Adenosine-5'-O-phosphorylated and adenosine-5'-O-phosphorothioylated polyols as strong inhibitors of (symmetrical) and (asymmetrical) dinucleoside tetraphosphatases. (1/14)Dinucleoside 5',5"'- P (1), P ( n )-polyphosphates, and particularly the diadenosine compounds, have been implicated in extracellular purinergic signalling and in various intracellular processes, including DNA metabolism, tumour suppression and stress responses. If permitted to accumulate, they may also be toxic. One approach to understanding their function is through the various specific degradative enzymes that regulate their levels. Eight adenosine-5'- O -phosphorylated polyols (derivatives of glycerol, erythritol and pentaerythritol) and 11 adenosine-5'- O -phosphorothioylated polyols (derivatives of glycerol, erythritol, pentaerythritol, butanediol and pentanediol) have been tested as inhibitors of specific diadenosine tetraphosphate (Ap(4)A) hydrolases. Of these two groups of novel nucleotides, the adenosine-5'- O -phosphorothioylated polyols were generally stronger inhibitors than their adenosine-5'- O -phosphorylated counterparts. 1,4-Di(adenosine-5'- O -phosphorothio) erythritol appeared to be the strongest inhibitor of ( asymmetrical ) Ap(4)A hydrolases (EC 18.104.22.168) from both lupin and human, with K (i) values of 0.15 microM and 1.5 microM respectively. Of eight adenosine-5'- O -phosphorylated polyols, 1,4-di(adenosine-5'- O -phospho) erythritol was the only compound that inhibited the lupin enzyme. Two derivatives of pentaerythritol, di(adenosine-5'- O -phosphorothio)-di(phosphorothio) pentaerythritol and tri(adenosine-5'- O -phosphorothio)-phosphorothio-pentaerythritol, proved to be the strongest inhibitors of the prokaryotic ( symmetrical ) Ap(4)A hydrolase (EC 22.214.171.124) so far reported. The estimated K (i) values were 0.04 microM and 0.08 microM respectively. All of these inhibitors were competitive with respect to Ap(4)A. These new selectively acting Ap(4)A analogues should prove to be valuable tools for further studies of Ap(4)A function and of the enzymes involved in its metabolism. (+info)
Efficient solubilization buffers for two-dimensional gel electrophoresis of acidic and basic proteins extracted from wheat seeds. (2/14)Plant tissues are made up of a broad range of proteins with a variety of properties. After extraction, solubilization of a diverse range of plant proteins for efficient proteomic analysis using two-dimensional electrophoresis is a challenging process. We tested the efficiency of 12 solubilization buffers in dissolving acidic and basic proteins extracted from mature seeds of wheat. The buffer containing two chaotropes (urea and thiourea), two detergents (3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate and N-decyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate), two reducing agents (dithiothreitol and tris (2-carboxyethyl) phosphine hydrochloride) and two types of carrier ampholytes (BioLyte pH 4-6 and pH 3-10) solubilized the most acidic proteins in the pH range between 4 and 7. The buffer made up of urea, thiourea, 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, DeStreak reagent (Amersham Biosciences, Uppsala, Sweden) and immobilized pH gradient buffer, pH 6-11 (Amersham Biosciences) solubilized the most basic proteins in the pH range between 6 and 11. These two buffers produced two-dimensional gels with high resolution, superior quality and maximum number of detectable protein (1425 acidic protein and 897 basic protein) spots. (+info)
Commercial ampholytes used for isoelectric focusing may interfere with bioactivity based purification of antimicrobial peptides. (3/14)BioRad's Rotofor system has been frequently used for the purification of proteins and smaller peptides such as bacteriocins. In this study, we report that some commercially available ampholytes used with the Rotofor isoelectric focusing system possess antimicrobial activity, which may interfere with the purification of bacteriocins and bacteriocin-like substances. (+info)
Infection with Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus t034. (4/14)(+info)
Effect of synthetic carrier ampholytes on saturation of human serum transferrin. (5/14)We have investigated the effect in solution of synthetic carrier ampholytes on the saturation of human serum transferrin. By spectrophotometric titrations of human serum transferrin with various Fe3+-carrier ampholyte solutions, we demonstrated that under these conditions carrier ampholytes behave as typical chelators, their binding curves being very similar to that obtained with disodium nitrilotriacetate. On performing titration experiments at three different pH values, carrier ampholytes act like nitrilotriacetate at pH 7.5, but the former are more effective iron donors at pH 8.4 and worse iron donors at pH 5.2. Spectrophotometric titrations of isolated C-terminal and N-terminal fragments obtained from human serum transferrin by thermolysin cleavage show no differences between them, and no differences with respect to the whole protein except that they contain half the number of binding sites. In order to determine a site-specificity of iron in the presence of ampholytes, the classical urea/polyacrylamide-gel-electrophoresis technique was adopted. Under saturating conditions carrier ampholyte solutions act mostly on the C-terminal site, whereas desaturating agents remove iron preferentially from the N-terminal site. Our findings support the hypothesis that Ampholine may chelate Fe3+ as well as many other compounds. (+info)
Resolution of alkaline phosphatase isoenzymes in serum by isoelectric focusing in immobilized pH gradients. (6/14)This new method for fractionation of serum alkaline phosphatase isoenzymes is based on isoelectric focusing on a mixed-type polyacrylamide support containing an immobilized pH gradient with a superimposed carrier-ampholyte gradient. All known forms of alkaline phosphatase are separated in an Immobiline pH 3.5-6.0 gradient, the sample being applied into pockets cast on a pH 8.0 plateau. Sharp zymogram bands are obtained by substituting alkaline-stable 5-bromo-4-chloro-3-indoxyl phosphate and tetrazolium salts for the standard 1- and 2-naphthyl phosphate-diazonium salt combinations. After hydrolysis of the phosphate group by the alkaline phosphatase the indoxyl moieties reduce tetrazolium salts to nearly insoluble and nondiffusible formazan precipitates. Normal sera show an array of about 10 isobands isoelectric between pH 3.9 and pH 4.79. In Paget's disease, two sharp isobands with pls of 4.97 and 5.09 are seen. Placental alkaline phosphatase overlaps with the higher pl bands of normal serum; however, upon heat destruction of the latter, it shows four sharp bands with the following pl's: 4.59, 4.62, 4.67 and 4.73. (+info)
Charge microheterogeneity of the major capsid protein of polyoma virus. (7/14)The behavior in isoelectric focusing of the major capsid polypeptide VPI of several strains of polyoma virus was studied. Two previously recognized phenomena were reexamined, namely, (i) the separation of the VP1 polypeptide into multiple subspecies differing only slightly from each other in apparent isoelectric point and (ii) strain differences in the overall apparent net charge of the family of VP1 subspecies. It was found that the pattern of subspecies was reproducible when focusing was initiated from either the basic or acidic region of the gel, keeping the ampholyte mixture constant. However, individual subspecies were unstable, and labeled polypeptide could be shifted dramatically by either refocusing of separated subspecies or by altering the concentration of ampholytes. These findings suggest that protein-protein and protein-ampholyte interactions play an important role in the generation of this charge heterogeneity. The basis for the overall charge difference between the VP1 of 3049 virus and several other strains (lpD, lpS, ts59, and A2) was studied, using recombinant viruses constructed of specific sequences derived from 3049 and lpD genomes. The portion of the VP1 polypeptide carrying the altered charge could be mapped to the body of the molecule 3' to the HindIII site at 45.0 map units (3,918 base pairs). This clearly segregates the VP1 charge phenotype from the cyc phenotype of 3049 in which capsid proteins are overproduced and accumulate in the cytoplasm of infected cells. (+info)
pH-dependent aggregation and electrofocusing of poliovirus. (8/14)Following isoelectric focusing, poliovirus can be detected at two pH values. The acidic form consists of poliovirus aggregates and the neutral form of single virions. (+info)
Ampholyte mixtures are a type of chemical compound that contain both acidic and basic groups within the same molecule. They are commonly used in a process called isoelectric focusing, which is a technique used to separate and purify proteins and other large molecules based on their isoelectric points (pI).
An ampholyte mixture typically contains a range of amphoteric compounds with different pI values, which create a pH gradient when subjected to an electric field. This gradient allows proteins and other large molecules to migrate to a position in the gradient where their net charge is zero (their isoelectric point), effectively separating them based on their pI.
Ampholyte mixtures are usually composed of a complex mixture of organic compounds, including polyamino polycarboxylic acids and their salts. They are available in various ranges of pH values to suit different applications.
Isoelectric focusing (IEF) is a technique used in electrophoresis, which is a method for separating proteins or other molecules based on their electrical charges. In IEF, a mixture of ampholytes (molecules that can carry both positive and negative charges) is used to create a pH gradient within a gel matrix. When an electric field is applied, the proteins or molecules migrate through the gel until they reach the point in the gradient where their net charge is zero, known as their isoelectric point (pI). At this point, they focus into a sharp band and stop moving, resulting in a highly resolved separation of the different components based on their pI. This technique is widely used in protein research for applications such as protein identification, characterization, and purification.
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- Two-dimensional gel electrophoresis (2DE) is an established technique for high-resolution profiling of complex protein mixtures. (thermofisher.com)
- 4 µl of the Cy2 solution is added to the internal standard protein mixture, which contains 12.5 µg protein from each sample. (expasy.org)
- The lower the ratio of these two components in the mixture, the higher the degree of crosslinking. (itwreagents.com)