scholarly journals Purification and characterization of a lysophosphatidic acid-specific phosphatase

1998 ◽  
Vol 336 (2) ◽  
pp. 483-489 ◽  
Author(s):  
Masami HIROYAMA ◽  
Tadaomi TAKENAWA
Keyword(s):  
Lysophosphatidic Acid ◽  
Inhibitory Effect ◽  
Bovine Brain ◽  
Reducing Conditions ◽  
Sds Page ◽  
Potent Inhibitory Effect ◽  
Sphingosine 1 Phosphate ◽  
Km Value ◽  
Triton X ◽  
Ceramide 1

Lysophosphatidic acid (LPA)-specific phosphatase was purified 3300-fold from bovine brain cytosol. The purification was achieved by (NH4)2SO4 fractionation and several chromatography steps, such as Q-Sepharose, DEAE-5PW, Superdex 200 and heparin–Sepharose. The final enzyme preparation showed a single band of molecular mass 44 kDa on SDS/PAGE under reducing conditions. The enzyme activity was completely dependent on the presence of detergents such as Triton X-100, CHAPS, cholate and octyl-β-glucoside. The activity was independent of Mg2+; other cations were inhibitory. The enzyme hydrolysed LPA specifically but not cardiolipin, tetraoleoyl-bisphosphatidic acid, ceramide 1-phosphate or sphingosine 1-phosphate, although phosphatidic acid was hydrolysed slightly. The purified enzyme hydrolysed 1-oleoyl LPA at a rate of 1.1 µmol/min per mg of protein when assayed with LPA as Triton X-100 mixed micelles. The Km value for LPA was 38 µM. NaF and N-ethylmaleimide markedly inhibited the activity, but propranolol had a less potent inhibitory effect. The LPA-specific phosphatase might have an important role in LPA elimination.

scholarly journals Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver

1995 ◽  
Vol 308 (3) ◽  
pp. 983-989 ◽  
Author(s):  
I N Fleming ◽  
S J Yeaman
Keyword(s):  
Phosphatidic Acid ◽  
Rat Liver ◽  
Lysophosphatidic Acid ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Sds Page ◽  
Chromatography Columns ◽  
Triton X ◽  
Second Peak

N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.

scholarly journals Effect of a membrane-targeted sphingosine kinase 1 on cell proliferation and survival

2005 ◽  
Vol 388 (3) ◽  
pp. 827-834 ◽  
Author(s):  
Farida SAFADI-CHAMBERLAIN ◽  
Li-Ping WANG ◽  
Shawn G. PAYNE ◽  
Chang-Uk LIM ◽  
Suzanne STRATFORD ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Plasma Membrane ◽  
Phorbol Esters ◽  
Cell Function ◽  
Sphingosine Kinase ◽  
Subcellular Location ◽  
Inhibitory Effect ◽  
Potent Inhibitory Effect ◽  
Sphingosine 1 Phosphate ◽  
Extracellular Stimuli

Numerous extracellular stimuli activate SK1 (sphingosine kinase type 1) to catalyse the production of sphingosine 1-phosphate, a bioactive lipid that functions as both an extracellular ligand for a family of G-protein-linked receptors and as a putative intracellular messenger. Phorbol esters, calcium or immunoglobulin receptors stimulate SK1 by promoting its translocation to the plasma membrane, which brings it into proximity both to its substrate (i.e. sphingosine) and to activating acidic phospholipids (e.g. phosphatidylserine). To evaluate the consequence of SK translocation, we generated an SK1-derivative tagged with a myristoylation sequence (Myr-SK1) on its N-terminus and overexpressed the construct in 3T3-L1 fibroblasts using recombinant retrovirus. Myr-SK1 overexpression increased SK activity by more than 50-fold in crude membranes, while only stimulating cytoplasmic SK activity by 4-fold. In contrast, the overexpression of WT-SK1 (wild-type SK1), as well as that of a construct containing a false myristoylation sequence (A2-Myr-SK1), markedly increased SK activity in both membrane and cytoplasmic compartments. Immunofluorescence confirmed that Myr-SK1 preferentially localized at the plasma membrane, whereas WT-SK1 and A2-Myr-SK1 partitioned in cytoplasmic/perinuclear cellular regions. Surprisingly, Myr-SK1 overexpression significantly decreased the rates of cell proliferation by delaying exit from G0/G1 phase. Moreover, expression of Myr-SK1 but not WT-SK1 or A2-Myr-SK1 protected cells from apoptosis induced by serum withdrawal. Collectively, these findings reveal that altering the subcellular location of SK1 has marked effects on cell function, with plasma membrane-associated SK having a potent inhibitory effect on the G1–S phase transition.

scholarly journals Purification and characterization of an α-galactosyltransferase from Trypanosoma brucei

1999 ◽  
Vol 338 (2) ◽  
pp. 545-551 ◽  
Author(s):  
Sabine PINGEL ◽  
Uta RHEINWEILER ◽  
Volker KOLB ◽  
Michael DUSZENKO
Keyword(s):  
Trypanosoma Brucei ◽  
Specific Binding ◽  
Broad Band ◽  
Glycosylation Site ◽  
Transferase Activity ◽  
Ph Optimum ◽  
Reducing Conditions ◽  
Sds Page ◽  
Molecular Masses ◽  
Triton X

A membrane-associated galactosyltransferase from Trypanosoma brucei was purified 34000-fold by affinity chromatography on UDP-hexanolamine–Sepharose™. Using SDS/PAGE under reducing conditions, the isolated enzyme ran as a relatively broad band with apparent molecular masses of 53 kDa and 52 kDa, indicative of glycosylation and the existence of two isoforms. N-Glycosylation of the enzyme was subsequently confirmed using Western blotting and either specific binding of concanavalin A or peptide-N4-(N-acetylglucosaminyl)asparagine amidase digestion. The de-N-glycosylated enzyme ran with apparent molecular masses of 51 kDa and 50 kDa, indicative of a single N-glycosylation site. The galactosyltransferase exhibited a pH optimum at 7.2 and had a pronounced requirement for Mn2+ ions (KM = 2.5 mM) for its action. The transferase activity was independent of the concentration of Triton X-100. The enzyme was capable of transferring galactose from UDP-galactose to a variety of galactose-based acceptors in α-glycosidic linkages. The apparent KM values for UDP-galactose and for the preferred acceptor substrate N-acetyl-lactosamine are 46 µM and 4.5 mM respectively. From these results we would like to suggest that the galactosyltransferase functions in the processing of terminal N-acetyl-lactosamine structures of trypanosomal glycoproteins.

scholarly journals Purification and Characterization of Peroxidase From Anthracnose Disease Infected Papaya (Carica papaya L.)

2014 ◽  
Vol 6 (2) ◽  
pp. 49-57 ◽  
Author(s):  
L Bari ◽  
P Hassan ◽  
N Absar ◽  
S Khatun ◽  
MI Hossain
Keyword(s):  
Ammonium Sulphate ◽  
Gel Filtration ◽  
Potassium Cyanide ◽  
Ammonium Sulphate Precipitation ◽  
Reducing Conditions ◽  
Sds Page ◽  
Peroxidase Enzyme ◽  
Km Value ◽  
Temperature Optima

Peroxidase enzyme was isolated and purified from the pulp of disease infected ripen papaya of local variety by 90% ammonium sulphate precipitation, chromatography on DEAEcellulose followed by hydrophobic chromatography on Phenyl Sepharose CL-4B and the purifications achieved was about 7.2 fold with 2.5% recovery. The purified enzyme was homogeneous as judged by polyacrylamide slab gel electrophoresis. The purified enzyme had a Mr of about 55,000 and 50 000 as determined by gel filtration on Sephadex G-100 and SDS-PAGE, respectively. The molecular mass of the enzyme was found to be very similar under both reducing and non-reducing conditions indicating that the enzyme contains no subunit. The enzyme has the following characteristics: pH optima at 6.0, temperature optima around 38°C, enzyme activity was found to be strongly inhibited in the presence of potassium cyanide and Fe+2 while the activity was found to be remarkably increased in the presence of ammonium sulphate. The Km value for the peroxidase obtained with pyrogallol as substrate was 0.027 mM. DOI: http://dx.doi.org/10.3329/bjmb.v6i2.17643 Bangladesh J Med Biochem 2013; 6(2): 49-57

Purification and Characterization of a Novel Metalloproteinase, Acurhagin, from Agkistrodon acutus Venom

2002 ◽  
Vol 87 (04) ◽  
pp. 641-650 ◽  
Author(s):  
Wen-Jeng Wang ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Aggregation ◽  
Molecular Mass ◽  
Hydrophobic Interaction Chromatography ◽  
Inhibitory Effect ◽  
High Molecular Mass ◽  
Dependent Manner ◽  
Peptide Fragments ◽  
Crude Venom ◽  
Reducing Conditions ◽  
Sds Page

SummaryAcurhagin, a high-molecular mass hemorrhagic metalloproteinase, was purified from the crude venom of Agkistrodon acutus using anionexchange and hydrophobic interaction chromatography. Acurhagin is a monomer with a molecular mass of 51.4 kDa under non-reducing conditions on SDS-PAGE and 48,133 Da by mass spectrometry. Partial amino acid sequence of its metalloproteinase domain is homologous to other high-molecular mass metalloproteinases from snake venoms. It preferentially cleaved Aα. chain of fibrinogen, followed by Bβ chain, while γ chains was minimally affected. Monitored by RP-HPLC, it extensively degraded fibrinogen into various peptide fragments. In aqueous solution, acurhagin autoproteolyzed to a 30 kDa fragment at 37° C. The N-terminal sequence of the 30 kDa fragment of acurhagin showed a high homology to those proteins consisting of disintegrinlike and cysteine-rich domains. Caseinolytic assay showed that the proteinase activity of acurhagin was slightly enhanced by Ca2+ and Mg2+, but completely inhibited by Zn2+. When treated with metal chelators, acurhagin was completely inactivated. Furthermore, acurhagin exerts an inhibitory effect on ADP-induced platelet aggregation of plateletrich plasma in an incubation-time dependent manner. It also impairs collagen- and ristocetin-induced platelet aggregation by cleaving collagen and vWF, respectively.

Effect of lysolecithin in solubilizing membrane-bound glycosyltransferases

1979 ◽  
Vol 57 (1) ◽  
pp. 66-71 ◽  
Author(s):  
Sailen Mookerjea
Keyword(s):  
Lysophosphatidic Acid ◽  
High Speed ◽  
Critical Concentration ◽  
Egg Yolk ◽  
Inhibitory Effect ◽  
Fatty Acyl ◽  
Supernatant Fraction ◽  
Sediment Fraction ◽  
Microsomal Membranes ◽  
Triton X

Microsomal membranes were solubilized by incubation with lysolecithin which caused considerable release of galactosyl- and N-acetylglucosaminyl-transferase into a high-speed supernatant fraction. With a critical concentration of lysolecithin (2.5 mg/10 mg protein in 1 mL microsome suspension), there was a maximal binding of radioactive lysolecithin to the sediment fraction obtained after high-speed centrifugation. Increase of lysolecithin concentration (above 2.5 mg/mL) in the incubation mixture caused a progressive release of the enzymes into the supernatant fraction.Lysolecithin binding to the membrane was greatly inhibited by 1 M NaCl, and high salt concentration also inactivated galactosyltransferase in the sediment, suggesting an electrostatic interaction between lysolecithin and membrane enzyme. In contrast, high NaCl concentration had no inhibitory effect on the enzyme activity in the sediment when the fraction was prepared by treatment with Triton X-100.Lysolecithin-treated microsomal sediment and supernatant galactosyltransferase was inactivated by oleoyllysophosphatidic acid but not by palmitoyllysophosphatidic acid or egg yolk lysophosphatidic acid. Triton X-100 treated microsomal fractions were also similarly affected by different species of lysophosphatidic acid. The results suggested a similarity of interactions of lysophosphatidic fatty acyl species with lysolecithin and Triton-treated galactosyltransferase.

scholarly journals Purification and characterization of a sialic acid-specific lectin from Tritrichomonas mobilensis

1994 ◽  
Vol 299 (2) ◽  
pp. 341-346 ◽  
Author(s):  
P Babál ◽  
F F Pindak ◽  
D J Wells ◽  
W A Gardner
Keyword(s):  
Monoclonal Antibodies ◽  
Sialic Acid ◽  
Haemagglutination Inhibition ◽  
Submaxillary Gland ◽  
Inhibitory Effect ◽  
Size Exclusion ◽  
Western Blots ◽  
Reducing Conditions ◽  
Sds Page ◽  
Exclusion Chromatography

New sialic acid-specific lectin has been isolated from culture supernatant of the protozoan Tritrichomonas mobilensis. It was purified by adsorption by erythrocytes or bovine submaxillary gland mucin (BSM)-Sepharose affinity chromatography. The T. mobilensis lectin (TML) does not require bivalent cations for activity and agglutinates all human erythrocytes. The lectin forms multimeric complexes with molecular mass 556 and 491 kDa as determined by size-exclusion chromatography. SDS/PAGE under reducing conditions disclosed a large band of 343 kDa and three bands of 246, 265 and 286 kDa which, after denaturation with urea, were split into three subunits of 56, 61 and 66 kDa; under non-reducing conditions there were two bands, of 360 and 260 kDa. Western blots performed with anti-TML monoclonal antibodies revealed bands identical with those in the silver-stained gels, suggesting homogeneity of the BSM -Sepharose-purified lectin. TML is a highly glycosylated protein with approx. 8% of N-linked glycosides found by protein-N-glycanase F treatment; the total amount of saccharides revealed by chemical deglycosylation was 20%. Haemagglutination-inhibition studies documented exclusive specificity for sialic acid (NeuAc). Both (alpha 2->6)- and (alpha 2->3)-linked and free NeuAc were eight times more potent inhibitors than N-glycolylneuraminic acid. The lectin does not require O-acetyl groups on NeuAc for recognition. A spectrum of mono- and oligo-saccharides other than sialic acid had no inhibitory effect at 200 mM. Anti-TML monoclonal antibodies strongly inhibited the lectin activity. TML was stable at temperatures below 4 degrees C and lyophilized with 3% (w/w) glycerol.

Purification and Characterization of Lupus Anticoagulant Like Protein from Agkistrodon Halys Brevicaudus Venom

1996 ◽  
Vol 76 (06) ◽  
pp. 0993-0997
Author(s):  
Zhao-Yan Li ◽  
Xiao-Wei Wu ◽  
Tie-Fu Yu ◽  
Eric C-Y Lian
Keyword(s):  
Amino Acid ◽  
Lupus Anticoagulant ◽  
Inhibitory Effect ◽  
Clotting Factor ◽  
Acid Oxidase ◽  
Crude Venom ◽  
Amino Acid Oxidase ◽  
Sds Page ◽  
The Individual ◽  
Reducing Condition

SummaryBy means of CM-Sephadex C-25, DEAE-Sephadex A-50, Sephadex G-200, and Sephadex G-75 chromatographies, a lupus anticoagulant like protein (LALP) from Agkistrodon halys brevicaudus was purified. On SDS-PAGE, the purified LALP had a molecular weight of 25,500 daltons under non-reducing condition and 15,000 daltons under reducing condition. The isoelectric point was pH 5.6. Its N terminal amino acid sequencing revealed a mixture of 2 sequences: DCP(P/S)(D/G)WSSYEGH(C/R)Q(Q/K). It was devoid of phospho-lipaseA, fibrino(geno)lytic, 5′-nucleotidase, L-amino acid oxidase, phosphomonoesterase, phosphodiesterase and thrombin-like activities, which were found in crude venom. In the presence of LALP, PT, aPTT, and dRVVT of human plasma were markedly prolonged and its effects were concentration-dependent but time-independent. The inhibitory effect of LALP on the plasma clotting time was enhanced by decreasing phospholipid concentration in TTI test. The individual clotting factor activity was not affected by LALP when higher dilutions of LALP-plasma mixture were used for assay. Russell’s viper venom time was shortened when high phospholipid confirmatory reagent was used. Therefore, the protein has lupus anticoagulant property.

ISOLATION of a AMYLASE INHIBITORS from MUNGBEAN and SOYBEAN and INHIBITORY EFFECT on HUMAN SALIVARY and PORCINE PANCREATIC AMYLASE

2013 ◽  
Vol 14 (1) ◽  
Author(s):  
Budiasih Wahyuntari ◽  
Martinus Nicoadi Tekol Tekol
Keyword(s):  
Inhibitory Effect ◽  
Pancreatic Amylase ◽  
Sds Page ◽  
Amylase Inhibitors

Penghambat alfa amilase adalah salah satu komponen dalam suplemen makanan yang telah lama digunakan untuk terapi kegemukan karena penghambat amilase mempengaruhi metabolisme karbohidrat dalam sistem pencernaan. Sejumlah peneliti melaporkan terdapat dua grup penghambat amilase, yaitu protein dan non protein. Penghambat protein dilaporkan terdapat dalam kelompok kacang-kacangan dan biji-bijian. Tujuan penelitian ini adalah untuk mengisolasi penghambat protein yang terdapat dalam kacang hijau dan kedele. Kacang hijau dan kedele merupakan kacang-kacangan yang penting dalam makanan popular di Indonesia. Penghambat protein diendapkan dengan konsentrasi bertingkat garam ammonium sulfat [(NH)4SO4] dari 30-70%. Penghambat protein diuji terhadap amilase saliva manusia (HSA) danamilase pankreas babi (PPA), serta kestabilannya terhadap pemanasan pada 100oC selama 30 menit. Hasil penelitian menunjukkan bahwa semua endapan jenuh dari semua konsentrasi amonium sulfat yang diuji menghambat PPA, tetapi tidak semua endapan jenuh tersebut dapatmenghambat HSA. Hanya semua endapan jenuh (NH)4SO4 dari kacang hijau yang dapat menghambat HSA, dan penghambatan tertinggi terhadap HSA adalah endapan jenuh (NH)4SO4 50%. Endapan jenuh (NH)4SO4 40 % dari kedele putih dan endapan jenuh (NH)4SO4 60% dari kedele hitam dengan masing-masing penghambatan 98.67; 26.86 and27.63%. Endapan jenuh (NH)4SO4 60-70% dari kacang hijau, 50% dari kedele putih dan 50% dari kedele hitam menghambat PPA 100%. Pemanasan penghambat pada 100oC selama 30 menit hampir tidak mempengaruhi penghambatannya terhadap PPA. Profil protein juga diamati menggunakan analisis SDS/PAGE.

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