Use of biotin-labeled nucleic acids for protein purification and agarose-based chemiluminescent electromobility shift assays

We have employed biotin-labeled RNA to serve two functions. In one, the biotin tethers the RNA to streptavidin-agarose beads, creating an affinity resin for protein purification. In the other, the biotin functions as a label for use in a modified chemiluminescent electromobility shift assay (EMSA), a technique used to detect the formation of protein-RNA complexes. The EMSA that we describe avoids the use not only of radioactivity but also of neurotoxic acrylamide by using agarose as the gel matrix in which the free nucleic acid is separated from protein-nucleic acid complexes. After separation of free from complexed RNA in agarose, the RNA is electroblotted to positively charged nylon. The biotin-labeled RNA is readily bound by a streptavidin-alkaline phosphatase conjugate, allowing for very sensitive chemiluminescent detection ( approximately 0.1-1.0 fmol limit). Using our system, we were able to purify both known iron-responsive proteins (IRPs) from rat liver and assess their binding affinity to RNA containing the iron-responsive element (IRE) using the same batch of biotinylated RNA. We show data indicating that agarose is especially useful for cases when large complexes are formed, although smaller complexes are even better resolved.     

Assay development and data analysis of receptor-ligand binding based on scintillation proximity assay

In this paper, we described the optimization of a generic binding assay to measure ligand-receptor interactions for peroxisome proliferator-activated receptors (PPARs). The assay is based on scintillation proximity assay, in which a protein is coated on scintillant-incorporated beads, and a radiolabeled ligand stimulates the beads to emit a signal by binding to the immobilized protein. An intrinsic binding affinity of unlabeled ligands is determined by competitive displacement of the radioligand. The protein coating and ligand binding are achieved in one step by simply mixing ligands, protein and beads in sequence. No additional steps of pre-coating and washing of beads are required. Protein is captured on beads effectively by electrostatic interactions, thus no affinity labeling of protein is required. In data analysis, ligands are grouped into two classes based on their binding affinities. For tight binding ligands, an equation is derived to accurately determine the binding affinity. Otherwise a general equation applies. This quantitative and high throughput assay provides a tool to screen a large library of molecules in search of potent ligands.     

Determination of the affinity of vitamin D metabolites to serum vitamin D binding protein using assay employing lipid-coated polystyrene beads.

We have developed an assay to measure the affinity of serum vitamin D binding protein for 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, and vitamin D3, using uniform diameter (6.4 microns) polystyrene beads coated with phosphatidylcholine and vitamin D metabolites as the vitamin D donor. The lipid metabolite coated beads have a solid core, and thus all of the vitamin D metabolites are on the bead surface from which transfer to protein occurs. After incubating these beads in neutral buffer for 3 h, essentially no 3H-labeled vitamin D metabolites desorb from this surface. Phosphatidylcholine/vitamin D metabolite-coated beads (1 microM vitamin D metabolite) were incubated with varying concentrations of serum vitamin D binding protein under conditions in which the bead surfaces were saturated with protein, but most of the protein was free in solution. After incubation, beads were rapidly centrifuged without disturbing the equilibrium of binding and vitamin D metabolite bound to sDBP in solution was assayed in the supernatant. All three vitamin D metabolites became bound to serum vitamin D binding protein, and after 10 min of incubation the transfer of the metabolites to serum vitamin D binding protein was time independent. The transfer followed a Langmuir isotherm, and the Kd for each metabolite binding to serum vitamin D binding protein was derived by nonlinear least-squares fit analysis. From this analysis the following values for the Kd were obtained: 5.59 x 10(-6) M, 25-hydroxyvitamin D; 9.45 x 10(-6) M, 1,25-dihydroxyvitamin D; and 9.17 x 10(-5) M, vitamin D. In conclusion, we have developed a method which avoids problems encountered in previous assays and allows the precise and convenient determination of binding affinities of vitamin D metabolites and serum vitamin D binding protein.     

Ca2+ bindings of pig cardiac myosin, subfragment-1, and g2 light chain.

Ca2+ binding to pig cardiac myosin, subfragment-1 (S-1), and g2 light chain were investigated by the equilibrium dialysis method. Two different S-1s, one of which can bind Ca2+ and another which cannot, were prepared. In order to calculate the free Ca2+ concentrations adequately, the amounts of Ca2+ included in various chemicals and proteins were measured by atomic absorption spectroscopy. Ca2+ contamination was greatest in KCl among the chemicals tested. In addition, the Ca2+ strongly bound to myosin and S-1 was released in the presence of Mg2+. When Mg2+ was not added, the Ca2+-binding constant of myosin was 4 x 10(5) M-1 and the maximum binding number was 1.8 mol per mol of myosin. Cooperativity between the 2 Ca2+ bindings could not be demonstrated. Mg2+ strongly inhibited the Ca2+ binding: at a free Ca2+ concentration of 1 x 10(-5) M, 1.3 mol Ca2+ was bound to myosin in the absence of Mg2+, but 0.6 and 0.2 mol were bound in the presence of 0.3 and 4.5 mM Mg2+, respectively. The Ca2+-binding constant of S-1, which contained a 15,000 dalton component, was 8.6 x 10(5) M-1, and the maximum binding number was 0.7 mol per mol of S-1. The 15,000 dalton component could be exchanged with extraneous g2. S-1 which lacked the 15,000 component could not bind Ca2+ at free Ca2+ concentrations less than 0.1 mM. The Ca2+ binding to free g2 light chain was about 100 times weaker than the binding to myosin, as indicated previously for skeletal myosin (Okamoto, Y. & Yagi, K. (1976) J. Biochem. 80, 111--120). The Ca2+-binding constant was obtained as 4.1 x 10(3) M-1 in the absence of added Mg2+. Phosphorylation of g2 light chain did not affect the Ca2+ binding to the free g2 light chain or to myosin. Ca2+ binding to cardiac native tropomyosin was also measured.     

Filter assay method for the estrogen receptor protein: Detection of both filled and unfilled estrogen binding sites

New filter assay methods are presented for quantitating both cytoplasmic and nuclear forms of the estrogen receptor protein. These methods exploit the strong adsorption of this protein to glass-fiber filters, which appears to occur without loss of steroid binding affinity. A “direct assay protocol” is described that detects only unfilled (nonliganded) estrogen binding sites. In addition, a convenient “exchange assay protocol” has been developed that detects, in addition, those receptors present whose binding sites have already bound nonradioactive estradiol. For the exchange assay, an extract containing receptor is adsorbed to a filter, which is washed free of unbound steroid and then equilibrated for a prolonged period with an excess volume of buffer containing radioactive estradiol. After brief washing in steroid-free buffer, the radioactivity adsorbed to the filter is measured to determine the amount of receptor present. These assays can be used at either 4 or 23°C, over a broad range of salt concentrations. The background of nonspecific binding is extremely low, due in part to the almost negligible affinity of free estradiol for the glass-fiber support.     

Molecular friction in an actomyosin molecular machine

In muscle contraction, it has been widely recognized that a binding state exists between myosin and actin in the presence of Mg-ATP. To estimate the magnitude of binding strength, I introduce a concept of frictional phenomena which occurs between two sliding bodies in contact each other. In such cases, the sliding speed can be formulated as a function of the actin-myosin bond strength. In order to validate this, the present theory is applied for the two movement assay systems with no external load; one movement assay of Phalloidin Rhodamine bound F-actin on a myosin coated hydrophobic cover glass and another assay of myosin coated beads along actin cables of Nitella. If a coefficient of 0.005 is applied to the kinetic friction, 1pN for the sliding force per cross-bridge and 10 microns sec-1 for the sliding speed, it is found that the bond strength between actin and one myosin head is about 200 pN in the contracting state.     

A radioassay for synaptic core complex assembly: screening of herbal extracts for effectors

Synaptic core complex formation between syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) on the plasma membrane and synaptobrevin on the vesicle membrane is responsible for membrane fusion and neurotransmitter release. A radiolabeled protein binding assay for synaptic core complex formation was developed. The components of this assay included recombinant glutathione S-transferase (GST)-syntaxin immobilized on glutathione agarose beads, SNAP25 and (125)I-labeled synaptobrevin. Reactions were performed in tubes containing filter inserts to facilitate removal of unbound protein. The radiolabeled protein bound was then quantified by gamma counter. A K(d) of 1.6 microM was determined for the GST-syntaxin/SNAP25/synaptobrevin complex, and a K(d) of 12 microM was determined for the GST-syntaxin/synaptobrevin complex. The assay was used to screen 14 herbal extracts for effectors of core complex formation. Herbs traditionally used to treat neurological conditions such as depression, anxiety, and stress were chosen. A Hypericum perforatum extract was found to have a nonspecific effect via protein complexation, whereas an Albizzia julibrissin extract was found to reduce the level of core complex formation. The assay was used to further investigate the effect of the A. julibrissin extract. The discovery of an inhibitor of core complex formation demonstrates the efficacy of the assay in screening natural products for substances that affect core complex formation.     

Solubilization and hydrophobic immobilization assay of a cAMP binding protein from Dictyostelium discoideum plasma membranes

A cAMP binding site present on isolated plasma membranes of aggregation-competent $\text{D.}\text{discoideum}$ cells has been solubilized with the nonionic detergent Emulphogene BC-720. An assay has been developed based on the principle of hydrophobic chromatography, in which the detergent solubilized cAMP binding protein is immobilized on alkyl-agarose beads at low detergent concentration. This allows the necessary rapid separation of bound and free [³H]-cAMP by filtration of the beads. The kinetics and nucleotide specificity of the detergent solubilized cAMP binding protein are comparable to those of the cAMP chemotactic receptor on intact cells and plasma membranes. The alkyl-agarose bead assay may have general utility for the assay of detergent solubilized membrane receptors.     

Preparation and characterization of an enzymatically active immobilized derivative of myosin.

Purified skeletal muscle myosin (EC 3.6.1.3) has been covalently bound to Sepharose 4B by the cyanogen bromide procedure. The resulting complex, Sepharose-Myosin, possesses adenosine triphosphatase activity and is relatively stable for long periods of time. Under optimal binding conditions, approximately 33% of the specific ATPase activity of the bound myosin is retained. Polyacrylamide gel electrophoresis of polypeptides released from denatured Sepharose-Myosin indicates that 85% of the myosin is attached to the agarose beads through the heavy chains and the remainder through the light chains, in agreement with predictions of binding and release based upon either the lysine contents or molecular weights of themyosin subunits. The adenosine triphosphatase of the immobilized myosin has been investigated under conditions of varying pH, ionic strength, and cation concentration. The ATPase profiles of immobilized myosin are quite similar to those for free myosin, however subtle differences are found. The Sepharose-Myosin ATPase is not as sensitive as myosin to alterations in salt concentration and the apparent KM is approximately two-fold higher than that of myosin. These differences are probably due to chemical modification in the region of the attachment site(s) to the agarose beads and hydration and diffusion limitations imposed by the polymeric agarose matrix.     

Effect of multiple phosphorylations of smooth muscle and cytoplasmic myosins on movement in an in vitro motility assay

The Nitella-based in vitro motility assay developed by Sheetz and Spudich (Sheetz, M.P., and Spudich, J. A. (1983) Nature 303, 31-35) is a quantitative assay for measuring the velocity of myosin-coated beads over an organized substratum of actin. We have used this assay to analyze the effect of phosphorylation of various sites on the 20,000-Da light chain of smooth muscle and cytoplasmic myosins. Phosphorylation by myosin light chain kinase at serine 19 on the 20,000-Da light chain subunit of smooth muscle myosin from turkey gizzard, bovine trachea and aorta, and of cytoplasmic myosin from human platelets was required for bead movement. The individual phosphorylated myosin-coated beads moved at characteristic rates under the same conditions (turkey gizzard myosin, 0.2 micron/s; aorta or trachea myosin, 0.12 micron/s; and platelet myosin, 0.04 micron/s; in contrast, rabbit skeletal muscle myosin, 2 micron/s). Myosin light chain kinase can also phosphorylate threonine 18 in addition to serine 19, and this phosphorylation resulted in an increase in the actin-activated MgATPase activity (Ikebe, M., and Hartshorne, D.J. (1985) J. Biol. Chem. 260, 10027-10031). Phosphorylation at this site had no effect on the velocity of smooth muscle myosin-coated beads. Protein kinase C (Ca2+/phospholipid-dependent enzyme) can also phosphorylate two to three sites on the 20,000-Da light chain, and this phosphorylation alone did not result in the movement of myosin-coated beads. When myosin that had been previously phosphorylated by myosin light chain kinase at serine 19 was also phosphorylated by protein kinase C, myosin-coated beads moved at the same velocity as beads coated with myosin phosphorylated by myosin light chain kinase alone. Tropomyosin binding to actin also had an activating effect on the actin-activated MgATPase activity through an effect on the Vmax and also resulted in an increase in the velocity of myosin-coated beads.     

Purification and characterization of a 200[emsp4 ]kDa fructosyllysine-specific binding protein from cell membranes of U937 cells

Amadori-modified proteins are bound by macrophages and monocytes via fructosyllysine-specific receptors. Detergent extracts from U937 cell membranes were used to purify the binding proteins by affinity purification on glycated polylysine coated magnetic beads followed by SDS-PAGE. Two proteins of 200 and 100[emsp4 ]kDa were isolated. MS-analysis of the 200[emsp4 ]kDa protein showed high homologies with cellular myosin heavy chain, type A. Both fructosyllysine specific binding proteins, cellular myosin heavy chain and nucleolin, are glycosylated.     

The 110-kD protein-calmodulin complex of the intestinal microvillus (brush border myosin I) is a mechanoenzyme

The 110-kD protein-calmodulin complex (110K-CM) of the intestinal brush border serves to laterally tether microvillar actin filaments to the plasma membrane. Results from several laboratories have demonstrated that this complex shares many enzymatic and structural properties with myosin. The mechanochemical potential of purified avian 110K-CM was assessed using the Nitella bead motility assay (Sheetz, M. P., and J. A. Spudich. 1983. Nature (Lond.). 303:31-35). Under low Ca2+ conditions, 110K-CM-coated beads bound to actin cables, but no movement was observed. Using EGTA/calcium buffers (approximately 5-10 microM free Ca2+) movement of 110K-CM-coated beads along actin cables (average rate of approximately 8 nm/s) was observed. The movement was in the same direction as that for beads coated with skeletal muscle myosin. The motile preparations of 110K-CM were shown to be free of detectable contamination by conventional brush border myosin. Based on these and other observations demonstrating the myosin-like properties of 110K-CM, we propose that this complex be named "brush border myosin I."     

The effect of mechanical stretching of the myosin rod component (fragment LMMMM S-2) on the ATPase activity of myosin.

The binding of myosin to nylon fiber gives immobilized myosin with a considerable ATPase activity. Treatment of immobilized enzyme with papain results in the entire ATPase activity (known to be concentrated in myosin heads, (fragment HMM S-1)) being replaced from the fiber into the solution; this means that myosin is chemically bound to the fiber via its rod part (fragment LMM+HMM S-2). When nylon fiber is mechanically stretched, the ATPase activity of myosin attached to it sharply decreases; after relaxation of the fiber the enzymatic activity returns to the initial level. The detailed study of this phenomenon has shown that reversible inactivation of myosin upon fiber stretching is not the result of an altered microenvironment of the enzyme. The discovered regulatory effect is ascribed to deformation of myosin molecules induced by support stretching. Thus deformation of the myosin tail (not indispensable for ATPase since its cleaving-off does not alter the enzymatic activity) leads to decrease in the ATPase activity of the enzyme. The possible role of the above phenomenon in the mechanism of muscle contraction is discussed.     

Characterization of intestinal microvillar membrane disks: detergent- resistant membrane sheets enriched in associated brush border myosin I (110K-calmodulin)

The actin bundle within each microvillus of the intestinal brush border (BB) is tethered laterally to the membrane by bridges composed of BB myosin I. Avian BB myosin I, formerly termed 110K-calmodulin, consists of a heavy chain with an apparent Mr of 110 kD and three to four molecules of calmodulin "light chains." Recent studies have shown that this complex shares many properties with myosin including mechanochemical activity. In this report, the isolation and characterization of a membrane fraction enriched in bound BB myosin I is described. This membrane fraction, termed microvillar membrane disks, was purified from ATP extracts of nonionic detergent-treated microvilli prepared from avian intestinal BBs. Ultrastructural analysis revealed that these membranes are flat, disk-shaped sheets with protrusions which are identical in morphology to purified BB myosin I. The disks exhibit actin-activated Mg-ATPase activity and bind and cross-link actin filaments in an ATP-dependent fashion. The mechanochemical activity of the membrane disks was assessed using the Nitella bead movement assay (Sheetz, M. P., and J. A. Spudich. 1983. Nature [Lond.]. 303:31-35). These preparations were shown to be free of significant contamination by conventional BB myosin. Latex beads coated with microvillar membrane disks move in a myosin-like fashion along Nitella actin cables at rates of 12-60 nm/s (average rate of 33 nm/s); unlike purified BB myosin I, the movement of membrane disk-coated beads was most reproducibly observed in buffers containing low Ca2+.     

A rapid filtration assay for cAMP

The receptor-binding assay for cAMP was improved by using polyethylenimine-treated glass filters. A polyethylenimine-treated glass filter has high protein binding capacity. This high capacity allows an increase in the amount of protein per assay tube and the use of a crude preparation, such as a beef heart extract, as specific binding protein instead of a purified protein, which has been used in the classical filtration assays involving cellulose ester filters. Since the time required for the separation of the protein-cAMP complex and the free nucleotide can be shortened by the use of polyethylenimine-treated filters, the dissociation of the bound ligand during the separation procedure, which is a serious problem with other modified assay methods involving charcoal adsorption, is minimized. Filtration through polyethylenimine-treated glass filters also gives low blanks and prevents the loss of protein or ligand due to breakage of the filters, which is often observed with fragile cellulose ester membranes. In consequence, this simple and rapid filtration assay allows more accurate and reproducible determinations.     

Erythrocytes attached to a wheat germ agglutinin coated surface display an altered phospholipid metabolism

Erythrocytes were bound to a lectin-coated surface; the multivalent attachment to this surface resulted in a severe deformation of the cells and an alteration in the cellular phospholipid metabolism. Human erythrocytes were allowed to bind for 20 min at 20 degrees C to polystyrene beads coated with wheat germ agglutinin (WGA beads). The bound erythrocytes were then lysed to produce stroma bound to WGA beads. Control stroma and stroma-WGA beads were incubated at 37 degrees C with gamma-32P-ATP to examine the phospholipid labeling patterns. The control stroma incorporated 32P-label into phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate, in agreement with earlier studies. However, the stroma-WGA beads showed incorporation of 32P-label into phosphatidic acid in addition to that in the phosphoinositides. The quantity of 32P-phosphatidic acid produced during the 20-min assay was 3.23 +/- 0.84 (n = 7) picomoles/micrograms stromal cholesterol; the amount synthesized, however, was dependent on the procedure used to prepare the stroma-WGA beads. If the erythrocytes were bound to the WGA beads at 0 degrees C instead of 20 degrees C, the quantity of 32P-phosphatidic acid produced during the subsequent 37 degrees C assay with gamma-32P-ATP was decreased 4.2 fold; the phosphoinositide labeling pattern was unchanged. In addition, when the time for binding of intact erythrocytes to the WGA beads was varied from 1 to 20 minutes, there was a time-dependent increase in the amount of 32P-phosphatidic acid produced. This induction of phosphatidic acid synthesis could not be duplicated with fluid phase WGA. Therefore, the multivalent binding of intact erythrocytes to WGA beads causes an alteration in phospholipid metabolism.     

两种富集方法相结合对蓖麻毒素进行SELEX筛选研究

为了获得能特异识别具有细胞毒性的蓖麻毒素蛋白寡核苷酸适配子,体外构建了含40个随机序列全长87nt的随机ssDNA文库,采用指数富集配基的系统进化(SELEX)技术方法,结合微孔板和亲和树脂两种分离、富集方法,经过数轮筛选,文库与蓖麻毒素的结合率达到了38．5％。结果表明,以亲和树脂为分离介质进行筛选,富集效果非常明显。     

PARTIAL CHARACTERIZATION OF A PUTATIVE 110kDa MYOSIN FROM THE GREEN ALGACHARA CORALLINABYIN VITROBINDING OF FLUORESCENT F-ACTIN

Using the binding of heterologous, rhodamine phalloidin-labelled F-actinin vitro, two F-actin binding proteins were identified in protein extracts from the green algaChara corallinaafter fractionation by anion exchange chromatography. The first protein, a putative myosin, released laterally bound F-actin at ATP-concentrations as low as 1μm ; equivalent concentrations of ADP were not effective. Binding of F-actin was inhibited by the sulfhydryl-alkylating agent N-ethylmaleimide (NEM). Binding of F-actin was also abolished by a monoclonal anti-myosin (J14) previously used for immunodetection and immunolocalization in internodal cells (Groliget al., 1988,Eur J Cell Biol 47 : 22–31). Immunoblotting with J14 detected a 110kDa polypeptide only in those protein fractions that had revealed ATP-sensitive binding of F-actin. The putative myosin bound with mediocre affinity to immobilized calmodulin and free Ca²⁺-concentration made no difference to this binding affinity. In contrast to the putative myosin, the second, less abundant protein revealed ATP-insensitive and end-wise binding to the microfilament and was not recognized by the anti-myosin antibody.     

A high-throughput assay for a human telomerase protein-human telomerase RNA interaction

The rapid rate at which cancer cells divide necessitates a mechanism for telomere maintenance, and in approximately 90% of all cancer types the enzyme telomerase is used to maintain the length of telomeric DNA. Telomerase is a multi-subunit enzyme that minimally contains a catalytic protein subunit, hTERT, and an RNA subunit, hTR. Proper assembly of telomerase is critical for its enzymatic activity and therefore is a requirement for the proliferation of most cancer cells. We have developed the first high-throughput screen capable of identifying small molecules that specifically perturb human telomerase assemblage. The screen uses a scintillation proximity assay to identify compounds that prevent a specific and required interaction between hTR and hTERT. Rather than attempting to disrupt all of the individual hTR-hTERT interactions, we focused the screen on the interaction of the CR4-CR5 domain of hTR with hTERT. The screen employs a biotin-labeled derivative of the CR4-CR5 domain of hTR that independently binds [(35)S]hTERT in a functionally relevant manner. The complex between hTERT and biotin-labeled RNA can be captured on streptavidin-coated scintillation proximity beads. Use of 96-well filter plates and a vacuum manifold enables rapid purification of the beads. After optimization, statistical evaluation of the screen generated a Z' factor of 0.6, demonstrating the high precision of the assay.     

Binding of brush border myosin I to phospholipid vesicles

The actin filament core within each microvillus of the intestinal epithelial cell is attached laterally to the plasma membrane by brush border (BB) myosin I, a protein-calmodulin complex belonging to the myosin I class of actin-based mechanoenzymes. In this report, the binding of BB myosin I to pure phospholipid vesicles was examined and characterized. BB myosin I demonstrated saturable binding to liposomes composed of anionic phospholipids, but did not associate with liposomes composed of only neutral phospholipids. The binding of BB myosin I to phosphatidylserine and phosphatidylglycerol vesicles reached saturation at 4-5 x 10(-3) nmol protein/nmol phospholipid, while the apparent dissociation constant was determined to be 1-3 x 10(-7) M. Similar to the free protein, membrane-associated BB myosin I bound F-actin in an ATP-sensitive manner and demonstrated actin-activated Mg-ATPase activity. Immunoblot analysis of peptides generated from controlled proteolysis of vesicle-bound BB myosin I provided structural information concerning the site responsible for the membrane interaction. Immunoblot staining with domain-specific mAbs revealed a series of COOH-terminal, liposome-associated peptides that were protected from digestion, suggesting that the membrane-binding domain is within the carboxy-terminal "tail" of the BB myosin I heavy chain.