scholarly journals Purification and characterization of phosphatidylinositol synthase from human placenta

1994 ◽  
Vol 297 (3) ◽  
pp. 517-522 ◽  
Author(s):  
B E Antonsson
Keyword(s):  
Human Placenta ◽  
Microsomal Fraction ◽  
Enzyme Reaction ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Sds Page ◽  
Phosphatidylinositol Synthase ◽  
Triton X

Phosphatidylinositol synthase (CDP-1,2-diacyl-sn-glycerol:myoinositol 3-phosphatidyltransferase, EC 2.7.8.11) was purified from the microsomal fraction of human placenta. The Triton X-100-extracted enzyme was purified 8300-fold over the microsomal fraction by affinity chromatography on CDP-diacylglycerol-Sepharose followed by ion-exchange chromatography on Mono Q. The purified enzyme had a molecular mass of 24,000 Da on SDS/PAGE. The enzyme had a pH optimum at 9.0, required Mn2+ or Mg2+, and was inhibited by Ca2+ and Zn2+. The Km for myo-inositol was determined to be 0.28 mM. Optimal activity was obtained at 0.2-0.4 mM CDP-diacylglycerol; higher concentrations of the lipid substrate inhibited the enzyme reaction. The enzyme was inhibited by nucleoside di- and tri-phosphates, Pi and PPi. CDP competitively inhibited the enzyme reaction with a Kis of 4 mM. The optimal temperature for the PtdIns synthase reaction was 50 degrees C.

scholarly journals Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles

1987 ◽  
Vol 244 (1) ◽  
pp. 219-224 ◽  
Author(s):  
J M Jacobs ◽  
N J Jacobs
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chlorophyll Synthesis ◽  
Yeast Mitochondria ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Haem Biosynthesis ◽  
Triton X ◽  
Polypeptide Band

The protoporphyrinogen-oxidizing enzyme from Triton X-100 extracts of the mitochondrial and etioplast fractions of etiolated barley was purified by using ion-exchange and hydroxyapatite chromatography. The purified enzyme from both organelle fractions exhibited a Km of 5 microM and was labile to mild heat and acidification. The pH optimum (5-6) and the substrate-specificity (mesoporphyrinogen was oxidized as rapidly as protoporphyrinogen) revealed properties very different from the protoporphyrinogen-oxidizing enzyme of rat liver or yeast mitochondria, which is specific for protoporphyrinogen as substrate. The purest fractions showed a polypeptide band corresponding to an Mr of approx. 36,000 on SDS/polyacrylamide-gel electrophoresis. This is the first purification and characterization of the enzyme from a plant, and indicates no readily detectable differences between the enzyme isolated from mitochondrial or etioplast fractions, although only the latter organelle has the capacity for both haem and chlorophyll synthesis.

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 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 A Boophilus microplus vitellin-degrading cysteine endopeptidase

Parasitology ◽  
2003 ◽  
Vol 126 (2) ◽  
pp. 155-163 ◽  
Author(s):  
A. SEIXAS ◽  
P. C. DOS SANTOS ◽  
F. F. VELLOSO ◽  
I. DA SILVA VAZ ◽  
A. MASUDA ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Boophilus Microplus ◽  
Hard Tick ◽  
Purification And Characterization ◽  
Basic Residue ◽  
Cysteine Endopeptidase ◽  
Sds Page

Here we describe the purification and characterization of a vitellin (VT) degrading cysteine endopeptidase (VTDCE) from eggs of the hard tick Boophilus microplus. A homogeneous enzyme preparation was obtained by chromatographic fractionation on ion-exchange and gel filtration columns and an autolysis step. This step consisted of incubation of a semipurified enzyme (after the first ion-exchange chromatography) at pH 4·0 that dissociated the enzyme from VT, to which VTDCE is naturally tightly associated. The enzyme purity was confirmed by capillary and native gel electrophoresis, and SDS–PAGE suggested the enzyme is a dimer of 17 and 22 kDa. VTDCE was active upon several synthetic substrates, with a preference for a hydrophobic or a basic residue in P1, and a hydrophobic residue in P2. VTDCE also hydrolysed haemoglobin, albumin, gelatin and vitellin. VTDCE is inactive in the absence of DTT and was totally inhibited by E-64, indicating it is a cysteine endopeptidase. Our results suggest that VTDCE is a major enzyme involved in yolk processing during B. microplus embryogenesis.

Purification and Characterization of Superoxide Dismutase from Martianus Dermestoides Chevrola

2013 ◽  
Vol 773 ◽  
pp. 336-341 ◽  
Author(s):  
Wen Ge Yu ◽  
Bei Bei Zhang ◽  
Yong Jian Shen ◽  
Ying Li ◽  
Yong Bin Tian ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Ph Value ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Exchange Chromatography ◽  
Enzyme Activation ◽  
Purification And Characterization ◽  
Sds Page ◽  
Optimum Reaction

In this paper, the superoxide dismutase fromMartianus dermestoidesis purified by the following methods: heat treatment, polyethylene concentration, Sephadex G-75 gel filtration, and DEAE-Sepharose FF ion exchange chromatography. The result shows that the purification multiple is 3.86, the activation yield is 21.89% and the specific activation of the enzyme is 447.6 U/mg. The purified SOD appears to be a sole protein on SDS-PAGE and the molecular weight is estimated to be 40.58 kDa. H2O2can obviously inhibit the enzyme activation and CHCl3-CH3CH2OH only demonstrates basically no inhibitory effect. The type of the dermestoides SOD might be Cu/Zn-SOD. After purification, some enzymatic characterizations of the SOD are studied. The optimum reaction temperature of purified SOD is 50°C. The optimum reaction pH value of purification is 6. The dermestoide SOD has a preferable stability below 50°C and at pH values between 5-8.

scholarly journals Purification and characterization of N-glycanase, a concanavalin A binding protein from jackbean (Canavalia ensiformis)

1998 ◽  
Vol 330 (1) ◽  
pp. 13-20 ◽  
Author(s):  
S. Philip SHELDON ◽  
N. Jeffrey KEEN ◽  
J. Dianna BOWLES
Keyword(s):  
Concanavalin A ◽  
Principal Components ◽  
Enzymic Activity ◽  
Purification And Characterization ◽  
Con A ◽  
Glycoprotein Precursor ◽  
Ph Optimum ◽  
Native Page ◽  
Sds Page

Removal of the N-glycan from the concanavalin A (Con A) glycoprotein precursor is a key step in its conversion into an active lectin. N-Glycanase (EC 3.5.1.52), the enzyme from jackbean catalysing this process, has been purified to homogeneity as judged by native PAGE. One of the purification steps is binding of the enzymic activity to Con A-Sepharose and its elution by methyl α-mannoside. On SDS/PAGE the principal components were found to be 78 kDa, 74 kDa, 54 kDa, 32 kDa and 30 kDa polypeptides. These did not react with Con A on an affinity blot. Cleveland mapping indicated that some of these polypeptides had related primary structures. The enzyme has a broad pH optimum in the region of 5.0.

scholarly journals Characterization of phospholipid methylation in rat brain myelin

1995 ◽  
Vol 307 (1) ◽  
pp. 239-244 ◽  
Author(s):  
V Tsvetnitsky ◽  
L Auchi ◽  
A Nicolaou ◽  
W A Gibbons
Keyword(s):  
Rat Brain ◽  
Affinity Purification ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Methyl Transferase ◽  
Sds Page ◽  
Triton X ◽  
Predominant Product

Highly purified rat brain myelin was solubilized in Triton X-100 and myelin phospholipid N-methyltransferase was characterized. The enzyme activities were separated by isoelectric focusing and ion-exchange chromatography. The phospholipid methyl-transferase has shown at least four peaks of activity with pIapp. values of 4.5, 5.2, 6.2 and 8.4. After affinity purification each of these activities revealed a close set of bands of approx. 65 kDa on SDS/PAGE. These data together with those from preparative SDS/PAGE separations suggested that rat brain myelin contains three acidic and at least one basic phospholipid-methylating isoenzymes and that the major isoenzyme in each case is approx. 65 kDa in size. While the predominant product of the reaction catalysed by all detected isoforms was monomethylated phosphatidylethanolamine, the least acidic isoform (pIapp. 6.2) also formed about 20% phosphatidylcholine, suggesting that these isoenzymes may play different roles in vivo.

scholarly journals Characterization of a salt-extractable phosphatidylinositol synthase from rat pituitary-tumour membranes

1989 ◽  
Vol 257 (3) ◽  
pp. 639-644 ◽  
Author(s):  
A B Cubitt ◽  
M C Gershengorn
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Pituitary Tumour ◽  
Ph Optimum ◽  
Low Concentrations ◽  
Rat Pituitary ◽  
Phosphatidylinositol Synthase ◽  
Triton X

Solubilization of phosphatidylinositol (PtdIns) synthase (CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) from rat pituitary (GH3) tumours was investigated. PtdIns synthase activity was partially extracted from crude membranes by 3 M-KCl. Prior separation of membranes revealed that a greater proportion of plasma-membrane PtdIns synthase activity was salt-extractable than was endoplasmic reticulum activity. The activity of the salt-extracted enzyme was maximized by low concentrations of 3-(3-cholamidopropyl) dimethylammonio-1-propanesulphonate (CHAPS; 0.5 mM), Triton X-100 (0.1 mM) or a phospholipid mixture (0.05 mg/ml), but higher concentrations of detergents were inhibitory. The activity of salt-extracted PtdIns synthase was 0.25 +/- 0.08 nmol/min per mg of protein. Salt-extracted PtdIns synthase activity was dependent on Mg2+ (maximal at 0.1 mM) and Mn2+ (maximal at 5 mM), and its pH optimum was in the range 7.0-7.5. The apparent Km for myo-inositol (in the presence of 0.1 mM-CDP-diacylglycerol) was 0.06 mM, and that for CDP-diacylglycerol (at 0.1 mM-myo-inositol) was 0.21 mM. Salt-extracted PtdIns synthase activity was potently inhibited by Ca2+ (50% inhibition at 1 microM), with over 90% inhibition at 10 microM-Ca2+. These data imply the existence of two forms of membrane-associated PtdIns synthase, namely salt-extractable and salt-resistant, with different intracellular localizations. The salt-extractable form of this enzyme may be a useful preparation for further characterization and purification of mammalian PtdIns synthase.

scholarly journals Purification and characterization of cysteine-S-conjugate N-acetyltransferase from pig kidney

1996 ◽  
Vol 317 (1) ◽  
pp. 213-218 ◽  
Author(s):  
Achim AIGNER ◽  
Martina JÄGER ◽  
Ralf PASTERNACK ◽  
Peter WEBER ◽  
Dirk WIENKE ◽  
...  
Keyword(s):  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Gel Filtration ◽  
Protein Band ◽  
Exchange Chromatography ◽  
Polyclonal Antiserum ◽  
Partial Purification ◽  
Ph Optimum ◽  
Sds Page

Microsomal cysteine-S-conjugate N-acetyltransferase catalyses the N-acetylation of various S-substituted cysteines in liver and kidney. We describe here the purification and more detailed characterization of this enzyme catalysing the final reaction of mercapturic acid biosynthesis, and thus playing a crucial role in the detoxicating metabolism of many xenobiotics. The solubilization of cysteine-S-conjugate N-acetyltransferase by deoxy-BIGCHAP [N,N´-bis-(3-d-gluconamidopropyl)deoxycholamide] was the prerequisite for partial purification by means of anion-exchange chromatography. The molecular mass of the enzyme was determined by gel filtration. A polyclonal antiserum was raised against the excised protein band from SDS/PAGE and purified antibodies were used for the complete purification of native cysteine-S-conjugate N-acetyltransferase by immunoaffinity chromatography. A dimeric form of the enzyme was sometimes detected on SDS/PAGE, depending on the degree of purification. For further characterization of cysteine-S-conjugate N-acetyltransferase, the stability of catalytic activity, the pH optimum and Km values were determined. The inhibitory effects of various agents were tested, revealing a substantial, yet not complete, loss of cysteine-S-conjugate N-acetyltransferase activity after treatment with cysteine proteinase inhibitors or probenecid under various conditions.

scholarly journals Purification and characterization of cephalosporin 7α-hydroxylase from Streptomyces clavuligerus

1991 ◽  
Vol 280 (2) ◽  
pp. 471-474 ◽  
Author(s):  
X Xiao ◽  
S Wolfe ◽  
A L Demain
Keyword(s):  
Gel Filtration ◽  
Streptomyces Clavuligerus ◽  
Exchange Chromatography ◽  
Cephalosporin C ◽  
Hydroxylase Activity ◽  
Purification And Characterization ◽  
Major Band ◽  
Sds Page ◽  
Optimum Ph

Cephalosporin 7 alpha-hydroxylase, which catalyses the conversion of cephalosporins into their 7 alpha-hydroxy derivatives, was purified nearly 390-fold from Streptomyces clavuligerus through ion-exchange chromatography, (NH4)2SO4 fractionation, gel filtration and dye chromatography, with the use of h.p.l.c. to monitor enzyme activity. The nearly pure enzyme migrates as a single major band, with an Mr of 32,000 in SDS/PAGE. Its optimum pH is in the range 7.3-7.7. Under our conditions the reaction was fastest at temperatures in the range 20-30 degrees C. The Km for cephalosporin C is 0.72 mM, and the Vmax. is 15.4 mumol of cephalosporin C hydroxylated/min per mg. Cephalosporin 7 alpha-hydroxylase did not show any deacetoxycephalosporin C synthase or deacetoxycephalosporin C hydroxylase activity.

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