scholarly journals Characterization of Affinity-Purified Isoforms ofAcinetobacter calcoaceticusY1 Glutathione Transferases

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Chin-Soon Chee ◽  
Irene Kit-Ping Tan ◽  
Zazali Alias
Keyword(s):  
Hydrogen Peroxide ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Ethacrynic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Acinetobacter Calcoaceticus ◽  
Glutathione Transferases ◽  
Glutathione S Transferase ◽  
Substrate Specificities

Glutathione transferases (GST) were purified from locally isolated bacteria,Acinetobacter calcoaceticusY1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, andtrans,trans-hepta-2,4-dienalwhile GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) ofAcinetobacter calcoaceticusstrain PHEA-2, respectively.

Prothrombin Metz : Purification and Characterization of a Variant of Human Prothrombin

1979 ◽  
Author(s):  
M Ribieto ◽  
J Elion ◽  
D Labie ◽  
F Josso
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Cleavage Pattern ◽  
Purification And Characterization ◽  
Double Immunodiffusion ◽  
The Family

For the purification of the abnormal prothrombin (Pt Metz), advantage has been taken of the existence in the family of three siblings who, being double heterozygotes for Pt Metz and a hypoprothrombinemia, have no normal Pt. Purification procedures included barium citrate adsorption and chromatography on DEAE Sephadex as for normal Pt. As opposed to some other variants (Pt Barcelona and Madrid), Pt Metz elutes as a single symetrical peak. By SDS polyacrylamide gel electrophoresis, this material is homogeneous and appears to have the same molecular weight as normal Pt. Comigration of normal and abnormal Pt in the absence of SDS, shows a double band suggesting an abnormal charge for the variant. Pt Metz exhibits an identity reaction with the control by double immunodiffusion. Upon activation by factor Xa, Pt Metz can generate amydolytic activity on Bz-Phe-Val-Arg-pNa (S2160), but only a very low clotting activity. Clear abnormalities are observed in the cleavage pattern of Pt Metz when monitored by SDS gel electrophoresis. The main feature are the accumulation of prethrombin l (Pl) and the appearance of abnormal intermediates migrating faster than Pl.

Purification and Characterization of a High-Molecular-Weight Insecticidal Protein Complex Produced by the Entomopathogenic BacteriumPhotorhabdus luminescens

1998 ◽  
Vol 64 (8) ◽  
pp. 3029-3035 ◽  
Author(s):  
David J. Bowen ◽  
Jerald C. Ensign
Keyword(s):  
Molecular Weight ◽  
Insecticidal Activity ◽  
Protein Complex ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Toxin ◽  
The Family ◽  
Toxin Complex

ABSTRACT Photorhabdus luminescens is a gram-negative enteric bacterium that is found in association with entomopathogenic nematodes of the family Heterorhabditidae. The nematodes infect a variety of soil-dwelling insects. Upon entering an insect host, the nematode releases P. luminescens cells from its intestinal tract, and the bacteria quickly establish a lethal septicemia. When grown in peptone broth, in the absence of the nematodes, the bacteria produce a protein toxin complex that is lethal when fed to, or injected into the hemolymph of, Manduca sexta larvae and several other insect species. The toxin purified as a protein complex which has an estimated molecular weight of 1,000,000 and contains no protease, phospholipase, or hemolytic activity and only a trace of lipase activity. The purified toxin possesses insecticidal activity whether injected or given orally. Analyses of the denatured complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed it to be composed of several protein subunits ranging in size from 30 to 200 kDa. The complex was further separated by native gel electrophoresis into three components, two of which retained insecticidal activity. The purified native toxin complex was found to be active in nanogram concentrations against insects representing four orders of the classInsecta.

Prothrombin Metz: Purification and Characterization of a Variant of Human Prothrombin

1979 ◽  
Author(s):  
M.J. Rabiet ◽  
J. Elion ◽  
D. Labie ◽  
F. Josso
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Cleavage Pattern ◽  
Purification And Characterization ◽  
Double Immunodiffusion ◽  
The Family

For the purification of the abnormal prothrombin (Pt Metz), advantage has been taken of the existence in the family of three siblings who, being double heterozygotes for Pt Metz and a hypoprothrombinemia, have no normal Pt. Purification procedures included barium citrate adsorption and chromatography on DEAE Sephadex as for normal Pt. As opposed to some other variants (Pt Barcelona and Madrid), Pt Metz elutes as a single symetrical peak. By SDS Polyacrylamide gel electrophoresis, this material is homogeneous and appears to have the same molecular weight as normal Pt. Comigration of normal and abnormal Pt in the absence of SDS, shows a double band suggesting an abnormal charge for the variant. Pt Metz exhibits an identity reaction with the control by double immunodiffusion.Upon activation by factor Xa, Pt Metz can generate amydolytic activity on Bz-Phe-Va1-Arg-pNa (S2160), but only a very low clotting activity. Clear abnormalities are observed in the cleavage pattern of Pt Metz when monitored by SDS gel electrophoresis. The main feature are the accumulation of prethrombin 1 (P1) and the appearance of abnormal intermediates migra-ti ng faster than P1.

Cloning, Expression and Characterization of β-Glucosidase from L. Delbrueckii Subsp. Delbrueckii in Escherichia coli

2011 ◽  
Vol 301-303 ◽  
pp. 347-351
Author(s):  
Xiu Hong Zhao ◽  
Jie Zeng ◽  
Hai Yan Gao ◽  
Chang Biao Li ◽  
Chang Jiang Liu
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Prokaryotic Expression ◽  
Expression Vector ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gene Encoding ◽  
Prokaryotic Expression Vector ◽  
Strain Bl21

Gene encoding β-glucosidase was amplified through PCR by using the genome DNA extracted from L .delbrueckii subsp. delbrueckii as a template. The gene encoding β-glucosidase was inserted into a prokaryotic expression vector pET-28a(+) and expressed in E.coli strain BL21(DE3). The gene encoding β-glucosidase was of 1380bp. The nucleotide sequence of the gene encoding β-glucosidase from L. delbrueckii subsp. delbrueckii showed as high as 97.9% homology comparing with that from L. delbrueckii subsp. bulgaricus indicating that the gene encoding β-glucosidase is highly conservative. The enzyme activity was about 34U/mg and the molecular weight of β-glucosidase is about 51 kDa analyzed by SDS-polyacrylamide gel electrophoresis.

Isolation, purification, and partial characterization of type V-A hemagglutinin from Escherichia coli GV-12, O1:H-

1982 ◽  
Vol 152 (2) ◽  
pp. 757-761
Author(s):  
V L Sheladia ◽  
J P Chambers ◽  
J Guevara ◽  
D J Evans
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Amino Terminus ◽  
N Terminus ◽  
Type V

A hemagglutinin which specifically agglutinates human type A erythrocytes (mannose resistant) was isolated from the growth medium of cultures of Escherichia coli GV-12, serotype O1:H-, and purified by chromatography on Bio-Gel A-1.5 and DEAE-Sephadex A-25. The purity of the hemagglutinin was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoelectrophoresis. N-terminus analysis indicated that only asparagine resides on the amino terminus. The native hemagglutinin is an aggregate exhibiting a sedimentation coefficient of 9.25, which corresponds to a molecular weight of approximately 200,000. The monomeric molecular weight was found to be approximately 16,300. Amino acid analysis indicated that the hemagglutinin consists of 131 residues, corresponding to a molecular weight of 13,400.

scholarly journals Purification and physical characterization of glutathione S-transferase K. Differential use of S-hexylglutathione and glutathione affinity matrices to isolate a novel glutathione S-transferase from rat liver

1986 ◽  
Vol 233 (3) ◽  
pp. 789-798 ◽  
Author(s):  
J D Hayes
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Structural Data ◽  
Glutathione S Transferase ◽  
Purification Scheme ◽  
Peptide Maps ◽  
Limited Sequence

A novel hepatic enzyme, glutathione S-transferase K, is described that, unlike previously characterized transferases, possesses little affinity for S-hexylglutathione-Sepharose 6B but can be isolated because it binds to a glutathione affinity matrix. A purification scheme for this new enzyme was devised, with the use of DEAE-cellulose, S-hexylglutathione-Sepharose 6B, glutathione-Sepharose 6B and hydroxyapatite chromatography. The final hydroxyapatite step results in the elution of three chromatographically interconvertible forms, K1, K2 and K3. The purified protein has an isoelectric point of 6.1 and comprises subunits that are designated Yk (Mr 25,000); during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it migrates marginally faster than the Ya subunit but slower than the pulmonary Yf monomer (Mr 24,500). Transferase K displays catalytic, immunochemical and physical properties that are distinct from those of other liver transferases. Tryptic peptide maps suggest that transferase K is a homodimer, or comprises closely homologous subunits. The tryptic fingerprints also demonstrate that, although transferase K is structurally separate from previously described hepatic forms, a limited sequence homology exists between the Yk, Ya and Yc polypeptides. These structural data are in accord with the immunochemical results presented in the accompanying paper [Hayes & Mantle (1986) Biochem. J. 233, 779-788].

Isolation and Partial Characterization of Adenylate Cyclase-Enriched Membranes from Human Platelets

1986 ◽  
Vol 55 (02) ◽  
pp. 178-183
Author(s):  
N Shimada ◽  
M Tsubokura ◽  
N Kimura
Keyword(s):  
Molecular Weight ◽  
Adenylate Cyclase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
High Specific Activity ◽  
Total Activity ◽  
Human Platelets ◽  
Protein Profiles

SummaryIsolation of adenylate cyclase-enriched membranes from human platelets was attempted using glycerol lysis technique followed by ultracentrifugation on discontinuous sucrose gradients composed of 24, 30, 34, 37, and 41% (w/w). Adenylate cyclase activity was enriched 4-fold in sample/24% sucrose interface, 7-fold in 24%/30% sucrose interface, and 4-fold in 30%/ 34% sucrose interface fractions with the recovery of 15-20% of the total activity. The enrichment and subcellular distribution of adenylate cyclase resembled in general those of phosphodiesterase and acid phosphatase with slight differences in each other. Protein profiles from SDS-polyacrylamide gel electrophoresis showed that the heavy chain of myosin (Mr = 200,000) was enriched in sample/24% sucrose interface and lower molecular weight proteins in 34%/37% sucrose interface and pellet. The interface fractions between 24 and 34% sucrose were, therefore, collected as adenylate cyclase-enriched membranes.Adenylate cyclase associated with the membranes displayed high specific activity (0.1 and 1-2 nmol/min/mg protein in the absence and presence of stimulants, respectively), and possessed sensitivities to prostaglandins (E1, I2, and D2) as well as cholera toxin. Activation of adenylate cyclase by these compounds required added GTP, indicating that the contamination of the membrane preparations with GTP-like substance (s) was minimal, if at all present.

Purification and Characterization of an Unusual Aminopeptidase From Pea Seeds

1980 ◽  
Vol 7 (2) ◽  
pp. 131 ◽  
Author(s):  
JB Caldwell ◽  
LG Sparrow
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sulfhydryl Group ◽  
Purification And Characterization ◽  
Apparent Homogeneity ◽  
Pea Seeds ◽  
Hydrolysis Of

An aminopeptidase with specificity for N-terminal glutamic and aspartic acid residues has been purified to apparent homogeneity from pea seeds (Pisum sativum cv. Greenfeast). It also catalyses the hydrolysis of the glutaryl-phenylalanine bond of the synthetic chymotrypsin substrate glutaryl- L-phenylalanine p-nitroanilide. The native enzyme, which has a molecular weight of approximately 500 000, gives a single band on polyacrylamide gel electrophoresis but two major bands when subjected to electrophoresis in the presence of sodium dodecyl sulfate after reduction. Its behaviour with various inhibitors suggests that a sulfhydryl group is important for its activity.

Purification and Characterization of Ginsenoside-Hydrolyzing β-Glucosidase from Wheat Bran

2013 ◽  
Vol 641-642 ◽  
pp. 906-909
Author(s):  
Chun Zhi Zhang ◽  
Ming Chen ◽  
Hai Chen Guo ◽  
Guo Ren Zu ◽  
Li Chen
Keyword(s):  
Molecular Weight ◽  
Wheat Bran ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Purification And Characterization ◽  
Enzyme Solution ◽  
Crude Enzyme Solution

The ginsenoside-hydrolyzing β-glucosidase that can converse the major ginsenosides into the minor ginsenosides was isolated from wheat bran, and the enzyme was purified and characterized. The crude enzyme solution extracted from wheat bran could hydrolyse the protopanaxadiol-type ginsenosides such as Rb1, Rc, Rd and Rg3, but could not hydrolyse the protopanaxatriol-type ginsenosides such as Re and Rg2. The enzyme fractionated on the DEAE-Cellulose DE-52 column was purified to one spot in SDS polyacrylamide gel electrophoresis, and the molecular weight of enzyme in the fraction 34, 47, and 61 was approximately 62 kDa, 62 kDa, and 68 kDa, respectively.

Sign in / Sign up

Export Citation Format

Share Document