scholarly journals Human placental β-galactosidase. Characterization of the dimer and complex forms of the enzyme

1992 ◽  
Vol 285 (3) ◽  
pp. 827-831 ◽  
Author(s):  
M Hubbes ◽  
R M D'Agrosa ◽  
J W Callahan
Keyword(s):  
Affinity Chromatography ◽  
Protein Composition ◽  
Autosomal Recessive Disorder ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Affinity Column ◽  
Major Protein ◽  
Ph Optimum ◽  
Single Polypeptide Chain ◽  
Total Beta

GM1 ganglioside beta-galactosidase (beta-Gal) is deficient in the autosomal recessive disorder GM1 gangliosidosis. A portion of the enzyme occurs in a complex with neuraminidase and an additional glycoprotein, protective protein, but the nature of the interactions conferring the stability of the complex is unknown. Affinity chromatography of beta-Gal on p-aminophenylthiogalactose-Sepharose (PATG-Sepharose) at pH 4.3, the pH optimum of beta-Gal, resulted in a 260-fold enrichment of beta-Gal, but the major protein in the fraction had an M(r) value of 74,000. Affinity chromatography on PATG-Sepharose at pH 5.2 showed substantial enrichment (4000-fold) of beta-Gal, and the mature form of the enzyme (M(r) 64,000) was the major protein in the preparation. Using h.p.l.c. molecular-sieve chromatography, we found that about 15% of the total beta-Gal occurred in a high-M(r) form (greater than 600,000), the presumptive complex, with 85% eluting at M(r) 150,000, suggestive of a dimer. This distribution was independent of both high (60 mg/ml) and low (5 mg/ml) protein concentration and the pH (pH 4.3 or 5.2) of the sample applied to the column. Furthermore, incubation for 90 min at 37 degrees C, conditions which had previously been suggested as optimal for formation of the complex, had no effect on this distribution. Further fractionation by anion-exchange chromatography and a second affinity column step yielded a beta-Gal preparation that contained a single polypeptide chain (M(r) 64,000), was devoid of neuraminidase and protective protein (absent carboxypeptidase activity), and when injected into rabbits gave rise to monospecific rabbit antisera. We conclude that the protein composition of the complex is variable (i.e. it is different when isolated at pH 4.3 and 5.2) and that the amount of beta-Gal tightly associated with the complex constitutes a small fraction of the total beta-Gal activity. The more prevalent form of the enzyme is a beta-Gal homodimer that is stable and devoid of either neuraminidase activity or protective protein.

scholarly journals Recombinant FIX/X-Bp from the Japanese Habu Snake Is a Universal Ligand for Purification of Highly Carboxylated Factor IX Variants and Factor X

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1081-1081
Author(s):  
Oblaise Mercury ◽  
Lucy Liu ◽  
Ayman Ismail ◽  
Ming Zhang ◽  
Qi Lu ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Culture Medium ◽  
Specific Activity ◽  
Protein S ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Hek293 Cells ◽  
Affinity Column ◽  
Factor X

Abstract Background: The purification of vitamin K-dependent clotting factors typically involves multiple chromatographic steps, including an ion exchange-based pseudo-affinity step to enrich for species with sufficiently high gamma-carboxyglutamic acid (Gla) content to achieve maximal specific activity. Variants of these factors have been engineered to improve their pharmacokinetic properties by appending or inserting a variety of elements, including the Fc domain of IgG, unstructured hydrophilic peptides of defined amino acid composition (XTEN), albumin, and polyethylene glycol (PEG). In most cases, however, such modification alters both the hydrodynamic and electrostatic properties of the resulting molecule relative to those of the predicate molecule, thereby complicating their purification, particularly with regard to Gla enrichment by pseudo-affinity chromatography. Factor IX (FIX)- and factor X (FX)-binding protein (FIX/X-bp) isolated from the venom of the Japanese Habu snake (T. flavoviridis) has been shown to bind with high affinity and specificity to both FIX and FX, and structural studies have demonstrated that FIX/X-bp binds to the highly carboxylated calcium-bound forms of the Gla domains of these proteins. We therefore reasoned that FIX/X-bp could serve as a novel affinity ligand for rapid and simple purification of variants of FIX and FX with high specific activity. Aims: To generate and purify recombinant FIX/X-bp (rFIX/X-bp) and assess its utility for the purification of FIX, FIX-XTEN, FIX-albumin, and FX with high Gla content. Methods: A two-chained rFIX/X-bp molecule in which a polyhistidine tag was appended to one chain was generated by stable co-transfection of Chinese hamster ovary (CHO) cells. Culture medium was concentrated by tangential-flow filtration (TFF), and rFIX/X-bp was purified by one of two methods: 1) immobilized metal ion affinity chromatography (IMAC), followed by anion-exchange chromatography, or 2) affinity chromatography on immobilized FIX in calcium-containing buffer and subsequent elution in EDTA-containing buffer. The potent anticoagulant activity of rFIX/X-bp was verified by prothrombin time (PT) and activated partial thromboplastin time (APTT) assays, and its ability to bind to human FIX, FX, factor VII (FVII), protein S, and prothrombin was evaluated by biolayer interferometry. The affinity of rFIX/X-bp for FIX and FX was determined by surface plasmon resonance (SPR). An affinity column was then generated by chemical conjugation of rFIX/X-bp to NHS-activated Sepharose. Recombinant FIX, FIX-albumin, and FIX-XTEN were first affinity purified on IXSelect resin from the culture medium of transiently transfected HEK293 cells, and the resulting protein preparations, which were heterogeneous with regard to Gla content, were then applied to the rFIX/X-bp affinity column in calcium- or magnesium-containing buffer and eluted with EDTA-containing buffer. Activity was assessed by APTT assay, and Gla content was determined by mass spectrometric peptide mapping. Recombinant FX was purified from the culture medium of transiently transfected HEK293 cells by sequential barium citrate adsorption, anion exchange chromatography, and affinity chromatography on a rFIX/X-bp column. Results: In the presence of calcium or magnesium ions, rFIX/X-bp binds to FIX and FX with high affinity (KD≈ 10 pM), to a lesser extent to protein S and prothrombin, but not to FVII. FIX and FIX-albumin that had been affinity purified on a rFIX/X-bp column had specific activities that were consistent with published data and greater than 11 Gla residues per molecule. The Gla content of FX that had been affinity purified on a rFIX/X-bp column was 10 Gla residues per molecule (out of 11 possible). Conclusions: rFIX/X-bp is a universal ligand for the purification of highly carboxylated FX and FIX variants, including FIX-albumin and FIX-XTEN. Disclosures Mercury: Biogen: Employment. Liu:Biogen: Employment. Ismail:Biogen: Employment. Zhang:Biogen: Employment. Lu:Biogen: Employment. Cameron:Biogen: Employment. Goodman:Biogen: Employment. Culyba:Biogen: Employment. Ravindran Nair:Biogen: Employment. Holthaus:Biogen: Employment. Kulman:Biogen: Employment. Peters:Biogen: Employment.

scholarly journals Mouse Uterine Alkaline Phosphatase: Improved Purification by Affinity Chromatography and Further Characterization of the Enzyme

1980 ◽  
Vol 33 (3) ◽  
pp. 279 ◽  
Author(s):  
RN Murdoch ◽  
Louise E Buxton ◽  
DJ Kay
Keyword(s):  
Alkaline Phosphatase ◽  
Affinity Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Anion Exchange Chromatography ◽  
Pregnant Mouse ◽  
Exchange Chromatography ◽  
Enzyme Preparations

An improved procedure for the purification of alkaline phosphatase from about 10 g of day 7 pregnant mouse uterine tissue is described. Following homogenization, the procedure involved solubilization and extraction with 0�8% (v/v) Triton X-lOO and 20% (v/v) n-butanol, ammonium sulfate precipitation, concanavalin A-Sepharose 4B affinity chromatography, DEAE-cellulose anion-exchange chromatography and Sephacryl S200 gel filtration. On subjecting 2162-fold purified enzyme preparations to polyacrylamide-gel electrophoresis, a single band of protein coincident with the zone of enzyme activity and having an apparent molecular weight of 205 OOO� lOOOO was identified. Affinity chromatography yielded the largest increase in purity of any step in the procedure and established the glycoprotein nature of the uterine enzyme.

scholarly journals A lectin from the exudate of the fruit of the vegetable marrow (Cucurbita pepo) that has a specificity for β-1,4-linked N-acetylglucosamine oligosaccharides

1979 ◽  
Vol 183 (1) ◽  
pp. 133-137 ◽  
Author(s):  
A K Allen
Keyword(s):  
Affinity Chromatography ◽  
Cucumis Melo ◽  
Cucurbita Pepo ◽  
Polypeptide Chain ◽  
Soya Bean ◽  
Major Protein ◽  
Phloem Exudate ◽  
Single Polypeptide Chain ◽  
Vegetable Marrow ◽  
Single Polypeptide

Lectins are present in the exudate (presumably from the phloem) of the fruits of three species of the Cucurbitaceae, namely vegetable marrow (Cucurbita pepo), melon (Cucumis melo) and cucumber (Cucumis sativus). They are all strongly inhibited in their activities by chitin oligosaccharides, but only weakly by N-acetylglucosamine. Glycopeptides from soya-bean agglutinin and fetuin are also strong inhibitors of Cucurbita pepo lectin, indicating that it interacts with internal N-acetylglucosamine residues. The lectin from Cucurbita pepo fruit was purified by affinity chromatography by using chitin oligosaccharides covalently attached to Sepharose. The lectin is not a glycoprotein, and it consists of a single polypeptide chain of about 20,000 mol.wt. It is a major protein (18% of the total) of the phloem exudate and it is postulated that it may have an anti-parasitic function.

scholarly journals The Modified β-Ketoadipate Pathway in Rhodococcus rhodochrous N75: Enzymology of 3-Methylmuconolactone Metabolism

1998 ◽  
Vol 180 (24) ◽  
pp. 6668-6673 ◽  
Author(s):  
Chang-Jun Cha ◽  
Ronald B. Cain ◽  
Neil C. Bruce
Keyword(s):  
Ammonium Sulfate ◽  
Gel Filtration ◽  
Sole Source ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Rhodococcus Rhodochrous ◽  
Natural Substrate ◽  
Ph Optimum ◽  
A Subunit ◽  
Hydrolysis Of

ABSTRACT Rhodococcus rhodochrous N75 is able to metabolize 4-methylcatechol via a modified β-ketoadipate pathway. This organism has been shown to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysis of the butenolide ring. A lactone-CoA synthetase is induced by growth of R. rhodochrous N75 on p-toluate as a sole source of carbon. The enzyme has been purified 221-fold by ammonium sulfate fractionation, hydrophobic chromatography, gel filtration, and anion-exchange chromatography. The enzyme, termed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a native M r of 128,000, and a subunitM r of 62,000, suggesting that the enzyme is homodimeric. The enzyme is very specific for its 3-methylmuconolactone substrate and displays little or no activity with other monoene and diene lactone analogues. Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA synthetase reaction with its natural substrate.

scholarly journals Purification and Characterization of Phenylalanine Ammonia Lyase from Trichosporon cutaneum

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Andrea Goldson-Barnaby ◽  
Christine H. Scaman
Keyword(s):  
Phenylalanine Ammonia Lyase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Hill Coefficient ◽  
Trichosporon Cutaneum ◽  
Amino Acid Residues ◽  
Ph Optimum ◽  
Ability To Act ◽  
Metal Cofactor ◽  
Intron Region

Trichosporon cutaneum phenylalanine ammonia lyase was selected as a model to investigate the dual substrate activity of this family of enzymes. Sequencing of the PAL gene identified an extensive intron region at the N-terminus. Five amino acid residues differing from a prior report were identified. Highest Phe : Tyr activities (1.6 ±0.3 : 0.4±0.1 μmol/h g wet weight) were induced by Tyr. The enzyme has a temperature optimum of 32°C and a pH optimum of 8–8.5 and shows no metal cofactor dependence. Michaelis-Menten kinetics (Phe, Km  5.0 ± 1.1 mM) and positive allostery (Tyr, K′  2.4 ± 0.6 mM, Hill coefficient 1.9±0.5) were observed. Anion exchange chromatography gave a purification fold of 50 with 20% yield. The His-Gln motif (substrate selectivity switch region) indicates the enzyme’s ability to act on both substrates.

scholarly journals Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: a reevaluation

1989 ◽  
Vol 86 (17) ◽  
pp. 6493-6497 ◽  
Author(s):  
J L Johnson ◽  
R E London ◽  
K V Rajagopalan
Keyword(s):  
Glucose Oxidase ◽  
Xanthine Oxidase ◽  
Metabolic Regulation ◽  
Bovine Milk ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Affinity Column ◽  
31P Nmr Spectroscopy ◽  
Highly Active ◽  
Covalently Bound

The reported presence of covalently bound phosphate residues in flavoproteins has significant implications with regard to the catalytic mechanisms and structural stability of the specific enzymes themselves and in terms of general cellular metabolic regulation. These considerations have led to a reevaluation of the presence of covalently bound phosphorus in the flavoproteins xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.1.3.22) and glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4). Milk xanthine oxidase purified by a procedure that includes anion-exchange chromatography is shown to contain three phosphate residues. All three are noncovalently associated with the protein, two with the FAD cofactor, and one with the molybdenum cofactor. Results of chemical analysis and 31P NMR spectroscopy indicate that enzyme purified by this method contains no phosphoserine residues. Xanthine oxidase preparations purified by chromatography on calcium phosphate gel in place of DEAE-Sephadex yielded higher phosphate-to-protein ratios, which could be reduced to the expected values by additional purification on a folate affinity column. Highly active, highly purified preparations of glucose oxidase are shown to contain only the two phosphate residues of the FAD cofactor. The covalently bound bridging phosphate reported by others may arise in aged or degraded preparations of the enzyme but appears not to be a constituent of functional glucose oxidase. These results suggest that the presence of covalent phosphate residues in other flavoproteins should be rigorously reevaluated as well.

scholarly journals Characterization of a chelator-resistant proteinase from Thermus strain Rt4A2

1993 ◽  
Vol 295 (2) ◽  
pp. 463-469 ◽  
Author(s):  
S A Freeman ◽  
K Peek ◽  
M Prescott ◽  
R Daniel
Keyword(s):  
Specific Activity ◽  
Sequence Similarity ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
High Sequence Similarity

The Thermus isolate Rt4A2 was found to produce an extracellular chelator-resistant proteinase. The proteinase was purified to homogeneity by (NH4)2SO4 precipitation, cation-exchange chromatography, gel-filtration chromatography, and weak anion-exchange chromatography. The Rt4A2 proteinase was found to have properties typical of an alkaline serine proteinase. It had a pH optimum of 9.0 and was specifically inhibited by phenylmethanesulphonyl fluoride. Its isoelectric point was greater than 10.25. Its molecular-mass was 31.6 kDa as determined by SDS/PAGE. N-terminal sequencing has shown it to have high sequence similarity with other serine proteinases from Thermus species. The proteinase hydrolysed a number of substrates including fibrin, casein, haemoglobin, collagen, albumin and the synthetic chromogenic peptide substrate Suc-Ala-Ala-Pro-Phe-NH-Np. The specific activity of the purified proteinase using azocasein as substrate was 313 units/mg. Substrate inhibition was observed above an azocasein concentration of 0.05% (w/v). Esterase activity was directed mainly towards those substrates containing the aliphatic or aromatic residues of alanine, glycine, tryptophan, tyrosine and phenylalanine. Thermostability half-lives of greater than 7 days at 70 degrees C, 43 h at 80 degrees C and 90 min at 90 degrees C were found in the presence of 5 mM CaCl2. At 90 degrees C increasing the CaCl2 concentration 100-fold (0.5 mM to 50 mM) caused a 4.3-fold increase in the half-life of the enzyme from 30 to 130 min. Half-lives of 19.4 min at 100 degrees C and 4.4 min at 105 degrees C were found in the presence of 50 mM CaCl2. The metal chelators EGTA and EDTA reduced the stability at higher temperatures but had no effect on the activity of the proteinase. Activity was not stimulated by common metal activators such as Ca2+, Mg2+ and Zn2+.

scholarly journals The human alpha 2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of alpha 2-macroglobulin.

1990 ◽  
Vol 110 (4) ◽  
pp. 1041-1048 ◽  
Author(s):  
J D Ashcom ◽  
S E Tiller ◽  
K Dickerson ◽  
J L Cravens ◽  
W S Argraves ◽  
...  
Keyword(s):  
Cell Surface ◽  
Affinity Chromatography ◽  
Anion Exchange Chromatography ◽  
Binding Activity ◽  
Exchange Chromatography ◽  
Surface Glycoprotein ◽  
Cell Surface Receptor ◽  
Affinity Matrix ◽  
Cell Surface Glycoprotein ◽  
A Cell

Ligand affinity chromatography was used to purify a cell surface alpha 2-macroglobulin (alpha 2M) receptor. Detergent extracts of human placenta were applied to an affinity matrix consisting of alpha 2M, previously reacted with methylamine, coupled to Sepharose. Elution with EDTA specifically released polypeptides with apparent molecular masses of 420 and 39 kD. In some preparations, small amounts of a 90-kD polypeptide were observed. The 420- and 39-kD polypeptides appear specific for the forms of alpha 2M activated by reaction with proteinases or methylamine and do not bind to an affinity matrix consisting of native alpha 2M coupled to Sepharose. Separation of these two polypeptides was accomplished by anion exchange chromatography, and binding activity was exclusively associated with the 420-kD polypeptide. The purified 420-kD protein binds to the conformationally altered forms of alpha 2M that are known to specifically interact with alpha 2M receptors and does not bind to native alpha 2M. Binding of the 420-kD polypeptide to immobilized wheat germ agglutinin indicates that this polypeptide is a glycoprotein. The cell surface localization of the 420-kD glycoprotein was confirmed by affinity chromatography of extracts from surface radioiodinated fibroblasts. These properties suggest that the 420-kD polypeptide is a cell surface receptor for the activated forms of alpha 2M.

A Combination of Cation Exchange and Ligand-Affinity Chromatography for Purification of Two Molecular Species of the Folate Binding Protein in Human Milk, One Equipped with a Hydrophobic Glycosyl Phosphatidylinositol Tail: Characterization of Hydrophobicity and Electrical Charge

2002 ◽  
Vol 22 (3-4) ◽  
pp. 443-454 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen ◽  
Mimi Høier-Madsen
Keyword(s):  
Human Milk ◽  
Affinity Chromatography ◽  
Cation Exchange ◽  
Large Scale ◽  
Gel Filtration ◽  
Molecular Size ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Affinity Column ◽  
Folate Binding Protein

Cation exchange chromatography combined with ligand (methotrexate) affinity chromatography on a column desorbed with a pH-gradient was used for separation and large scale purification of two folate binding proteins in human milk. One of the proteins, which had a molecular size of 27 kDa on gel filtration and eluted from the affinity column at pH 5–6 was a cleavage product of a 100 kDa protein eluted at pH 3–4 as evidenced by identical N-terminal amino acid sequences and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidyl-inositol tail that inserts into Triton X-100 micelles. Chromatofocusing showed that both proteins possessed multiple isoelectric points within the pH range 7–9. The 100 kDa protein exhibited a high affinity to hydrophobic interaction chromatographic gels, whereas this was only the case with unliganded forms of the 27 kDa protein indicative of a decrease in the hydrophobicity of the protein after ligand binding.

Sign in / Sign up

Export Citation Format

Share Document