scholarly journals Characterization of Multiple forms of Human Thrombin

1977 ◽  
Author(s):  
J.J. Gorman ◽  
P.A. Castaldi
Keyword(s):  
Specific Activity ◽  
Peptide Mapping ◽  
Multiple Forms ◽  
Bovine Thrombin ◽  
Molecular Weights ◽  
Human Thrombin ◽  
Affinity Resin ◽  
C 50 ◽  
High Degree

Human thrombin was obtained by activation of partially purified human prothrombin with venom of the Australian Taipan (oxyuranus scutellatus scutellatus).The crude thrombin was precipitated with ammonium sulphate and subsequently purified by chromatography on Sephadex G-75 CM-Sephadex C-50 and the affinity resin am inobenzamidine-CH-Sepharose. The final preparation had a specific activity of 1700 units per absorbance unit (A| cm 280n m Was herterogenous as shown by urea-acrylamide gel electrophoresis at acid pH and by isoelectric focusing. SDS-acrylamide electrophoresis revealed molecular weights of 39,000, 28,000, 25-23,000 and 15-12,000 for these proteins. The 39,000 dalton species predominated (greater than 90%) when the enzyme was inhibited with phenyImethanesuI phony I fluoride prior to dialysis against 0.02M sod i urn phosphate (pH 8.0) containing 0.1% SDS. Lack of such inhibition reduced the amount of the 39,000 dalton species to less than 60% with concomitant increase in the smaller species. Increase in the smaller species also occurred during incubation in 0.IM NaCI-0.I M Tris buffer (pH 8.0).Peptide mapping studies indicated that the smaller species were structurally related to the 39,000 dalton species. Amino acid compositions of tryptic peptides indicated a high degree of homology with bovine thrombin.It has been established that human thrombin can exist in at least two secondary structural forms of different molecular weights, probably due to autolytic degradation of the largest (39,000 dalton) protein species.

scholarly journals Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Vol 81 (1) ◽  
Author(s):  
Rahma R. Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M. S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

scholarly journals Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants (Camellia sinensis (L.) O. Kuntze)

2005 ◽  
Vol 37 (6) ◽  
pp. 363-370 ◽  
Author(s):  
Ye-Yun Li ◽  
Chang-Jun Jiang ◽  
Xiao-Chun Wan ◽  
Zheng-Zhu Zhang ◽  
Da-Xiang Li
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Optimum Activity ◽  
Development Of Resistance ◽  
Sds Page ◽  
C 50 ◽  
Tea Plants

Abstractβ-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity, with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50 °C and was stable at temperatures lower than 40 °C. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.

scholarly journals Plasmid-mediated trimethoprim-resistance in Staphylococcus aureus. Characterization of the first gram-positive plasmid dihydrofolate reductase (type S1)

1987 ◽  
Vol 243 (1) ◽  
pp. 309-312 ◽  
Author(s):  
H K Young ◽  
R A Skurray ◽  
S G B Amyes
Keyword(s):  
Staphylococcus Aureus ◽  
Dihydrofolate Reductase ◽  
Specific Activity ◽  
Gram Positive ◽  
Heat Stable ◽  
Dihydrofolate Reductases ◽  
High Degree ◽  
Moderately Resistant ◽  
Identification And Characterization

The trimethoprim-resistance gene located on plasmid pSK1, originally identified in a multi-resistant Staphylococcus aureus from Australia, encodes the production of a dihydrofolate reductase (type S1), which confers a high degree of resistance to its host and is quite unlike any plasmid-encoded dihydrofolate reductase hitherto described. It has a low Mr (19,700) and has a higher specific activity than the constitutive Gram-negative plasmid dihydrofolate reductases. The type S1 enzyme is heat-stable and has a relatively low affinity for the substrate, dihydrofolate (Km 10.8 microM). It is moderately resistant to trimethoprim, and is competitively inhibited by this drug with an inhibitor-binding constant of 11.6 microM. This is the first identification and characterization of a plasmid-encoded trimethoprim-resistant dihydrofolate reductase derived from a Gram-positive species.

scholarly journals Purification and characterization of fat body lipase from the Greater Wax Moth Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Author(s):  
Rahma R.Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M.S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

AbstractLipid mobilization and transport in insects is under investigation, especially lipases and lipophorin because of their roles in energy production and transport of lipids at flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from last larval instar of Galleria mellonella. Purification methods by combination of ammonium sulfate precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633±0.000551 mg/ml and 1.5754±0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore biochemical characterization of fat body lipase was carried out through testing its activities against several factors such as; different temperatures, pH ranges, metal ions and inhibitors ending by determination of their kinetic parameters with the use of p-Nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35-37°C and 37-40°C and pH ranges of 7-9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+ and Na+ metal ions indicating that fat body lipase is metalloproteinase. Additionally, lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfony fluoride (PMSF), ethylene-diaminetetractic acid (EDTA) and ethylene glycoltetraacetic acid (EGTA) providing an evidence of presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 301.95mM Km and 0.316 Umg−1 Vmax. By considering the purification of fat body lipase from larvae and using some inhibitors especially ion chelating agents, it is suggested to develop this study by using lipase inhibitors to reach a successful control of Galleria mellonella in the near future.

Transgenically Produced Human Antithrombin: Structural and Functional Comparison to Human Plasma–Derived Antithrombin

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4561-4571 ◽  
Author(s):  
Tim Edmunds ◽  
Scott M. Van Patten ◽  
Julie Pollock ◽  
Eric Hanson ◽  
Richard Bernasconi ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Human Plasma ◽  
High Performance ◽  
Specific Activity ◽  
Peptide Mapping ◽  
Goat Milk ◽  
Glycosylation Site ◽  
Thrombin Inhibition ◽  
High Degree

Abstract Recombinant human antithrombin (rhAT) produced in transgenic goat milk was purified to greater than 99%. The specific activity of the rhAT was identical to human plasma–derived AT (phAT) in an in vitro thrombin inhibition assay. However, rhAT had a fourfold higher affinity for heparin than phAT. The rhAT was analyzed and compared with phAT by reverse phase high-performance liquid chromatography, circular dichroism, fluorophore-assisted carbohydrate electrophoresis (FACE), amino acid sequence, and liquid chromatography/mass spectrography peptide mapping. Based on these analyses, rhAT was determined to be structurally identical to phAT except for differences in glycosylation. Oligomannose structures were found on the Asn 155 site of the transgenic protein, whereas only complex structures were observed on the plasma protein. RhAT contained a GalNAc for galactose substitution on some N-linked oligosaccharides, as well as a high degree of fucosylation. RhAT was less sialylated than phAT and contained both N-acetylneuraminic and N-glycolylneuraminic acid. We postulate that the increase in affinity for heparin found with rhAT resulted from the presence of oligomannose-type structures on the Asn 155 glycosylation site and differences in sialylation.

scholarly journals Purification and characterization of an L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)-specific suppressor factor from genetic responder mice.

1983 ◽  
Vol 158 (4) ◽  
pp. 1034-1047 ◽  
Author(s):  
C M Sorensen ◽  
C W Pierce ◽  
D R Webb
Keyword(s):  
Specific Activity ◽  
Reversed Phase ◽  
Biologically Active ◽  
Reversed Phase Hplc ◽  
Purification And Characterization ◽  
Suppressor Factor ◽  
Chemical Homogeneity ◽  
T Cell Factor ◽  
Molecular Weights

A hybridoma-derived, GAT-specific suppressor T cell factor (GAT-TsFR) from responder C57BL/10 mice has been purified to apparent chemical homogeneity using reversed phase HPLC techniques. 40 l of starting material yielded approximately 880 micrograms protein with a specific activity of 28.4 X 10(3) S50 U/ng protein representing a purification factor of 4.2 X 10(6). Purified GAT-TsFR is a hydrophobic protein with a minimum molecular weight of 18,000 that is capable of forming biologically active aggregates with molecular weights of 28,000, 64,000 and approximately 84,000 and has a pI of 6.4. GAT-TsFR is a glycoprotein that binds GAT and GT, but not GA, and bears determinants encoded by the I-J subregion of the H-2 complex. This GAT-TsFR derived from an H-2b responder haplotype to GAT is compared with GAT-TsF derived from the nonresponder H-2q haplotype on the basis of biochemical and some serological properties.

Purine catabolism in man: characterization of placental microsomal 5′-nucleotidase

1976 ◽  
Vol 54 (5) ◽  
pp. 462-469 ◽  
Author(s):  
Irving H. Fox ◽  
Pamela J. Marchant
Keyword(s):  
Specific Activity ◽  
Ph Optimum ◽  
Electrophoretic Variants ◽  
Molecular Weights ◽  
Wide Range ◽  
Stokes Radius ◽  
Michaelis Constants ◽  
Light Form ◽  
Molecular Weight Form

Human placental microsomal 5′-nucleotidase (EC 3.1.3.5) was prepared free of alkaline phosphatase by isoelectric focusing. A total of seven electrophoretic variants were isolated during the preparation of six placentas. Only three to six variants were found in a single placenta. The isoelectric pH's were 6.70, 6.44, 6.23, 6.02, 5.76, 5.63 and 5.44. These were found to be composed of variable quantities of a large, medium and low molecular weight form. The apparent molecular weights of the medium and light form of the enzyme were 86 500 and 43 500, respectively, as estimated from Stokes radius and sedimentation velocity determinations. The electrophoretic variants were not distinguishable with respect to specific activity and Michaelis constants for AMP, GMP or CMP or inhibition by ATP, CTP or adenosine. These electrophoretic variants appeared to be pseudoisozymes based upon different states of aggregation of a common primary sequence.There was a wide range of substrate specificity among nucleoside 5′-monophosphates which included 2-deoxyribose compounds. With AMP as 100, substrate activity was: CMP, 122; NMN, 74; GMP, 68; IMP, 63; XMP, 28 and UDP–glucose, 68. The Michaelis constants for AMP, GMP and CMP ranged from 12–18 μM, from 33–67 μM and from 170–250 μM, respectively. Although 5′-nucleotidase was active in the absence of divalent cation, 5 mM MgCl2 stimulated the enzyme activity to 234% of control and shifted the pH optimum of 9.8 to a plateau from pH 7.4–9.8.

Transgenically Produced Human Antithrombin: Structural and Functional Comparison to Human Plasma–Derived Antithrombin

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4561-4571
Author(s):  
Tim Edmunds ◽  
Scott M. Van Patten ◽  
Julie Pollock ◽  
Eric Hanson ◽  
Richard Bernasconi ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Human Plasma ◽  
High Performance ◽  
Specific Activity ◽  
Peptide Mapping ◽  
Goat Milk ◽  
Glycosylation Site ◽  
Thrombin Inhibition ◽  
High Degree

Recombinant human antithrombin (rhAT) produced in transgenic goat milk was purified to greater than 99%. The specific activity of the rhAT was identical to human plasma–derived AT (phAT) in an in vitro thrombin inhibition assay. However, rhAT had a fourfold higher affinity for heparin than phAT. The rhAT was analyzed and compared with phAT by reverse phase high-performance liquid chromatography, circular dichroism, fluorophore-assisted carbohydrate electrophoresis (FACE), amino acid sequence, and liquid chromatography/mass spectrography peptide mapping. Based on these analyses, rhAT was determined to be structurally identical to phAT except for differences in glycosylation. Oligomannose structures were found on the Asn 155 site of the transgenic protein, whereas only complex structures were observed on the plasma protein. RhAT contained a GalNAc for galactose substitution on some N-linked oligosaccharides, as well as a high degree of fucosylation. RhAT was less sialylated than phAT and contained both N-acetylneuraminic and N-glycolylneuraminic acid. We postulate that the increase in affinity for heparin found with rhAT resulted from the presence of oligomannose-type structures on the Asn 155 glycosylation site and differences in sialylation.

scholarly journals Purification and Characterization of a Novel Thermostable α-l-Arabinofuranosidase from a Color-Variant Strain of Aureobasidium pullulans

1998 ◽  
Vol 64 (1) ◽  
pp. 216-220 ◽  
Author(s):  
Badal C. Saha ◽  
Rodney J. Bothast
Keyword(s):  
Column Chromatography ◽  
Metal Ion ◽  
Aureobasidium Pullulans ◽  
Specific Activity ◽  
Gel Filtration ◽  
Variant Strain ◽  
Color Variant ◽  
C 50 ◽  
Hydrolysis Of

ABSTRACT A color-variant strain of Aureobasidium pullulans (NRRL Y-12974) produced α-l-arabinofuranosidase (α-l-AFase) when grown in liquid culture on oat spelt xylan. An extracellular α-l-AFase was purified 215-fold to homogeneity from the culture supernatant by ammonium sulfate treatment, DEAE Bio-Gel A agarose column chromatography, gel filtration on a Bio-Gel A-0.5m column, arabinan-Sepharose 6B affinity chromatography, and SP-Sephadex C-50 column chromatography. The purified enzyme had a native molecular weight of 210,000 and was composed of two equal subunits. It had a half-life of 8 h at 75°C, displayed optimal activity at 75°C and pH 4.0 to 4.5, and had a specific activity of 21.48 μmol · min−1· mg−1 of protein againstp-nitrophenyl-α-l-arabinofuranoside (pNPαAF). The purified α-l-AFase readily hydrolyzed arabinan and debranched arabinan and released arabinose from arabinoxylans but was inactive against arabinogalactan. TheKm values of the enzyme for the hydrolysis of pNPαAF, arabinan, and debranched arabinan at 75°C and pH 4.5 were 0.26 mM, 2.14 mg/ml, and 3.25 mg/ml, respectively. The α-l-AFase activity was not inhibited at all byl-arabinose (1.2 M). The enzyme did not require a metal ion for activity, and its activity was not affected byp-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM).

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