell walls.