scholarly journals Vacuolar Acidification in Citrus Fruit: Comparison between Acid Lime (Citrus aurantifolia) and Sweet Lime (Citrus limmetioides) Juice Cells

2002 ◽  
Vol 127 (2) ◽  
pp. 171-177 ◽  
Author(s):  
Andreas Brune ◽  
Mathias Müller ◽  
Lincoln Taiz ◽  
Pedro Gonzalez ◽  
Ed Etxeberria
Keyword(s):  
Atpase Activity ◽  
Gel Filtration ◽  
Citrus Fruit ◽  
Partial Purification ◽  
Ph Gradients ◽  
Permeable Membrane ◽  
Citrus Aurantifolia ◽  
Sweet Lime ◽  
Vacuolar Acidification ◽  
Acid Lime

Vacuolar acidification was investigated in `Palestine' sweet (Citrus limmetioides Tanaka) and `Persian' acid lime [(Citrus aurantifolia (Christm.) Swingle] (vacuolar pHs of 5.0 and 2.1, respectively) using tonoplast vesicles isolated from juice cells. The ATPase activity of tonoplast-enriched vesicles from sweet limes was strongly inhibited by bafilomycin A1 and NO3-, but was unaffected by vanadate. In contrast, the ATPase activity in acid lime membranes was only slightly inhibited by bafilomycin A1 and NO3- and was strongly inhibited by high concentrations of vanadate. The vacuolar origin of the acid lime vesicles was confirmed by immunoblotting. After solubilization and partial purification of the two enzymes by gel filtration, their inhibitor profiles were largely unchanged. Based on equal ATPase activities, vesicles from sweet and acid limes were able to generate similar pH gradients. However, in tonoplast vesicles from sweet limes, the maximum ΔpH was reached four times faster than in those from acid limes. Addition of ethylenediamine tetraacetic acid (EDTA) to chelate Mg+2 after the maximal ΔpH was attained resulted in collapse of the pH gradient in vesicles from sweet limes, whereas no change in ΔpH was observed in vesicles from acid limes, indicating a less H+ permeable membrane. Vacuolar ATPases from both cultivars exhibited identical pH optima and showed similar Mg+2 dependence, but only the acid lime ATPase activity was inhibited by Ca+2. These data confirm that the vanadate-sensitive form of the V-ATPase found in lemon and acid limes is specific to hyperacidifying tissues rather than to citrus juice cells. Sweet lime vacuoles bear the classical V-ATPase also found in vegetative plant tissues.

scholarly journals Partial Purification and Characterisation of Pectinase Produced by Aspergillus niger LFP-1 Grown on Pomelo Peels as a Substrate

2021 ◽  
Vol 32 (1) ◽  
pp. 1-22
Author(s):  
Mohd Taufiq Mat Jalil ◽  
◽  
Darah Ibrahim ◽  
◽  
Keyword(s):  
Aspergillus Niger ◽  
Column Chromatography ◽  
Metal Ion ◽  
Specific Activity ◽  
Gel Filtration ◽  
Citrus Fruit ◽  
Crude Enzyme ◽  
Partial Purification ◽  
Anion Exchange Column ◽  
C 50

In the present study, pectinase was produced by local fungal isolate, Aspergillus niger LFP-1 grown on pomelo peels as a sole carbon source under solid-state fermentation (SSF). The purification process begins with the concentration of crude enzyme using ammonium sulfate precipitation and followed by purification using anion-exchange column chromatography (DEAE-Sephadex) and subsequently using gel filtration column chromatography (Sephadex G-100). On the other hand, the molecular weight of the purified enzyme was determined through SDS-PAGE. The findings revealed the crude enzyme was purified up to 75.89 folds with a specific activity of 61.54 U/mg and the final yield obtained was 0.01%. The molecular mass of the purified pectinase was 48 kDa. The optimum pH and temperature were 3.5 and 50°C, respectively. This enzyme was stable at a range of pH 3.5 to 4.5 and a relatively high temperature (40°C–50°C) for 100 min. The Km and Vmax were found to be 3.89 mg/mL and 1701 U/mg, respectively. Meanwhile, pectin from citrus fruit and the metal ion (Co2+) were the best substrate and inducer to enhance pectinase yield, respectively.

Studies on an Inhibitor of Plasminogen Activation in Human Serum

1973 ◽  
Vol 30 (02) ◽  
pp. 414-424 ◽  
Author(s):  
Ulla Hedner
Keyword(s):  
Ion Exchange ◽  
Human Serum ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Specific Adsorption ◽  
Partial Purification ◽  
Plasminogen Activation ◽  
Preparative Electrophoresis

SummaryA procedure is described for partial purification of an inhibitor of the activation of plasminogen by urokinase and streptokinase. The method involves specific adsorption of contammants, ion-exchange chromatography on DEAE-Sephadex, gel filtration on Sephadex G-200 and preparative electrophoresis. The inhibitor fraction contained no antiplasmin, no plasminogen, no α1-antitrypsin, no antithrombin-III and was shown not to be α2 M or inter-α-inhibitor. It contained traces of prothrombin and cerulo-plasmin. An antiserum against the inhibitor fraction capable of neutralising the inhibitor in serum was raised in rabbits.

Plasminogen-Activator in Human Early Milk: Its Partial Purification and Characterization

1981 ◽  
Vol 45 (02) ◽  
pp. 121-126 ◽  
Author(s):  
Utako Okamoto ◽  
Noboru Horie ◽  
Yoko Nagamatsu ◽  
Jun-Ichiro Yamamoto
Keyword(s):  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Partial Purification ◽  
Order Kinetic ◽  
Diisopropyl Fluorophosphate ◽  
Step Procedure ◽  
Activity Band ◽  
C 50 ◽  
Gel Isoelectric Focusing

SummaryMilk plasminogen-activator was partially purified from human transitional milk collected at about 10 days after delivery, by a five-step procedure involving chloroform treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-150, CM Sephadex C-50 and DEAE Sephadex A-50. This gave milk-activator with a maximum purification factor of about 2,400-fold with respect to the skimmed milk. The CM Sephadex-step preparation showed, on polyacrylamide gel electrophoresis, a single plasminogen-activator activity band located between the bands of albumin and prealbumin of human serum. This preparation exhibited no kinin forming activity. The activator hydrolyzed acetyl-glycyl-L-lysine methyl ester with similar order kinetic constants to urokinase, and was inhibited strongly by diisopropyl-fluorophosphate. The molecular weight of the activator as estimated by gel filtration was approximately 86,000, the isoelectric points as estimated by gel isoelectric focusing were pH 7.2, 6.9 and 6.6, and the activator activity was not quenched by antiurokinase globulin, indicating that the milk-activator is a different entity from urokinase.

scholarly journals Hepatic endosome fractions contain an ATP-driven proton pump

1985 ◽  
Vol 225 (1) ◽  
pp. 51-58 ◽  
Author(s):  
T Saermark ◽  
N Flint ◽  
W H Evans
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Atpase Activity ◽  
Proton Pump ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Ph Gradients ◽  
Subcellular Organelle ◽  
Liver Homogenates

Endosome fractions were isolated from rat liver homogenates on the basis of the subcellular distribution of circulating ligands, e.g. 125I-asialotransferrin internalized by hepatocytes by a receptor-mediated process. The distribution of endocytosed 125I-asialotransferrin 1-2 min and 15 min after uptake by liver and a monensin-activated Mg2+-dependent ATPase activity coincided on linear gradients of sucrose and Nycodenz. The monensin-activated Mg2+-ATPase was enriched relative to the liver homogenates up to 60-fold in specific activity in the endosome fractions. Contamination of the endosome fractions by lysosomes, endoplasmic reticulum, mitochondria, plasma membranes and Golgi-apparatus components was low. By use of 9-aminoacridine, a probe for pH gradients, the endosome vesicles were shown to acidify on addition of ATP. Acidification was reversed by addition of monensin. The results indicate that endosome fractions contain an ATP-driven proton pump. The ionophore-activated Mg2+-ATPase in combination with the presence of undegraded ligands in the endosome fractions emerge as linked markers for this new subcellular organelle.

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

1990 ◽  
Vol 10 (2) ◽  
pp. 131-139
Author(s):  
Oyewole Adeyemo ◽  
E. O. Okegbile ◽  
O. O. Olorunsogo
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Boar Sperm ◽  
Sperm Membrane ◽  
Triton X

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.

scholarly journals Partial purification and characterization of an endo-α-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242

1992 ◽  
Vol 288 (2) ◽  
pp. 475-482 ◽  
Author(s):  
I Ishii-Karakasa ◽  
H Iwase ◽  
K Hotta ◽  
Y Tanaka ◽  
S Omura
Keyword(s):  
Enzyme Activity ◽  
Culture Medium ◽  
Gel Filtration ◽  
Partial Purification ◽  
Gastric Mucus ◽  
Purification And Characterization ◽  
Streptomyces Sp ◽  
Optimum Ph ◽  
New Type

For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151, 422-428], a method for assaying enzyme activity was established. Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than Gal beta 1-3GalNAc were analysed by h.p.1.c. The crude enzyme of endo-GalNAc-ase-S was prepared as an 80% (w/v) ammonium sulphate precipitate from the concentrated culture medium. The enzyme was partially purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography. Endo-enzyme activity eluted around pI 4.8 on a gel chromatofocusing column and eluted with 0.19-0.25 M-NaCl on a DEAE-Toyopearl column. In the enzyme fraction obtained, no exo-glycosidases or proteases could be detected. The molecular mass of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 5.5. Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser (Thr) in 3H-labelled and unlabelled asialofetuin, liberating both the disaccharide (Gal beta 1-3GalNAc) and the tetrasaccharide [Gal beta 1-3 (Gal beta 1-4GlcNAc beta 1-6)GalNAc]. When endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNac-ase-A) was incubated with 3H-labelled and unlabelled asialofetuin, only the disaccharide (Gal beta 1-3GalNAc) was liberated.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

scholarly journals Cardioactive Peptides from the CNS of a Caterpillar, the Tobacco Hornworm, Manduca Sexta

1985 ◽  
Vol 114 (1) ◽  
pp. 397-414
Author(s):  
Nicholas Platt ◽  
Stuart E. Reynolds
Keyword(s):  
Manduca Sexta ◽  
Specific Activity ◽  
Gel Filtration ◽  
Corpora Allata ◽  
Performic Acid ◽  
Partial Purification ◽  
Relative Molecular Mass ◽  
Two Factors ◽  
Frontal Ganglion ◽  
Manduca Sexta Larvae

1. A semi-isolated caterpillar heart bioassay was used to detect the presence of endogenous cardioactive material in the CNS of Manduca sexta larvae. 2. Cardioactivity was detected in all nervous tissue examined. Most activity (about 70% of the total in the CNS) was in the ganglia of the abdominal nerve cord (ANC). Cardioactivity was also detected in the abdominal transverse nerves, the proctodeal nerves and the corpora cardiaca/corpora allata. The source with the highest specific activity was the frontal ganglion. 3. Two factors, separable by Sephadex gel filtration, were distinguished in extracts of ANC: CAF 1, which has an estimated relative molecular mass (Mr) of about 4000, and CAF2 for which Mr is probably less than 1000. Both factors are apparently peptides. Neither is similar to any known insect cardioaccelerator. 4. Both CAF 1 and CAF 2 are able to cause cardioacceleration when injected into tetrodotoxin-paralysed caterpillars. 5. CAF 2 is present in both larvae and in adults. CAF 1 is present only in the caterpillar. The larval heart responds to both factors; the adult heart responds only to CAF 2. 6. Partial purification of CAF 1 and CAF 2 by reverse-phase HPLC gives a single peak of bioactivity in each case. 7. The biological activity of CAF 1 is destroyed by α-chymotrypsin, but not by trypsin. CAF 2 is not attacked by trypsin or by α-chymotrypsin. Treatment with performic acid or cyanogen bromide destroys the activity of both CAF 1 and CAF 2.

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