scholarly journals Purification and characterization of two forms of microsomal carbonyl reductase in guinea pig liver

1984 ◽  
Vol 223 (3) ◽  
pp. 697-705 ◽  
Author(s):  
S Usui ◽  
A Hara ◽  
T Nakayama ◽  
H Sawada
Keyword(s):  
Carbonyl Compounds ◽  
Gel Filtration ◽  
Carbonyl Reductase ◽  
Cofactor Specificity ◽  
Major Form ◽  
Aldehydes And Ketones ◽  
Minor Form ◽  
Triton X ◽  
Guinea Pig Liver

Two forms of microsomal carbonyl reductase, solubilized in Triton X-100, were purified to homogeneity from the liver of male guinea pigs, primarily by affinity, DEAE-Sephacel, gel-filtration and hydroxyapatite chromatography. The major form was a tetrameric glycoprotein of single subunits of Mr 32000 and a pI value of 7.0; another minor form was a monomeric protein with Mr 34000 and a pI value of 7.8. The enzymes were immunologically distinct. Although the enzymes showed similar substrate specificity for exogenous aldehydes and ketones and apparently absolute cofactor specificity for NADPH, their specificity for natural carbonyl compounds differed. The major form irreversibly reduced 5 alpha- and 5 beta-dihydrotestosterones, menadione and lauryl aldehyde with low Km values of 10-70 microM, whereas the minor form not only reduced 17-oxosteroids, of which 3 alpha-hydroxy-5 beta-androstan-17-one was the best substrate, but also oxidized 17-hydroxysteroids in the presence of NADP+. The two forms of carbonyl reductase also exhibited different sensitivity to heavy metal ions, dicoumarol, tetramethyleneglutaric acid, phenobarbitone and corticosteroids.

scholarly journals Characterization of a Novel Plasma Membrane Protein, Expressed in the Midgut Epithelia of Bombyx mori, That Binds to Cry1A Toxins

2004 ◽  
Vol 70 (8) ◽  
pp. 4604-4612 ◽  
Author(s):  
Delwar M. Hossain ◽  
Yasuyuki Shitomi ◽  
Kenta Moriyama ◽  
Masahiro Higuchi ◽  
Tohru Hayakawa ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Brush Border ◽  
Membrane Vesicle ◽  
Gel Filtration ◽  
Resistance Mechanism ◽  
Binding Assays ◽  
Toxin Resistance ◽  
Cry Toxin ◽  
Triton X

ABSTRACT We describe the properties of a novel 252-kDa protein (P252) isolated from brush border membranes of Bombyx mori. P252 was found in a Triton X-100-soluble brush border membrane vesicle fraction, suggesting that it may be a component of the midgut epithelial cell membrane. P252 was purified to homogeneity, and the amino acid sequence of two internal peptides was determined, but neither of the peptides matched protein sequences in the available databases. The apparent molecular mass of the purified protein was estimated by denaturing gel electrophoresis to be 252 kDa, and it migrated as a single band on native gels. However, gel filtration chromatography indicated an apparent mass of 985 kDa, suggesting that P252 may exist as a homo-oligomer. The associations of P252 with Cry1Aa, Cry1Ab, and Cry1Ac were specific, and Kd constants were determined to be 28.9, 178.5, and 20.0 nM, respectively. A heterologous competition assay was also done. P252 did not exhibit Leu-pNA hydrolysis activity, and binding to the Cry1A toxins was not inhibited by GalNAc. Binding assays of P252 with various lectins indicated the presence of three antennal N-linked high-mannose-type as well as O-linked mucin-type sugar side chains. While the function of P252 is not yet clear, we propose that it may function with Cry1A toxins during the insecticidal response and/or Cry toxin resistance mechanism.

scholarly journals Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver

1995 ◽  
Vol 308 (3) ◽  
pp. 983-989 ◽  
Author(s):  
I N Fleming ◽  
S J Yeaman
Keyword(s):  
Phosphatidic Acid ◽  
Rat Liver ◽  
Lysophosphatidic Acid ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Sds Page ◽  
Chromatography Columns ◽  
Triton X ◽  
Second Peak

N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.

scholarly journals Solubilization and characterization of pellet-associated human brain α-L-fucosidase activity

1985 ◽  
Vol 229 (3) ◽  
pp. 679-685 ◽  
Author(s):  
R L Hopfer ◽  
J A Alhadeff
Keyword(s):  
Human Brain ◽  
Isoelectric Focusing ◽  
Human Liver ◽  
Gel Filtration ◽  
Compelling Evidence ◽  
Supernatant Fluid ◽  
Ph Optimum ◽  
Triton X ◽  
Soluble Supernatant

The pellet-associated portion of human brain alpha-L-fucosidase (which represents approx. 20% of the homogenate activity) was solubilized with 0.5% (w/v) Triton X-100, characterized with regard to several properties and compared with the corresponding properties of the soluble supernatant-fluid enzyme in an attempt to find a second alpha-L-fucosidase in human brain. The solubilized and soluble alpha-L-fucosidase activities exhibited complete stability after storage at 2-4 degrees C for up to 29 days, comparable thermostability after preincubation at 50 degrees C, comparable apparent Km values (0.07-0.08 mM) for 4-methylumbelliferyl alpha-L-fucopyranoside, comparable hydrophobicity, comparable isoelectric-focusing profiles (six major forms, with pI values between 4.5 and 5.8) and comparable immunoprecipitation curves (with the IgG fraction of antisera prepared against human liver alpha-L-fucosidase). Differences in three properties were found between solubilized and soluble alpha-L-fucosidase activities: the solubilized activity was less stable to storage at −20 degrees C, had a 0.5-pH-unit neutral shift in its pH optimum (6.0) and had smaller Mr forms after gel filtration on Sephadex G-200. The overall results indicate that the pellet-associated and soluble portions of human brain alpha-L-fucosidase are quite similar in most of their properties. Thus there is still no compelling evidence for the existence of a second mammalian alpha-L-fucosidase.

SOLUBILIZATION AND PARTIAL CHARACTERIZATION OF THYROID MEMBRANE TSH BINDING PROTEINS

1979 ◽  
Vol 92 (3) ◽  
pp. 512-521 ◽  
Author(s):  
B. Czarnocka ◽  
J. Nauman ◽  
G. Adler ◽  
W. Kiełczyński
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Gel Filtration ◽  
Soluble Proteins ◽  
The Other ◽  
High Affinity ◽  
Triton X ◽  
Maximal Capacity

ABSTRACT Crude plasma membranes obtained from bovine thyroids were found to possess one class of high affinity, low capacity binding sites for TSH with average association constant (Ka) of 1.301 × 109 m−1 and maximal capacity 8.76 × 10−10 m/mg of protein. Treatment of crude membranes fraction with 0.1 % Triton X-100 and the subsequent sonication in ultrasonic disintegrator resulted in solubilization of membranes proteins with mean recovery of 40.0 ± 6.2 %. Soluble proteins retained the property to bind [125I]TSH, but the binding of the hormone was decreased. The removal of the detergent from the solubilizate by gel filtration on Sephadex LH-20 increased the binding of TSH well above that demonstrated for crude thyroid membranes. The chromatography of soluble proteins on Ultrogel AcA-44 revealed the presence of two TSH binding proteins, one with the molecular weight (m.w.) above 130 000 daltons and the other with the m.w. approximately 30 000 daltons. The electrofocusing of solubilizate on Ampholine resulted in two protein peaks, one at pH 4.0–4.1 and the other at pH 4.4–4.6. The latter peak was shown to bind [125I]TSH specifically. The present results have confirmed the heterogeneous character of solubilized TSH receptor preparation and have shown that the hormone binding sites belong to acid proteins.

A new anthraquinoid ligand for the iron-catalyzed hydrosilylation of carbonyl compounds at room temperature: new insights and kinetics

2018 ◽  
Vol 47 (21) ◽  
pp. 7272-7281 ◽  
Author(s):  
Álvaro Raya-Barón ◽  
Carmen P. Galdeano-Ruano ◽  
Pascual Oña-Burgos ◽  
Antonio Rodríguez-Diéguez ◽  
Robert Langer ◽  
...  
Keyword(s):  
Carbonyl Compounds ◽  
Room Temperature ◽  
Synthesis And Characterization ◽  
Highly Active ◽  
Aldehydes And Ketones

The synthesis and characterization of a highly active Fe(ii) catalyst for the hydrosilylation of aldehydes and ketones have been described.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

The Immunological Characterization of Human Antibodies to Factor VIII Isolated by Immuno-Affinity Chromatography

1981 ◽  
Vol 45 (01) ◽  
pp. 060-064 ◽  
Author(s):  
M L Kavanagh ◽  
C N Wood ◽  
J F Davidson
Keyword(s):  
Factor Viii ◽  
Affinity Chromatography ◽  
Gel Filtration ◽  
Immunological Characterization ◽  
Human Antibodies ◽  
Heavy Chains ◽  
Light Chain Type ◽  
Antibody Preparations ◽  
Chain Type

SummaryNine human antibodies to factor VIII were isolated from haemophilic plasmas by affinity chromatography and gel filtration and six were subsequently subjected to immunological characterization. Three partially purified preparations were similarly characterized. Eight of the antibodies were characterized as being exclusively IgG and one preparation was found to contain IgM. Seven of the antibodies contained only a single light chain type, four being of type lambda and three of type kappa. Two antibody preparations contained both kappa and lambda light chains. In four of the preparations, only a single heavy chain sub-class could be demonstrated, three of IgG3 and one of IgG4. Of the remainder, three were a mixture of IgG3 and IgG4 sub-classes and one contained both IgG2 and IgG4. IgG sub-classification could not be achieved with the IgM-containing preparation. These results demonstrate a restricted heterogeneity of light and heavy chains in human antibodies to factor VIII.

Preparation and Characterization of NH2-Terminal Fibrinogen Bβ Fragments from N-DSK of Human Fibrinogen

1984 ◽  
Vol 51 (01) ◽  
pp. 016-021 ◽  
Author(s):  
S Birken ◽  
G Agosto ◽  
B Lahiri ◽  
R Canfield
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Human Fibrinogen ◽  
Human Volunteers ◽  
Cnbr Cleavage ◽  
Two Peaks

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

1982 ◽  
Vol 47 (03) ◽  
pp. 197-202 ◽  
Author(s):  
Kurt Huber ◽  
Johannes Kirchheimer ◽  
Bernd R Binder
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Gel Filtration ◽  
Chain Structure ◽  
The Other ◽  
Single Chain ◽  
Apparent Homogeneity ◽  
Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

Purification and Characterization of Tissue-Type Plasminogen Activator in Maxillary Mucosa with Chronic Inflammation

1985 ◽  
Vol 54 (02) ◽  
pp. 485-489 ◽  
Author(s):  
Yukiyoshi Hamaguchi ◽  
Masuichi Ohi ◽  
Yasuo Sakakura ◽  
Yasuro Miyoshi
Keyword(s):  
Plasminogen Activator ◽  
Chronic Inflammation ◽  
Gel Filtration ◽  
Potassium Acetate ◽  
Tissue Type ◽  
Purification And Characterization ◽  
Tissue Type Plasminogen Activator ◽  
Urokinase Inhibitor ◽  
Type Plasminogen Activator

SummaryTissue-type plasminogen activator (TPA) was purified from maxillary mucosa with chronic inflammation and compared with urokinase. Purification procedure consisted of the extraction from delipidated mucosa with 0.3M potassium acetate buffer (pH 4.2), 66% saturation of ammonium sulfate, zinc chelate-Sepharose, concanavalin A-Sepharose and Sephadex G-100 gel filtration chromatographies.The molecular weight of the TPA was approximately 58,000 ± 3,000. Its activity was enhanced in the presence of fibrin and was quenched by placental urokinase inhibitor, but not quenched by anti-urokinase antibody. The TPA made no precipitin line against anti-urokinase antibody, while urokinase did.All these findings indicate that the TPA in maxillary mucosa with chronic inflammation is immunologically dissimilar to urokinase and in its affinity for fibrin.

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