Evaluation of immobilized boronates for studies of adenine and pyridine nucleotide metabolism

Three different immobilized boronates were compared with respect to their possible utility in studies of adenine and pyridine nucleotide metabolism. These included boronate derivatives of polyacrylamide, Sepharose, and the cation-exchange resin, Bio-Rex 70. The relative binding affinities, binding capacities, and elution properties were compared. Under the conditions utilized, the Sepharose and Bio-Rex 70 derivatives selectively retained nucleotides containing two or more sets of 1,2 cis-diol groups. Since the bulk of cellular nucleic acids contain but a single set of 1,2 cis-diol groups or less, these boronate derivatives are very useful for the isolation of the acid-soluble nucleotides, NAD and diadenosine 5',5"'-tetraphosphate, and for the acid-insoluble polymer, poly(ADP-ribose). The Bio-Rex 70 derivative had a particularly useful combination of binding selectivity, capacity, and elution characteristics. Specific applications of this resin for studies of NAD and poly(ADP-ribose) metabolism are presented.     

Inhibition of mouse embryonic development by calmodulin antagonists

Two-cell mouse embryos were incubated in the presence of calmodulin inhibitors to determine their effect on embryonic development to the blastocyst stage. Calmodulin, a Ca²⁺-dependent regulatory protein, has been localized in the cytoplasm and has been implicated in regulation of many cellular events, such as mitosis. Several concentrations of either commercial or synthesized calmodulin inhibitors were tested. Several phenothiazine sulfoxide derivatives were more effective than the three naphthalene sulonic acid derivatives tested; 2-chloro-10-aminopropyl phenothiazine and 10-aminopropyl phenothiazine were the most potent phenothiazines to inhibit embryonic development at the two-cell stage. The interesting aspect of this study is that phenothiazine sulfoxide derivatives are not potent inhibitors of calmodulin, however, they were successful in inhibiting embryonic development. Potent inhibitors of calmodulin apparently did not penetrate the embryonic membranes because they had no effect.     

Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Small organic ligands.

The usefulness of affinity chromatography for the purification of aminoacyl-tRNA synthetases was explored by using column ligands derived from the corresponding amino acid and aminoalkyladenylate, a non-labile analogue of the aminoacyladenylate reaction intermediate. Four modes of attachment of the aminoalkyladenylate to Sepharose were studied. The interaction between amino acid derivatives and the corresponding aminoacyl-tRNA synthetases is too weak to allow their use as ligands for affinity chromatography. Attachment of the aminoalkyladenylate via the alpha-nitrogen atom of the amino acid or via C-8 of the nucleotide abolishes synthetase binding, and immobilization via the oxidized ribose ring is only marginally useful. However, attachment of the aminoalkyladenylate to the matrix via N-6 of the nucleotide allows strong and specific synthetase binding, and the use of such columns permits the isolation of homogeneous synthetase from crude mixtures. The effect of non-specific adsorption and the utility of pre-columns and of specific substrate elution are investigated and discussed.     

Preparation and application of an affinity matrix for phosphatidylinositol-specific phospholipase C

A non-hydrolyzable phosphonate analogue of phosphatidyl inositol, racemic myo-inosityl-(1)-5-oxa-16-trifluoroacetamidohexadecyl phosphonate, was synthesized. This phosphonate inhibited the activity of phosphatidyl inositol-specific phospholipase C (PI-PLC) from Bacillus cereus with an IC50 of approximately 10 mM. Removal of the trifluoroacetyl blocking group followed by covalent binding of the phosphonate to cyanogen bromide activated Sepharose 4B via the amino group produced an affinity matrix specific for the PI-PLC from B. cereus. This affinity matrix was used to purify the phospholipase C from a complex mixture of proteins in a single step. Competition experiments with myo-inositol in the elution medium indicated that specific binding of the enzyme to the matrix most likely involves the enzyme active site. The inositol phosphonate derivatized matrix was stable over several months in neutral and alkaline media and was used repeatedly without loss of binding capacity. These results show that affinity matrices employing myo-inositol phosphonate ligands are useful for isolation and binding studies of PI-PLC and possibly of other enzymes interacting with phosphoinositides or myo-inositol phosphate derivatives.     

Purine nucleoside phosphorylase: purification using an ether-linked formycin B/sepharose 6B resin with unusual properties

Formycin B [9-deazainosine] was reacted with epoxy-activated Sepharose 6B to form an affinity resin for purine nucleoside phosphorylase (PNPase). This resin had a large capacity (7,600 units/ml) for the enzyme from Escherichia coli. Enzyme retention was dependent on high ionic strength. Although this property is reminiscent of hydrophobic interaction chromatography, analogous resins prepared with pseudouridine or monoethanolamine instead of with formycin B, did not retain the enzyme even at high ionic strength. Furthermore, hypoxanthine facilitatted elution of the enzyme from the resin. It appeared, therefore, that the enzyme was not bound simply by hydrophobic interactions. A simple two-step purification procedure for PNPase from Escherichia coli was devised using this resin. Overall recovery was 50%, and purity of the final preparation was greater than 95%. This resin was also useful in the purification of PNPase from human erythrocytes. The ether linkage between formycin B and Sepharose 6B, together with the carbon-to-carbon linkage between the pentose and heterocyclic moieties of formycin B, provided stability to both chemical and enzymatic degradation. After 5 years of use and exposure to a variety of biological preparations, the resin showed no detectable decrease in its ability to bind PNPase.     

Calmodulin antagonists inhibit the mitochondrial pyruvate dehydrogenase complex

Calmodulin antagonists, including phenothiazine, sulfonamide, butyrophenone, and imidazolium derivatives, were in vitro inhibitors of pea mitochondrial pyruvate dehydrogenase complex activity. Inhibition was observed both during direct assay of the partially purified complex and during assay of pyruvate oxidation by isolated, intact mitochondria. When tested against the purified complex, the sulfonamide compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) was a competitive inhibitor with respect to coenzyme A and an uncompetitive inhibitor with respect to NAD and pyruvate. Inhibition of a process as crucial as mitochondrial respiration should serve to emphasize the care necessary in interpretation of whole-organism calmodulin antagonist studies.     

Synthesis and in vitro evaluation of α-synuclein ligands

Accumulation of misfolded α-synuclein in Lewy bodies and Lewy neurites is the pathological hallmark of Parkinson's disease (PD). To identify ligands having high binding potency toward aggregated α-synuclein, we synthesized a series of phenothiazine derivatives and assessed their binding affinity to recombinant α-synuclein fibrils using a fluorescent thioflavin T competition assay. Among 16 new analogues, the in vitro data suggest that compound 11b has high affinity to α-synuclein fibrils (K(i)=32.10 ± 1.25 nM) and compounds 11d, 16a and16b have moderate affinity to α-synuclein fibrils (K(i)≈50-100 nM). Further optimization of the structure of these analogues may yield compounds with high affinity and selectivity for aggregated α-synuclein.     

A review of: “Membrane Mimetic Chemistry J. H. Fendler John Wiley and Sons,New York, 1982 Hardcover, 522 pages, $59.95”

Formycin B [9-deazainosine] was reacted with epoxy-activated Sepharose 68 to form an affinity resin for purine nucleoside phosphorylase (PNPase). This resin had a large capacity (7,600 units/ml) for the enzyme from Escherichia coli. Enzyme retention was dependent on high ionic strength. Although this property is reminiscent of hydrophobic interaction chromatography, analogous resins prepared with pseudouridine or monoethanolamine instead of with formycin B, did not retain the enzyme even at high ionic strength. Furthermore, hypoxanthine facilitated elution of the enzyme from the resin. It appeared, therefore, that the enzyme was not bound simply by hydrophobia interactions. A simple two-step purification procedure for PNPase from Escherichia coli was devised using this resin. Overall recovery was 50%, and purity of the final preparation was greater than 95%. This resin was also useful in the purification of PNPase from human erythrocytes. The ether linkage between formycin B and Sepharose 6B, together with the carbon-to-carbon linkage between the pentose and heterocyclic moieties of formycin B, provided stability to both chemical and enzymatic degradation. After 5 years of use and exposure to a variety of biological preparations, the resin showed no detectable decrease in its ability to bind PNPase.     

Aromatic amino acids and their derivatives as ligands for the isolation of aspartic proteinases

Affinity chromatography was used to study an interaction of aspartic proteinases with immobilized aromatic amino acids and their derivatives. The following ligands were used: L-tyrosine, 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine, L-phenylalanine, p-iodo-L-phenylalanine and N-acetyl-L-phenylalanine. With the exception of the last one, ligands were coupled directly to divinyl sulfone activated Sepharose 4B. For the preparation of immobilized N-acetyl-L-phenylalanine, divinyl sulfone activated Sepharose 4-B with linked ethylene diamine was used. Porcine pepsin was used for the evaluation of the capacity of the prepared affinity carriers. The capacity of the immobilized amino acid derivatives significantly increased in comparison with the non-derivatized amino acids. The prepared immobilized ligands were further used for the separation of human pepsinogens.     

Calmodulin-dependent collagenase and proteoglycanase activities in chondrocytes from human osteoarthritic cartilage.

Chondrocyte metalloproteinases appear to play a major role in the development of osteoarthritis. The intracellular post-traductional mechanisms regulating collagenase and proteoglycanase are not known. Calmodulin antagonists including phenothiazine and sulfonamide derivatives significantly increased proteoglycanase activity and decreased collagenase activity. H-7, a specific inhibitor of protein kinase C, had no effect on the two metalloproteinase activities, and calmodulin was ineffective in in vitro assays upon metalloproteinase activities. We postulate that collagenase and proteoglycanase activities are controlled by calmodulin-dependent regulation.     

Purification of recombinant proteins based on the interaction between a phenothiazine-derivatized column and a calmodulin fusion tail.

A method to purify proteins by fusing them to the Ca2+-dependent protein calmodulin is described by using glutathione-S-transferase (GST) from Schistosoma japonicum as a model. Glutathione-S-transferase was genetically fused to calmodulin (CaM). The designed GST-CaM fusion protein has a selective factor Xa cleavage site located between the C-terminus of GST and the N-terminus of CaM. The recombinant fusion protein was expressed in Escherichia coli, and the crude cell extract was loaded onto a phenothiazine affinity column in the presence of Ca2+. Calmodulin was used as an affinity tail to enable binding of the fusion protein to the phenothiazine column. Removal of Ca2+ with a calcium-complexing solution causes elution of the fusion protein. The GST-CaM fusion protein was then digested with factor Xa, and the target protein GST was isolated. The purity of the isolated GST was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).     

The molecular forms of gamma-glutamyl transferase in bile and serum of icteric rats

Calmodulin coupled to Sepharose has provided a rapid and sensitive means of isolating a cyclic nucleotide phosphodiesterase activity which is stimulated by the calmodulin-Ca2+ complex, from rat parotid gland. Initial experiments established that phosphodiesterase activity sensitive to calmodulin and Ca2+ could not be demonstrated in crude extracts of rat parotid gland or after partial purification of rat parotid phosphodiesterase over DEAE-cellulose. However, it was possible to readily demonstrate the presence of a cyclic nucleotide phosphodiesterase activity regulated by calmodulin if the extracts were first purified by batch ion-exchange chromatography over DEAE-cellulose followed by affinity chromatography with calmodulin coupled to Sepharose. The batch ion-exchange chromatography step removed the major portion of free parotid calmodulin which could compete with calmodulin-coupled Sepharose for the proteins regulated by calmodulin. Thus, by employing an initial chromatography step over DEAE-cellulose to separate phosphodiesterase activity from calmodulin, it was possible to increase the recovery of calmodulin-sensitive phosphodiesterase after affinity chromatrography with calmodulin coupled to Sepharose. This approach should be useful for demonstrating the presence of and for purifying other parotid proteins regulated by calmodulin.     

Detection of a calmodulin-sensitive cyclic nucleotide phosphodiesterase in rat parotid gland

Calmodulin coupled to Sepharose has provided a rapid and sensitive means of isolating a cyclic nucleotide phosphodiesterase activity which is stimulated by the calmodulin-Ca²⁺ complex, from rat parotid gland. Initial experiments established that phosphodiesterase activity sensitive to calmodulin and Ca²⁺ could not be demonstrated in crude extracts of rat parotid gland or after partial purification of rat parotid phosphodiesterase over DEAE-cellulose. However, it was possible to readily demonstrate the presence of a cyclic nucleotide phosphodiesterase activity regulated by calmodulin if the extracts were first purified by batch ion-exchange chromatography over DEAE-cellulose followed by affinity chromatography with calmodulin coupled to Sepharose. The batch ion-exchange chromatography step removed the major portion of free parotid calmodulin which could compete with calmodulin-coupled Sepharose for the proteins regulated by calmodulin. Thus, by employing an initial chromatography step over DEAE-cellulose to separate phosphodiesterase activity from calmodulin, it was possible to increase the recovery of calmodulin-sensitive phosphodiesterase after affinity chromatography with calmodulin coupled to Sepharose. This approach should be useful for demonstrating the presence of and for purifying other parotid proteins regulated by calmodulin.     

Influence of type of linkage and spacer on the interaction of beta-galactoside-binding proteins with immobilized affinity ligands

Affinity chromatography provides a powerful tool for isolation of carbohydrate-binding proteins. However, the choice of the ligand and spacer has an important impact on effectiveness. The influence of several different ligands on qualitative and quantitative aspects of the purification of two beta-galactoside-specific lectins has been evaluated. Sepharose was modified by coupling four types of neoglycoproteins (galactosylated or lactosylated bovine serum albumin with increasing sugar content) and two naturally occurring asialoglycoproteins at similar densities. Carbohydrate ligands at essentially equal density were made accessible to the lectins by seven commonly used methods. The yield of mistletoe lectin was high when lactosylated neoglycoproteins were used for separation. For these resins the sugar incorporation exceeded 10 sugar groups per protein carrier molecule. The yield was similarly high with the asialoglycoproteins and with lactose; the sugar was coupled to the resin as a p-aminophenyl derivative or by means of divinyl sulfone activation. An epoxy group in linkages of galactose or lactose decreased the binding capacity. A quantitatively similar degree of protein yields was obtained for the beta-galactoside-binding protein of bovine heart, although different proteins were obtained when neoglycoproteins were used as ligand. The nature of the affinity ligand in lectin purification can increase the yield and may also influence the profile of the carbohydrate-binding proteins.     

Affinity chromatography of porcine pepsin and pepsinogen using immobilized ligands derived from the specific substrate for this enzyme

Affinity chromatography of porcine protease and its zymogen was carried out on immobilized components of specific substrate used for the pepsin determination. For the immobilization of N-acetyl-L-phenylalanine and iodinated derivative of L-tyrosine, divinyl sulfone activated Sepharose was used. Ligands with blocked amino group and free carboxyl one were linked to Sepharose via ethylene diamine spacer using carbodiimide reaction. Conditions of affinity chromatography of porcine pepsin and pepsinogen on the prepared carriers were optimized: the effect of pH, ionic strength and a nature of the buffers used on adsorption of the enzyme and zymogen to an affinity carrier, as well as their elution was studied. The following parameters were taken into consideration: capacity of the prepared affinity matrices, reproducibility of experiments and the enzyme stability. Pepsin was adsorbed to both immobilized ligands at pH 3.5-4.0; for the elution of the enzyme it was necessary to increase ionic strength (up to 0.5 M). For the adsorption of pepsinogen pH 5.2 was found to be optimum, for its desorption, an increase of ionic strength was used.     

A new method for affinity chromatography of heme-binding proteins: Synthesis and characterization of hematin- and hematoporphyrin-agarose

Two new affinity columns, using hematin and hematoporphyrin as ligands, have been prepared. Both were made by first attaching 1,6-diaminohexane to Sepharose 4B by the cyanogen bromide procedure and then coupling the porphyrins to the free amino groups of this arm with carbodiimide. This second reaction was done in dimethylformamide to increase the solubility of the porphyrins at pH 4.7. This resin was then washed extensively with dimethylformamide to remove all of the unreacted porphyrin. The new affinity columns are able to bind apoglobin, albumin, and hemopexin, which demonstrates their ability to purify heme-binding proteins. The proteins could be removed by washing the column with a deforming buffer, such as 8 m urea or 0.1 m sodium citrate buffer at pH 4. Neither lysozyme nor hemoglobin bound to these resins demonstrating that the absorbants are specific for apo heme-binding proteins.     

Ligand polarizability contributes to tau fibril binding affinity

Whole brain imaging of tau-bearing neurofibrillary lesions has the potential to improve the premortem diagnosis and staging of Alzheimer's disease. Diverse compounds with high affinity for tau aggregates have been reported from high-throughput screens, but the affinity driving features common among them have not been determined. To identify these features, analogs of compounds discovered by high-throughput screening, including phenothiazine, triarylmethine, benzothiazole, and oxindole derivatives, were tested for their ability to displace fluorescent thioflavin dyes from filaments made from recombinant tau protein or authentic paired helical filaments purified from Alzheimer's disease tissue. When representative members of all scaffolds were assayed, the rank order of binding affinity determined for synthetic and authentic filaments correlated strongly, indicating that synthetic filaments have predictive utility for ligand development. Within individual scaffold families, binding affinity was found to correlate with compound polarizability, consistent with a role for dispersion forces in mediating ligand binding. Overall, the data indicate that polarizability is an important commonality among structurally diverse tau binding ligands, and that affinity for tau aggregates can be maximized by integrating formal assessment of this parameter into ligand discovery efforts.     

Lactation-associated redistribution of the glial fibrillary acidic protein within the supraoptic nucleus

Summary Three clones of myeloproliferative virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and morphology were transplanted to athymic nude mice. Presence of carbohydrate-binding proteins was inferred by fluorescence microscopy using fluorescent, glycosylated markers. Salt and detergent extracts of tumors from this model system were fractionated under identical conditions on different sets of Sepharose columns, to which lactose, asialofetuin, melibiose, mannan and fucose had been covalently linked. Successive elution by chelating reagent and specific sugar resulted in isolation of the different Ca²⁺-dependent and Ca²⁺-independent endogenous carbohydrate-binding proteins that were assayable as agglutinins. In comparison, the different tumors displayed a pattern with qualitative and quantitative alterations. Since protein-carbohydrate interaction mediated by carbohydrate-binding proteins (lectins) is of importance for cognitive processes, it is remarkable that the pattern of membrane glycoproteins, isolated by affinity chromatography on resins with immobilized plant lectins, had also been found to reveal certain individual properties for receptors specific for peanut agglutinin (PNA) and Ulex europaeus agglutinin (UEA). These demonstrated differences within the system of protein-carbohydrate interaction suggest that endogenous lectins and their ligands have potential significance as markers defining a certain phenotype within this tumor model system.Dedicated to Prof. Dr. W. Lamprecht on the occasion of his 60th birthday     

Affinity chromatography of arabinogalactan-proteins

Arabino-(1→3), (1→6)-β-d-galactan-proteins (AGPs) and related compounds from Lolium multiflorum (ryegrass) endosperm cell suspension culture, wheat endosperm, larchwood and Gladiolus stigma extract were shown to bind selectively at neutral pH to a column of Sepharose to which the anti-galactan myeloma protein J539 had been covalently linked, Elution was achieved with buffer at pH 3 or with a pulse of p-nitrophenyl-β-d-galactopyranoside at neutral pH. These observations formed the basis for an affinity chromatographic purification of AGPs from natural sources. Some heterogeneity in a ryegrass AGP preparation was indicated by its incomplete elution by d-galactose.     

Synthesis of 3-alkyl naphthalenes as novel estrogen receptor ligands

A series of estrogen receptor ligands based on a 3-alkyl naphthalene scaffold was synthesized using an intramolecular enolate-alkyne cycloaromatization as the key step. Several of these compounds bearing a C6-OH group were shown to be high affinity ligands. All compounds had similar ERalpha and ERbeta binding affinity ranging from micromolar to low nanomolar.