scholarly journals The transporting proteins of cholecalciferol and 25-hydroxycholecalciferol in serum of chicks and other species. Partial purification and characterization of the chick proteins

1973 ◽  
Vol 135 (3) ◽  
pp. 417-426 ◽  
Author(s):  
S. Edelstein ◽  
D. E. M. Lawson ◽  
E. Kodicek
Keyword(s):  
Binding Proteins ◽  
Binding Protein ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Molecular Weights ◽  
Filtration Step ◽  
25 Hydroxycholecalciferol

Chick serum contains two cholecalciferol-binding proteins, one of which binds mainly cholecalciferol (cholecalciferol-binding protein) and the other binds 25-hydroxycholecalciferol (25-hydroxycholecalciferol-binding protein). By means of Cohn fractionation, (NH4)2SO4 precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephadex and an additional gel-filtration step on Sephadex G-100, these two binding proteins were purified. Both proteins possess β-globulin mobility on analytical polyacrylamide-disc-gel electrophoresis, a sedimentation coefficient of 3.5S and approximate molecular weights of 60000 for the cholecalciferol-binding protein and 54000 for the 25-hydroxycholecalciferol-binding protein. Sera obtained from rat, pig, human and monkey were shown to contain a single binding protein that is responsible for the transport of both cholecalciferol and 25-hydroxycholecalciferol. In the toad the lipoproteins are used for the transport of these two steroids.

Characterization and partial purification of a broad spectrum antibiotic AS-48 produced by Streptococcus faecalis

1986 ◽  
Vol 32 (10) ◽  
pp. 765-771 ◽  
Author(s):  
A. Gálvez ◽  
M. Maqueda ◽  
E. Valdivia ◽  
A. Quesada ◽  
E. Montoya
Keyword(s):  
Broad Spectrum ◽  
Gel Filtration ◽  
Basal Medium ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Gram Negative Bacteria ◽  
Broad Spectrum Antibiotic ◽  
Streptococcus Faecalis ◽  
Group D

Streptococcus faecalis S-48 produces a broad spectrum antibiotic, active against Gram-positive and Gram-negative bacteria. This substance is produced in solid and liquid media and also in a defined basal medium. It is sensitive to protease, pronase, or trypsin, heating at 70 °C, and alkaline pH, but resistant to treatment with lipase, lysozyme, alkaline phosphatase, DNAase, RNAase, acidic or neutral pHs, and also lower temperatures (60 °C). Several organic solvents cause precipitation, but not inactivation. This antibiotic has been partially purified by gel filtration and further ion-exchange chromatography. Its molecular weight has been estimated close to 2000. The biological activity of this antagonistic substance against the selected indicator strains, Streptococcus faecalis S-47 and Escherichia coli U-9, is bactericidal. The characterization of this substance, initially classified as a bacteriocin, indicates that it is an antibiotic of peptidic nature. The significance of antibiotic occurrence in group D of the genus Streptococcus is also discussed.

scholarly journals Partial purification of two lithocholic acid-binding proteins from rat liver 100000g supernatants

1977 ◽  
Vol 165 (3) ◽  
pp. 425-429 ◽  
Author(s):  
R C Strange ◽  
R Cramb ◽  
J D Hayes ◽  
I W Percy-Robb
Keyword(s):  
Ion Exchange ◽  
Binding Proteins ◽  
Lithocholic Acid ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Anion Binding ◽  
Partial Purification ◽  
Transferase Activity ◽  
Glutathione S Transferase

1. The partial purification of two lithocholic acid-binding proteins from liver 100 000g supernatants is described. 2. Gel-filtration, (NH4)2SO4 fractionation, Ca3(PO4)2 fractionation and ion-exchange chromatography were used. 3. Both proteins exhibited glutathione S-transferase activity; one may be the non-specific anion-binding protein ligandin. 4. Glutathione S-transferase activity of one of the binding proteins was inhibited by lithocholic acid.

scholarly journals Cadmium-binding proteins of rat testes. Apparent source of the protein of low molecular mass

1984 ◽  
Vol 220 (3) ◽  
pp. 819-824 ◽  
Author(s):  
M P Waalkes ◽  
S B Chernoff ◽  
C D Klaassen
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Amino Acid Analysis ◽  
Binding Proteins ◽  
Binding Protein ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Breakdown Product ◽  
Heat Stable ◽  
Metal Binding Protein

Fractionation of rat testicular cytosolic proteins by gel filtration indicates three major metal-binding proteins, or groups of proteins, termed testicular metal-binding protein (TMBP) 1, 2 and 3 by order of elution. The major heat-stable, metal-binding proteins in testes is TMBP-2, which has an Mr of approx. 25000. In most tissues, metallothionein (MT) is the major heat-stable, metal-binding protein, but it has an Mr of 6000. This testicular protein (TMBP-2) is much larger than MT, and since polymeric forms of MT have been previously reported, further characterization of TMBP-2 was performed. TMBP-2 was separated into two forms by DEAE-Sephadex A-25 anion-exchange chromatography. Amino acid analysis of both forms of TMBP-2 revealed that they differed markedly from MT, having particularly low cysteine contents. However, amino acid analysis showed that TBMP-2 was strikingly similar to TMBP-3, with an approximate stoichiometric relationship of 4:1. Therefore, experiments were conducted to determine if TMBP-3 could be a breakdown product of TMBP-2. Heat treatment of testicular cytosol in room air before gel filtration resulted in a marked increase in TMBP-3 and loss of TMBP-2. Storing intact testes at −20 degrees C for 2 weeks before processing for gel filtration also resulted in an increase in TMBP-3 and a loss of TMBP-2. Addition of a reducing agent (dithiothreitol) or proteinase inhibitor (N-ethylmaleimide) in processing of samples before gel filtration inhibited the appearance of TMBP-3. Results suggest that the low-Mr Cd-binding protein (TMBP-3) of rat testes results from either proteolytic or oxidative breakdown of a higher-Mr species, or from a combination of such factors.

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.

scholarly journals Identification and characterization of a calcium-binding protein in the mouse chorioallantoic placenta

1986 ◽  
Vol 233 (1) ◽  
pp. 41-49 ◽  
Author(s):  
R S Tuan ◽  
S T Cavanaugh
Keyword(s):  
Calcium Binding ◽  
Binding Protein ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Calcium Binding Protein ◽  
Chorionic Villi ◽  
Chorioallantoic Placenta ◽  
Identification And Characterization

Mouse chorioallantoic placenta contains a specific calcium-binding protein (MCaBP). A procedure involving gel filtration and ion-exchange chromatography was developed to purify the MCaBP. The MCaBP activity increased as a function of embryonic gestation and was highly specific for Ca2+. The MCaBP is a monomeric protein of Mr 57000, with pI 4.7. Specific antibodies were prepared against the MCaBP and were used to localize the MCaBP to syncytiotrophoblasts of the chorionic villi of mouse chorioallantoic placenta. These properties suggest that the MCaBP may be involved in transplacental calcium transport.

Purification and characterization of chymosin and pepsin from kid

2006 ◽  
Vol 73 (1) ◽  
pp. 49-57 ◽  
Author(s):  
Ekaterini E Moschopoulou ◽  
Ioannis G Kandarakis ◽  
Efstathios Alichanidis ◽  
Emmanouil M Anifantakis
Keyword(s):  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Temperature Dependent ◽  
Molecular Weights ◽  
Indigenous Breed ◽  
Increased Sensitivity ◽  
Pepstatin A

The objective of this work was to study the characteristics of the gastric aspartic proteinases chymosin and pepsin which are constituents of the kid rennet. The two enzymes were extracted from abomasal tissue of one kid from a local indigenous breed, separated from each other by DEAE-cellulose chromatography and then were purified by gel filtration and anion-exchange chromatography. The molecular weights of the purified kid chymosin and pepsin as determined by gel filtration were 36 kDa and 40 kDa respectively. The isoelectric point of kid chymosin was as multiple forms of 3–6 zones at pH 4·6–5·1, while that of kid pepsin was at pH [les ]3·0. Kid pepsin contained 0·37 molecules phosphorous per molecule and was totally inhibited by 5 μM pepstatin A, being more sensitive than kid chymosin. Both enzymes were almost equally as proteolytic as calf chymosin on total casein at pH 5·6. Kid pepsin activity was more pH and temperature dependent than kid chymosin activity. In comparison with the calf chymosin temperature sensitivity, the order of increased sensitivity was: calf chymosin <kid chymosin <kid pepsin.

Peroxidase-Polyphenol Oxidase Association in Dioscorea esculenta

1998 ◽  
Vol 53 (11-12) ◽  
pp. 957-960 ◽  
Author(s):  
J. Okpuzor ◽  
O. Omidiji
Keyword(s):  
Polyphenol Oxidase ◽  
Binding Protein ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Exchange Chromatography ◽  
Crude Enzyme Extract ◽  
Anionic Peroxidase ◽  
Molecular Weights ◽  
Protein Peak ◽  
Main Protein

Abstract A crude enzyme extract from Dioscorea esculenta var. fasiculata tissue subjected to ion exchange chromatography on DEAE-Sephadex A-50 column. This proced ure resolved the extract into two main protein peaks one of which eluted through the column relatively unbound while the other protein peak which remained bound to the column was eluted with 1.0 ᴍ NaCl. Both protein peaks contained polyphenol oxidase (PPO) and peroxidase (POD) activities. The non-binding protein peak was resolved by gel filtration on Sephadex G-200 into distinct PPO and POD activities and by virtue of their apparent molecular weights of 95.5 Kd and 38.0 Kd for PPO and POD respectively were determined to be the typical enzymes. The PPO activity was completely inhibited invitro by 5 mᴍ polyvinyl pyrrolidone (PVP). The binding protein peak was not resolved by gel filtration. It contained PPO activity which was not inhibited by PVP and a POD activity which was completely inhibited by dithiothreitol (DTT) This ionic protein peak contained 60% of total POD in the tissue, has an apparent molecular weight of 56 Kd and is suggested to be a strongly anionic peroxidase which also exhibits polyphenol oxidase activity.

Isolation and partial characterization of cytoplasmic and wall-bound acid phosphatases from wheat roots

1976 ◽  
Vol 54 (10) ◽  
pp. 1163-1169 ◽  
Author(s):  
Yoichi Hasegawa ◽  
K. R. Lynn ◽  
W. James Brockbank
Keyword(s):  
Sodium Dodecylsulfate ◽  
Gel Filtration ◽  
Creatine Phosphate ◽  
Total Activity ◽  
Exchange Chromatography ◽  
Acid Phosphatases ◽  
Wheat Roots ◽  
Molecular Weights ◽  
Ph Range

In wheat (Triticum aestivum) roots, about 67% of the total activity of acid phosphatase was associated with the cell wall debris, and 35% of it was released from the wall by incubation with 0.8 M NaCl overnight. Three major cytoplasmic and two major wall-bound acid phosphatases were then separated by gel filtration on Sephadex G-75 and ion-exchange chromatography on carboxymethyl cellulose, and some of their characteristics were compared. They showed maximum activities in the same pH range (4.7–5.0) and had broad substrate specificities. They all showed high activity toward ADP. followed by ATP, glucose-6-phosphate, and creatine phosphate and much less activity toward AMP, glucose-1-phosphate, fructose-1,6-diphosphate, and ribose-5-phosphate. The wall-bound enzymes were more active to ADP and ATP than those of the cytoplasm. Mg2+, Ni2+, NaCN, and EDTA had no appreciable effects on the enzymatic activities. However, all enzymes were strongly inhibited by Hg2+ and Fe3+ and, to varied degrees, by Cu2+, Zn2+, Co2+, NaF. p-chloromercuribenzoate, and urea in that order. The molecular weights, estimated by sodium dodecylsulfate gel electrophoresis, ranged from 28 000 to 64 000.

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