The mechanism of oleanolic acid monoglycosides transport into vacuoles isolated from Calendula officinalis leaf protoplasts

The uptake of [3-³H]-oleanolic acid 3-O-monoglucoside and 3-O-monoglucuronide into vacuoles isolated from Calendula officinalis leaf protoplasts was investigated under various conditions. The transport of monoglucoside was stimulated by ATP and pyrophosphate, and sensitive to protein-modifying agent as well as dissipaters of the membrane potential. On the contrary, the transport of monoglucuronide was not dependent on tonoplast energization and was affected only by a protein-modifying factor. Moreover, the kinetics of tonoplast transport of both monoglycosides was characterized as concentration-dependent with saturation phase. The obtained results indicate that oleanolic acid monoglucoside is transported to isolated vacuoles by an active, carrier-mediated and energy-dependent mechanism, whereas the transport of monoglucuronide is a passive, carrier-mediated process.     

The metabolism of [3-H]oleanolic acid and its monoglycosides in cytoplasm and vacuole of protoplasts isolated from Calendula officinalis leaves

Isolated protoplasts from C. officinalis leaves were supplied with [3-³H]oleanolic acid, its 3-O-monoglucoside and 3-O-monoglucuronide. Transformations of these compounds into two series of oleanolic acid glycosides, i.e. glucosides (derivatives of 3-O-monoglucoside) and glucuronides (derivatives of 3-O-monoglucuronide) in the extravacuolar space and the vacuole were investigated. In the cytoplasm free oleanolic acid is glycosylated to both monoglycosides and to higher glycosides. Monoglycosides are partly hydrolysed to free oleanolic acid and partly glycosylated to higher derivatives. The vacuole contains the same radioactive compounds as the extravacuolar space. However, it seems most likely that these compounds are transported there from the sites of their synthesis in the cytoplasm.     

High-performance liquid chromatographic separation of bilirubin conjugates

A fast sensitive method for the isolation and quantitation of biliary bile pigments by reverse-phase high-performance liquid chromatography has been developed. Nine conjugates of bilirubin as well as unconjugated bilirubin and an internal standard, unconjugated mesobilirubin IX alpha, were all separated to baseline by gradient elution. The following sequence of eluted compounds was chemically identified by separating their ethyl anthranilate derivatives by thin-layer chromatography and by their enzymatic formation with UDP-bilirubin transferase and cosubstrate: bilirubin diglucuronide, bilirubin monoglucuronide monoglucoside, bilirubin monoglucuronide monoxyloside, bilirubin monoglucuronide (C-8, C-12), bilirubin diglucoside, bilirubin monoglucoside monoxyloside, bilirubin dixyloside, bilirubin monoglucoside (C-8, C-12), and bilirubin monoxyloside. The use of the commercially available mesobilirubin IX alpha as an internal standard was found to facilitate quantitation of the bilirubin conjugates.     

Distribution of oleanolic acid glycosides in vacuoles and cell walls isolated from protoplasts and cells of Calendula officinalis leaves

The contents of oleanolic acid and its 3-0-glucuronide derivatives as well as of 3-0-glucoside derivatives were determined in vacuoles prepared from protoplasts and cell walls obtained from cells of Calendula officinalis leaves. In both cell compartments studied 37% of total cellular oleanolic acid were accumulated, 0.6% occurring as free oleanolic acid (only in vacuoles). Glucuronides accounted for 31.1% (20.7% in vacuoles and 10.4% in cell walls), and glucosides for 5.3% (2.6% in vacuoles and 2.7% in cell walls).     

Sucrose uptake into vacuoles of sugarcane suspension cells

Uptake of sucrose into vacuoles of suspension cells of Saccharum sp. (sugarcane) was investigated using a vacuole-isolation method based on osmotic- and pH-dependent lysis of protoplasts. Vacuoles took up sucrose at high rates without the influence of tonoplast energization on sucrose transport. Neither addition of ATP or pyrophosphate nor dissipation of the membrane potential or the pH gradient by ionophores changed uptake rates appreciably. Generation of an ATP-dependent pH gradient across the tonoplast was measured in vacuoles and tonoplast vesicles by fluorescence quenching of quinacrine. No H⁺ efflux could be measured by addition of sucrose to energized vacuoles or vesicles so that there was no evidence for a sucrose/H⁺ antiport system. Uptake rates of glucose and other sugars were similar to those of sucrose indicating a relatively non-specific sugar uptake into the vacuoles. Sucrose uptake was concentration-dependent, but no clear saturation kinetics were found. Strict dependence on medium pH and inhibition of sucrose transport by p-chloromercuriphenylsulfonic acid (PCMBS) indicate that sucrose uptake into sugarcane vacuoles is a passive, carrier-mediated process.Abbreviations FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - Mes 2-(N-morpholino)ethanesulfonic acid - Mops 3-(N-morpholino)propanesulfonic acid - PCMBS p-chloromercuriphenylsulfonic acid - PPi pyrophosphate This research was supported by the Deutsche Forschungsgemeinschaft. The technical assistance of H. Schroer is gratefully acknowledged.     

The enzyme-catalyzed formation of bilirubin diglucuronide by a solubilized preparation from cat liver microsomes

When bilirubin monoglucoronide is incubated with a preparation from the 105 000 × g-supernatant of deoxycholate-treated cat liver microsomes, bilirubin diglucuronide is formed. This is an UDPglucuronate-dependent reaction whereby bilirubin IXα monoglucuronide is stoichiometrically converted into bilirubin IXα diglucuronide.The pH optimum for the conversion of bilirubin into bilirubin monoglucuronide lies between pH 8.0 and pH 8.8. For the conversion of mono- into diglucuronide two optima were found, one at about pH 6.5 and another at pH 8.1.When incubation was performed at pH 6.5 and the enzyme protein concentration was lowered, bilirubin monoglucuronide started to isomerise. As a result of this isomerisation bilirubin diglucuronide is also formed. Diglucuronide formation according to this mechanism however, can be clearly differentiated from the enzyme-catalyzed diglucuronide formation.By the formation of bilirubin monoglucuronide, one monoglucuronide isomer is preferentially synthesized.The alkaline-labile bilirubin conjugates in the bile of cats and rats have mainly the IXα isomeric structure. This suggests that in these animals bilirubin diglucuronide is formed enzymically as the bilirubin moiety of diglucuronide, formed by means of the isomerisation reaction, has predominantly the XIIα structure.     

The Transport of Oleanolic Acid in Calendula officinalis

The transport of oleanolic acid synthesized from ¹⁴CO₂ in the individual leaves of Calendula officinalis plants was investigated. It was proved that the rate and the direction of the transport depend on the stage of vegetation and the position of the leaf on the plant. The greatest transport rates of oleanolic acid were observed from the upper level leaves towards the inflorescence during the flowering period. The overblowing period was characterized by a slower migration of oleanolic acid from the leaf supplied with radioactive precursor. The migration was directed mainly to the root especially from the low level leaves. Using CH₃¹⁴COONa as precursor, it was established that the root is unable to synthesize oleanolic acid.     

The transport of S-adenosyl-L-methionine in isolated yeast vacuoles and spheroplasts.

1. The properties of S-adenosyl-L-methionine accumulating system for both vacuoles and spheroplasts are described. Yeast vacuoles were obtained by a modified metabolic lysis procedure from spheroplasts of Saccharomyces cerevisiae. 2. Isolated vacuoles accumulate S-adenosyl-L-methionine by means of a highly specific transport system as indicated by competition experiments with structural analogs of S-adenosyl-L-methionine. The S-adenosyl-L-methionine transport system shows saturation kinetics with an apparent Km of 68 muM in vacuoles and 11 muM in spheroplasts. 3. S-Adenosyl-L-methionine accumulation into vacuoles does not require glucose, phosphoenolpyruvic acid, ATP, ADP nor any other tri- or di-phosphorylated nucleotides. It is insensitive to azide and 2,4-dinitrophenol which strongly inhibit the glucose-dependent accumulation of S-adenosyl-L-methionine in spheroplasts. 4. The transport of S-adenosyl-L-methionine into vacuoles is optimal at pH 7.4 and is insensitive to nystatin while the uptake of S-adenosyl-L-methionine into spheroplasts is optimal at pH 5.0 and is strongly sensitive to nystatin. On this basis it has thus been possible to measure both the intracytoplasmic and the intravacuolar pool of S-adenosyl-L-methionine. 5. Our results indicate the existence of a highly specific S-adenosyl-L-methionine transport system in the vacuolar membrane which is clearly different from the one present in the plasma membrane of yeast cells.     

The metabolism of [3−3H]oleanolic acid-3-O-mono-[14C]glucoside in isolated cells from Calendula officinalis leaves

The radioactive precursor, [3?³H]oleanolic acid-3-O-mono-[¹⁴C]glucoside was administrated to isolated cells obtained from the leaves of Calendula officinalis. The radioactivity of the precursor was incorporated into fractions containing free oleanolic acid, individual glucosides, glucuronide F and other glucuronides. The ratio of ³H: ¹⁴C radioactivity in these fractions indicated that glucosides were formed in a process involving direct glycosylation of the precursor, whereas the glucuronides were formed from oleanolic acid released by hydrolysis of the precursor. Dynamics curves showed that glucoside II formed by direct glycosylation of the precursor was intensively transformed to other derivatives.     

Isolation of protoplasts and vacuoles from cell suspension cultures of Coleus blumei Benth.

In order to study the accumulation and transport of rosmarinic acid in suspension cells of Coleus blumei we established an efficient method to isolate protoplasts and vacuoles. Protoplasts were disrupted by an osmotic shock in a medium with basic pH containing ethylenediamine tetraacetic acid. The resulting vacuoles were purified on a two-step Ficoll gradient. The comparison of the rosmarinic acid contents of cells, protoplasts and vacuoles showed that the depside is localized in the vacuole. Data concerning the yield and purity of the vacuoles are presented. In addition we show that at the physiological pH of the cytoplasm rosmarinic acid is present almost exclusively as an anion and cannot pass a membrane by simple diffusion. We therefore propose a carrier system for the transport of rosmarinic acid into the vacuole.Abbreviations EDTA ethylenediamine tetraacetic acid - HEPES 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethane sulfonic acid - HPLC high performance liquid chromatography - MES morpholinoethane sulfonic acid - NADP⁺ ß-nicotinamide adenine dinucleotide phosphate - PEG polyethylene glycol - RA rosmarinic acid - Tris Tris(hydroxymethyl)aminomethane     

Tonoplast stability and survival of isolated vacuoles in different buffers

The mechanism of sucrose transport into vacuoles isolated from leaf tissue has been studied only in barley (Hordeum vulgare) mesophyll cells. In this tissue, sucrose transport was reported to be a facilitated diffusion. We have observed a facilitated diffusion of sucrose into vacuoles isolated from this tissue. However, no pH dependence was observed. Evidence is presented indicating that the pH dependence of sucrose uptake into vacuoles may be an artifact, reflecting tonoplast instability and survival of isolated vacuoles in different buffers. Apparently vacuoles do not withstand exposure to some commonly used buffers.     

Calcium transport into the vacuole of oat roots. Characterization of H+/Ca2+ exchange activity

Calcium (Ca2+) is sequestered into vacuoles of oat root cells through a H+/Ca2+ antiport system that is driven by the proton-motive force of the tonoplast H+-translocating ATPase. The antiport has been characterized directly by imposing a pH gradient in tonoplast-enriched vesicles. The pH gradient was imposed by diluting K+-loaded vesicles into a K+-free medium. Nigericin induced a K+/H+ exchange resulting in a pH gradient of 2 (acid inside). The pH gradient was capable of driving 45Ca2+ accumulation. Ca2+ uptake was tightly coupled to H+ loss as increasing Ca2+ levels progressively dissipated the steady state pH gradient. Ca2+ uptake displayed saturation kinetics with a Km(app) for Ca2+ of 10 microM. The relative affinity of the antiporter for transport of divalent cations was Ca2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. La3+ or Mn2+ blocked Ca2+ uptake possibly by occupying the Ca2+-binding site. Ruthenium red (I50 = 40 microM) and N,N'-dicyclohexylcarbodiimide (I50 = 3 microM) specifically inhibited the H+/Ca2+ antiporter. When driven by pH jumps, the H+/Ca2+ exchange generated a membrane potential, interior positive, as shown by [14C]SCN accumulation. Furthermore, Ca2+ uptake was stimulated by an imposed negative membrane potential. The results support a simple model of one Ca2+ taken up per H+ lost. The exchange transport can be reversed, as a Ca2+ gradient (Ca2+in greater than Ca2+out) was effective in forming a pH gradient (acid inside). We suggest that the H+/Ca2+ exchange normally transports Ca2+ into the vacuole; however, under certain conditions, Ca2+ may be released into the cytoplasm via this antiporter.     

The uptake of acylated anthocyanin into isolated vacuoles from a cell suspension culture of Daucus carota

Anthocyanin-containing vacuoles were isolated from protoplasts of a cell suspension culture of Daucus carota. The vacuoles were stable for at least 2 h as demonstrated by the fact that they showed no efflux of anthocyanin. The uptake of radioactively labelled anthocyanin was time-dependent with a pH optimum at 7.5, and could be inhibited by the protonophore carbonylcyanide m-chlorophenylhydrazone. Furthermore, the transport was specific, since vacuoles from other plant species showed no uptake of labelled anthocyanin, and strongly depended on acylation with sinapic acid, as deacylated glycosides were not taken up by isolated vacuoles. Hence, it is suggested that the acylation of anthocyanin, which is also required for the stabilization of colour in vacuoles, is important for transport, and that acylated anthocyanin is transported by a selective carrier and might be trapped by a pH-dependent conformational change of the molecule inside the acid vacuolar sap.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - PVP polyvinylpyrrolidone - TLC thin-layer chromatography     

Characterization of a specific transport system for arginine in isolated yeast vacuoles.

The transport of L-arginine was studied in isolated vacuoles of Saccharomyces cerevisiae. A centrifugation method allowed rapid separation of the fragile vacuoles from the incubation media so that initial uptake rates of [14C]arginine could be measured. Labelled arginine added to the medium was accumulated in the isolated vacuoles; it was found to exchange specifically with the arginine already present in the vacuoles. Such an exchange did not take place in intact spheroplasts. The pH dependence of the arginine transport in the vacuoles was tested. As the vacuoles are unstable in the pH range of optimal transport activity (pH above 7.0), the pH optimum of the transport reaction could not be determined. From the temperature dependence, the apparent energy of activation was calculated to be 9800 cal/mol. Arginine transport shows saturation kinetics with an apparent Km of 30 muM in the isolated vacuoles, and of 1.5 muM in the spheroplasts. Competition experiments with amino acids and arginine analogues demonstrated that the arginine transport in both vacuoles and spheroplasts, is highly specific. The two systems, however, were shown to have distinct specificities. The inhibition of vacuolar L-arginine transport by D-arginine, L-histidine, and L-canavanine was competitive with apparent Ki values of 60 muM, 400 muM and 600 muM respectively.     

Rational design and synthesis of topoisomerase I and II inhibitors based on oleanolic acid moiety for new anti-cancer drugs

Semisynthetic reactions were conducted on oleanolic acid, a common plant-derived oleanane-type triterpene. Ten rationally designed derivatives of oleanolic acid were synthesized based on docking studies and tested for their topoisomerase I and IIα inhibitory activity. Semisynthetic reactions targeted C-3, C-12, C-13, and C-17. Nine of the synthesized compounds were identified as new compounds. The structures of these compounds were confirmed by spectroscopic methods (1D, 2D NMR and MS). Five oleanolic acid analogues (S2, S3, S5, S7 and S9) showed higher activity than camptothecin (CPT) in the topoisomerase I DNA relaxation assay. Four oleanolic acid analogues (S2, S3, S5 and S6) showed higher activity than etoposide in a topoisomerase II assay. The results indicated that the C12–C13 double bond of the oleanolic acid skeleton is important for the inhibitory activity against both types of topoisomerases, while insertion of a longer chain at either position 3 or 17 increases the activity against topoisomerases by various degrees. Some of the synthesized compounds act as dual inhibitors for both topoisomerase I and IIα.     

Enzymatic conversion of bilirubin monoglucuronide to diglucuronide by rat liver plasma membranes.

Formation of bilirubin monoglucuronide from unconjugated bilirubin requires a microsomal enzyme, UDP-glucuronate glucuronyltransferase (EC 2.4.1.17). Conversion of bilirubin monoglucuronide to bilirubin diglucuronide, the major bilirubin conjugate in bile, was studied in subcellular fractions of rat liver. The highest specific activity for bilirubin diglucuronide formation occurred in a fraction highly enriched in plasma membranes. Studies of reaction stoichiometry and utilization of UDP-D-[14C]glucuronic acid revealed that conversion of bilirubin monoglucuronide to bilirubin diglucuronide is not catalyzed by UDP-glucuronyltransferase, and results from transglucuronidation of bilirubin monoglucuronide, with formation of bilirubin diglucuronide and unconjugated bilirubin. When unconjugated bilirubin was infused intravenously into rats at rates exceeding the maximal hepatic excretory capacity, bilirubin monoglucuronide accumulated in serum and bilirubin diglucuronide was found exclusively in bile as the predominant bilirubin metabolite. These results suggest that formation of bilirubin diglucuronide occurs at the surface membrane of the liver cell. Conversion of bilirubin monoglucuronide to bilirubin diglucuronide may play a role in the transport of bilirubin glucuronides from liver to bile.     

The structure of sialyl-glycopeptides of the O-glycosidic type, isolated from sialidosis (mucolipidosis I) urine

Plants performing crassulacean acid metabolism show a large nocturnal accumulation of malic acid in the vacuole of the photosynthetic cells. It has been postulated that an H+-translocating ATPase energizes the transport of malic acid across the tonoplast into the vacuole. In the present work we have characterized the ATPase activity associated with vacuoles of the crassulacean-acid-metabolism plant Kalancho? daigremontiana and compare it with other phosphohydrolases. Vacuoles were isolated by polybase-induced lysis of mesophyll-cell protoplasts. The vacuoles had a high activity of unspecific acid phosphatase (pH optimum 5.3). The acid phosphatase was strongly inhibited by ammonium molybdate (with 50% inhibition at about 0.5 mmol m-3), but was not completely inhibited even at much higher ammonium-molybdate concentrations. In contrast, the vacuolar ATPase activity, assayed in the presence of 100 mmol m-3 ammonium molybdate, had a pH optimum of 8.0. ATP was the preferred substrate, but GTP, ITP and ADP were hydrolyzed at appreciable rates. The mean ATPase activity at pH 8.0 was 14.5 nmol h-1 (10(3) vacuoles)-1, an average 13% of which was attributable to residual acid-phosphatase activity. Inorganic-pyrophosphatase activity could not be demonstrated unambiguously. The vacuolar ATPase activity was Mg2+-dependent, had an apparent Km for MgATP2- of 0.31 mol m-3, and was 32% stimulated by 50 mol m-3 KCl. Of the inhibitors tested, oligomycin slightly inhibited the vacuolar ATPase activity and diethylstilbestrol and NO-3 were both markedly inhibitory. Dicyclohexylcarbodiimide and tributyltin were also strongly inhibitory. Tributyltin caused a 50% inhibition at about 0.3 mmol m-3. This is taken as evidence that the vacuolar ATPase might function as an H+-translocating ATPase. It is shown that the measured activity of the vacuolar ATPase would be of the right order to account for the observed rates of nocturnal malic-acid accumulation in K. daigremontiana.     

Isolation and characterization of autophagic vacuoles from rat kidney cortex

The number of autophagic vacuoles in the proximal tubule cells of the rat kidney increased considerably after 3 h of vinblastine treatment. This increase was paralleled by stimulated proteolysis in an homogenate prepared from the cortex. We have taken advantage of this expansion in autophagic vacuoles in an effort to isolate these organelles from rat kidney cortex on a discontinuous Metrizamide gradient. Autophagic vacuoles have recently been purified from liver but not from other tissues. The purity of the isolated fraction was 95% of which 55% consisted of typical intact autophagic vacuoles containing sequestered organelles and 45% of other types of secondary lysosome. On plane section many of these displayed one or several intramatrical vesicles or flap like processes forming apparent vesicles at the pole of the organelles, which occasionally contained pinocytosed membranous material. These lysosomes were designated microautophagic vacuoles. It is suggested that the microautophagic vacuoles could be the morphological expression of uptake into lysosomes of small portions of cytosol. The isolated autophagic vacuole fraction was enriched in lysosomal enzymes (acid phosphatase and cathepsin D activities) and displayed high proteolytic rates, especially at acid pH.     

Proton Gradient-Driven Nickel Uptake by Vacuolar Membrane Vesicles of Saccharomyces cerevisiae

A vacuolar H⁺-ATPase-negative mutant of Saccharomyces cerevisiae was highly sensitive to nickel ion. Accumulation of nickel ion in the cells of this mutant of less than 60% of the value for the parent strain arrested growth, suggesting a role for this ATPase in sequestering nickel ion into vacuoles. An artificially imposed pH gradient (interior acid) induced transient nickel ion uptake by vacuolar membrane vesicles, which was inhibited by collapse of the pH difference but not of the membrane potential. Nickel ion transport into vacuoles in a pH gradient-dependent manner is thus important for its detoxification in yeast.     

Isolation and partial characterization of vacuoles from tobacco protoplasts

Protoplasts from suspension-cultured cells of Nicotiana glutinosa L. were lysed in 0.3 molar sorbitol in 2 millimolar ethylenediaminetetraacetate-tris(hydroxymethyl) aminomethane (pH 7.5) to release intact vacuoles. The vacuoles were purified by centrifugation in a Ficoll step gradient. About 11% of the vacuoles and 13% of the acid phosphatase activity was recovered in the purified vacuole fraction, suggesting that the vacuole is the major site for acid phosphatase in these cells. NADH-cytochrome c reductase, malate dehydrogenase, and cytochrome c oxidase activities were reduced during vacuole purification. The majority of the adenosine 5′-triphosphate (ATP) hydrolytic activity of purified vacuoles was associated with nonspecific acid phosphatase and not with a transport ATPase. As judged by acid phosphatase distribution and electron microscopy, the effective density of vacuoles in a sucrose gradient was low (less than 1.1 grams per cubic centimeter), although an unequivocal estimate of the vacuole or tonoplast density was not possible from the experiments conducted.