Isolation of brain tubulin by affinity chromatography

An affinity chromatographic procedure for the isolation of tubulin from brain is described. The yield is good and the method rapid and gentle. Criteria of purity are colchicine binding, SDS acrylamide gel electrophoresis, and velocity sedimentation.     

Separation of cobalt binding proteins by immobilized metal affinity chromatography

BAY 43-9006 is a selective Raf-1 kinase inhibitor with antitumor activity against a variety of human cancers. A highly sensitive HPLC method for determination of BAY 43-9006 in small volumes of serum (30 microl) was developed. Sample preparation involved a liquid-liquid extraction procedure with tolnaftate as internal standard followed by linear gradient elution at a reversed phase C18 column and UV detection. The method was selective and the calibration curves were linear over the concentration range of 80-2000 ng/ml. The intra-day accuracy ranged from 99.9 to 107.6% and the inter-day accuracy from 94.6 to 115%. The lower limit of quantitation (LOQ) was 80 ng/ml with an accuracy of 105.8%. Thus, this method has been validated and can be applied for the drug monitoring or pharmacokinetic studies of BAY 43-9006 in small volumes of serum samples.     

Identification of S-100 proteins and S-100-binding proteins in a detergent-resistant EDTA/KCl-extractable fraction from bovine brain membranes

The Triton X-100-resistant residue of brain membranes contains appreciable amounts of S-100 proteins. This fraction of S-100 can be solubilized by high concentrations of EDTA plus or minus high concentrations of KCl. Whereas KCl (0.6 M) extracts the detergent-resistant S-100, NaCl (1 M) does not. Endogenous Ca2+ is required and is sufficient for S-100 to remain associated with the detergent-resistant residue. However, 0.6 M KCl extracts a further fraction of Triton X-100-resistant S-100. In contrast, the Triton X-100-extractable fraction of S-100 resists the action of EDTA. These data suggest that Ca2+ regulates the extent of association of S-100 with Triton X-100-resistant components in brain membranes, whereas the association of S-100 with the lipid bilayer of brain membranes and/or with some intrinsic membrane proteins is less Ca2+-regulated. Several S-100-binding proteins are identified in the detergent-resistant residue of brain membranes by an overlay procedure.     

S-100 proteins

S-100 is a group of closely related, small, acidic Ca2+-binding proteins (S-100a0, S-100a and S-100b, which are alpha alpha, alpha beta, and beta beta in composition, respectively). S-100 is structurally related to calmodulin and other Ca2+-binding proteins. S-100 is abundant in the brain and is contained in well defined cell types of both neuroectodermal and non-neuroectodermal origin, as well as in their neoplastic counterparts. In the mammalian brain, S-100a and S-100b are confined to glial cells, while S-100a0 is neuronal in localization. Single S-100 isoforms bind Ca2+ with nearly the same affinity. K+ antagonizes the binding of Ca2+ to high affinity sites on S-100. S-100 binds Zn2+ with high affinity. S-100 is found in a soluble and a membrane-bound form and has the ability to interact with artificial and natural membranes. S-100 has no enzymatic activity. S-100 has been involved in several activities including memory processes, regulation of diffusion of monovalent cations across membranes, modulation of the physical state of membranes, regulation of the phosphorylation of several proteins, control of the assembly-disassembly of microtubules. Some of these effects are strictly Ca2+-dependent, while other are not. S-100 is being secreted or released to the extracellular space. In some cases, this event is hormonally regulated. Several S-100 binding proteins are being described.     

Isolation and purification of cytokinin binding protein from tobacco leaves by affinity column chromatography

Cytokinin binding protein from tobacco leaves was isolated and purified to a single protein by means of affinity chromatography on benzyladenine-linked Sepharose column combined with polyacrylamide gel electrophoresis. In vitro binding of this protein to [¹⁴C] benzyladenine was inhibited remarkably by cold benzyladenine and kinetin and slightly by adenine, but not adenosine. The molecular weight of the protein was determined to be about 4,000 daltons by gel filtration and SDS polyacrylamide gel electrophoresis.     

Partial purification of rat liver glucocorticoid binding proteins by affinity chromatography

Dexamethasone (9-fluoro-16α-methyl-116,17,21-trihydroxy-1,4-pregnadiene-3, 20-dione) binding proteins from rat liver cytosol were purified approximately 6470 fold by the use of an affinity column in which deoxycorticosterone was linked to CH-Sepharose 4B through a disulfide linkage. The receptor proteins were eluted from the column by washing with β-mercaptoethanol. A preliminary Sephadex G-200 filtration step of the cytosol was necessary in order to separate the dexamethasone binding proteins from other glucocorticoid receptors.     

Screening for beta-poly(L-malate) binding proteins by affinity chromatography

Poly(beta-L-malic acid) is a cell type-specific polymer of myxomycetes (true slime molds) with the physiological role to organize mobility of certain proteins over the giant multinucleated plasmodia. We have developed an affinity chromatography employing 1,6-diamino-n-hexane-Sepharose-coupled poly(malic acid) to identify such proteins in cellular extracts of Physarum polycephalum. Molecular masses were measured by SDS-PAGE and non-denaturing PAGE after silver staining and/or Western blotting. Protein complexes/subunits were detected by 2-dimensional non-denaturing PAGE/SDS-PAGE. A simplified gel shift experiment displayed binding to fragmented calf thymus DNA. Nuclei were richest in poly(malate) binding proteins followed by cytoplasm and membranes. A protein of 370 kDa dissociated into 11 subunits of 11-29 kDa, indicative of a highly complex protein. This and other proteins displayed binding to nucleic acid in gel shift experiments. Poly(malate) is considered a structural and functional equivalent of long contiguous aspartate repeats in proteins of eukaryotes.     

Isolation of 3,4-dihydroxyphenylalanine-containing proteins using boronate affinity chromatography

A rapid procedure for the isolation of 3,4-dihydroxyphenylalanine-containing proteins has been developed in which the protein is selectively bound to a m-phenylboronate agarose column, and eluted with 1.0 M ammonium acetate, pH 3.0. The method is based on the affinity of boronates for diols including catechol. The chromatography is carried out in the absence of oxygen to prevent oxidation of the catechol. Other proteins are eluted beforehand with 0.25 M ammonium acetate, pH 8.5, or for glycoproteins with a Tris buffer containing 0.2 M sorbitol, pH 8.5.     

Calcium-sensitivity of brain microtubule proteins in the presence of S-100 proteins

In the presence of the usual 0.1 M Mes buffer, pH 6.7, mM free Ca2+ levels are required for half-maximal decrease in the rate and extent of brain microtubule protein (MTP) assembly in the absence of ox brain S-100, while microM free Ca2+ levels are sufficient in the presence of S-100. At the same pH 6.7, but in the presence of 0.12 M KCl, as low as 1.5 microM free Ca2+ is sufficient for S-100 to produce half-maximal reduction in the rate of assembly, while as high as 0.5 mM free Ca2+ is required in the absence of S-100. Similar results are obtained with rat brain S-100 (S-100b), indicating that single S-100 iso forms are equipotent in affecting the MTP assembly. At pH 7.5, MTPs are remarkably resistant to Ca2+ in the absence of S-100. In the presence of S-100, not only is the free Ca2+ concentration required for complete inhibition of assembly at least one order of magnitude smaller than that required in the absence of S-100, but significant S-100-dependent inhibition of assembly occurs in the absence of Ca2+. Under the two conditions where S-100 is particularly effective in inhibiting the assembly, i.e. at pH 6.7 in the presence of KCl and at pH 7.5, S-100 increases the disassembly rate even in the presence of microM Ca2+ levels. Our results suggest that the free Ca2+ concentration regulates the way S-100 disassembles microtubules (MTs): at microM Ca2+ levels, S-100 sequesters tubulin with concomitant increase in the disassembly rate; at mM Ca2+ levels, the S-100-Ca2+ complex probably interacts with MTs producing endwise disassembly.     

Isolation of ADP-ribosyltransferase by affinity chromatography

An affinity adsorbent for ADP-ribosyltransferase (EC 2.4.2.30) has been synthesized by coupling 3-aminobenzamide to Sepharose 4B. Using this material, ADP-ribosyltransferase from human placenta has been purified from crude extract to homogeneity within a few hours. The enzyme has an apparent Km for NAD+ of 52 microM. Its molecular mass is 115,000 as determined by gel electrophoresis. The enzyme is DNA dependent and stimulated by histone, its temperature optimum is at 25 degrees C, and its pH optimum is around pH 9. alpha-NAD+, thymidine, caffeine, theophylline, theobromine, 3-methoxybenzamide, and nicotinamide inhibit the enzyme. Purification of ADP-ribosyltransferases from horse, rat, and chicken liver was also achieved with the method described.     

Immobilized-biomembrane affinity chromatography for binding studies of membrane proteins

Analyses of specific interactions between solutes and a membrane protein can serve to characterize the protein. Frontal affinity chromatography of an interactant on a column containing the membrane protein immobilized in a lipid environment is a simple and robust approach for series of experiments with particular protein molecules. Regression analysis of the retention volumes at a series of interactant concentrations shows the affinity of the protein for the interactant and the amount of active binding sites. The higher the affinity, the fewer sites are required to give sufficient retention. Competition experiments provide the affinities of even weakly binding solutes and the non-specific retention of the primary interactant. Hummel and Dreyer size-exclusion chromatography allows complementary analyses of non-immobilized membrane materials. Analyses of the human facilitative glucose transporter GLUT1 by use of the inhibitor cytochalasin B (radioactively labeled) and the competitive substrate D-glucose (non-labeled) showed that GLUT1 interconverted between two states, exhibiting one or two cytochalasin B-binding sites per two GLUTI monomers, dependent on the membrane composition and environment. Similar analyses of a nucleoside transporter, a photosynthetic reaction center, nicotinic acetylcholine receptors and a P-glycoprotein, alternative techniques, and immobilized-liposome chromatographic approaches are presented briefly.     

Isolation and identification of actin-binding proteins in Plasmodium falciparum by affinity chromatography

The invasion of the erythrocyte by Plasmodium falciparum depends on the ability of the merozoite to move through the membrane invagination. This ability is probably mediated by actin dependent motors. Using affinity columns with G-actin and F-actin we isolated actin binding proteins from the parasite. By immunoblotting and immunoprecipitation with specific antibodies we identified the presence of tropomyosin, myosin, a-actinin, and two different actins in the eluate corresponding to F-actin binding proteins. In addition to these, a 240-260 kDa doublet, different in size from the erythrocyte spectrin, reacted with an antibody against human spectrin. All the above mentioned proteins were metabolically radiolabeled when the parasite was cultured with 35S-methionine. The presence of these proteins in P. falciparum is indicative of a complex cytoskeleton and supports the proposed role for an actin-myosin motor during invasion.     

Specific adsorbents for affinity chromatography of benzodiazepine binding proteins

The use of affinity chromatography as means of isolating/purifying proteins which have an affinity for benzodiazepine is described. Three such drugs are employed: chlorazepate, clonazepam and delorazepam. The results presented in this paper indicate that the proposed technique only works for chlorazepate and delorazepam. In fact these benzodiazepine-Sepharose derivatives are able to retain specifically proteins from human serum and rat kidney, lung, skeletal muscle and brain.     

Isolation of two novel adenosine binding proteins from bovine brain

Adenosine plays many significant roles both as a metabolic precursor and cell communicator. This report describes the preliminary characterization of two adenosine binding proteins isolated from bovine brain membranes. By using N6-9-aminononane adenosine labeled Sepharose 4B two major affinity bound proteins were purified having apparent molecular weights of 16 and 35 kDa. Either or both of the proteins could be selectively eluted from the affinity column with N6-9-aminononane adenosine, adenosine, cAMP, AMP, ADP, ATP, R-/S-phenylisopropyladenosine and NAD(H). By contrast, no proteins were eluted with caffeine, adenine, deoxyadenosine, 2',3'-AMP, inosine, IMP, xanthine, XMP, GMP, GTP or 5'-N-ethylcarboxamideadenosine. The selectivity of elution and lack of apparent enzymatic activity suggests that these proteins are novel membrane bound adenosine binding proteins.     

Rapid purification of calmodulin and S-100 protein by affinity chromatography with melittin immobilized to sepharose

Melittin-Sepharose was prepared for Ca2+-dependent affinity chromatography of calmodulin and S-100 protein. This matrix exhibits extremely high capacity (approximately 10 mg calmodulin/ml gel), low nonspecific binding, and excellent recovery (greater than 90%) under optimal conditions. Recovery of calmodulin from melittin-Sepharose was related to the degree of saturation of column capacity with lower yields when only partial saturation was achieved. Large-scale, simultaneous purification of calmodulin and S-100 protein from brain was carried out using selective adsorption to organomercurial agarose followed by melittin-Sepharose chromatography; yields were 250-300 mg of calmodulin and 200-300 mg of S-100 per kg tissue. Calmodulin also was purified in a single step from bovine testis supernatant using melittin-Sepharose in yields comparable to those from brain.     

Isolation of two cholic acid-binding proteins from rat liver cytosol using affinity chromatography

Using an affinity matrix coupled with cholic acid, two proteins that recognise bile acids were isolated from rat liver cytosol. One protein of molecular weight 68 000 was immunologically identical to rat albumin. The other protein was of molecular weight 46 000. On discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis the 46 000 molecular weight protein dissociated to a single band with an RF value identical to the Yb subunit of the bromosulphophthalein-binding fraction (Y-fraction) of whole liver cytosol. The monomers of purified ligandin under these conditions resolved into two bands which corresponded to the Ya and Yc subunits of liver cytosol Y-fraction. Anti-serum to the purified ligandin reacted monospecifically with purified ligandin and whole liver cytosol, but did not cross-react with the Yb dimer eluted from the affinity column. The Yb dimer was shown to possess glutathione-S-transferase activity with a substrate specificity distinct from ligandin but similar to glutathione-S-transferase C. Cholic acid inhibited the catalytic activity of the transferase.     

Isolation of an organic anion binding protein from rat liver plasma membrane fractions by affinity chromatography

As part of a study of hepatic organic anion transport, solubilized liver plasma membrane proteins were subjected to affinity chromatography on bilirubin- and sulfobromophthalein-labeled agarose columns. Both columns retained a Sudan Black and PAS negative protein of molecular weight 60,000 daltons, which cochromatographed with [³⁵S]sulfobromophthalein on Sephadex G-75, and reversibly bound [³⁵S]sulfobromophthalein in vitro with high affinity (K_a ? 10⁷ M^?1) and a valence of 2. Erythrocyte ghost membranes did not contain this protein. Sulfobromophthalein-agarose retained two additional smaller proteins which did not cochromatograph with [³⁵S]sulfobromophthalein. Their significance is unclear. This study supports the hypothesis that liver cell plasma membranes participate in the hepatic transport of organic anions.     

Isolation and characterization of the two active fractions from porcine brain phosphofructokinase purified by affinity chromatography

Phosphofructokinase was purified from porcine brain by the successive affinity chromatographies of Blue-Sepharose and ATP-agarose. The purified enzyme was found to contain three subunits, namely, the L-, M- and C-type by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme separated into the two active fractions through gel filtration (FIs and FIIs). SDS-PAGE revealed that the FIs contained the L- and M-type subunits, while the FIIs contained the M- and C-type. The FIIs preparation was more sensitive to ATP inhibition, ADP activation and citrate inhibition than the FIs.