scholarly journals Purification of a lysophosphatidic acid-hydrolysing lysophospholipase from rat brain

1994 ◽  
Vol 300 (2) ◽  
pp. 457-461 ◽  
Author(s):  
F J Thompson ◽  
M A Clark
Keyword(s):  
Rat Brain ◽  
Lysophosphatidic Acid ◽  
Cellular Localization ◽  
Gel Filtration ◽  
Degree Of Saturation ◽  
Biological Responses ◽  
Octyl Glucoside ◽  
Sds Page ◽  
Membrane Bound ◽  
Hydrolysis Of

A lysophosphatidic acid (LPA)-hydrolysing lysophospholipase was purified from rat brain and characterized. This membrane-bound lysophospholipase was solubilized by using n-octyl glucoside and purified by sequential cation, hydrophobic and gel-filtration chromatography. The purified protein has a mass of 80 kDa as assayed by SDS/PAGE. This lysophospholipase catalysed the hydrolysis of a variety of lysophosphatidic acids, but with different rates, depending on the length and degree of saturation of the sn-1 acyl group (1-oleoyl-LPA approximately 1-stearoyl-LPA > 1-palmitoyl-LPA > 1-myristoyl-LPA). This enzyme had no-measurable catalytic activity when other lysophospholipids, monoacylglycerol or phosphatidic acid were used as substrates. On the basis of its chromatographic properties, substrate specificity and cellular localization, we conclude that this lysophospholipase differs from those previously purified and speculate that it has an important function in terminating biological responses to LPA.

scholarly journals Purification of a phosphatidic-acid-hydrolysing phospholipase A2 from rat brain

1995 ◽  
Vol 306 (1) ◽  
pp. 305-309 ◽  
Author(s):  
F J Thomson ◽  
M A Clark
Keyword(s):  
Catalytic Activity ◽  
Phosphatidic Acid ◽  
Phospholipase A2 ◽  
Rat Brain ◽  
Lysophosphatidic Acid ◽  
Catalytic Properties ◽  
Gel Filtration ◽  
Gel Filtration Chromatography ◽  
Ph Optimum ◽  
Sds Page

A phosphatidic-acid-hydrolysing phospholipase A2 was purified from rat brain and characterized. This phospholipase A2 was purified by sequential cation, hydrophobic, heparin and gel-filtration chromatography. The purified protein had a mass of approximately 58 kDa as assayed by SDS/PAGE, had a pH optimum of 6.0, and was Ca(2+)-independent. This enzyme was apparently phosphatidic-acid-selective and had little measurable catalytic activity when phosphatidylcholine, phosphatidylethanolamine or diacylglycerol was used as substrate. On the basis of its physical and catalytic properties, we conclude that this phospholipase A2 is unique from those previously purified, and we speculate that it may be important for the production of the bioactive lipid lysophosphatidic acid.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

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 Activation of a phospholipase Cβ2 deletion mutant by limited proteolysis

1998 ◽  
Vol 330 (1) ◽  
pp. 461-468 ◽  
Author(s):  
Petra SCHNABEL ◽  
Montserrat CAMPS
Keyword(s):  
Deletion Mutant ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Cell System ◽  
Heterotrimeric G Proteins ◽  
Sds Page ◽  
Catalytically Active ◽  
Heterodimeric Complex ◽  
High Degree ◽  
Hydrolysis Of

All phosphoinositide-specific phospholipases C (PLC) identified until today exhibit a high degree of similarity within two regions of 170 and 260 residues, respectively, which are designated regions X and Y. The PLCβ family, including four members designated PLCβ1, PLCβ2, PLCβ3 and PLCβ4, is regulated by heterotrimeric G proteins. In order to investigate structure-function relationships of PLCβ2, we expressed PLCβ2Δ, a deletion mutant of PLCβ2 which lacks most of the sequence downstream of region Y, in the baculovirus/insect cell system. The mutant was present in both soluble and particulate fractions of Sf9 cells and was demonstrated to be catalytically active and sensitive to βγ-subunits. Sf9 cytosol containing this mutant was subjected to limited proteolysis by trypsin and S. aureus protease V8, respectively. Immunochemical analysis revealed that both proteases cleaved the enzyme between the regions X and Y. Most interestingly, proteolytic cleavage at this site by both proteases stimulated the catalytic activity of PLC2β2Δ. The proteolytically activated enzyme was still sensitive to βγ-subunits and showed an unchanged concentration dependence on Ca2+. Gel filtration chromatography indicated that the fragments generated by cleavage between the regions X and Y were still connected and formed a functional heterodimeric complex. In order to visualize all fragments generated by protease V8, PLCβ2Δ was purified to homogeneity from Sf9 cytosol. Limited proteolysis of the purified enzyme by S. aureus protease V8 and subsequent SDS/PAGE and silver staining revealed that several cuts take place between the regions X and Y and that the resulting fragments remain intact. We hypothesize that the activating proteolytic cut induces a conformational change of the enzyme which might facilitate hydrolysis of the phospholipid substrate.

scholarly journals Purification and characterization of an extracellular (1 → 6)-β-glucanase from the filamentous fungus Acremonium persicinum

1996 ◽  
Vol 316 (3) ◽  
pp. 841-846 ◽  
Author(s):  
Stuart M. PITSON ◽  
Robert J. SEVIOUR ◽  
Barbara M. McDOUGALL ◽  
Bruce A. STONE ◽  
Maruse SADEK
Keyword(s):  
Molecular Mass ◽  
Filamentous Fungus ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Eisenia Bicyclis ◽  
Ph Optimum ◽  
Hydrolysis Products ◽  
Sds Page ◽  
Hydrolysis Of

An endo-(1 → 6)-β-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 → 6)-β-glucans (pustulan and lutean), initially yielding a series of (1 → 6)-β-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as β-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent hydrolysis of (1 → 6)-β- and some (1 → 3)-β-linkages in this substrate. Km values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Regent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

scholarly journals Biochemical Characteristics of Penicillium crustosum FP 11 Xylanase II and an Assessment of the Properties of Xylanases Produced by the Genus Penicillium

2020 ◽  
pp. 64-75
Author(s):  
Jorge W. F. Bittencourt ◽  
Vanessa C. Arfelli ◽  
Jaina C. Lunkes ◽  
Carla L. D. Torre ◽  
Jose L. C. Silva ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Fermentable Sugars ◽  
Optimal Temperature ◽  
Recovery Yield ◽  
Biochemical Characteristics ◽  
Beechwood Xylan ◽  
Sds Page ◽  
Penicillium Crustosum ◽  
Hydrolysis Of

  Penicillium crustosum FP 11 produces two extracellular xylanase, which are designated xylanase I and II, and are induced by corn stover. In this work, xylanase II was purified 40-fold with a recovery yield of 9.2% using DEAE-Sephadex and Sephadex G-75 gel filtration, and the biochemical characteristics of the enzyme were compared with other xylanases produced by the genus Penicillium. Xylanase II exhibited a single band on SDS–PAGE, and had an apparent molecular mass of 28 kDa. The optimal temperature and pH of xylanase II activity were 50ºC and 5.5, respectively. Xylanase II had activities of 61, 53 and 55% in the presence of Mg2+, DTT and β-mercaptoethanol, respectively; however, the enzyme was strongly inhibited by 5 mM Cu2+, EDTA, and SDS. Hydrolysis of beechwood xylan released mainly xylose and short-chain xylo-oligosaccharides as final products. Thus, an assessment of the enzymatic properties of xylanase II showed that its biochemical characteristics are best suited for the saccharification of lignocellulosic biomass into fermentable sugars.

scholarly journals Pemurnian Parsial dan Karakterisasi Enzim Xilanase dari Bakteri Laut Bacillus safencis strain LBF P20 Asal Pulau Pari Jakarta

2017 ◽  
Vol 37 (1) ◽  
pp. 31
Author(s):  
Fitria Fitria ◽  
Nanik Rahmani ◽  
Sri Pujiyanto ◽  
Budi Raharjo ◽  
Yopi Yopi
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Centrifugal Filter ◽  
Sds Page ◽  
Enzyme Specific Activity ◽  
Hydrolysis Of

Enzyme xylanase (EC 3.2.1.8) is widely used in various industrial  fields for the hydrolysis of xylan (hemicellulose) into xylooligosaccharide and xylose. The aims of this study were to  conduct partial purification and characterization of xylanase from marine Bacillus safencis strain LBF P20 and to obtain the  xylooligosaccharide types from xylan hydrolysis by this enzyme.  Based on this research, the optimum time for enzyme production  occurred at 96 hours with the enzyme activity of 6.275 U/mL and  enzyme specific activity of 5.093 U/mg. The specific activities were  obtained from precipitation by amicon® ultra-15 centrifugal filter devices, gel filtration chromatography and anion exchange chromatography that were increased by 15.07, 34.7, and 96.0  U/mg. The results showed that the highest activity at pH 7, temperature of 60 °C, and stable at 4 °C. Type of  xylooligosaccharide produced by this study were xylohexoses, xylotriose, and xylobiose. SDS-PAGE analysis and zimogram  showed that the molecular weight of xylanase protein were about  25 kDa. ABSTRAKEnzim xilanase (EC 3.2.1.8) digunakan dalam hidrolisis xilan  (hemiselulosa) menjadi xilooligosakarida dan xilosa. Penelitian  ini bertujuan untuk melakukan purifikasi parsial dan karakterisasi xilanase dari bakteri laut Bacillus safencis strain LBF P20 serta uji  hidrolisis untuk mengetahui jenis xilooligosakarida yang  dihasilkan oleh enzim tersebut. Berdasarkan hasil penelitian, waktu optimum untuk produksi enzim terjadi pada jam ke 96  dengan aktivitas enzim sebesar 6,275 U/mL dan aktivitas spesifik enzim sebesar 5,093 (U/mg). Aktivitas spesifik enzim hasil  pemekatan dengan amicon® ultra-15 centrifugal filter devices,  kromatografi filtrasi gel dan kromatografi penukar anion  mengalami peningkatan berturut-turut sebesar 15,1; 34,7 dan96,0 U/mg. Hasil karakterisasi menunjukkan aktivitas  tertinggi pada pH 7, suhu 60 °C dan stabil pada suhu 4 °C. Analisis SDS-PAGE dan zimogram menunjukkan berat molekul protein xilanase berkisar 25 kDa. Jenis gula reduksi yang  dihasilkan yaitu xiloheksosa, xilotriosa, dan xilobiosa.

scholarly journals Purification and characterization of membrane-bound semicarbazide-sensitive amine oxidase (SSAO) from bovine lung

1998 ◽  
Vol 331 (1) ◽  
pp. 69-78 ◽  
Author(s):  
José M. LIZCANO ◽  
Keith F. TIPTON ◽  
Mercedes UNZETA
Keyword(s):  
Amine Oxidase ◽  
Gel Filtration ◽  
Integral Membrane Protein ◽  
Significant Activity ◽  
Bovine Plasma ◽  
Sds Page ◽  
Catalytically Active ◽  
Cibacron Blue 3Ga ◽  
Membrane Bound ◽  
Triton X

Semicarbazide-sensitive amine oxidase (SSAO) has been purified from bovine lung microsomes in a form which is catalytically active and stable to storage. The enzyme, an integral membrane protein, was solubilized with Triton X-100 and purification was achieved, in the presence of detergent, by chromatography with Cibacron Blue 3GA-agarose, hydroxylapatite, Lens culinaris-agarose, Resource Q-FPLC and gel filtration on Superdex 200 HR-FPLC. This is the first reported procedure for the extensive purification of a membrane-bound SSAO. The purified enzyme had an apparent Mr of 400000 but exhibited microheterogeneity with SDS/PAGE and isoelectric focusing, probably as a result of its glycoprotein nature. It behaved as a tetramer with subunits with apparent Mr values of 100. Antibodies raised towards the purified enzyme cross-reacted with the enzymes from human lung and bovine plasma. Redox-cycling staining and reaction with carbonyl reagents were consistent with the presence of a quinone cofactor, possibly topa quinone. The enzyme was also shown to contain two mol of Cu/mol of enzyme and removal of half of this bound copper resulted essentially in complete inhibition of enzyme activity. In contrast to the reported behaviour of the SSAO enzymes from plasma, the bovine lung enzyme was relatively insensitive to inhibition by cyanide, copper-chelating agents and amiloride. The specificity of the bovine lung enzyme was also narrower than reported for soluble SSAO. It catalysed the oxidative deamination of benzylamine, methylamine, 2-phenylethylamine and histamine but had no significant activity towards dopamine, 5-hydroxytryptamine, tryptamine or tyramine.

scholarly journals Purification and Characterization of an Alkaline Phosphatase Induced by Phosphorus Starvation in Common Bean (Phaseolus vulgaris L.) Roots

2017 ◽  
Vol 56 (1) ◽  
Author(s):  
Lorena Morales ◽  
Natalia Gutiérrez ◽  
Vanessa Maya ◽  
Carmen Parra ◽  
Eleazar Martínez-Barajas ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Phaseolus Vulgaris ◽  
Common Bean ◽  
Divalent Cations ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Phosphate Deficiency ◽  
Ionic Exchange ◽  
Sds Page ◽  
Hydrolysis Of

Two phosphatase isoforms from roots of the common bean (<em>Phaseolus vulgaris</em> L.) showed an increase in activity in response to phosphate deficiency. One of them (APIII) was chosen for further purification through ionic exchange chromatography and preparative electrophoresis. The estimated molecular mass of APIII was 35 kDa by both SDS-PAGE and gel filtration analyses, suggesting a monomeric form of the active enzyme. The phosphatase was classified as an alkaline phosphatase based on the requirement of pH 8 for optimum catalysis. It not only exhibited broad substrate specificity, with the most activity against pyrophosphate, but also effectively catalyzed the hydrolysis of polyphosphate, glucose-1-phosphate and phosphoenolpyruvate. Activity was completely inhibited by molybdate, vanadate and phosphate but was only partially inhibited by fluoride. Although divalent cations were not essential for the pyrophosphatase activity of this enzyme, the hydrolysis of pyrophosphate increased substantially in the presence of Mg<sup>2+</sup>.

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