H-colchicine binding : Failure to detect any binding to soluble proteins from various lower organisms

No ³H-colchicine binding could be detected to post-ribosomal supernatants from Schizosaccharomyces pombe, Chlamydomonas reinhardii, Tetrahymena pyriformis, pea root tips, or Zea mays coleoptiles. However, under the same conditions, ³H-colchicine bound to supernatants from mouse brain and unfertilised sea urchin eggs. A protein with similar characteristics to sea urchin and mouse brain tubulins in terms of its molecular weight and elution properties from DEAE-cellulose columns has been detected in supernatants from the yeast S. pombe. Similar results (unpublished) have been obtained for both Zea mays and Tetrahymena. This protein is tentatively identified as tubulin. The lack of binding of ³H-colchicine by supernatants from lower organisms is consistent with the variable sensitivity of nuclear division to colchicine, and with the possible evolution of the colchicine-binding site of tubulin.     

Properties of purified colchicine-binding protein from a cultured carrot cell extract

Colchicine-binding protein (CBP) was purified from a cultured carrot cell extract by DEAE-Sephacel, phosphocellulose and Sephadex G200 column chromatographies. The purified CBP separated into three bands on SDS-polyacrylamide gel electrophoresis. One of them reacted with a monoclonal antibody against chick brain alpha-tubulin and the other two with that against beta-tubulin. Colchicine-binding activity of the purified protein was enhanced by tartrate and inhibited little by an excess of podophyllotoxin. It decayed following first order kinetics, but was more stable than the CBP in the crude extract. The binding constant of the purified CBP for colchicine was 0.57 microM-1 and the number of binding sites of colchicine per mg protein was about 2 nmol. This binding constant is about ten times lower than that of porcine brain tubulin under identical conditions.     

Characterization of colchicine-binding activity in Dictyostelium discoideum.

A colchicine-binding component was detected in vegetative amoebae of Dictyostelium discoideum by using a Millipore-filter assay. The colchicine-binding activity is temperature-and time-dependent, maximum binding occurring at 22-35 degrees C after 60 min incubation. Further increases in temperature are without effect on the extent of binding, but bound colchicine is released with increased time of incubation. Furthermore, colchicine-binding activity itself decreased in the high-speed supernatant from D. discoideum, with half the activity being lost in approx. 2.5h. Several lines of evidence, including the saturation kinetics of colchicine binding, enhancement of colchicine binding by tartrate, insensitivity to lumicolchicine, precipitation of the binding protein by vinblastine and behaviour of the binding protein on DEAE-cellulose and Sephadex resins, suggest that the colchicine-binding protein may be tubulin.     

Characterization of 3H-AVP binding sites in particulate preparations of rat brain

Specific binding sites for vasopressin (AVP) were located in subcellular particulate fractions of rat brain with tritiated vasopressin of high specific activity, 22.5 Ci/mmol. Rat brain tissue was dissected, placed in cold 0.32 M sucrose containing proteolytic inhibitors, homogenized and fractionated into a crude nuclear fraction (1K pellet), crude mitochondrial fractions (12K pellet), and plasma membranes and microsomes (100K pellet). Specific binding of vasopressin was found in the 12K and 100K pellets in the presence of a divalent metal ion with Ni greater than Co greater than Mg greater than Mn greater than no metal ion at pH 7.4 in 50 mM Tris-Maleate buffer. Maximum specific binding of 16 nM AVP was located in the 100K anterior cortex fraction which bound 350 fmoles/mg protein; striatum, midbrain/thalamus, cerebellum, and medulla oblongata and pons bound specifically about 200 fmoles/mg protein and frontal poles and parietal cortex about 100 fmoles/mg protein in the 100K pellet. In all of the brain regions studied, except hippocampus and septum, the 100K pellet bound specifically 2 to 4 times more 3H-AVP than the 12K pellet. In the hippocampus with 16 nM AVP, the 12K pellet bound specifically 150 fmoles/mg protein; the septum, 75 fmoles/mg protein. Little or no binding to the 100K pellet was present in these regions. Bound AVP could be dissociated rapidly from the membranes by the addition of EDTA. The 12K hippocampal pellet was further fractionated into myelin, mitochondria, and synaptosomes; purification was confirmed by marker enzyme assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

A histidine binding protein ofEscherichia coli: a component of cystine binding protein ofEscherichia coli

Summary Commercially obtained cystine binding protein (CBP), an osmotic shock protein ofEscherichia coli, was studied in an effort to determine its binding characteristics. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) analysis of commercially obtained CBP showed three protein bands. N-terminal amino acid microsequencing and subsequent computer search revealed that the sequence of one of these proteins (25-kDa) was nearly identical to histidine binding protein (HisJ) ofSalmonella typhimurium. Purification of CBP by HPLC yielded four protein peaks, of which one bound histidine exclusively. Binding was maximal at pH 5.0 to 6.0, at 4°C, did not require calcium or magnesium ions and was not inhibited by reduction of CBP disulfide bonds. Amino acids other than histidine or cystine did not bind to CBP. These data show that commercially available CBP is not a homogenous protein; it contains a histidine as well as a cystine binding component.     

CBP60g and SARD1 play partially redundant critical roles in salicylic acid signaling

Arabidopsis thaliana calmodulin binding protein 60g (CBP60g) contributes to production of salicylic acid (SA) in response to recognition of microbe‐associated molecular patterns (MAMPs) such as flg22, a fragment of bacterial flagellin. Calmodulin binding is required for the function of CBP60g in limiting growth of the bacterial pathogen Pseudomonas syringae pv. maculicola (Pma) ES4326 and activation of SA synthesis. Here, we describe a closely related protein, SARD1. Unlike CBP60g, SARD1 does not bind calmodulin. Growth of Pma ES4326 is enhanced in sard1 mutants. In cbp60g sard1 double mutants, growth of Pma ES4326 is greatly enhanced, and SA levels and expression of PR‐1 and SID2 are dramatically reduced. Expression profiling placed the CBP60g/SARD1 node between the PAD4/EDS1 and SA nodes in the defense signaling network, and indicated that CBP60g and SARD1 affect defense responses in addition to SA production. A DNA motif bound by CBP60g and SARD1, GAAATTT, was significantly over‐represented in promoters of CBP60g/SARD1‐dependent genes, suggesting that expression of these genes is modulated by CBP60g/SARD1 binding. Gene expression patterns showed a stronger effect of cbp60g mutations soon after activation of a defense response, and a stronger effect of sard1 mutations at later times. The results are consistent with a model in which CBP60g and SARD1 comprise a partially redundant protein pair that is required for activation of SA production as well as other defense responses, with CBP60g playing a more important role early during the defense response, and SARD1 to playing a more important role later.     

Specific affinity labelling of tubulin with bromocolchicine

Bromocolchicine, synthesized by substituting tho N-acetyl moiety of colchicine with a reactive bromoacetyl group, was found to be an affinity label for tubulin. Binding of [³H]colchicine to tubulin was competitively and irreversibly inhibited by bromocolchicine with a K_i value of 2.3 × 10^?5m. The affinity label could not be displaced by precipitating the protein with trichloroacetic acid and is thus covalently bound. Autoradiographs of brain high-speed supernatant proteins after their electrophoretic separation on sodium dodecyl sulphate/polyacrylamide gels showed that [³H]bromocolchicine reacted with four proteins, of which tubulin was one.Labelling of two of these proteins could be prevented by pretreatment of the brain extracts with α-bromoacetic acid, after which 70% of the covalently bound label was specifically located in the tubulin band. Up to 1.6 mol of affinity label could be bound per mol of tubulin, while under our experimental conditions 1 mol of protein bound irreversibly only 0.2 mol of [³H]colchicine. Autoradiography of sodium dodecyl sulphate/urea-polyacrylamide gels, which separate the subunits of tubulin, showed about 30% [³H] bromocolchicine bound to the α-subunit of tubulin and 70% to tho β-subunit.The irreversible binding site of colchicine was localized to the α-subunit, as labelling of only this subunit was inhibited by colchicine at high affinity label concentrations. At lower concentrations, colchicine inhibited the labelling of both subunits.Bromoacetic acid did not inhibit the reaction of the affinity label with the tubulin subunits, but increased the inhibition of [³H]bromocolchicine binding at lower concentrations of the affinity label in brain extracts preincubated with cold colchicine. This is interpreted to show a conformational change which takes place in the two subunits of tubulin upon binding of colchicine and results in the exposure of some of the binding sites of [³H]bromocolchicine to bromoacetic acid.     

Assay of DNA-RNA hybrids by S1 nuclease digestion and adsorption to DEAE-cellulose filters.

A fast and accurate assay procedure for DNA-RNA hybrids is described in which exhaustive digestion of unhybridized DNA with S1 nuclease is followed by binding of hybrids to filter discs of DEAE-cellulose. The digested DNA can be efficiently washed from the filters so that background levels of 0.1-0.2% of input tracer DNA can be achieved, in contrast to the much higher (approximately 1-5%) backgrounds obtained using TCA precipitation procedures. Short duplexes, as small as 36 nucleotides in length, which are inefficiently bound to hydroxyapatite, are quantitatively bound to the DEAE-cellulose filters.     

Membrane-bound tubulin in brain and thyroid tissue.

Brain and thyroid tissue contain membrane-bound colchicine-binding activity that is not due to contamination by loosely bound cytoplasmic tubulin. This activity can be solubilized to the extent of 80 to 90% by treatment with 0.2% Nonidet P-40 with retention of colchicine binding. Extracts so obtained contain a prominent protein band in disc gel electrophoresis that co-migrates with tubulin. Membranes, and the solubilized protein therefrom, exhibit ligand binding properties like tubulin; for colchicine the KA is approximately 1 X 10(6) M-1 in brain and approximately 0.6 X 10(6) M-1 in thyroid; for vinblastine the KA is approximately 8 X 10(6) M-1 for both tissues; and for podophyllotoxin the Ki is approximately 2 X 10(-6) M for both tissues. Displacement by analogues of colchicine is of the same order as for soluble tubulin. Although membrane-bound colchicine-binding activity shows greater thermal stability and a higher optimum binding temperature (54 degrees versus 37 degrees) than soluble tubulin, this appears to be the result of the membrane environment since the solubilized binding activity behaves like the soluble tubulin. Antibody against soluble brain tubulin reacts with membranes and solubulized colchicine-binding activity from both brain and thyroid gland. We conclude that brain and thyroid membrane preparations contain firmly bound tubulin or a very similar protein.     

The chemokine‐binding protein encoded by the poxvirus orf virus inhibits recruitment of dendritic cells to sites of skin inflammation and migration to peripheral lymph nodes

Orf virus (ORFV) is a zoonotic parapoxvirus that induces acute pustular skin lesions in sheep and humans. ORFV can reinfect its host and the discovery of several secreted immune modulatory factors that include a chemokine‐binding protein (CBP) may explain this phenomenon. Dendritic cells (DC) are professional antigen presenting cells that induce adaptive immunity and their recruitment to sites of infection in skin and migration to peripheral lymph nodes is critically dependent on inflammatory and constitutive chemokine gradients respectively. Here we examined whether ORFV‐CBP could disable these gradients using mouse models. Previously we established that ORFV‐CBP bound murine inflammatory chemokines with high affinity and here we show that this binding spectrum extends to constitutive chemokines CCL19 and CCL21. Using cell‐based chemotaxis assays, ORFV‐CBP inhibited the movement of both immature and mature DC in response to these inflammatory and constitutive chemokines respectively. Moreover in C57BL/6 mice, intradermally injected CBP potently inhibited the recruitment of blood‐derived DC to lipopolysaccharide‐induced sites of skin inflammation and inhibited the migration of ex vivo CpG‐activated DC to inguinal lymph nodes. Finally we showed that ORFV‐CBP completely inhibited T responsiveness in the inguinal lymph nodes using intradermally injected DC pulsed with ovalbumin peptide and transfused transgenic T cells.     

Podophyllotoxin as a probe for the colchicine binding site of tubulin.

The binding of [3H]podophyllotoxin to tubulin, measured by a DEAE-cellulose filter paper method, occurs with an affinity constant of 1.8 X 10(6) M-1 (37 degrees at pH 6.7). Like colchicine, approximately 0.8 mol of podophyllotixin are bound per mol of tubulin dimer, and the reaction is entropy-driven (43 cal deg-1 mol-1). At 37 degrees the association rate constant for podophyllotoxin binding is 3.8 X 10(6) M-1 h-1, approximtaely 10 times higher than for colchicine; this is reflected in the activation energies for binding which are 14.7 kcal/mol for podophyllotoxin and 20.3 kcal/mol for colchicine. The dissociation rate constant for the tubulin-podophyllotoxin complex is 1.9 h-1, and the affinity constant calculated from the ratio of the rates is close to that obtained by equilibrium measurements. Podophyllotxin and colchicine are mutually competitive inhibitors. This can be ascribed to the fact that both compounds have a trimethoxyphenyl ring and analogues of either compound with bulky substituents in their trimethoxyphenyl moiety are unable to inhibit the the binding of either of the two ligands. Tropolone, which inhibits colchicine binding competitively, has no effect on the podophyllotoxin/tubulin reaction. Conversely, podophyllotoxin does not influence tropolone binding. Moreover, the tropolone binding site of tubulin does not show the temperature and pH lability of the colchicine and podophyllotoxin domains, hence this lability can be ascribed to the trimethoxyphenyl binding region of tubulin. Since podophyllotoxin analogues with a modified B ring do not bind, it is concluded that both podophyllotoxin and colchicine each have at least two points of attachment to tubulin and that they share one of them, the binding region of the trimethoxyphenyl moiety.     

High-affinity microtubule protein-higher organism DNA complexes. Many-fold enrichment in repetitive mouse DNA sequences comprised of satellite DNAs

We have examined aspects of the interaction of cycled microtubule protein preparations with ³⁵S-labeled mouse DNA tracer in a competition system with unlabelled competitor E. coli or mouse DNA. The nitrocellulose filter binding assay was used to measure interaction by scintillation counting. DNA molecular weight affected the levels of filter retained ³⁵S-labelled mouse tracer DNA. Filter retention levels increased if ³⁵S-labelled mouse DNA tracer size was increased, and the filter binding level decreased if competitor DNA size was increased. There was a sizeable, reproducible difference in the ³⁵S-labelled mouse DNA tracer binding level of about 1% when E. coli or mouse DNA competitors were compared. Mouse DNA more effectively competed with ³⁵S-labelled mouse DNA for microtubule protein binding than did E. coli DNA, suggesting that a small class of higher-organism DNA sequences interacts very strongly with microtubule protein. From other studies we know this to be the MAP fraction (Marx, K.A. and Denial, T. (1984) in The Molecular Basis of Cancer (Rein, R., ed.), Alan R. Liss, New York, in the press; and Villasante, E., Corces, V.G., Manso-Martinez, R. and Avila, J. (1981) Nucleic Acids Res. 9, 895–908). We find that this difference in competitor DNA strength is qualitatively similar under high-stringency conditions (0.5 M NaCl, high competitor [DNA]) we developed for examining high-affinity complexes. Under high-stringency conditions we isolated 1.2% and 0.6% of ³⁵S-labelled mouse DNA at 4200 and 350 bp respective sizes as nitrocellulose filter bound DNA-protein complexes. At both molecular weights these high-affinity DNA sequences, isolated from the filters, were shown to be significantly enriched in repetitive DNA sequences by S₁ nuclease solution reassociation kinetics. The kinetics are consistent with about a 4-fold mouse satellite DNA enrichment as well as enrichment in other repetitious DNA sequence classes. The high molecular weight filter-bound DNA samples were sedimented to equilibrium in CsCl buoyant density gradients and found to contain primarily mouse satellite DNA density sequences (1.691 g/cm³) with some minor fractions at other density positions (1.670, 1.682, 1.705, 1.740, 1.760 g/cm³) similar to those observed by our laboratory in previous investigations of micrococcal nuclease-resistant chromatin (Marx, K.A. (1977) Biochem. Biophys. Res. Commun. 78, 777–784). That the high-affinity microtubule-bound DNA was some 3–5-fold enriched in mouse satellite sequences was demonstrated by its characteristic BstNI restriction enzyme cleavage pattern     

Interaction of 1-propargyl-5-chloropyrimidin-2-one (a metahalone) with rat brain tubulin

The mitotic inhibitor 1-propargyl-5-chloropyrimidin-2-one (a metahalone) was found to bind to DEAE-cellulose purified rat brain tubulin. A decrease in the fluorescence of 1-propargyl-5-chloropyrimidin-2-one was seen when the drug was incubated in the presence of increasing tubulin concentrations. The decrease in metahalone fluorescence was not affected by the addition of GTP, indicating drug interaction at other portions of the tubulin molecule than the nucleotide binding sites. Scatchard plot analysis following incubation of tubulin with 1-propargyl-5-chloro-[2-14C]pyrimidin-2-one revealed that 1 mol of metahalone bound to 1 mol of tubulin dimer with a measured association constant of 8.0 X 10(3) M-1. Double reciprocal plots of vincristine and colchicine binding to tubulin in the presence of 1-propargyl-5-chloropyrimidin-2-one showed that the metahalone competitively inhibited colchicine binding to tubulin but had no influence on vincristine binding. This conclusion was supported by gel filtration chromatography where an increase in unbound colchicine was measured when 1-propargyl-5-chloropyrimidin-2-one was present in an incubation mixture containing colchicine and tubulin. In the presence of 5 mM 1-propargyl-5-chloropyrimidin-2-one, tubulin self-aggregated into crystalline structures. The binding of 1-propargyl-5-chloropyrimidin-2-one to tubulin at or near the colchicine binding site may be responsible for the metaphase arresting characteristics of this drug.     

CBP1 Associates with the Dictyostelium Cytoskeleton and Is Important for Normal Cell Aggregation under Certain Developmental Conditions

In cells of the eukaryotic microorganism Dictyostelium discoideum, at least eight small, four-EF-hand Ca²⁺-binding proteins of unknown function are expressed at specific times during development. One of these proteins, calcium-binding protein 1 (CBP1), first appears just prior to cell aggregation and then is present at relatively constant levels throughout development. To determine a role for CBP1 during development, the protein was used as bait in a yeast two-hybrid screen to reveal putative CBP1-interacting proteins. Two proteins identified in this screen were the actin-binding proteins, protovillin and EF-1α. Using an in vitro binding assay, both of these proteins were found to interact with CBP1 in the absence of Ca²⁺, but the interaction of CBP1 with EF-1α was increased substantially by Ca²⁺. CBP1 was also shown by fluorescence microscopy and by binding assays to associate with the actin cytoskeleton of Dictyostelium cells during development, and these interactions were partially Ca²⁺-dependent. cbpA-null cells grew normally, but under certain developmental conditions, cell aggregation was prolonged and irregular. This defect in aggregation appeared to be related to a general reduction in cell motility rather than to a decrease in the ability of the cells to respond to the chemoattractant cAMP. Together, these results suggest that CBP1 might function to help regulate the reorganization of the Dictyostelium actin cytoskeleton during cell aggregation.     

Cytokinin-binding proteins from tobacco callus share homology with osmotin-like protein and an endochitinase

To study the signal transduction of cytokinins, we characterized cytokinin-binding proteins (CBPs) isolated from tobacco callus Nicotiana tabacum. Two high-affinity CBPs, CBP1 and CBP2, were isolated from the soluble fraction of tobacco callus BY-2 cells by anion exchange chromatography on a DEAE-cellulose column and affinity chromatography on a benzyladenine (BA)-linked Sepharose 4B column. Cytokinin-binding activity was determined by the equilibrium dialysis method. The degree of purification of CBP1 and CBP2 was 270 and 600-fold, respectively. These proteins had molecular masses of 34 kDa and 26 kDa, and to bind benzyladenine (BA) with dissociation constants (Kd) of 8.9 x 10(-6) M and 1.1 x 10(-6) M, respectively. Binding of BA to CBP2 was inhibited by zeatin and kinetin but not by adenine, adenosine, ATP or IAA. The optimum pH for binding of BA to CBP1 and CBP2 was approximately pH 6.5 and 7.5, respectively. CBP1 showed significant homology (90%) with endochitinase and CBP2 with osmotin-like protein (OLP). These findings and the results of immunoblotting analysis and cytokinin-binding assay of recombinant OLP indicated that CBP2 is OLP, a stress protein.     

Purification and characterization of chitin-binding proteins from the hemolymph of sweet potato hornworm, Agrius convolvuli

Three chitin-binding proteins (CBPs: CBP9, CBP15, CBP66) were identified from the larval hemolymph of sweet potato hornworm, Agrius convolvuli.Two (CBP9 and CBP15) of them have been isolated and purified by gel filtration (Superdex HR 75), cation-exchange chromatography (Mono S), and reverse-phase chromatography (μRPC PC 2.1/3). In experiments to detect CBPs in hemolymph, we examined whether ionic strength and existence of bovine serum albumin in the incubation solution influenced binding affinity of CBPs to chitin. The N-terminal sequences of three CBPs were determined by the automated Edman degradation and showed the sequence homology in basic local alignment search tool search. CBP15 and CBP66 were quite similar to lysozymes and bovine serum albumins, respectively. In contrast, CBP9 is not similar to any other known protein, as judged from databank comparisons. Therefore, we concluded that CBP9 is a novel protein with binding capacity to chitin that is a component of the fungal cell wall. CBP9 has no antibacterial activity against Escherichia coli and Micrococcus luteus, and also showed negative response in hemagglutination assay. CBP9 is confirmed as a monomer with a molecular mass of 9.14 kDa by electron spray ionization and matrix-assisted laser desorption ionization mass spectrometry.     

Colchicine-binding activity in particulate fractions of mouse brain

Both particulate and soluble fractions of brain homogenates bound [³H]colchicine. Approximately one-half of the total colchicine-binding activity in mouse brain was found in the particulate fraction. Of the particulate fractions, the microsomal and nerveending subfractions which sediment at the 1·0–1·2 m interface on sucrose gradients were richest in colchicine-binding activity. Intact microtubules were not found in these fractions, but colchicine-binding activity of these fractions may be related to the presence of microtubular protein.     

Effects of vinblastine, oncodazole, procarbazine, chlorambucil, and bleomycin in vivo on colchicine binding activity of tubulin.

Five chemotherapeutic agents which inhibit mitosis caused an $\text{in vivo}$ effect on the colchicine bound to the isolated tubulin from mouse lymphoma L5178Y cells. These effects were examined over a time course of 4, 12, 24, 48, and 72 hours after a single administration of each drug. Vinblastine, oncodazole, and bleomycin decreased the amount of colchicine bound per mg protein; procarbazine and chloroambucil increased the amount bound. All of the drugs except procarbazine required more than 4 hours to cause an effect on colchicine binding and in the case of bleomycin and oncodazole some recovery occurred after 48 hours. The mitotic index was affected by 4 hours by all drugs: procarbazine, chlorambucil and bleomycin caused a decrease; vinblastine and oncodazole, an increase.     

Glutamine synthetase cascade: enrichment of uridylyltransferase in Escherichia coli carrying hybrid ColE1 plasmids

Colchicine-binding properties of the total cytoplasmic pool of tubulin from rat liver were evaluated in tubulin-stabilizing (TS) supernates. Microtubules were separated from free tubulin using a microtubule-stabilizing solution (MTS) and ultracentrifugation. [³H]Colchicine-binding properties of microtubule-derived tubulin were investigated in supernates prepared after resuspension of MTS pellets in TS. In TS buffer at 37 °C the colchicine-binding activity of the total cytoplasmic pool of tubulin decayed with $\text{T}\text{1}\text{2}$ of 3.39 h. Resuspended pellet tubulin decayed much more rapidly under the same conditions with a $\text{T}\text{1}\text{2}$ of 0.72 h. This rapid time decay of microtubule-derived tubulin was found to be at least partially attributable to prior microtubule-stabilizing solution exposure. Since tartrate has been reported to increase the rate of colchicine binding to tubulin, sodium tartrate (150 mm) was added to our colchicine-binding system. This addition increased the detectable [³H]colchicine binding by 10% in the total cytoplasmic preparation and by 85% in the resuspended pellet preparation. Addition of tartrate (150 mm) also resulted in a 105% increase in the $\text{T}\text{1}\text{2}$ for total cytoplasmic tubulin and a 412% increase for microtubule derived tubulin. Total cytoplasmic supernates of liver bound [³H]colchicine linearly over a wide range of tissue concentrations. However, resuspended microtubule-stabilizing solution pellet supernates in tubulin-stabilizing solution showed some increase in colchicine binding per tissue weight in the more dilute samples. Our data which demonstrate differences in colchicine-binding properties for total cytoplasmic and microtubule-derived pools of tubulin suggest that present assays for hepatic tubulin polymerization which assume identical binding properties should be interpreted with caution.