Purification of Tetragalloylglucose 4-O-Galloyltransferase and Preparation of Antibodies against This Key Enzyme in the Biosynthesis of Hydrolyzable Tannins

2000 ◽  
Vol 55 (7-8) ◽  
pp. 582-587 ◽  
Author(s):  
Petra Grundhöfer ◽  
Georg G. Gross
Keyword(s):  
Quercus Robur ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Pedunculate Oak ◽  
Hydrolyzable Tannins ◽  
Apparent Homogeneity ◽  
Key Enzyme ◽  
Rhus Typhina

Abstract The enzyme, β-glucogallin: 1,2,3,6-tetra-O-galloyl- β-ᴅ-glucose 4-O-galloyltransferase, which catalyzes the last common step in the biosynthesis of the two subclasses of hydrolyzable tannins, i.e. gallotannins and ellagitannins, was purified 868-fold from leaves of pedunculate oak ( Quercus robur, syn. Q. pedunculata) to apparent homogeneity. Polyclonal antibodies against this pivotal enzyme were raised in rabbits and purified by protein-A chromatography, gel-filtration and affinity complexation. They were found to react specifically with acyltransferase from oak, displaying no cross-reactivity towards analogous enzymes from other plants synthesizing hydrolyzable tannins along the same biogenetic route, e.g. Rhus typhina or Tellima grandiflora.

scholarly journals Purification and characterization of protein disulphide-isomerase from the unicellular green alga Chlamydomonas reinhardii. A 120 kDa dimer antigenically distinct from the vertebrate enzyme

1990 ◽  
Vol 268 (1) ◽  
pp. 63-68 ◽  
Author(s):  
D D Kaska ◽  
K I Kivirikko ◽  
R Myllylä
Keyword(s):  
Green Alga ◽  
Polyclonal Antibodies ◽  
Active Form ◽  
Protein A ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Chlamydomonas Reinhardii ◽  
Protein Disulphide Isomerase ◽  
Unicellular Green Alga ◽  
A Subunit

Protein disulphide-isomerase (PDI) has been isolated from the unicellular green alga Chlamydomonas reinhardii and purified by (NH4)2SO4 precipitation, gel filtration and DEAE-Sephacel, hydroxyapatite and f.p.l.c. chromatography. The active algal enzyme is a 120 kDa dimer with a subunit molecular mass of 60 kDa when determined by SDS/PAGE. Although similar in size to the previously isolated vertebrate PDIs, the algal enzyme is antigenically distinct, polyclonal antibodies against the algal PDI showing no cross-reactivity with the vertebrate enzyme on immunoblots, and vice versa. The anti-(algal PDI) antiserum did not inhibit algal PDI activity, and C. reinhardii PDI could be immobilized on anti-PDI-Protein A-Sepharose in active form. In contrast with the situation in vertebrates, where PDI functions as a subunit of prolyl 4-hydroxylase, the C. reinhardii PDI is not associated with the algal prolyl 4-hydroxylase.

scholarly journals A novel whey protein synthesized only in late lactation by the mammary gland from the tammar (Macropus eugenii)

1987 ◽  
Vol 241 (3) ◽  
pp. 899-904 ◽  
Author(s):  
K R Nicholas ◽  
M Messer ◽  
C Elliott ◽  
F Maher ◽  
D C Shaw
Keyword(s):  
Mammary Gland ◽  
Amino Acid ◽  
Whey Protein ◽  
Sequence Data ◽  
Protein A ◽  
Gel Filtration ◽  
Tammar Wallaby ◽  
Exchange Chromatography ◽  
Macropus Eugenii ◽  
Apparent Homogeneity

A major whey protein which appears in milk from the tammar wallaby (Macropus eugenii) only during the second half of lactation (late lactation protein-A, LLP-A) was purified to apparent homogeneity by ion-exchange chromatography and gel filtration. An Mr of 21,600 +/- 2000 was calculated from its amino acid composition. A computer-based comparison of the sequence of the first 69 amino acid residues with the Atlas of Protein Sequence data base showed no significant homology with known proteins. Antiserum to LLP-A was prepared in rabbits, and single radial immunodiffusion was used to measure the amounts of LLP-A in milk during the first 40 weeks of lactation. LLP-A was first detected at 26 weeks; thereafter its concentration increased abruptly, to reach a maximum of 26 g/l at approx. 36 weeks of lactation. Explants prepared from mammary gland biopsies at 20 and 35 weeks of lactation were exposed to [3H]amino acids for 8 h; immunoprecipitation of tissue extracts showed that, whereas the rate of casein synthesis was the same at both stages of lactation, LLP-A was synthesized only by the 35-week mammary gland.

scholarly journals Purification, Characterization, and Cloning of a Eubacterial 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase, a Key Enzyme Involved in Biosynthesis of Terpenoids

1999 ◽  
Vol 181 (4) ◽  
pp. 1256-1263 ◽  
Author(s):  
Shunji Takahashi ◽  
Tomohisa Kuzuyama ◽  
Haruo Seto
Keyword(s):  
Amino Acid ◽  
Coenzyme A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Hmg Coa Reductase ◽  
Apparent Homogeneity ◽  
Key Enzyme ◽  
Coa Reductase

ABSTRACT The eubacterial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34 ) was purified 3,000-fold fromStreptomyces sp. strain CL190 to apparent homogeneity with an overall yield of 2.1%. The purification procedure consisted of (NH4)2SO4 precipitation, heat treatment and anion exchange, hydrophobic interaction, and affinity chromatographies. The molecular mass of the enzyme was estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 100 to 105 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of around 7.2, with apparent Km values of 62 μM for NADPH and 7.7 μM for HMG-CoA. A gene from CL190 responsible for HMG-CoA reductase was cloned by the colony hybridization method with an oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The amino acid sequence of the CL190 HMG-CoA reductase revealed several limited motifs which were highly conserved and common to the eucaryotic and archaebacterial enzymes. These sequence conservations suggest a strong evolutionary pressure to maintain amino acid residues at specific positions, indicating that the conserved motifs might play important roles in the structural conformation and/or catalytic properties of the enzyme.

scholarly journals Macroprolactinemia: Diagnostic, Clinical, and Pathogenic Significance

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Akira Shimatsu ◽  
Naoki Hattori
Keyword(s):  
Middle Age ◽  
Molecular Form ◽  
Protein A ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Precipitation Method ◽  
Binding Studies ◽  
Rat Pituitary ◽  
Peg Precipitation Method

Macroprolactinemia is characterized by a large molecular mass of PRL (macroprolactin) as the main molecular form of PRL in sera, the frequent elevation of serum PRL (hyperprolactinemia), and the lack of symptoms. Macroprolactin is largely a complex of PRL with immunoglobulin G (IgG), especially anti-PRL autoantibodies. The prevalence of macroprolactinemia is 10–25% in patients with hyperprolactinemia and 3.7% in general population. There is no gender difference and a long-term followup demonstrates that macroprolactinemia develops before middle age and is likely a chronic condition. Polyethylene-glycol- (PEG-) precipitation method is widely used for screening macroprolactinemia, and gel filtration chromatography, protein A/G column, andI125-PRL binding studies are performed to confirm and clarify its nature. The cross-reactivity of macroprolactin varies widely according to the immunoassay systems. The epitope on PRL molecule recognized by the autoantibodies is located close to the binding site for PRL receptors, which may explain that macroprolactin has a lower biological activity. Hyperprolactinemia frequently seen in macroprolactinemic patients is due to the delayed clearance of autoantibody-bound PRL. When rats are immunized with rat pituitary PRL, anti-PRL autoantibodies are produced and hyperprolactinemia develops, mimicking macroprolactinemia in humans. Screening of macroprolactinemia is important for the differential diagnosis of hyperprolactinemia to avoid unnecessary examinations and treatments.

scholarly journals Structural comparison of two esterases from Drosophila mojavensis isolated by immunoaffinity chromatography

1986 ◽  
Vol 238 (3) ◽  
pp. 691-699 ◽  
Author(s):  
J Pen ◽  
J Van Beeumen ◽  
J J Beintema
Keyword(s):  
Gel Electrophoresis ◽  
High Efficiency ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Gel Filtration ◽  
Immunoaffinity Chromatography ◽  
Cross Reactivity ◽  
Drosophila Mojavensis ◽  
Structural Comparison ◽  
Final Purification

Antibodies raised against esterase-4 and esterase-5 from Drosophila mojavensis were coupled to Protein A-Sepharose CL-4B to prepare high-efficiency immunomatrices used for their purification. Final purification was achieved by anion-exchange h.p.l.c., in the case of esterase-5 followed by gel-filtration h.p.l.c. The resultant esterase preparations were homogeneous, as judged by gel-filtration h.p.l.c., SDS/polyacrylamide-gel electrophoresis and non-denaturing gel electrophoresis. Esterase-4 and esterase-5 are the products of a duplicated gene. They are differently localized in the insect's body and expressed in different periods during development. Although both enzymes exhibit little immunological cross-reactivity, their amino acid compositions show few significant differences and their N-terminal sequences are largely identical, which clearly show their common origin.

scholarly journals Purification and characterization of cytosolic aldolase from carrot storage root

1990 ◽  
Vol 269 (1) ◽  
pp. 133-139 ◽  
Author(s):  
G B Moorhead ◽  
W C Plaxton
Keyword(s):  
Specific Activity ◽  
Polyclonal Antibodies ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Storage Roots ◽  
Carrot Root ◽  
Daucus Carota L ◽  
Native Molecular Mass ◽  
Spinach Leaf ◽  
Protein Staining

A single fructose-1,6-bisphosphate (FBP) aldolase has been detected in extracts from carrot storage roots (Daucus carota L.). The enzyme was purified 850-fold to electrophoretic homogeneity and a final specific activity of 26.3 mumols of FBP utilized/min per mg of protein. SDS/PAGE of the final preparation revealed a single protein-staining band of 40 kDa. The native molecular mass was determined by analytical gel filtration to be 159 kDa, indicating that the enzyme is a homotetramer. Denaturing isoelectric focusing revealed two predominant protein-staining bands, with pI values of 5.6 and 5.7. The enzyme is a class I aldolase, since EDTA or metal ions had no effect on its activity. The enzyme was relatively heat-stable, had an activation energy (Ea) of 68.3 kJ.mol-1, and had an absorption coefficient of 8.08 x 10(4) M-1.cm-1 at 280 nm. Km values for FBP and sedoheptulose 1,7-bisphosphate (SBP) were both determined to be 6 microM (pH optima 7.4). The specificity constant with FBP was 2.6 times that obtained with SBP. Ribose 5-phosphate, 6-phosphogluconate, MgAMP, glucose 1-phosphate and phosphoenolpyruvate (PEP) were inhibitors. PEP was a mixed-type inhibitor with respect to FBP (Ki = 3.2 mM, K′i = 5.1 mM). No activators were found. Rabbit anti-(carrot aldolase) polyclonal antibodies immunoprecipitated the activity of both carrot root aldolase and spinach leaf cytosolic aldolase, but not that of spinach leaf plastid aldolase. Western-blot analysis also revealed cross-reactivity with cytosolic, but not plastid, spinach leaf aldolase, indicating that the single carrot root aldolase is cytosolic.

scholarly journals IMMUNOLOGICAL COMPARISON OF PECTINESTERASE ISOENZYMES FROM TOMATO

HortScience ◽  
1990 ◽  
Vol 25 (9) ◽  
pp. 1085e-1085
Author(s):  
Floyd M. Woods ◽  
Russell Pressey
Keyword(s):  
Polyclonal Antibodies ◽  
Tomato Fruit ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Cherry Tomato ◽  
Ripe Fruit ◽  
Molecular Weights ◽  
Cation Exchangers ◽  
Immunological Comparison ◽  
Tomato Cultivars

Pectinesterase is present in green tomato fruit and increases several-fold during ripening. Several isoenzymes of pectinesterase can be separated by chromatography of tomato extracts on DEAE-Sephadex A-50. The predominant isoenzyme in most tomato cultivars including Better Boy has been designated PE IV. This isoenzyme accounts for most of the increase in total pectinesterase during ripening of these cultivars. The fruit of some cherry tomato cultivars such as Pixie and Short Red contain some PE IV, but the major isoenzyme is PE III which occurs only in these cultivars. PE III and PE IV were isolated from ripe fruit of Short Red and Better Boy, respectively, to further characterize differences between the isoenzymes. PE III binds more strongly to cation exchangers, indicating that it is more basic than PE IV, The molecular weights were estimated by gel filtration to be 26,900 and 25, 100 for PE III and PE IV, respectively. Polyclonal antibodies were raised against the two enzymes. Cross reactivity of the enzymes with the antibodies indicates that PE III and PE IV are immunologically identical.

scholarly journals Detection of Aspergillus Flavus in Wheat Grains Using Anti-Mannoprotein (MP1) and Spore Proteins Polyclonal Antibodies

2021 ◽  
Author(s):  
Ranjana Kumari ◽  
Ananta Ghosh
Keyword(s):  
Aspergillus Flavus ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Fungal Species ◽  
Cross Reactivity ◽  
Soluble Form ◽  
Minimum Concentration ◽  
Wheat Grains ◽  
Stored Wheat ◽  
Spore Proteins

Abstract Cell wall mannoprotein (MP1) gene of an aflatoxigenic strain of Aspergillus flavus, isolated from stored wheat grains, was cloned and sequenced. MP1 protein was expressed in E. coli in soluble form and purified. Polyclonal antibodies were raised against recombinant MP1 protein and inactivated spores of this fungus in rabbit, and purified by ammonium sulphate precipitation, Protein A sepharose and antigen affinity chromatography. The minimum concentration of purified mycelial or spore proteins that could be detected by ELISA was determined as 100 ng using 2 µg of these antibodies. The anti-MP1 antibody was found more sensitive than anti-spore protein antibody. Western blot and immunofluorescence analysis showed reactivity of these antibodies to various proteins (30 kDa to 200 kDa) distributed throughout the surface of mycelia and spore of A. flavus. Cross reactivity of these antibodies was detected with fungi belonging to different Aspergillus, Rhizopus and Alternaria species out of fourteen different fungal species tested. In fungal contaminated wheat grains these antibodies could detect presence of as low as 1 µg mycelia or 103 spores per gram of wheat grains using ELISA. The results suggest that the developed antibodies could be successfully applied for the detection of predominant fungal infestation in stored wheat grains.

Phenol degradation by an enterobacterium: aKlebsiellastrain carries a TOL-like plasmid and a gene encoding a novel phenol hydroxylase

1999 ◽  
Vol 45 (2) ◽  
pp. 162-171 ◽  
Author(s):  
Kerstin Heesche-Wagner ◽  
Thomas Schwarz ◽  
Michael Kaufmann
Keyword(s):  
Klebsiella Oxytoca ◽  
Phenol Degradation ◽  
Gel Filtration ◽  
Phenol Hydroxylase ◽  
Gene Encoding ◽  
Kinetic Measurements ◽  
Apparent Homogeneity ◽  
Steady State Kinetic ◽  
Sds Page ◽  
Key Enzyme

Although phenol catabolism is described for many different microorganisms, there is no example for such a pathway in an enterobacterial strain. Here we characterize a Klebsiella oxytoca strain that grows on phenol as the only source of carbon and energy. As the key enzyme of phenol degradation, phenol hydroxylase was purified to apparent homogeneity. Compared with other phenol hydroxylases, the Klebsiella enzyme differs with respect to several properties: (i) SDS-PAGE and gel-filtration analysis of the purified protein revealed that the enzyme is a monomer with a molecular mass of 156 kDa; (ii) steady-state kinetic measurements resulted in a Kmvalue of 0.22 mM for phenol; and (iii) the enzyme is both dependent on NADPH/FAD and sensitive to EDTA. Further degradation of catechol, the reaction product of phenol hydroxylase, may occur via the effective meta-fission pathway often located on TOL or TOL-like plasmids. Such a plasmid was prepared from the Klebsiella strain and further characterized. The given data demonstrate that the isolated strain exhibits all characteristics of an efficient phenol-degrading microorganism.Key words: phenol metabolism, Klebsiella oxytoca, phenol hydroxylase, TOL plasmids.

scholarly journals Purification and characterization of glucosidase I involved in N-linked glycoprotein processing in bovine mammary gland

1987 ◽  
Vol 247 (3) ◽  
pp. 555-562 ◽  
Author(s):  
K Shailubhai ◽  
M A Pratta ◽  
I K Vijay
Keyword(s):  
Mammary Gland ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Gel Filtration ◽  
Mammary Tissue ◽  
Ph Optimum ◽  
Reducing Conditions ◽  
Solubilized Enzyme ◽  
Biological Substrates

Glucosidase I, the first enzyme involved in the post-translational processing of N-linked glycoproteins, was purified to homogeneity from the lactating bovine mammary tissue. The enzyme was extracted by differential treatment of the microsomal fraction with Triton X-100 and Lubrol PX. The solubilized enzyme was subjected to affinity chromatography on Affi-Gel 102 with N-5-carboxypentyldeoxynojirimycin as ligand and DEAE-Sepharose CL-6B chromatography. Purified glucosidase I shows a molecular mass of 320-330 kDa by gel filtration on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis under reducing conditions indicates a single band of approx. 85 kDa, indicating that the native enzyme is probably a tetrameric protein. Several criteria, including pH optimum of 6.6-7.0, specific hydrolytic action towards Glc3Man9GlcNAc2, to release the terminally alpha-1,2-linked glucosyl residue, and total lack of activity towards Glc1Man9GlcNAc2 and Glc2Man9GlcNAc2 saccharides, which are the biological substrates for processing glucosidase II, and 4-methylumbelliferyl alpha-D-glucopyranoside show the non-lysosomal origin and the processing-specific role of the purified enzyme. The enzyme does not require any metal ions for its activity. Hg2+, Ag+ and Cu2+ are potent inhibitors of the enzyme; this inhibition can be reversed by adding an excess of dithiothreitol. Among the saccharides tested, kojibiose (Glc alpha 1----2Glc) was inhibitory to the enzyme. Polyclonal antibodies raised against the enzyme in rabbit were found to be specific for glucosidase I, as revealed by Western-blot analysis and by immunoadsorption with Protein A-Sepharose. Anti-(glucosidase I) antibodies were cross-reactive towards a similar antigen in solubilized microsomal preparations from liver, mammary gland and heart from the bovine, guinea pig, rat and mouse.

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