scholarly journals Affinity-chromatography purification of alkaline phosphatase from calf intestine

1975 ◽  
Vol 151 (2) ◽  
pp. 291-296 ◽  
Author(s):  
O Brenna ◽  
M Perrella ◽  
M Pace ◽  
P G Pietta
Keyword(s):  
Alkaline Phosphatase ◽  
Affinity Chromatography ◽  
Specific Activity ◽  
Diazonium Salt ◽  
Competitive Inhibitor ◽  
Affinity Column ◽  
Arsanilic Acid ◽  
Nitrophenyl Phosphate ◽  
Derivatives Of ◽  
Phenylarsonic Acid

A crude preparation of alkaline phosphatase (EC 3.1.3.1) from calf intestinal mucosa was purified by affinity chromatography on Sepharose-bound derivatives of arsanilic acid, which was found to be a competitive inhibitor of the enzyme. Three biospecific adsorbents were prepared for the chromatography, and the best results were obtained with a tyraminyl-Sepharose derivative coupled with the diazonium salt derived from 4-(p-aminophenylazo)phenylarsonic acid. Alkaline phosphatase was the only enzyme retained by the affinity column in the absence of Pi. The enzyme eluted by phosphate buffer had a specific activity of about 1200 units per mg of protein at pH 10.0, with 5.5mM-p-nitrophenyl phosphate as the substrate.

scholarly journals Affinity chromatography and inhibition of chorismate mutase-prephenate dehydrogenase by derivatives of phenylalanine and tyrosine

1977 ◽  
Vol 165 (1) ◽  
pp. 121-126 ◽  
Author(s):  
G D Smith ◽  
D V Roberts ◽  
A Daday
Keyword(s):  
Affinity Chromatography ◽  
Competitive Inhibitor ◽  
Chorismate Mutase ◽  
Prephenate Dehydrogenase ◽  
Step Procedure ◽  
One Step ◽  
High Degree ◽  
Sepharose 4B ◽  
Derivatives Of

Several derivatives of phenylalanine and tyrosine were prepared and tested for inhibition of chorismate mutase-prephenate dehydrogenase (EC 1.3.1.12) from Escherichia coli K12 (strain JP 232). The best inhibitors were N-toluene-p-sulphonyl-L-phenylalanine, N-benzenesulphonyl-L-phenylalanine and N-benzloxycarbonyl-L-phenylalanine. Consequently two compounds, N-toluene-sulphonyl-L-p-aminophenylalanine and N-p-aminobenzenesulphonyl-L-phenylalanine, were synthesized for coupling to CNBr-activated Sepharose-4B. The N-toluene-p-sulphonyl-L-p-aminophenylalanine-Sepharose-4B conjugate was shown to bind the enzyme very strongly at pH 7.5. The enzyme was not eluted by various eluents, including 1 M-NaCl, but could be quantitatively recovered by washing with buffer of pH9. Elution was more effective in the presence of 10 mM-1-adamantaneacetic acid, a competitive inhibitor of the enzyme. This affinity-chromatography procedure results in a high degree of purification of the enzyme and can be used to prepare the enzyme in a one-step procedure from the bacterial crude extract. Such a procedure may therefore prove useful in studying this enzyme in a state that closely resembles that in vivo.

Purification by Affinity Chromatography of Glutathione Reductase (EC 1.6.4.2) from Escherichia coli and Characterization of such Enzyme

1984 ◽  
Vol 39 (9-10) ◽  
pp. 908-915 ◽  
Author(s):  
Anna M. Mata ◽  
M. Carmen ◽  
Juan López-Barea
Keyword(s):  
Escherichia Coli ◽  
Affinity Chromatography ◽  
Glutathione Reductase ◽  
Specific Activity ◽  
Total Yield ◽  
Coli Strain ◽  
Affinity Column ◽  
Heat Stable ◽  
Activity Loss ◽  
Fast Procedure

Abstract The glutathione reductase from Escherichia coli strain S33 was purified to homogeneity by a simple and fast procedure consisting of two affinity chromatography steps. After 40-80% ammonium sulfate fractionation, the enzyme was adsorbed to an N6-2′.5′-ADP-Sepharose affinity column from which it was specifically eluted by a 0 - 10 mᴍ NADP+ linear gradient. The enzyme was finally purified to homogeneity after a second affinity chromatography step in a C8-ATPR-Sepharose column, from which it was eluted by means of the same NADP+ gradient. Starting from 182 g of E. coli cells. 6.9 mg of pure enzyme was obtained after a 2632-fold purifi­cation, with a total yield of 63%. The pure enzyme showed a specific activity of 361 U/mg, and its absorption spectrum was characteristic of a flavoprotein. with an A272A450 of 7.84. The enzyme was a dimer with a molecular weight 109 000 and 40 Å hydrodynamic radius. The optimum pH were 7.5 and 4.5 with NADPH and NADH. respectively, as reductants. Apparent K′m values of 16, 377, and 66 μᴍ were determined at pH 7.5 for NADPH, NADH, and GSSG, respectively. Upon storage the enzyme was stable at pH values ranging from 7.5 to 9.5, being additionally stabilized by FAD. NADP+, dithiothreitol, or glycerol. The pure enzyme was quite heat stable, denaturing signifi­cantly only after 10 min at 70 0C. A marked activity loss was observed however, even at 0 °C, in the presence of 20 μᴍ NADPH. The enzyme was inactivated by low concentrations of para- hydroximercuribenzoate: the sensitivity towards such mercurial was greatly enhanced after reduction of the enzyme by NADPH.

scholarly journals Purification of human placental alkaline phosphatase. Salt effects in affinity chromatography

1974 ◽  
Vol 141 (1) ◽  
pp. 103-112 ◽  
Author(s):  
George J. Doellgast ◽  
William H. Fishman
Keyword(s):  
Alkaline Phosphatase ◽  
Affinity Chromatography ◽  
Binding Site ◽  
Specific Binding ◽  
Placental Alkaline Phosphatase ◽  
Aminobenzoic Acid ◽  
Human Placental Alkaline Phosphatase ◽  
High Concentrations ◽  
Triton X ◽  
Derivatives Of

Human placental alkaline phosphatase was chromatographed on Sepharose derivatives of d- and l-phenylalanine, l-leucine, glycine, aniline and p-aminobenzoic acid in high concentrations of (NH4)2SO4. Retention on these columns was greatest at the highest concentrations of (NH4)2SO4. By using decreasing concentrations and changing the types of salts, elution was effected from each of the columns. The (NH4)2SO4-mediated retention appeared to be related to the hydrophobic character of the substituted Sepharose, rather than to any specific binding site of the enzyme. It is suggested that this provides a way of controlling hydrophobic affinity chromatography. By use of chromatography on l-phenylalanine–Sepharose and of DEAE-Sephadex chromatography in the presence of Triton X-100 detergent, a preparation of highly purified (1000-fold) human placental alkaline phosphatase was obtained in 22% yield.

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 Characterization of angiotensin-converting enzyme in canine testis

Reproduction ◽  
2001 ◽  
pp. 139-146 ◽  
Author(s):  
K Sabeur ◽  
AT Vo ◽  
BA Ball
Keyword(s):  
Enzyme Activity ◽  
Angiotensin Converting Enzyme ◽  
Affinity Chromatography ◽  
Ace Inhibitor ◽  
Specific Activity ◽  
Normal Activity ◽  
Seminiferous Tubules ◽  
Affinity Column ◽  
Converting Enzyme ◽  
Ace Activity

The aim of this study was to characterize angiotensin-converting enzyme (ACE) in canine testis. Detergent-extracted canine testes were sonicated in the presence of protease inhibitors and purified on an affinity column with the ACE inhibitor, lisinopril, as an affinity ligand for ACE. The fractions recovered were assessed for ACE enzyme activity via an enzyme kinetic microplate assay (at 330 nm) based on the hydrolysis of Fa-Phe-Gly-Gly (FAPGG) at pH 7.5 during an 8 min incubation. The specific activity of ACE in the starting testicular extracts was 3.53 +/- 0.99 mU mg(-1) protein with a 1588 times enrichment in ACE activity after lisinopril affinity chromatography (4239 +/- 2600 mU mg(-1) protein). The recovery efficiency of ACE after lisinopril affinity chromatography was 71.2%. The ACE activity in the detergent extracts and the purified fractions was inhibited significantly by 10 micromol captopril l(-1), a specific ACE inhibitor, and was restored to 88% of normal activity by the addition of the thiol-alkylating agent N-ethylmaleimide (0.5 mmol l(-1)) in the detergent extracts and the purified fractions incubated with captopril. The treatment of testicular extracts with 10 mmol EDTA l(-1) reduced the ACE activity significantly (5.40 +/- 1.26 versus 0.58 +/- 0.23 mU mg(-1)). The ACE activity was restored fully in the presence of zinc (5.28 +/- 0.70 mU mg(-1)). The anti-ACE antibody (raised against a 70 kDa protein from the periacrosomal plasma membrane of equine spermatozoa) recognized a 65-70 kDa protein in the detergent-extracted testes as well as in the affinity-purified fractions. This antibody also recognized a protein of similar molecular mass in ejaculated spermatozoa. ACE was localized in the periacrosomal area of the ejaculated spermatozoa and in spermatids in the seminiferous tubules. The results of this study demonstrate that ACE is present in canine testis and retains its enzyme activity after purification with lisinopril affinity chromatography. Activity of canine ACE is inhibited by captopril and EDTA and is restored in the presence of N-ethylmaleimide and zinc.

scholarly journals Periodate-oxidized AMP as a substrate, an inhibitor and an affinity label of human placental alkaline phosphatase

1981 ◽  
Vol 199 (2) ◽  
pp. 281-287 ◽  
Author(s):  
G G Chang ◽  
S C Wang ◽  
F Pan
Keyword(s):  
Alkaline Phosphatase ◽  
Inorganic Phosphate ◽  
Competitive Inhibitor ◽  
Binary Complex ◽  
Placental Alkaline Phosphatase ◽  
Phosphate Binding ◽  
Complete Inactivation ◽  
Nitrophenyl Phosphate ◽  
Saturation Kinetics ◽  
Human Placental Alkaline Phosphatase

Human placental alkaline phosphatase (EC 3.1.3.1) was inactivated by periodate-oxidized AMP. The inactivation showed saturation kinetics and could be partially prevented by the substrate AMP or the product inhibitor inorganic phosphate. Oxidized AMP was itself a substrate for this enzyme, with an apparent Km of 0.67 mM. The hydrolytic products of oxidized AMP were identified as oxidized adenosine hemiacetals. Oxidized AMP was also found to be a non-competitive inhibitor with respect to p-nitrophenyl phosphate, with identical Kis and Kii values of 0.15 mM. Our results indicate that oxidized AMP could combine with the enzyme to form a binary complex, followed by reaction with the proximal lysyl amino group to yield a Schiff base. The latter was reduced with NaBH4 and identified by t.l.c. The incorporation of only 1.5 molecules of oxidized [14C]AMP per enzyme subunit resulted in a complete inactivation of the enzyme. The modified enzyme showed higher apparent Km for the substrates and higher Ki for inorganic phosphate, but lower [32P]phosphate incorporation, than the native enzyme. These results support the conclusion that a lysine residue is involved in the phosphate-binding site of human placental alkaline phosphatase.

scholarly journals Human liver alkaline phosphatase purified by affinity chromatography, ultracentrifugation and polyacrylamide-gel electrophoresis

1976 ◽  
Vol 159 (3) ◽  
pp. 697-705 ◽  
Author(s):  
A L Latner ◽  
A W Hodson
Keyword(s):  
Alkaline Phosphatase ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Human Liver ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Nitrophenyl Phosphate ◽  
Hydrolysis Of

A method is presented for the preparation of human liver alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1). The method gives a purification factor of 12.5 × 10(3) over the initial aq. butan-1-ol extract, a recovery of 6.0% and a specific activity for the preparation of 1450-1550 units/mg of protein, 1 unit being defined as the amount of enzyme catalysing the hydrolysis of 1mumol of p-nitrophenyl phosphate/min at 35 degrees C in 0.1 M-2-amino-2-methylpropan-1-ol/HCl buffer, pH 10.5, containing 10mM-p-nitrophenyl phosphate. Homogeneity was studied by ultracentrifugation, by immunoelectrophoresis and by polyacrylamide-gel electrophoresis. A single contaminating protein was present which was less than 5% of the total. Ultracentrifugation and equilibrium-gradient-pore electrophoresis techniques indicated a mol.wt. of 156000 and 160000 respectively. Equilibrium-gradient-pore electrophoresis indicated that the alkaline phosphatase molecule is possibly a dimer, comprising two subunits of about 80000 mol.wt. Amino acid analysis proved remarkably similar to that for alkaline phosphatase from other sources, regardless of species.

Purification and Properties of Alkaline Phosphatase with Protein Phosphatase Activity from Saccharomyces cerevisiae

2000 ◽  
Vol 55 (7-8) ◽  
pp. 588-593 ◽  
Author(s):  
Danka Galabova ◽  
Borijana Tuleva ◽  
Evgenia Vasileva-Tonkova ◽  
Nelly Christova
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Specific Activity ◽  
Specific Enzyme ◽  
Optimum Activity ◽  
Purification And Properties ◽  
Nitrophenyl Phosphate ◽  
Dimeric Protein

Abstract An alkaline phosphatase (ALPase) from Saccharomyces cerevisiae strain 257 was purified 345-fold with specific activity of 54 533 nmol × min−1 × mg protein−1 . It was shown to be a dimeric protein (apparent mol. wt. approx. 130 kDa) with optimum activity at pH 8.6 - 8.8 and good stability at 50 °C. The ALPase was a non-specific enzyme hydrolyzing a wide variety of monophosphate esters. The enzyme showed protein phosphatase activity and this activity was not Mg2+ - dependent in contrast to p-nitrophenyl phosphate (pNPP) activity. The Km value for pNNP hydrolysis was determined to be 2.2 × 10−5 м. Orthophosphate inhibited the enzyme in a competitive mode with the Ki of 2.3 x 10−4 м. Phosphate transfer of the ALPase is almost zero with all alcohols tested except for Tris.

Liver- and bone-derived isoenzymes of alkaline phosphatase in serum as determined by high-performance affinity chromatography

1990 ◽  
Vol 36 (2) ◽  
pp. 240-246 ◽  
Author(s):  
D J Anderson ◽  
E L Branum ◽  
J F O'Brien
Keyword(s):  
Alkaline Phosphatase ◽  
Affinity Chromatography ◽  
High Performance ◽  
Bone Alkaline Phosphatase ◽  
Phosphate Solution ◽  
Serum Samples ◽  
High Performance Affinity Chromatography ◽  
Nitrophenyl Phosphate ◽  
On Line ◽  
Two Peaks

Abstract To separate liver and bone alkaline phosphatase (ALP) isoenzymes in human serum, we used high-performance affinity chromatography (HPAC) on a column of wheat-germ lectin conjugated to 7-microns-diameter silica particles and an eluent containing N-acetyl-D-glucosamine (NAG). On-line spectrophotometric detection of ALP involved pumping diethanolamine-buffered p-nitrophenyl phosphate solution post-column. Bone and liver isoenzymes could be separated into two peaks with only 10% overlap when an exponential gradient was used. A linear-step gradient separated 80.9% of liver ALP and 91.6% of bone ALP in two distinct peaks. True bone and liver ALP peak areas for the linear-step gradient were determined by using correction factors, because each peak contained a co-eluted portion of the other ALP isoenzyme. The detection limit improved 10-fold over those of other techniques for ALP isoenzymes, owing to the relatively large sample that could be applied to the column. Correlation with a urea-inactivation procedure was reasonable for patients' serum samples (r = 0.98 and 0.79 for liver ALP and bone ALP, respectively).

Synthesis and Antimicrobial Activity of Adamantyl Substituted Pyridoxine Derivatives

2019 ◽  
Vol 16 (12) ◽  
pp. 1360-1369 ◽  
Author(s):  
Rail Khaziev ◽  
Nikita Shtyrlin ◽  
Roman Pavelyev ◽  
Raushan Nigmatullin ◽  
Raylya Gabbasova ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Lipid Membranes ◽  
Specific Activity ◽  
Tuberculosis H37rv ◽  
Microbial Pathogens ◽  
Adamantane Derivatives ◽  
Carbon Cage ◽  
H37rv Strain ◽  
Derivatives Of

Background: Adamantane derivatives possess multiple pharmacological activities such as antiviral, anticancer, antimycobacterial, antidiabetic, antiparkinsonian and others. The interest of medicinal chemists in adamantane compounds is due to their unique spatial structure, high lipophilicity, and carbon cage rigidity. As a result, these molecules can easily penetrate biological lipid membranes and often have unique target-specific activity profile. Another pharmacophore studied in this work is pyridoxine (vitamin B6). Pyridoxine plays highly important roles in living cells as a key cofactor of many enzymes. On the other hand, its molecular scaffold is a valuable structural platform which has led to the development of several launched drugs (Pyritinol, Pirisudanol, Cycletanine, Mangafodipir) and a wide number of preclinical and clinical drug candidates. Objective: The objective of this study is a synthesis of pyridoxine-adamantane and pyridoxinecyclooctane dipharmacophore molecules. The underlying idea was to assess the antibacterial and antiviral potential of such dipharmacophores, based on multiple examples of promising antiinfective agents which have in their structures adamantane and pyridoxine moieties. Another specific reason was to explore the ability of pyridoxine pharmacophore to suppress the potential of microbial pathogens to develop resistance to drug molecules. Methods: In this study, a series of pyridoxine-adamantane and pyridoxine-cyclooctane dipharmacophore molecules were synthesized based on reactions of three different cycloalkyl amines with the corresponding electrophilic derivatives of pyridoxine aldehydes, chlorides and acetates. All synthesized compounds have been tested for their in vitro activity against M. tuberculosis H37Rv strain and H3N2 (A/Aichi/2/68) influenza virus. Results: Series of pyridoxine-adamantane and pyridoxine-cyclooctane dipharmacophore molecules were synthesized based on reactions of three different cycloalkylamines with the corresponding electrophilic derivatives of pyridoxine aldehydes, chlorides and acetates. Reaction of cycloalkylamines with pyridoxine derivatives, in which meta-hydroxyl and ortho-hydroxymethyl groups are protected by acetyl groups, represents a useful alternative to reductive amination of aldehydes and nucleophilic substitution of alkyl halides. According to a tentative mechanism, it proceeds via paraand ortho-pyridinone methides which readily react with nucleophiles. None of the synthesized dipharmacophore compounds showed activity against M. tuberculosis H37Rv strain. At the same time, three compounds demonstrated some antiviral activity against H3N2 (A/Aichi/2/68) influenza virus (EC50 52-88 µg/mL) that was comparable to the activity of Amantadine, though lower than the activity of Rimantadine. The results of this work can be useful in the design of physiologically active derivatives of pyridoxine and adamantane. Conclusion: The results of this work can be useful in the design of physiologically active derivatives of pyridoxine and adamantane.

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