Lipolytic activity of ricin from Ricinus sanguineus and Ricinus communis on neutral lipids

2001 ◽  
Vol 358 (3) ◽  
pp. 773-781 ◽  
Author(s):  
Sophie LOMBARD ◽  
Mohamed E. HELMY ◽  
Gérard PIÉRONI
Keyword(s):  
Surface Pressure ◽  
Specific Activity ◽  
Neutral Lipids ◽  
Ricinus Communis ◽  
Lipolytic Activity ◽  
Fatty Acid Esters ◽  
Lipid Monolayer ◽  
Serine Esterase ◽  
Order Of Magnitude ◽  
Esterase Inhibitor

The present study was carried out with a view of determining ricin lipolytic activity on neutral lipids in emulsion and in a membrane-like model. Using 2,3-dimercapto-1-propanol tributyrate (BAL-TC4) as substrate, the lipolytic activity of ricin was found to be proportional to ricin and substrate concentrations, with an apparent Km (Km,app) of 2.4mM, a kcat of 200min−1 and a specific activity of 1.0unit/mg of protein. This work was extended to p-nitrophenyl (pNP) fatty acid esters containing two to twelve carbon atoms. Maximum lipolytic activity was registered on pNP decanoate (pNPC10), with a Km,app of 3.5mM, a kcat of 173min−1 and a specific activity of 3.5units/mg of protein. Ricin lipolytic activity is pH and galactose dependent, with a maximum at pH7.0 in the presence of 0.2M galactose. Using the monolayer technique with dicaprin as substrate, ricin showed a lipolytic activity proportional to the ricin concentration at 20mN/m, which is dependent on the surface pressure of the lipid monolayer and is detectable up to 30mN/m, a surface pressure that is of the same order of magnitude as that of natural cell membranes. The methods based on pNPC10 and BAL-TC4 hydrolysis are simple and reproducible; thus they can be used for routine studies of ricin lipolytic activity. Ricin from Ricinus communis and R. sanguineus were treated with diethyl p-nitrophenylphosphate, an irreversible serine esterase inhibitor, and their lipolytic activities on BAL-TC4 and pNPC10, and cytotoxic activity, were concurrently recorded. A reduction in lipolytic activity was accompanied by a decrease in cytotoxicity on Caco2 cells. These data support the idea that the lipolytic activity associated with ricin is relevant to a lipase whose activity is pH and galactose dependent, sensitive to diethyl p-nitrophenylphosphate, and that a lipolytic step may be involved in the process of cell poisoning by ricin. Both colorimetric tests used in this study are sensitive enough to be helpful in the detection of possible lipolytic activities associated with other cytotoxins or lectins.

scholarly journals Lipolytic activity of ricin from Ricinus sanguineus and Ricinus communis on neutral lipids

2001 ◽  
Vol 358 (3) ◽  
pp. 773 ◽  
Author(s):  
Sophie LOMBARD ◽  
Mohamed E. HELMY ◽  
Gérard PIÉRONI
Keyword(s):  
Neutral Lipids ◽  
Ricinus Communis ◽  
Lipolytic Activity

Poly(β-L-malate) hydrolase from Plasmodia of Physarum polycephalum

1995 ◽  
Vol 41 (13) ◽  
pp. 192-199 ◽  
Author(s):  
Christian Korherr ◽  
Michael Roth ◽  
Eggehard Holler
Keyword(s):  
Hydrophobic Interaction ◽  
Malic Acid ◽  
Physarum Polycephalum ◽  
Culture Medium ◽  
Specific Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Reduced Form ◽  
Serine Esterase ◽  
Hydroxybutyric Acid ◽  
Ph Optimum

A 68-kDa extracellular glycoprotein from Physarum polycephalum that hydrolyses specifically poly(β-L-malic acid) by removing monomers of L-malic acid in an exolytic manner has been purified and characterized. The enzyme was purified 1740-fold from the culture medium by ammonium sulfate precipitation, hydrophobic interaction chromatography on butyl-Toyopearl, and gel permeation chromatography on Superdex 200 to a specific activity of 9.0 μmol∙min−1∙mg−1. The hydrolase was also purified from the cytosol, which contained 1 mg in 43 g cells in contrast to 1 mg extracellular enzyme in 28 L of culture medium. The pH optimum was pH 3.5 as a result of the effect of an acidic side chain on Vmax and the preferred binding of poly(β-L-malate) in the ionized form. Intracellular hydrolase was only marginally active on [14C]poly(β-L-malate) that had been injected into plasmodia. Poly(L-aspartate), poly(L-glutamate), poly(vinyl sulfate), and poly(acrylate) were neither bound nor degraded by the hydrolase. Poly(β-hydroxybutyric acid), which was considered the reduced form of poly(β-L-malate), was not a substrate. The enzyme is neither a metallo- nor a serine-esterase, and is distinct from poly(3-hydroxybutyric acid) depolymerases. It is related to a glucosidase with respect to hydrophobic interaction chromatography, the pH-activity dependence, and its inhibition with mercuribenzoate, N-bromosuccinimide, and D-gluconolactone, but not the use of the substrates.Key words: poly(β-L-malate), polymalatase, Physarum polycephalum, biodegradative polymer.

scholarly journals Re-evaluation of the interaction of malonyl-CoA with the rat liver mitochondrial carnitine palmitoyltransferase system by using purified outer membranes

1990 ◽  
Vol 267 (1) ◽  
pp. 85-90 ◽  
Author(s):  
M P Kolodziej ◽  
V A Zammit
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Mitochondria ◽  
Subsequent Treatment ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Outer Membranes ◽  
High Affinity ◽  
Malonyl Coa ◽  
Order Of Magnitude

1. The interaction of malonyl-CoA with the outer carnitine palmitoyltransferase (CPT) system of rat liver mitochondria was re-evaluated by using preparations of highly purified outer membranes, in the light of observations that other subcellular structures that normally contaminate crude mitochondrial preparations also contain malonyl-CoA-sensitive CPT activity. 2. In outer-membrane preparations, which were purified about 200-fold with respect to the inner-membrane-matrix fraction, malonyl-CoA binding was largely accounted for by a single high-affinity component (KD = 0.03 microM), in contrast with the dual site (low- and high-affinity) previously found with intact mitochondria. 3. There was no evidence that the decreased sensitivity of CPT to malonyl-CoA inhibition observed in outer membranes obtained from 48 h-starved rats (compared with those from fed animals) was due to a decreased ratio of malonyl-CoA binding to CPT catalytic moieties. Thus CPT specific activity and maximal high-affinity [14C]malonyl-CoA binding (expressed per mg of protein) were increased 2.2- and 2.0-fold respectively in outer membranes from 48 h-starved rats. 4. Palmitoyl-CoA at a concentration that was saturating for CPT activity (5 microM) decreased the affinity of malonyl-CoA binding by an order of magnitude, but did not alter the maximal binding of [14C]malonyl-CoA. 5. Preincubation of membranes with either tetradecylglycidyl-CoA or 2-bromopalmitoyl-CoA plus carnitine resulted in marked (greater than 80%) inhibition of high-affinity binding, concurrently with greater than 95% inhibition of CPT activity. These treatments also unmasked an effect of subsequent treatment with palmitoyl-CoA to increase low-affinity [14C]malonyl-CoA binding. 6. These data are discussed in relation to the possible mechanism of interaction between the malonyl-CoA-binding site and the active site of the enzyme.

scholarly journals Dynamics of Polysaccharides and Neutral Lipids during Anther Development in Castor (Ricinus communis)

2015 ◽  
Vol 140 (4) ◽  
pp. 356-361 ◽  
Author(s):  
Dongmei Wei ◽  
Huimin Xu ◽  
Ruili Li
Keyword(s):  
Neutral Lipids ◽  
Ricinus Communis ◽  
Starch Content ◽  
Single Layer ◽  
Pollen Grains ◽  
Middle Layer ◽  
Anther Development ◽  
Anther Wall ◽  
Starch Grains ◽  
Tapetal Cells

Anthers contain starch and neutral lipids, which have key roles in microspore ontogeny and gametophyte development. In this study, we observed the dynamic changes in starch and neutral lipids in the anther developmental processes of castor (Ricinus communis) by cytochemical methods. Starch grains and neutral lipids presented a regular dynamic distribution during anther development. In young anthers, some neutral lipids accumulated in sporogenous cells, whereas neutral lipids disappeared with microspore growth. At the late microspore stage, starch grains began to accumulate in microspores, and the starch content of bicellular pollen significantly increased after microspore mitosis. At anthesis, starch grains and neutral lipids accumulated in the mature pollen grains. Visible changes occurred in anther wall cells. The epidermis, middle layer, and tapetum were degenerated, and only a single layer of endothecium remained at anthesis. The dynamic variation of starch grains and neutral lipids in tapetal cells was consistent with the changes in microspores and pollen during anther development. All these findings demonstrated that tapetal cells directly interacted with the developing gametophytes. The tapetal cells play an important role in supplying nutritional substances for microspore absorption. Moreover, the endothecium protects the pollen and contributes to anther dehiscence. The results of this study provide a foundation for the further research on sexual reproduction in angiosperms.

scholarly journals A high-performance oxygen evolution catalyst in neutral-pH for sunlight-driven CO2 reduction

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Li Qin Zhou ◽  
Chen Ling ◽  
Hui Zhou ◽  
Xiang Wang ◽  
Joseph Liao ◽  
...  
Keyword(s):  
Oxygen Evolution ◽  
High Performance ◽  
Specific Activity ◽  
Co2 Reduction ◽  
Poor Performance ◽  
Electrolysis Cell ◽  
Neutral Ph ◽  
Highly Active ◽  
Order Of Magnitude ◽  
Neutral Conditions

Abstract The efficiency of sunlight-driven reduction of carbon dioxide (CO2), a process mimicking the photosynthesis in nature that integrates the light harvester and electrolysis cell to convert CO2 into valuable chemicals, is greatly limited by the sluggish kinetics of oxygen evolution in pH-neutral conditions. Current non-noble metal oxide catalysts developed to drive oxygen evolution in alkaline solution have poor performance in neutral solutions. Here we report a highly active and stable oxygen evolution catalyst in neutral pH, Brownmillerite Sr2GaCoO5, with the specific activity about one order of magnitude higher than that of widely used iridium oxide catalyst. Using Sr2GaCoO5 to catalyze oxygen evolution, the integrated CO2 reduction achieves the average solar-to-CO efficiency of 13.9% with no appreciable performance degradation in 19 h of operation. Our results not only set a record for the efficiency in sunlight-driven CO2 reduction, but open new opportunities towards the realization of practical CO2 reduction systems.

scholarly journals Regulation of Fibrinolysis Through Activation and Inhibition of Activation of a Plasminogen Proactivator (Preurokinase).

1977 ◽  
Author(s):  
M.B. Bernik ◽  
E.P. Oiler
Keyword(s):  
Tissue Culture ◽  
Dose Response ◽  
Antithrombin Iii ◽  
Specific Activity ◽  
Esterase Inhibitor

Purified preparations of a plasminogen proactivator, preurokinase (preUK), the enzyme plasmin and thrombin and native inhibitors of these enzymes (C’l-esterase inhibitor and antithrombin III) were used to study factors and events which may participate in the regulation of fibrinolysis by facilitating or hindering conversion of preUK to UK.PreUK, expressed in CTA units of generated UK, was purified from supernates of kidney cultures by chromatography on Sephadex G-100 columns to specific activity of 20,000 CTA U/mg protein and was studied by immunodiffusion and immunoelectrophoresis with antisera to highly purified urinary and tissue culture UK. In assays on fibrin plates, generation of UK activity (3-4 CTA U/ml) from pre UK occurred with traces of plasmin (0.01 CTA< U/ml) or trypsin (10 BAEE U/ml) but not with thrombin (0.01-100 NIH U/ml). Inhibition of UK generation occurred in two ways: 1) by inhibition of the activating enzymes, and 2) by inactivation of pre UK itself. The former was demonstrated with C’l-esterase inhibitor which inhibited the fibrinolytic as well as preUK-activating properties of plasmin and trypsin, and the latter with thrombin which inactivated preUK progressively in a dose-response fashion without being itself consumed.Thus, 3 CTA U/nl preUK were inactivated in 20 min or less at 37°C by 2.5 NIH U/ml thrombin and in more than 3 hr by 0.01 NIH U/ml. Thrombin-induced preUK inactivation was inhibited by antithrombin III indicating that while thrombin acted as an inhibitor of fibrinolysis by inactivating preUK, antithrombin III facilitated fibrinolysis indirectly by protecting preUK from thrombin.

scholarly journals Persistent Activities of Extracellular Enzymes Adsorbed to Soil Minerals

2020 ◽  
Vol 8 (11) ◽  
pp. 1796
Author(s):  
Folasade K. Olagoke ◽  
Klaus Kaiser ◽  
Robert Mikutta ◽  
Karsten Kalbitz ◽  
Cordula Vogel
Keyword(s):  
Enzyme Activities ◽  
Active Sites ◽  
Extracellular Enzymes ◽  
Specific Activity ◽  
High Specific Activity ◽  
Enzyme Release ◽  
Soil Mineral ◽  
Specific Enzyme ◽  
Soil Minerals ◽  
Order Of Magnitude

Adsorption of extracellular enzymes to soil minerals is assumed to protect them against degradation, while modifying their activities at the same time. However, the persistence of the activity of adsorbed enzymes remains poorly understood. Therefore, we studied the persistence of cellulase and α-amylase activities after adsorption to soil amended with various amounts (+1, +5, and +10 wt.%) of three typical soil minerals, montmorillonite, kaolinite, and goethite. Soil without mineral addition (pure soil), pure minerals, and pure dissolved enzymes were used as references. Soil mineral–enzyme complexes were prepared and then incubated for 100 days; temporal changes in enzyme activities were analyzed after 0, 0.1, 1, 10, and 100 days. The specific enzyme activities (activities normalized to protein content) and their persistence (activities relative to activities at day 0) were compared to enzyme activities in solution and after sorption to the control soil. Amylase adsorption to pure minerals increased in the following order: montmorillonite > kaolinite > goethite. That of cellulase increased in the following order: goethite > montmorillonite > kaolinite. Adsorption of enzymes to soils did not increase in the same order of magnitude as the addition of reactive binding sites. Based on inverse relationships between the amount of enzyme adsorbed and the specific enzyme activity and their persistency, we showed that a limited availability of sorption sites is important for high specific activity and persistence of the enzymes. This is probably the consequence of less and weaker bonds, as compared to a high availability of sorption sites, resulting in a smaller impact on the active sites of the enzyme. Hence, we suppose that the soil mineral phase supports microorganisms in less-sorptive environments by saving energy on enzyme production, since small enzyme release could already result in sufficient activities to degrade respective target carbon substrates.

scholarly journals Production by Human Lung Cells of Trypsin and Plasmin Inhibitor(s) Differing from a1-Antitrypsin, a2-Macroglobulin, C1-Esterase Inhibitor and Inter-a-Trypsin Inhibitor

1977 ◽  
Author(s):  
Maria B. Bernik
Keyword(s):  
Trypsin Inhibitor ◽  
Inhibitory Activity ◽  
Human Lung ◽  
Specific Activity ◽  
Cross Reaction ◽  
Lung Cells ◽  
C1 Esterase Inhibitor ◽  
Human Lung Cells ◽  
Wide Range ◽  
Esterase Inhibitor

Human lung cells in primary culture and serial subculture were used to study the production of inhibitory activity and to isolate and identify inhibitor(s) of trypsin and plasmin produced and released by the cells into the supernatant medium. Assays of inhibitory activity were performed on fibrin, and casein substrate and results expressed in BAEE units of trypsin inhibited on the former substrate. Inhibitory activity against plasmin and trypsin accumulated progressively in serum free supernates of cultures to concentrations of 80–150 BAEE units/ml and was isolated from the supernates by concentration with Amicon PM 30 membranes, gel filtration on Sephadex G—100 or G-200 columns and polyacrilamide gel electrophoresis. On calibrated columns inhibitory activity eluted in the range of 75,000 mol wt substances. On immunodiffusion, performed using a wide range of concentrations of chromatographed inhibitor preparations with specific activity of about 1,300 BAEE units/mg protein there was no cross-reaction with antiserum to a1-antitrypsin, a2-macroglobulin, C1-esterase inhibitor or inter-a-trypsin inhibitor. There was also no cross-reaction with antiserum to antithrombin III or antichymotrypsin. Immunoelectrophoresis showed no immunoreactive material with the antisera. These observation indicate the production in lung of inhibitor(s) differing from the major protease inhibitors and derivatives or subunits of these inhibitors described to date.

Lipid metabolism in rabbit lungs

1970 ◽  
Vol 48 (2) ◽  
pp. 170-177 ◽  
Author(s):  
B. M. J. Wolfe ◽  
B. Anhalt ◽  
J. C. Beck ◽  
D. Rubinstein
Keyword(s):  
Lipid Metabolism ◽  
Intravenous Injection ◽  
Specific Activity ◽  
Neutral Lipids ◽  
Rabbit Lung ◽  
Alveolar Cell ◽  
Methylation Pathway ◽  
Alveolar Tissue ◽  
Specific Activities ◽  
Rapid Drop

The incorporation of palmitate-3H into phospholipids and neutral lipids by alveolar slices from rabbit lung rose with increasing medium palmitate concentration, but glycerol-U-14C incorporation was inadequate to account for all of the palmitate-3H esterified. The highest specific activities with respect to palmitate-3H were found in the triglycerides, diglycerides, and phosphatidylcholine fractions, although that of the phosphatidyl-N,N-dimethylethanolamine was greatest in the early stages of incubation. Incubation of slices with the labelled precursors for 5 min followed by a chase of 175 min resulted in a rapid drop in the specific activity of the phosphatidyl-N,N-dimethylethanolamine and diglycerides with concomitant increases in phosphatidylcholine and triglycerides. However, the radioactivity of the lecithin was too great and the specific activity of the phosphatidylethanolamine too low for the methylation pathway to be of importance in the synthesis of lecithin.Lysophosphatidylcholine could be converted to phosphatidylcholine by lung slices but its addition did not increase the esterification of palmitate. Methionine-methyl-14C was incorporated into phospholipids only slightly, phosphatidylserine having the highest specific activity.Following the intravenous injection of the labelled precursors, half the radioactivity in the lung was found in the phosphatidylcholine fraction of the alveolar tissue, little radioactivity being found in the bronchiolar tissue and the alveolar cell-free saline washings. The highest specific activities were found in the triglycerides and phosphatidylcholine fractions in all three tissue components.

scholarly journals Activity of key enzymes involved in glucose and triglyceride catabolism during bovine oocyte maturation in vitro

Reproduction ◽  
2002 ◽  
pp. 675-681 ◽  
Author(s):  
P Cetica ◽  
L Pintos ◽  
G Dalvit ◽  
M Beconi
Keyword(s):  
Enzymatic Activity ◽  
Pentose Phosphate Pathway ◽  
In Vitro Maturation ◽  
Specific Activity ◽  
Lipolytic Activity ◽  
Cumulus Cells ◽  
Pentose Phosphate ◽  
Key Enzymes ◽  
Glucose 6 Phosphate Dehydrogenase

Little is known about the metabolic profile of cumulus-oocyte complexes (COCs) during maturation. The aim of this study was to determine the differential participation of enzymatic activity in cumulus cells and the oocyte during in vitro maturation of bovine oocytes, by measuring the activity of key enzymes involved in the regulation of glycolysis (phosphofructokinase), the pentose phosphate pathway (glucose-6-phosphate dehydrogenase) and lipolysis (lipase). COCs were matured in medium 199 plus 10% (v/v) steer serum for 22-24 h at 39 degrees C in 5% CO(2):95% humidified air. Phosphofructokinase, glucose-6-phosphate dehydrogenase and lipase activities were measured in immature and in vitro matured COCs, denuded oocytes and cumulus cells, respectively. Phosphofructokinase and glucose-6-phosphate dehydrogenase activities (enzymatic units) remained constant during in vitro maturation of COCs, but there was a significant decrease in lipase activity (units) (P < 0.05), as activity in cumulus cells decreased significantly (P < 0.05). For the three enzymes studied, enzyme activity (units) remained unchanged in the oocyte during in vitro maturation. Specific activity increased in the oocyte (P < 0.05) and decreased in cumulus cells as a result of maturation (P < 0.05). In cumulus cells, phosphofructokinase was the most abundant of the three enzymes followed by glucose-6-phosphate dehydrogenase and then lipase (P < 0.05), whereas in the denuded oocyte this order was reversed (P < 0.05). Thus, the metabolism of cumulus cells is adapted to control the flow of metabolites toward the oocyte, which maintains its enzymatic activity even when dissociated from cumulus cells during maturation. The high activity of phosphofructokinase in cumulus cells indicates that glucose is metabolized mainly via the glycolytic pathway in these cells. The greater relative activity of glucose-6-phosphate dehydrogenase recorded in the oocyte indicates that glucose uptake could be directed mainly toward the pentose phosphate pathway. The marked lipolytic activity concentrated in the oocyte indicates an active participation in lipid catabolism during maturation.

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