Solubilization and partial purification of GABAB receptor from bovine brain

gamma-Aminobutyric acid (GABA)B receptor has been solubilized and partially purified by an affinity column chromatography. GABAB receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of asolectin. The solubilized GABAB receptor was adsorbed on baclofen-coupled epoxy-activated Sepharose 6B. The affinity matrix adsorbed 80% of the solubilized [3H]GABA binding activity to GABAB receptor, and approximately 75% of the adsorbed activity could be eluted with 1 M KC1. GABAB receptor binding in the fraction eluted from affinity column was displaced by GABA, baclofen and 2-hydroxy saclofen in a dose-dependent manner. Furthermore, the purified GABAB receptor showed approximately 2800-fold purification as compared with the original solubilized fraction and possessed the specific binding activity of 17.68 p mol/mg of protein. This binding consisted of a single binding site with a dissociation constant of 64.4 nM. The present results indicate that affinity column chromatographic procedures using baclofen-coupled epoxy-activated Sepharose 6B are suitable for the partial purification of GABAB receptor from cerebral tissues.     

Simple procedure for purification of diphtheria toxin and separation of its fragments by hydrophobic chromatography

Diphtheria toxin and fragment B bind to hydrocarbon-coated agaroses. Fragment A of the toxin is not adsorbed to such resins. Using Seph-C₄, the toxin and fragment B can be eluted from the column after adsorption by increasing the ionic strength of the eluent. The toxin is also eluted from the Seph-C₆ column, but fragment B is eluted only in the denatured form. Purification of the toxin can be achieved simply by passing the growth medium supernatant through a small size Seph-C₆ column and eluting the toxin by 0.1 m NaCl. The fragments of diphtheria toxin obtained after mild trypsin treatment can be separated purely on a Seph-C₄ column. The hydrophobic chromatography system may thus serve as a tool for purification of the toxin and its fragments: it may also be useful in large-scale preparations.     

A simple procedure for regeneration of an organomercurial agarose column

Organomercurial agarose has been used in the purification of various thiol compounds including enzymes (1). Thiol compounds are first adsorbed on a column of organomercurial agarose, and then eluted with a second thiol compound, e.g., 2-mercaptoethanol (2-ME)¹ and cysteine. Although this column can be used repeatedly, a usual method for regeneration of the column is to remove the second thiol by HgCl₂. It would be desirable to regenerate the column without using HgCl₂, since it is biohazardous. In the study of the purification of a thiol-containing enzyme, we found that organomercurial agarose, which had previously been treated with 2-ME, could adsorb the enzyme and that the enzyme was eluted with 2-ME. This finding led us to examine whether the column can be used repeatedly without the regeneration using HgCl₂.     

Determination of phosphorylase kinase activity in crude homogenates by affinity chromatography on 5′-AMP Sepharose

A sensitive method for measuring phosphorylase kinase activity by the incorporation of ³²P from [γ-³²]ATP into phosphorylase in the presence of other phosphorylation reactions is described. The kinase reaction is carried out in a crude homogenate. After stopping the reaction, a portion of the reaction mixture is withdrawn for assay of phosphorylase conversion and the rest is applied on a 5′-AMP Sepharose column. Phosphorylase in both forms is retained on the column while other phosphorylated proteins and [γ-³²P]ATP are washed out. The phosphorylase is then eluted by 10 mm AMP and the radioactivity incorporated is counted.     

Isolation of an arabinogalactan protein by lectin affinity chromatography on tridacnin-sepharose 4B.

An arabinogalactan protein from the style canal of Gladiolus has been isolated by a one-step procedure involving lectin affinity chromatography, using the galactose-binding lectin from the small giant clam, Tridacna maxima, coupled to Sepharose 4B. The lectin binding is calcium-ion dependent, so that the arabinogalactan protein which is bound to the column in the presence of calcium can be eluted by washing the column with a calcium-free buffer. This method has a general utility for the purification of polysaccharides and glycoproteins containing β-linked galactosyl residues.     

Simultaneous microdetermination of homovanillic acid and 5-hydroxyindoleacetic acid in brain tissue using Sephadex G-10 and QAE-Sephadex A-25

Homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in animal brains were simultaneously purified by two steps of column chromatography on Sephadex G-10 and QAE-Sephadex A-25. Perchloric acid extracts of brain tissue were directly passed through a column of Sephadex G-10. The gel retained both HVA and 5-HIAA, thereby separating them from Cl0^?₄ which interferes with subsequent purification process and from endogenous substances which give blank fluorescence. HVA was loosely adsorbed on the gel and was easily desorbed with dilute acetic acid. This effluent was successively passed onto a column of QAE-Sephadex A-25 placed beneath the G-10 column and the adsorbed HVA was eluted with 0.1 M Na₂HPO₄. The 5-HIAA remaining on the Sephadex G-10 without being desorbed by acetic acid was eluted with dilute ammonia. The recovery of both acid metabolites by this column procedure was more than 90%. Thus, it is possible to determine the levels of HVA and 5-HIAA in single brains of small rodents.     

General aspects of hydrophobic chromatography. Adsorption and elution characteristics of some skeletal muscle enzymes.

If the degree of substitution of Sepharose 4 B with alpha-alkylamines is varied gels of different hydrophobicity are produced. Proteins can be adsorbed when a critical hydrophobicity (ca. 10-12 alkyl residues/Sepharose sphere) is reached. The enzymes phosphorylase kinase, phosphorylase phosphatase, 3',5'-cAMP dependent protein kinase, glycogen synthetase, and phosphorylase b are successively adsorbed as the hydrophobicity of the Sepharose is increased. The capacity of the gels for these enzymes and protein in general increases exponentially reaches plateau values as a function of the degree of substitution. There is no indication of a restriction of the hydrophobic centers for a given protein. The critical hydrophobicity needed to adsorb proteins can either be otained in the above manner or by elongation of the employed alkylamine at a constant degree of substitution. Additonally, as the hydrophobicity of a gel is increased higher binding forces result and desorption of proteins requires an augmentation of the salt concentration in the elution buffer. Elution of proteins from a hydrophobic matrix can be described in terms of salting-in phenomena since desorption is dependent on the type of salt employed and not on the ionic strength alone. This also rules out ionic interactions as a major factor in adsorption per se. By rationally controlling the hydrophobicity of a Sepharose gel the adsorption and elution of a protein may be thus establised that its purification or elimination can be optimally performed.     

The cytokinin activities of 6-α-alkylbenzyloxypurines

The homologous series of 6-α-n-alkylbenzyloxypurines with alkyl groups (—(CH₂)_nCH₃) from n = 0 to n = 11 have been prepared and examined in three tests for cytokinin activity. The parent benzyloxypurine was also included in the tests. Substituting methyl (n = 0) into the methylene group of 6-benzyloxypurine removed activity almost completely; thereafter, increasing the size of the alkyl group (n = 1–5) gave compounds which were active in all tests, the butyl (n = 3) and pentyl (n = 4) derivatives being more active than 6-benzyloxypurine itself. Activity then fell as the series was ascended; the higher homologues (n = 8–11) being completely inactive. The results are discussed in relation to steric and other considerations.     

Affinity chromatography of Pseudomonas salicylate hydroxylase

In order to facilitate the purification of salicylate hydroxylase (salicylate 1-monooxygenase, EC 1.14.13.1) from Pseudomonas sp. RPP (ATCC 29351), an affinity chromatography procedure was developed employing immobilized salicylate as the affinity ligand. The immobilization was achieved by reacting p-aminosalicylate with the N-hydroxysuccinimide ester of Sepharose 4B-6-aminohexanoic acid. When the bacterial crude extract was chromatographed with this affinity column, salicylate hydroxylase was absorbed to the gel while the bulk of protein freely passed through. The absorbed enzyme was subsequently eluted from the affinity column by applying a 0–60 mm sodium salicylate gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzymatically most active fraction of the affinity effluent revealed salicylate hydroxylase was by far the most predominant protein but there were also small amounts of contaminating proteins. However, a virtually homogeneous enzyme preparation was obtained when the crude extract was first fractionated with a DE-52 anion-exchange column followed by the affinity step. The enzyme preparation obtained by this two-step procedure showed a specific activity of 14.9 units/mg and an A₄₅₀:A₃₇₂:A₂₈₀ of 1.01:1:10.23. Because most of the enzymes belonging to the class of external flavoprotein monooxygenase utilize salicylate analogs as substrates and share many other common properties, there is a strong possibility that the salicylate column may be useful for the purification of other member monooxygenases.     

Affinity chromatography of lactic acid dehydrogenase on N-(6-aminohexyl)oxamate-sepharose

A competitive inhibitor (K_i = 10⁻⁴–10⁻⁵m) for lactic acid dehydrogenase (LDH), N-(6-aminohexyl)oxamate, was synthesized from diethyl oxalate and 1,6-diaminohexane. The affinity column prepared by attachment of this compound to CNBr-activated Sepharose 4B enabled efficient purification of LDH from the marine teleost, Fundulus heteroclitus (Lin.). A similar affinity absorbent prepared by the method of O'Carra and Barry (FEBS Lett. (1972) 21, 281) gave a less satisfactory purification because the LDH was contaminated with phosphorylase a. The reason for this contamination was traced to an incomplete acylation of amino groups on the modified Sepharose beads by the carbodiimide-mediated reaction. Immobilization of the presynthesized affinant, N-(6-aminohexyl)oxamate, assured complete absence of these interfering groups. An alternate synthetic scheme, involving an active ester of ethyl oxalate, was also found to eliminate this problem.     

Purification of estradiol-17 beta dehydrogenase from human placenta by affinity chromatography

Estriol 16-hemisuccinate has been synthesized and covalently attached to Sepharose through 1,5-diaminopentane. A crude preparation of estradiol-17β dehydrogenase from human placenta was adsorbed on the gel. After extensive washing, the enzyme was eluted by $\text{M}$ hydroxylamine in 0.1 $\text{M}$ potassium phosphate buffer (20–50% glycerol), pH 7, at room temperature. An apparently homogeneous enzyme with a specific activity of 7.2 U/mg (82% recovery) was obtained. It is stable for weeks in the eluting buffer. The hydroxylamine can be removed by passing the enzyme solution over a Sephadex G-100 column or by dialyzing it against 0.1 $\text{M}$ potassium phosphate buffer containing 20% glycerol. This one-step process makes purification of the enzyme simple and easy.     

Extracellular Lytic Enzymes of Myxococcus virescens II. Purification of Three Bacteriolytic Enzymes and Determination of their Molecular Weights and Isoelectric Points

The bacteriolytic enzymes produced by Myxococcus virescens and previously concentrated and separated from most of the non-bacteriolytic proteins have been further separated and purified. The bacteriolytic enzyme solution was concentrated by lyo-philization. When applied to a Sephadex G-100 column, three peaks of bacteriolytic activity were eluted. Polyacrylamide gel electrophoresis showed that all the three enzyme fractions were contaminated with at least four non-bacteriolytic proteins. In the first enzyme fraction the bacteriolytic enzymes could be freed from the contaminating proteolytic activity by adsorption on a hydroxylapatite column. The bacteriolytic enzymes could then be adsorbed on a CM-cellulose column. The remaining contaminating proteins passed the column un-adsorbed while the bacteriolytic enzymes could be eluted with a gradient of 0.02–0.10 M ammonium hydrogen carbonate solution. The second enzyme fraction was adsorbed on a CM-cellulose column and then eluted with 0.03–0.15 M NH4 HCO₃. After rechromatography on a new column under the same conditions, all of the contaminating proteins had disappeared. For purification of the third enzyme fraction chro-matography on one single CM-cellulose column was sufficient. The elution of the adsorbed enzymes was performed with a gradient of 0.15–0.30 M NH₄HCO₃. The recovery of activity for each of the ion-exchange chromatography separations was at least 90%. The purity of the enzymes was tested by polyacrylamid gel electrophoresis. Each of the purified enzymes gave only one coloured band which coincided with the enzyme activity assayed in sliced gels. The molecular weights of the enzymes were determined by electrophoresis on acryl-amide gels containing sodiumdodecylsulphate. The molecular weights determined in this way (about 40,000, 30,000 and 20,000, respectively) were about 10,000 daltons higher than those obtained by gel chromatography on Sephadex G-100. This discrepancy seems to depend on interactions between the enzymes and the dextran molecules probably caused by the strongly basic nature of the enzymes or by formation of enzyme-substrate complexes.     

Purification of long helical capsid of newcastle disease virus from Escherichia coli using anion exchange chromatography

NP_Δc375 is a truncated version of the nucleocapsid protein of Newcastle disease virus (NDV) which self‐assembles into a long helical structure. A packed bed anion exchange chromatography (PB‐AEC), SepFastTM Supor Q pre‐packed column, was used to purify NP_Δc375 from clarified feedstock. This PB‐AEC column adsorbed 76.2% of NP_Δc375 from the clarified feedstock. About 67.5% of the adsorbed NP_Δc375 was successfully eluted from the column by applying 50 mM Tris‐HCl elution buffer supplemented with 0.5 M NaCl at pH 7. Thus, a recovery yield of 51.4% with a purity of 76.7% which corresponds to a purification factor of 6.5 was achieved in this PB‐AEC operation. Electron microscopic analysis revealed that the helical structure of the NP_Δc375 purified by SepFast^TM Supor Q pre‐packed column was as long as 490 nm and 22–24 nm in diameter. The antigenicity of the purified NP_Δc375 was confirmed by enzyme‐linked immunosorbent assay. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 564–567, 2013     

The activation of glycogen synthase in hepatocytes from rats with a glycogen storage disorder (gsd/gsd)

A rapid high resolution method of purification of the Trp-containing S100 proteins (S100a, S100a′) and of the S100b protein has been developed. The principle of this method is based on the fact that S100b protein becomes highly hydrophobic upon Zn²⁺ binding, whereas S100a and S100a′ are not affected. On an affinity chromatography of phenyl—Sepharose column, S100b is selectively bound in presence of zinc, whereas the Trp-containing S100 patients are quickly eluted. The S100b protein is further eluted with a buffer containing EDTA.     

Purification of histidine-tagged nucleocapsid protein of Nipah virus using immobilized metal affinity chromatography

Nucleocapsid (N) protein of Nipah virus (NiV) is a potential serological marker used in the diagnosis of NiV infections. In this study, a rapid and efficient purification system, HisTrap? 6 Fast Flow packed bed column was applied to purify recombinant histidine-tagged N protein of NiV from clarified feedstock. The optimizations of binding and elution conditions of N protein of NiV onto and from Nickel Sepharose? 6 Fast Flow were investigated. The optimal binding was achieved at pH 7.5, superficial velocity of 1.25 cm/min. The bound N protein was successfully recovered by a stepwise elution with different concentration of imidazole (50, 150, 300 and 500 mM). The N protein of NiV was captured and eluted from an inlet N protein concentration of 0.4 mg/ml in a scale-up immobilized metal affinity chromatography (IMAC) packed bed column of Nickel Sepharose? 6 Fast Flow with the optimized condition obtained from the method scouting. The purification of histidine-tagged N protein using IMAC packed bed column has resulted a 68.3% yield and a purification factor of 7.94.     

Human Brain-Type Glycogen Phosphorylase: Quantitative Localization in Human Tissues Determined with an Immunoassay System

Abstract: Glycogen phosphorylase (EC 2.4.1.1) from human brain tissue was purified to homogeneity. Antisera were developed in rabbits with purified phosphorylase as the immunogen. Antibodies were first affinity-purified with a column of brain phosphorylase-coupled Sepharose, and then the antibody fraction was adsorbed with a column of muscle phosphorylase-coupled Sepharose to remove antibodies reactive also with muscle phosphorylase. By using the specific antibodies, a sandwich-type immunoassay system for measurement of brain phosphorylase was prepared. The assay system consisted of polystyrene balls with immobilized antibrain phosphorylase F(ab')₂ fragments and the same antibody Fab' fragments labeled with β-D-galactosidase from Escherichia coli. The assay was sensitive and specific to the brain phosphorylase. The minimum detection limit of the assay was 0.1 ng/assay tube, and the cross-reactivity of the assay with muscle phosphorylase was <1%. Tissue concentrations of immunoreactive brain-type phosphorylase were estimated. The phosphorylase was present in the heart at as high a level as in the brain. The immunoreactivity for brain phosphorylase was distributed widely at a significant concentration in various peripheral tissues, such as the digestive tract, bladder, aorta, liver, and testis. Immunohistochemical localization of brain phosphorylase in the CNS revealed that the enzyme is present in most astrocytes and amyloid bodies, as well as in some neurons in the cerebral cortex and Golgi cells in the cerebellar cortex.     

Hydrophobic chromatography in the purification of the histidine-binding protein J from Salmonella typhimurium

Upon increasing the length of the side chains in a homologous series of ω-aminoalkyl agaroses (Seph—C_n—NH₂), more and more proteins become adsorbed onto the column and it is thus possible to achieve purification by selective exclusion of a desired protein. This principle is illustrated in the purification of the histidine-binding protein j from Salmonella typhimurium, vising ω-aminodecyl or ω-aminododecyl agarose columns. The protein was purified 7- to 10-fold in one step from either the shock fluid or the crude extract of the bacteria.Since protein j has an isoelectric pH of 5.5 and yet does not bind to the positively charged columns at pH 7, it seems that this protein is not likely to have available hydrophobic pockets and may thus be remarkably hydrophilic, compared with the other proteins in the shock fluid.     

Large-scale purification of plasmid DNA by fast protein liquid chromatography using a Hi-Load Q Sepharose column.

The large-scale purification of plasmid DNA was achieved using fast protein liquid chromatography on a Hi-Load Q Sepharose column. This method allows for the purification of plasmids starting from crude plasmid DNA, prepared by a simple alkaline lysis procedure, to pure DNA in less than 5 h. In contrast to the previously described plasmid purification methods of CsCl gradient centrifugation or high-pressure liquid chromatography, this method does not require the use of any hazardous or expensive chemicals. More than 100 plasmids varying in size from 3 to 15 kb have been purified using this procedure. A Mono Q Sepharose column was initially used to purify plasmids smaller than 8.0 kb; however, a Hi-Load Q Sepharose column proved more effective with plasmids larger than 8 kb. The loading of plasmids larger than 8 kb on the Mono Q column resulted in a high back pressure and the plasmid DNA could not be eluted from the column. Thus, for routine purification we utilize the Hi-Load Q Sepharose column. Plasmids purified by this method had purity, yield, and transfection efficiency in mammalian cells similar to those of plasmids purified by CsCl density gradient centrifugation.     

Yeast Glycogen Phosphorylase: Characterization of the Dimeric Form and Its Activation

The purification of yeast glycogen phosphorylase [EC 2.4.1.1] was improved by ethanol precipitation and affinity chromatography on a glycogen-Sepharose column. The purified enzyme gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had a subunit molecular mass of 100 kDa. Gel electrophoresis also showed that the major activity of native phosphorylase was ascribed to a dimer of 203 kDa, which was agreed with the value obtained by gel filtration on Sephadex G-200. The yeast phosphorylase showed a high affinity for AMP- Sepharose, whereas the enzyme was specifically inhibited by AMP. This inhibition was competitive with respect to the substrate glucose 1-phosphate and gave a Ki value of 9.3 mm. Activation of the crude extract by phosphorylation with an endogenous phosphorylase kinase indicated that the yeast phosphorylase occurred in a mixture of phosphorylated and non-phosphorylated forms.     

A Procedure for Human Pregnancy Zone Protein (and Human α2-Macroglobulin) Purification Using Hydrophobic Interaction Chromatography on Phenyl–Sepharose CL-4B Column

In the present work we describe a procedure for the purification of human pregnancy zone protein (PZP) from pooled late pregnancy plasma by using hydrophobic interaction chromatography (HIC) on a phenyl–Sepharose column. The HIC step allowed the complete isolation of haptoglobins and the partial separation of human α₂-macroglobulin (α₂-M) from a protein fraction containing PZP previously obtained by a DEAE-Sephacel chromatography. Pure and native PZP, with a recovery of nearly 25% and biological activity of protease-binding, was obtained by two definitive final steps consisting of zinc-chelate and size-filtration chromatographies. Moreover, we further present an alternative procedure for the purification of α₂-M from the same pregnancy plasma, based on the differential elution of PZP and α₂-M from the HIC. This purification step gave rise to a highly purified product with a recovery of 10%. This differential elution could be explained by differences in surface hydrophobicity observed between both proteins. In addition, considering the different hydrophobic properties exhibited by native PZP and PZP–protease complexes, HIC on phenyl–Sepharose column could also be used for separating both conformational states of PZP.