Monoclonal antibodies against the plant cytokinin isopentenyl adenosine

Sixteen monoclonal antibodies against isopentenyl adenosine (iPA) were developed and characterized for reactivity towards this cytokinin and structurally related molecules by use of a competition fluorescence enzyme immunoassay. Antibodies with suitable affinity and specificity were used in an immunoassay to detect and quantify isopentenyl adenosine. Logit/log plots of fluorescence vs pmol of the cytokinin indicated that the assay had a measurement range of 0.03–256 pmol (10–8500 pg) with high linearity (r =–0.98). This competition fluorescence enzyme immunoassay showed that three antibodies cross-reacted with N-6-benzyladenine and its riboside: cross-reactivity with dihydrozeatin and its riboside, cis -zeatin, trans -zeatin riboside, adenine and adenosine was minor. When an immunoaffinity-HPLC technique was used to measure the cross-reactivity of two of the antibodies in the presence of known quantities of multiple cytokinins, iPA was bound in preference to the other cytokinins. One of the antibodies was used to quantify this cytokinin in developing wheat ( Triticum aestivum L. cv. Stephens) seeds and in wheat seeds spiked with known amounts of certain other cytokinins. The use of these antibodies in immunoassay in combination with HPCL for quantification of iPA in plant extracts is discussed.     

The specificity of the chemical modification of N6-delta 2-(isopentenyl)adenosine in purified yeast tRNA Ser.

The specific modification of N6-delta 2-(isopentenyl)adenosine in purified tRNA Ser yeast by mild treatment with KMnO4 and I2 was studied. N6-delta 2-(isopentenyl)adenosine in tRNA SER is specifically modified by iodination, providing us with a suitable method for the quantitative determination of N6-delta 2-(isopentenyl)adenosine in tRNA was found to contain 114 +/- 8 pmol/A260nm unit of N6-delta 2-(isopentenyl)adenosine and gave three labelled fractions on an RPC-5 column. The product obtained after KMnO4 treatment of tRNA Ser was not homogeneous. The enzymatic "reisopentenylation" of KMnO4-treated tRNA Ser resulted in the regeneration of only traces of the original molecule(s). Most of them had been damaged either by the KMnO4 treatment or in the incubation mixture used for "reisopentenylation".     

Lack of pokeweed mitogen-induced IgE formation in vitro by human peripheral blood mononuclear cells: detection of cross-reacting idiotypic determinants on polyclonal Ig by antibodies to a single IgE myeloma protein

The ability of human peripheral blood mononuclear cells to synthesize IgE in vitro in response to pokeweed mitogen (PWM) is controversial. To determine whether the conflicting results obtained by different laboratories could be due to inherent qualitative differences in the anti-IgE antibodies used to measure low concentrations of IgE in culture supernatants, we compared the specificities of anti-IgE reagents prepared by various methods. Immunoadsorbent-purified antibodies were isolated from a goat antiserum to the lambda, IgE myeloma protein PS and a rabbit antiserum to the kappa, IgE protein Bed in three ways: 1) antibodies to IgE PS (anti-PS) were isolated from the goat antiserum by affinity chromatography with PS coupled to Sepharose 4B; these antibodies consisted of anti-epsilon chain-specific and anti-idiotypic antibodies to protein PS; 2) antibodies specific for the epsilon-chain (anti-epsilon) were purified by affinity chromatography with IgE myeloma proteins that were not used for immunization; and 3) antibodies to idiotypic determinants of proteins PS (anti-id PS) and Bed (anti-id Bed) were isolated on affinity columns with the respective myeloma proteins after absorption of the epsilon-chain-specific antibodies. These three types of antibodies were then used in a solid phase radioimmunosorbent test to quantitate the amount of "IgE" synthesized by peripheral blood mononuclear cells from nonatopic and atopic donors cultured for 7 days in the presence and absence of PWM. ANTI-PS antibodies detected a PWM-induced "IgE formation" in cell culture supernatants of both non-atopic and atopic donors. (ABSTRACT TRUNCATED AT 250 WORDS)     

Characierization of monoclonal antibodies specific for isopentenyl adenosine derivatives occurring in transfer RNA

A family of isopentenyl adenosine derivatives are naturally occurring components of transfer RNA and are involved in several different functional roles in the cell. To facilitate the study of the biochemistry of these modified nucleosides we have raised monoclonal antibodies to N⁶-(Δ²-isopentenyl)adenosine and N⁶-(4-hydroxy-3-methyl-but-2-enyl)adenosine. The antibodies show considerable specificity and three characteristic types are distinguishable. The first type have the hydroxylated derivative as the preferred antigen, the second type have isopentenyl adenosine as the preferred antigen and a third type show a specificity for all isopentenyl-containing derivatives.     

Apoptosis induced in neuronal cells by C-terminal amyloid ß-fragments is correlated with their aggregation properties in phospholipid membranes

Rat brain proteins presenting high-affinity binding of S-adenosyl-L-homocysteine were solubilized and purified. Extraction of binding protein was carried out in the presence of Triton X-100 and 1 M NaCl; this solubilized fraction exhibits similar kinetic properties than the membrane proteins. Purification was performed using affinity chromatography on S-adenosyl-L-homocysteine carboxyhexyl Sepharose 48 conjugate. The analysis of the affinity gel eluate by SDS-PAGE showed high purification ratios for two proteins exhibiting 54 and 68 kDa. Three activity peaks were separated when solubilized membrane proteins were submitted to isoelectric focusing; the activity peaks corresponded to proteins of pH, 6.0, 6.5, and 7.2. SDS-PAGE separation of proteins contained in each peak showed protein aggregation; a 54-kDa subunit was present in each aggregate. Solubilized membrane proteins were labeled by photoaffinity labeling with tritiated S-adenosyl-L-homocysteine; the 54-and 68-kDa proteins were found among the specifically labeled proteins. Finally, according to the previous data from the literature, the purified S-adenosyl-L-homocysteine binding proteins do not seem to be the same as adenosine receptors or phosphatidylethanolamine-N-methyltransferase.     

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.     

腺苷转运体亲和层析分离

本文合成了一种腺苷亲和层析凝胶,并采用亲和层析法从牛脑细胞膜上分离出了几种膜上结合的腺苷结合蛋白质。这些蛋白质在ＳＤＳ－ＰＡＧＥ电泳凝胶上为单一或主要的蛋白带,分子量分别为６４ｋｄ,４５ｋｄ,３５ｋｄ。腺苷转运体抑制剂潘生丁和ＮＢＭＰＲ对６４ｋｄ蛋白与＾３ｈ－腺苷的结合抑制作用远强于腺苷受体的激动剂ＮＥＣＡ和Ｒ－ＰＩＡ;这表明６４ｋｄ蛋白为牛脑细胞膜上结合的腺苷转运体。     

Reductive alkylation with oxidized nucleotides. Use in affinity labeling or affinity chromatography

A study has been made of the products of a reaction of oxidized ribonucleotides with a primary amine. As a model reaction, periodate-oxidized adenosine was combined with glycine in the presence of NaCNBH3. The purified major product of this reaction, adenine 9,2'-(4'-carboxymethyl-6'-hydroxymethylmorpholine), was characterized by 13C and 1H NMR spectroscopy, ultraviolet spectroscopy, and thin layer chromatography. When used to generate affinity columns, oxidized adenosine or oxidized ATP formed stable products with immobilized diaminohexane when treated with NaCNBH3. Failure to treat with NaCNBH3 yielded an unstable affinity matrix. These results are used in the interpretation of differing results when oxidized nucleotides have been used as affinity labels for different proteins.     

One-step purification of hybrid proteins which have beta-galactosidase activity

A one-step purification method of hybrid proteins exhibiting beta-galactosidase activity, based on affinity chromatography in the presence of high salt concentration, is described. Starting from crude bacterial extracts, several milligrams of near-homogeneous proteins can be obtained in a few hours with an overall yield of 85 to 95%. The purified hybrid proteins can be used to obtain antibodies against the foreign portion of the protein fusion.     

重组酶Exo的纯化及多克隆抗体制备

目的:纯化Exo重组酶融合蛋白并制备相应抗体。方法:用阴离子交换柱对蛋白进行初步纯化,然后用Ni-NTA介质填充的层析柱分离纯化含His标签的融合蛋白,用谷胱甘肽琼脂糖4B介质填充的层析柱分离纯化GST融合蛋白;二次纯化的蛋白利用硝酸纤维素膜结合法制备抗原蛋白并免疫实验动物。结果:ELISA结果显示血清抗体效价可达到1∶12 800,说明通过Western免疫印迹自制的多克隆抗体能特异地与Exo重组蛋白相互作用。结论:该蛋白纯化方法操作简单,制备的抗原纯度高,多克隆抗体特异性好。     

兔抗五种鱼免疫球蛋白抗体的制备和HRP标记

目的制备兔抗鱼类免疫球蛋白抗体并进行辣根过氧化酶标记,为鱼类血清学检测系统的建立提供工具。方法利用proteinA亲和层析的方法纯化鱼血清免疫球蛋白,免疫大耳白兔制备抗血清,利用免疫双扩散来检测抗血清的效价,并用Protein A亲和层析的方法来纯化兔抗鱼免疫球蛋白的抗体,采用简易过碘酸钠法对纯化的兔抗鱼免疫球蛋白的抗体进行HRP标记,通过ELISA方法测定标记抗体的效价,并利用Western blotting来考察标记抗体与其他常见鱼类血清蛋白间的交叉反应。结果纯化了鳙、鲤、乌鳢、黄鳝、鲈五种鱼血清免疫球蛋白,免疫双扩散法测定兔抗这五种鱼免疫球蛋白的抗血清效价均达到1∶32,并对纯化的兔抗鳙、鲤、乌鳢、黄鳝、鲈五种鱼类免疫球蛋白的抗体进行了HRP标记,ELISA测定标记抗体的效价达到1∶10000左右,Western blots显示标记抗体与部分其他鱼类免疫球蛋白之间存在不同程度的交叉反应。结论成功制备了HRP标记的兔抗鱼类免疫球蛋白抗体,为鱼类血清学检测体系的建立提供了工具。     

A1 adenosine receptor of rat testis membranes. Purification and partial characterization

Purification of an A1 adenosine receptor of rat testes was performed using a newly developed affinity chromatography system (Nakata, H. (1989) Mol. Pharmacol. 35, 780-786). The A1 adenosine receptor was solubilized with digitonin from rat testicular membranes and then purified more than 25,000-fold by sequential use of affinity chromatography on xanthine amine congener-immobilized agarose, hydroxylapatite chromatography, re-affinity chromatography on xanthine amine congener-agarose, and finally gel permeation chromatography on TSK-3000SW. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final preparation showed a single broad band of Mr 41,000 by autoradiography after radioiodination. This Mr 41,000 peptide was also specifically labeled with an A1 adenosine receptor affinity labeling reagent. A high affinity A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-[3H]dipropylxanthine, bound saturably to the purified receptor with a KD of approximately 1.4 nM. The purified receptor also showed essentially the same specificity for adenosine agonists and antagonists as the unpurified receptor preparations, although the affinities of the purified adenosine receptor for agonists were significantly low compared to those of unpurified receptor preparations indicating that the purified A1 adenosine receptor exists as a low agonist-high antagonist affinity state. Deglycosylation of the purified testis adenosine A1 receptors with endoglycosidase F produced an increase in the mobility of the receptor protein to an apparent Mr 30,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, similar to that of deglycosylated A1 adenosine receptors of rat brain membranes. Peptide maps of the purified testis and brain A1 adenosine receptors using trypsin and V8 protease suggest that these receptors show some structural homologies.     

Utilization of antipeptide antibodies as affinity ligands in immunoaffinity purification

Anti-peptide antibodies against the C-terminal regions of chimeric α-amylase, recombinant CD2 and insulin B-chain were obtained by using peptides corresponding to the C-terminal regions as immunogens. These anti-peptide antibodies adsorbed the native proteins, as well as the antigen peptides. The proteins were purified to high purity using the anti-peptide antibodies as affinity ligands. These ligands could discriminate the target proteins having different C-terminal regions. The adsorbed proteins were specifically eluted by the eluents containing the antigen peptides.     

Antibodies against p-aminophenyl-beta-D-galactopyranoside-containing proteins

The antibodies were prepared from antisera of rabbits immunized with bovine serum albumin containing covalently bound p-aminophenyl-beta-D-galactopyranoside (APG) and purified by affinity chromatography on APG-containing ovalbumin immobilized by BrCN-activated Sepharose 4B. The antibodies possessed a selective specificity for APG and interacted with different APG-containing proteins, including APG-containing lysosomal alpha-glucosidase. The purified antibodies are immunoglobulins of G type as was determined from the molecular weights of native and dissociated antibodies and from the immunochemical assays with antibodies against rabbit IgG.     

Monoclonal antibodies to adenosine receptor by an auto-anti-idiotypic approach

Monoclonal antiadenosine receptor antibodies have been raised by an auto-anti-idiotypic approach. BALB/c mice were immunized with adenosine 6-aminocaproyl-bovine serum albumin. Hybridoma cell lines were raised and lines that secreted antibodies that bound to rabbit antiadenosine antibodies were obtained. Two such monoclonal antibodies, AA18 and AA21, were studied in detail and found to be directed at adenosine receptors by the following criteria. They inhibited the binding of [3H] adenosine to rabbit antiadenosine antibodies that had binding characteristics similar to those of adenosine receptors. They bound to rat brain membranes and binding could be inhibited by N6-cyclohexyladenosine and L-N6-phenylisopropyladenosine, both adenosine receptor agonists. They also inhibited the binding of [3H]L-N6-phenylisopropyladenosine to rat brain membranes. In functional assays, they inhibited adenylate cyclase of rat brain membranes, but had no effect on adenylate cyclase of rat hepatic membranes, indicating that they mimic agonists of the A1 receptor, therefore, carrying an "internal image" of the adenosine molecule. When adenosine receptors of rat brain membranes were solubilized with 1% cholic acid, partially purified on an adenosine 6-aminocaproyl AH-Sepharose column and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, both AA18 and AA21 recognized a 62,000 band under nonreducing conditions, and a major band of 36,000 under reducing conditions. We conclude that the auto-anti-idiotypic route has yielded specific antibodies that recognize the A1 adenosine receptor.     

Purification and specificity of antibodies to adenosine.

Antibodies to adenosine were elicited in rabbits by immunization with bovine serum albumin-adenosine conjugate. The antibodies were purified and fractionated on two affinity columns (Sepharose-oligo(A) and Sepharose-AMP). Two families of antibodies have been obtained. The antibodies purified on the Sepharose-oligo(A) column react with poly(A) while those purified on the Sepharose-AMP column do not, as shown by gel diffusion. The association constants for the binding of Fab fragments or IgG purified on the Sepharose-oligo(A) column and several haptens were deduced from dialysis equilibrium, fluorescence quenching and displacement of AMP-fluorescein conjugate. The antibodies mainly recognize adenine, and the ribose or the phosphate group of (or AMP derivatives) do not play a critical role in the interaction. Thermodynamic parameters for adenosine-Fab fragments complexes have been determined deltaH degrees = 16 kcal/mole and deltaS degrees = - 15 cal. degree-1 mole-1. Circular dichroism studies indicate that about three nucleotide residues penetrate the binding site of Fab fragments.     

N-6-(delta-2-isopentenyl) adenosine: hydrolysis by a mucleosidase isolated from Lactobacillus acidophilus cells.

A nucleosidase activity has been isolated from Lactobacillus acidophilus which rapidly hydrolyses N-6 (delta-2-isopentenyl) adenosine to its corresponding base, N-6(delta-2-isopentenyl) adenine. The activity can be distinguished from the spleen exzyme (EC. 2.4.2.1), a purine nucleoside transferase, on the basis of its substrate specificity, electrophoretic behavior, and nondependence on phosphate. The bacterial enzyme hydrolyzes both inosine and isopentenyl adenosine, giving Km values of 63.3muM and 177 muM respectively. The presence of this enzyme in bacteria counts for the rapid conversion of the parent nucleoside to isopentenyl adenine, which has been observed in these cells. The enzyme thus assumes importance as one of the catabolic activities available to the cell for metabolizing the cytokinin, N-6-(delta-2-isopentenyl) adenosine.     

Purification and partial sequence of proteins involved in the cholic acid transport into rat liver hepatocytes

Two proteins, in previous work labeled by affinity markers derived from taurocholic acid, were purified and partially sequenced. Antibodies were raised against purified proteins, and cross-reactions were carefully checked. The influence of these antibodies upon taurocholic acid import into vesicles from rat liver plasma membranes was measured, and showed a distinct inhibition of transport in the case of the 54 kD protein.     

Prenyltransferase from Gossypium hirsutum

A protein fraction has been purified from Gossypium hirsutum var. Coker 413 which synthesized all four geometrical isomers of farnesyl pyrophosphate from isopentenyl pyrophosphate alone, from isopentenyl pyrophosphate and geranyl or neryl pyrophosphate. Electrophoretic analysis showed that this protein fraction consisted of three proteins. One of these proteins contained isopentenyl pyrophosphate /ag dimethylallyl pyrophosphate isomerase activity. The other two proteins were insufficiently pure to characterize. Estimation of molecular weights by electrophoresis of the three proteins revealed values in the order of 3 × 10⁴ to 1.3 × 10⁵. However the same protein fraction eluted as one peak from Sepharose 6B molecular sieve columns, indicative of a larger protein component as could be accounted for by the electrophoretic molecular weight estimation. From these results and from the different products synthesized it is proposed that isopentenyl pyrophosphate /ag dimethylallyl pyrophosphate isomerase and prenyltransferase (farnesyl pyrophosphate synthetase) exists as a multiprotein complex in G. hirsutum.     

Intracellular adenosine 3',5'-phosphate binding proteins in Dictyostelium discoideum: partial purification and characterization in aggregation competent cells

Three adenosine 3',5'-phosphate (cAMP) binding proteins were separated and partially purified from cytoplasmic extracts of Dictyostelium discoideum cells developed to aggregation competence. Two species, A and B, representing respectively 50% and 20% of the total activity, bind cAMP with very rapid kinetics and high specificity. Species A (Kd = 7.5 nM) is a monomeric protein of 36 000 daltons with a sedimentation coefficient of 2.3 S. Species B, which binds cAMP with positive cooperativity, also displays a high affinity for the ligand (Kd = 3.2 nM). This protein is present in the extracts as an equilibrium between monomeric, dimeric, and tetrameric forms with respective sedimentation coefficients of 2.4, 4.5, and 6.5 S; binding of cAMP to the monomer induces the appearance of the multimeric forms. A third cAMP binding protein (species C, Kd - 9.5 nM) was characterized as a larger protein (Mr 190 000, sedimentation coefficient of 9.2 S) which also binds adenosine and adenosyl derivatives. Species C represents 30% of the activity in the extracts and resemble the "adenosine analogue binding proteins" described in mammalian cells. The relevance of the properties of these proteins to the developmental process of D. discoideum amoebas is discussed.