Purification and Characterization of Activation Fragments F1 and F2 from Human Prothrombin

1975 ◽  
Author(s):  
A. P. Ball ◽  
J. Fenton ◽  
D. L. Aronson ◽  
R. B. Franza ◽  
A. M. Young
Keyword(s):  
Gel Electrophoresis ◽  
Large Scale ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Scale Production ◽  
Purification And Characterization ◽  
Large Scale Production ◽  
Human Thrombin ◽  
A Chain

Considerable quantities of the non-thrombin portions of human prothrombin (II) have become available as a byproduct of the large-scale production of human thrombin (Biochim. Biophys. Acta 229, 26). Components not adsorbed on CG-50 are further purified by DEAE-cellulose chromatography and gel filtration, yielding the NH2-terminal fragment (F1) and the inner fragment (F2) which are homogeneous by SDS gel electrophoresis. SDS gel electrophoresis of reduced F1 indicates variable amounts of a two-chain derivative, F1’, with one chain migrating just ahead of F1 and one just ahead of the thrombin A-chain. The F1 → F1’ conversion is catalyzed by thrombin with the creation of a new MH2-terminal threonine. Ultracentrifugal patterns of human F1 and F2 closely resemble those of the bovine fragments. NH2-terminal residues were found to be alanine (± threonine) for F1 and serine for F2. Minor deviations from the reported amino acid compositions of bovine F1 and F2 were observed, primarily in the acidic residues. Other properties include:Immunization of rabbits with F1 gave a precipitating antibody to F1 which cross-reacts with II, but native F2 does not appear to be immunogenic. 3H-F1 is rapidly cleared from the blood of rabbits (T 1/2 20 min), with a major portion detectable in the urine.

scholarly journals Purification and characterization of acid phosphatase from a germinating black gram (Vigna mungo L.) seedling

2011 ◽  
Vol 63 (3) ◽  
pp. 747-756 ◽  
Author(s):  
A.K.M. Asaduzzaman ◽  
Habibur Rahman ◽  
Tanzima Yeasmin
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Black Gram ◽  
Km Value

An acid phosphatase has been isolated and purified from an extract of a germinating black gram seedling. The method was accomplished by gel filtration of a germinating black gram seedling crude extract on sephadex G-75 followed by ion exchange chromatography on DEAE cellulose. The acid phosphatase gave a single band on SDS-polyacrylamide slab gel electrophoresis. The molecular weight of the acid phosphatase determined by SDS-polyacrylamide slab gel electrophoresis was estimated to be 25 kDa. The purified enzyme showed maximum activity at pH 5 and at temperature of 55?C. Mg2+, Zn2+ and EDTA had an inhibitory effect on the activity of the acid phosphatase. Black gram seedling acid phosphatase was activated by K+, Cu2+ and Ba2+. The Km value of the enzyme was found to be 0.49 mM for pNPP as substrate.

scholarly journals Large-scale production and physicochemical characterization of human immune interferon.

1979 ◽  
Vol 26 (1) ◽  
pp. 36-41 ◽  
Author(s):  
M P Langford ◽  
J A Georgiades ◽  
G J Stanton ◽  
F Dianzani ◽  
H M Johnson
Keyword(s):  
Large Scale ◽  
Physicochemical Characterization ◽  
Scale Production ◽  
Immune Interferon ◽  
Large Scale Production ◽  
Human Immune

Purification and Characterization of Two Proteins from Culture Filtrates of Mycobacterium tuberculosis H 37 Ra Strain

1970 ◽  
Vol 1 (2) ◽  
pp. 164-168
Author(s):  
Thomas M. Daniel ◽  
Lavenia E. Ferguson
Keyword(s):  
Molecular Weight ◽  
Mycobacterium Tuberculosis ◽  
Skin Testing ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Purification And Characterization ◽  
Culture Filtrates ◽  
Zone Electrophoresis

Two proteins have been purified from culture filtrates of Mycobacterium tuberculosis , H 37 Ra strain by a procedure combining gel filtration, diethylaminoethyl (DEAE)-cellulose chromatography, and zone electrophoresis. The two proteins are similar in molecular weight but differ slightly in charge. The faster migrating protein, designated a 1 , is not antigenic. The slower migrating protein, designated a 2 , is antigenic both with respect to antisera and as a skin-testing antigen.

Purification and Characterization of an Acetyl Xylan Esterase from Bacillus pumilus

1998 ◽  
Vol 64 (2) ◽  
pp. 789-792 ◽  
Author(s):  
Giuliano Degrassi ◽  
Benedict C. Okeke ◽  
Carlo V. Bruschi ◽  
Vittorio Venturi
Keyword(s):  
Gel Electrophoresis ◽  
Bacillus Pumilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Acetyl Xylan Esterase ◽  
Purification And Characterization ◽  
Michaelis Constant ◽  
Naphthyl Acetate

ABSTRACT Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55°C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis constant (Km ) andV max for α-naphthyl acetate were 1.54 mM and 360 μmol min−1 mg of protein−1, respectively.

scholarly journals Further purification and characterization of the acid α-glucosidase

1968 ◽  
Vol 108 (2) ◽  
pp. 161-167 ◽  
Author(s):  
F. Auricchio ◽  
C. B. Bruni ◽  
V. Sica
Keyword(s):  
Rat Liver ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Human Kidney ◽  
Purification And Characterization ◽  
Preliminary Purification ◽  
Kidney Enzyme ◽  
Michaelis Constants ◽  
Final Degree

1. Centrifugation of rat liver acid glucosidase, which had been purified by adsorption on dextran gel, on a density gradient of sucrose showed the enzyme to be impure. 2. Preliminary purification of the enzyme before the gel filtration improved the final degree of purity of this preparation. Disc gel electrophoresis of this preparation showed a single band of protein. 3. The sedimentation co-efficient and the molecular weight determined on a sucrose gradient were 4·9–5·1s and 76000–83000 respectively for the rat liver enzyme, and 5·6s and 97000 for the acid α-glucosidase purified by means of the same procedure from the human kidney. 4. The Michaelis constants of rat liver and human kidney enzyme were 4·7×10−3m and 13·6×10−3m respectively with maltose as substrate. 5. The enzyme from both tissues was inhibited by tris and by erythritol. The inhibition of the rat liver acid glucosidase by erythritol was competitive.

scholarly journals Purification and characterization of histidine decarboxylase from mouse kidney

1986 ◽  
Vol 234 (2) ◽  
pp. 349-354 ◽  
Author(s):  
S A M Martin ◽  
J O Bishop
Keyword(s):  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Histidine Decarboxylase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Mouse Kidney ◽  
Purification Procedure ◽  
Purification And Characterization ◽  
A Subunit

Histidine decarboxylase was purified 800-fold from the kidneys of thyroxine-treated mice. The purification procedure included precipitation of protein from a crude supernatant after heating it to 55 degrees C at pH 5.5, fractionation with (NH4)2SO4, phosphocellulose column chromatography, chromatofocusing, DEAE-Sepharose column chromatography, gel filtration on Sephacryl S-300 and preparative polyacrylamide-gel electrophoresis. The native enzyme had an estimated Mr of 113 000. The protein was analysed in SDS/10%-polyacrylamide gels and formed a single band corresponding to a subunit Mr of 55 000, indicating that it is a dimer. Three forms of the enzyme were resolved on isoelectrofocusing gels, with pI 5.3, 5.5 and 5.7.

scholarly journals Preclinical Characterization of Naturally Occurring Polyketide Cyclophilin Inhibitors from the Sanglifehrin Family

2011 ◽  
Vol 55 (5) ◽  
pp. 1975-1981 ◽  
Author(s):  
Matthew A. Gregory ◽  
Michael Bobardt ◽  
Susan Obeid ◽  
Udayan Chatterji ◽  
Nigel J. Coates ◽  
...  
Keyword(s):  
Large Scale ◽  
Scale Production ◽  
Subgenomic Replicon ◽  
Isomerase Activity ◽  
Lead Compounds ◽  
Naturally Occurring ◽  
Large Scale Production ◽  
Pharmacokinetic Properties ◽  
Biosynthetic Engineering

ABSTRACTCyclophilin inhibitors currently in clinical trials for hepatitis C virus (HCV) are all analogues of cyclosporine (CsA). Sanglifehrins are a group of naturally occurring cyclophilin binding polyketides that are structurally distinct from the cyclosporines and are produced by a microorganism amenable to biosynthetic engineering for lead optimization and large-scale production by fermentation. Preclinical characterization of the potential utility of this class of compounds for the treatment of HCV revealed that the natural sanglifehrins A to D are all more potent than CsA at disrupting formation of the NS5A-CypA, -CypB, and -CypD complexes and at inhibition of CypA, CypB, and CypD isomerase activity. In particular, sanglifehrin B (SfB) was 30- to 50-fold more potent at inhibiting the isomerase activity of all Cyps tested than CsA and was also shown to be a more potent inhibitor of the 1b subgenomic replicon (50% effective concentrations [EC50s] of 0.070 μM and 0.16 μM in Huh 5-2 and Huh 9-13 cells, respectively). Physicochemical and mouse pharmacokinetic analyses revealed low oral bioavailability (F< 4%) and low solubility (<25 μM), although the half-lives (t1/2) of SfA and SfB in mouse blood after intravenous (i.v.) dosing were long (t1/2> 5 h). These data demonstrate that naturally occurring sanglifehrins are suitable lead compounds for the development of novel analogues that are less immunosuppressive and that have improved metabolism and pharmacokinetic properties.

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