Partial Purification and Characterization of an ADP Phosphohydrolase from Human Plasma

1971 ◽  
Vol 26 (01) ◽  
pp. 177-191 ◽  
Author(s):  
I Holmsen ◽  
H Holmsen
Keyword(s):  
Substrate Concentration ◽  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Partial Purification ◽  
Low Concentrations ◽  
Isotope Technique ◽  
High Concentrations ◽  
Hydrolysis Of ◽  
Plasma Adenosine

SummaryPlasma contains enzymes capable of dephosphorylating ADP, ATP and AMP (adenosine di-, tri- and monophosphate). In platelet-rich plasma these enzymes are important for the regulation of the levels of (platelet-aggregating) ADP and (aggregation-inhibitory) adenosine.Plasma ADPase and ATPase were studied at 1 (µM substrate concentration using an isotope technique. Both enzymes were precipitated from plasma at 45-65% saturation with (NH4)2S04 and emerged together by gel filtration on Sephadex G-200 and from DEAE-Sephadex (0.12-0.20 M Cl-, pH 8.2). In combination these procedures gave 1,500-1,800 times purification of ADPase relative to plasma. The purest fraction contained ATPase, ADPase and AMPase in a 0.17:1.00:2.92 proportion, quite different from their 5.34:1.00:5.34 proportion in plasma. Adenosine deaminase and adenylate kinase were not present in the purest fraction, whereas nucleoside diphosphokinase appeared to be present.The purified ADPase was stimulated by low concentrations of Mg2+ and Mn2+, whereas high concentrations were inhibitory. This inhibition could not be explained by an increase in the ionic strength. Ca2+ and Zn2+ were inhibitory at all concentrations used (0-3 mM). Lineweaver-Burke plots were linear for both ADPase and ATPase in the 0−4 x 10-5 M substrate range, and both enzymes had Km = 1.1 x 10−5 M. Increase of the substrate concentration above 4 x 10−5M gave deviation from MichaelisMenten kinetics, and Eadie-Hofstee plots indicated the presence of “high-Km” ADPase and ATPase. The latter enzymes were not studied.Déphosphorylation of 3H-ADP by purified “low-Km” ADPase was reduced by nonradioactive diphosphates of guanosine, inosine, cytidine and uridine in the same way as when nonradioactive ADP was used. Nonradioactive AMP also reduced dephosphorylation of 3H-ADP, whereas nonradioactive ATP did not.Cyanide, cysteine and tartrate inhibited “low-Km” ADPase whereas p-chloromercuribenzoate, p-chloromercuribenzoesulphonate and N-ethylmaleimide had no effect. EDTA inhibited the enzyme activity in a way that could not be abolished by excess Mg2+. Purified plasma “low-Km” ADPase thus appears to be an unspecific enzyme, as one and the same active site does not seem to distinguish between the base moiety of nucleoside diphosphates, and catalyzes hydrolysis of phosphate esters as well as pyrophosphate bonds. The relation between plasma ADPase and ATPase remains unclear.

scholarly journals The cellulolytic enzymes of Botryodiplodia theobromae Pat. Separation and characterization of cellulases and β-glucosidases

1979 ◽  
Vol 177 (1) ◽  
pp. 9-19 ◽  
Author(s):  
Gabriel M. Umezurike
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Cellulolytic Enzymes ◽  
Botryodiplodia Theobromae ◽  
Molecular Weights ◽  
Low Concentrations ◽  
High Concentrations

1. Filtrates from cultures of different ages of Botryodiplodia theobromae Pat. were fractionated by gel filtration, ion-exchange chromatography and polyacrylamide-gel electrophoresis. 2. Five cellulases (C1, C2, C3, C4 and C5) were found, and their molecular weights, estimated by gel filtration, were 46000–48000 (C1), 30000–35000 (C2), 15000–18000 (C3), 10000–11000 (C4) and 4800–5500 (C5). 3. Cellulase C5 was absent from old culture filtrates. 4. Cellulase C1 had little or no activity on CM-cellulose (viscometric assay), but degraded cotton flock and Whatman cellulose powder to give cellobiose only. 5. The other components (C2–C5) produced cellobiose and smaller amounts of glucose and cellotriose from cellulosic substrates and were more active in lowering the viscosity of CM-cellulose. 6. The ratio of activities assayed by viscometry and by the release of reducing sugars from CM-cellulose increased with decrease in the molecular weights of cellulases C2–C5. 7. Cellobiose inhibited the activities of the cellulases, but glucose stimulated at low concentrations although it inhibited at high concentrations. 8. A high-molecular-weight β-glucosidase (component B1, mol.wt. 350000–380000) predominated in filtrates from young cultures, but a low-molecular-weight enzyme (B4, mol.wt. 45000–47000) predominated in older filtrates. 9. Intermediate molecular species of β-glucosidase (B2, mol.wt. 170000–180000; B3, mol.wt. 83000–87000) were also found. 10. Cellulases C2–C5 acted in synergism with C1, particularly in the presence of β-glucosidase.

Post-proline endopeptidase. Partial purification and characterization of the enzyme from pig kidneys

1982 ◽  
Vol 47 (4) ◽  
pp. 1139-1148 ◽  
Author(s):  
Karel Hauzer ◽  
Linda Servítová ◽  
Tomislav Barth ◽  
Karel Jošt
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Hydrolysis Of ◽  
Prolyl Leucyl Glycinamide

Post-proline endopeptidase was isolated from pig kidneys and partially purified. The procedure consisted of fractionation with ammonium sulphate, ion exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-200 and rechromatography on DEAE-Sephadex A-50. The preparation had 55 times higher specific activity than the crude extract and did not contain any contaminating enzymic activities. The enzyme cleaved a number of proline-containing peptides and was strictly specific in catalyzing the hydrolysis of the peptide bond on the carboxyl side of the proline residue. The optimum pH for the hydrolysis of the synthetic peptides benzyl-oxycarbonylglycyl-prolyl-leucyl-glycinamide and benzyloxycarbonyl-glycyl-proline β-naphtylamide was 7.8-8.0 and, in the case of benzyloxycarbonylglycyl-proline p-nitroanilide, 7.2 to 7.5. For the hydrolysis of the tetrapeptide benzyloxycarbonylglycyl-prolyl-leucyl-glycinamide, the Km value of 75 μ mol l-1 was obtained.

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.

Isolation and characterization of ferritin from the liver of the rainbow trout (Salmo gairdneri R.)

1991 ◽  
Vol 69 (10-11) ◽  
pp. 735-741 ◽  
Author(s):  
J. L. Miguel ◽  
M. I. Pablos ◽  
M. T. Agapito ◽  
J. M. Recio
Keyword(s):  
Rainbow Trout ◽  
Gel Filtration ◽  
Heat Extraction ◽  
Salmo Gairdneri ◽  
Neutral Sugar ◽  
Isolation And Characterization ◽  
Low Concentrations ◽  
Decreasing Ph ◽  
High Concentrations

A method for the purification of ferritin from rainbow trout liver by heat extraction and gel filtration is described. The number of iron atoms varied from 500 to 2000 in purified ferritin. The neutral sugar composition detected was 86 mol of glucose, 24 mol of fucose, 12 mol of galactose, and 8 mol of mannose per mol of ferritin and apoferritin. Release of iron was achieved using low molecular weight chelating agents. The order of effectiveness of chelators was nitrilotriacetate > EDTA > citrate. Removal of the iron does not imply reduction of Fe3+. The rate of release of iron increased with decreasing pH. The slowest release was at pH 7.5. The endogenous chelator is not only sulphydrylic but seems to include carbohydrates that participate in the binding of Fe2+. Trout ferritin exhibits heterogeneity upon isoelectric focusing; four isoferritins with pI values of 4.5 to 4.85 were detected. This heterogeneity represents polymorphic, not polymer, forms. The amino acid composition differs from that of ferritins from other species. High concentrations of glutamic and aspartic acids, alanine, leucine, glycine, and lysine were detected along with low concentrations of methionine and cysteine.Key words: ferritin, isoferritin, rainbow trout.

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.

LOCALIZATION AND CHARACTERIZATION OF CHOLINESTERASE IN SUBCELLULAR FRACTIONS OF RAT BRAIN AND BEEF PITUITARY

1961 ◽  
Vol 39 (9) ◽  
pp. 1335-1345 ◽  
Author(s):  
Surendra S. Parmar ◽  
Morley C. Sutter ◽  
Mark Nickerson
Keyword(s):  
Rat Brain ◽  
Cholinesterase Activity ◽  
Succinic Dehydrogenase ◽  
Anterior Lobe ◽  
Subcellular Fractions ◽  
Posterior Pituitary ◽  
Low Concentrations ◽  
Pituitary Glands ◽  
Hydrolysis Of

Fresh rat brains and fresh anterior and posterior pituitary glands of beef were separated by differential centrifugation into subcellular fractions, characterized on the basis of sedimentation and succinic dehydrogenase activity. Cholinesterase activity was measured by both manometric and colorimetric methods, the results of which were comparable. Cholinesterase activity of rat brain was found mainly in the microsome and supernatant fractions. It was quite uniformly distributed in all subcellular fractions of both anterior and posterior pituitary. Comparisons of the relative rates of hydrolysis of acetylthiocholine and butyrylthiocholine, and of inhibition by eserine, indicated that brain contains a much higher percentage of acetylcholinesterase than do both lobes of the pituitary, which contain relatively low concentrations of the specific enzyme. Total cholinesterase activity and its sensitivity to inhibition by eserine in the posterior pituitary were found to be midway between those of the anterior lobe and of the brain, from which the posterior pituitary was derived during embryological development.

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 Effects of a Polyoxyethylene Detergent on Platelet Function

1977 ◽  
Author(s):  
P.G. Barton
Keyword(s):  
Lactate Dehydrogenase ◽  
Platelet Rich Plasma ◽  
Secondary Phase ◽  
Human Platelets ◽  
Brij 58 ◽  
Low Concentrations ◽  
High Concentrations ◽  
Glass Surfaces ◽  
Induced Aggregation ◽  
Membrane Destabilization

Low concentrations of a polyoxyethylene detergent, Brij 58, inhibited the secondary phase of platelet aggregation induced by ADP in human citrated platelet rich plasma but had no effect on primary aggregation.Thrombin-induced aggregation of washed human platelets suspended in Tyrode’s buffer was inhibited after incubation of cells with 4.5 × 10-6M detergent. Development of prothrombin-converting activity and efflux of [14C]-serotonin, 45Ca2+ ions and labile endoperoxides were abolished concomitantly. Aggregation of washed platelets by collagen or sodium arachidonate and the attachment of cells to clean glass surfaces were also inhibited by the same concentration of Brij 58 that inhibited thrombin aggregation. This concentration of Brij 58 did not itself produce any release of a cytoplasmic marker, lactate dehydrogenase, from platelets. Higher concentrations of Brij 58, exceeding 10-4 M, lysed the cells liberating all of their serotonin, Ca2+ and lactate dehydrogenase. These results suggest that low concentrations of Brij 58 stabilize a membrane conformation against the action of platelet stimulatory agents while high concentrations produce membrane destabilization and cell lysis. The presence of albumin (BSA) in the suspending fluid increased by tenfold the concentrations of detergent required to “elicit these effects and this could be attributed to competitive binding of the detergent to albumin, demonstrated with [14C]-acetylated Brij 58.

scholarly journals Partial purification and properties of a β-N-acetylglucosaminidase from the fungus Sclerotinia fructigena

1973 ◽  
Vol 131 (2) ◽  
pp. 381-388 ◽  
Author(s):  
F. Reyes ◽  
R. J. W. Byrde
Keyword(s):  
Nitrogen Source ◽  
Cellulose Acetate ◽  
Isoelectric Focusing ◽  
Culture Filtrate ◽  
Gel Filtration ◽  
Partial Purification ◽  
Conflicting Evidence ◽  
Purification And Properties ◽  
Km Value ◽  
Hydrolysis Of

1. As cultures of the fungus Sclerotinia fructigena autolysed, the filtrates contained increasing quantities of a β-N-acetylglucosaminidase. 2. The enzyme was purified up to 42-fold by a combination of isoelectric focusing and gel filtration. 3. It ran as a single band in cellulose acetate strip electrophoresis and in isoelectric focusing (pI3.76). 4. The enzyme did not readily hydrolyse chitin or a glycopeptide with terminal N-acetylglucosamine residues, but rapidly degraded the N-acetylglucosamine dimer NN′-diacetylchitobiose; the monomer was readily utilized by the fungus as a nitrogen source. The Km value for hydrolysis of p-nitrophenyl β-2-acetamido-2-deoxy-d-glucopyranoside at 37°C was 2.0mm. The Sclerotinia enzyme was generally less susceptible to inhibition by 2-acetamido-2-deoxygluconolactone and other related sugars than the corresponding enzyme from other sources. Inhibition by excess of substrate was observed. 5. The culture filtrate also contained N-acetylgalactosaminidase activity; conflicting evidence was obtained as to whether the same enzyme was responsible for both hexosaminidase activities.

scholarly journals Ultrastructural and Partial Biochemical Characterization of Platelets from Chediak-Higashi Cattle

1977 ◽  
Author(s):  
K. M. Meyers ◽  
C. I. Seacord ◽  
G. Hopkins ◽  
H. Holmsen
Keyword(s):  
Electron Micrographs ◽  
Biochemical Characterization ◽  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Human Platelets ◽  
Additional Information ◽  
Storage Pool ◽  
Calcium And Magnesium ◽  
Chediak Higashi Syndrome

To provide additional information on the platelet defect which is associated with the Chediak-Higashi syndrome (CHS), platelet rich plasma from normal and CHS cattle was incubated with 14C-adenine. Platelets were then isolated by gel filtration and treated with thrombin. Both the resting amount and extent of secretion of ATP, ADP, several acid hydrolasis, serotonin, calcium and magnesium was determined. Nucleotide profiles and electron micrographs of resting and thrombin treated platelets were also obtained. The markedly reduced secretion of nucleotides, serotonin, and metals demonstrate that CHS cattle have a storage pool defect. Furthermore, there appears to be significant differences in both the resting amount and extent of secretion of several of these measured substances between normal cattle and human platelets.

Sign in / Sign up

Export Citation Format

Share Document