Elution Of Components Research Articles

Biochemical Studies on the Mode of Action of Cytochalasin B: CYTOCHALASIN B BINDING TO RED CELL MEMBRANE IN RELATION TO GLUCOSE TRANSPORT

Abstract Cytochalasin B binds rapidly and reversibly to human red blood cells. Scatchard plot analysis of binding data reveals a class of high affinity binding sites (dissociation constant ≅10-7 m; 3 x 105 sites per cell) and a class of low affinity binding sites (dissociation constant ≧10-5 m). All of the high affinity binding sites are in the plasma membrane (i.e. ghost). Variation of ionic strength and of pH in the range of 5.0 to 9.0 has only a small effect on the ability of the membrane to bind cytochalasin B with high affinity. The high affinity binding is not affected when intact cells are treated with pronase or with trypsin, but is lost when ghosts are similarly treated under conditions where the inner surface of the membrane is accessible to these enzymes. This binding decreases by more than 90% when either intact cells or ghosts are treated with p-chloromercuribenzoate. d-Glucose inhibits the high affinity binding by 80 to 90%, whereas l-glucose has very little effect. The effectiveness of eight different sugars to inhibit high affinity binding of cytochalasin B to ghosts is related to their affinity for the sugar transport system. All of these results indicate that at least 80% of the high affinity cytochalasin B binding sites are intimately related to this transport system. As judged by competition experiments, cytochalasin A and B have the same affinity for the high affinity binding sites, whereas dihydrocytochalasin B and the γ-lactone derivative have a much lower affinity. Selective elution of spectrin and components 5 and 6 has no effect on the high affinity binding of cytochalasin B to the membrane.

Liquid scintillation counting of 14C- and 3H-labeled samples on solid supports: A general solution of the problem

The data presented demonstrate the potential of surfactant-fortified scintillation cocktails in overcoming many of the problems encountered in the quantitation of radioactivity on solid supports. Using a broad range of representative ionic and polar biochemical compounds, the in vial elution and quantitative recovery of 3H and 14C-labeled components from a wide assortment of common solid support media has been demonstrated. This methodology in combination with zero elution counting systems and in some circumstances gelled suspension counting should combine to overcome most of the problems associated with determination of isotopic activity on such solid support media.

Trypsin Inhibitors of Corn (Zea mays)

Trypsin inhibitors of three genotypic sources of corn were iso lated. Gel electrophoresis after chromatography on Sephadex G75 and CM-cellulose resolved the inhibitors of normal, opaqu*2, and floury-2 corns into 2, 3, and 4 or more bands, respectively. All inhibitors were slowly inactivated by heat, but that of normal corn was appreciably more stable than the others. Normal and opaqub2 inhibitors were competitive iIl action, while flourn,r-2 was noncompetitive. The data indicate that corn trypsin inhibitor may consist of multiple components, the number depending on the genetic compOSitiOrI of the corn. Trans. Kans. Acad. Sci., 76 (4), 1973. The grain of corn possesses antitryptic activity (10), with most of the activity located in the endosperm (5). Inhibitory activity of endosperm of corn containing the opaque-2 gene is much greater than that of normal corn endosperm, while that of floury-2 is least (5). The trypsin inhibitor of immature sweet corn was isolated and shown to consist of two inhibitory components (1). Mies and Hymowitz (8) used disc electrophoresis of crude soybean extracts to show that some species contained two inhibitory components while others contained three. Hence, some of the differences between inhibitory activity of normal, opaque-2, and floury-2 corn may result from variation in inhibitory components. The problem was studied by isolating and examining the inhibitors of the endosperm of a normal, hard-starch corn and of opaque-2 and floury-2 strains. Melhods One kilogram of corn seed was finely ground and extracted several times with acetone to remove lipids. The powder was extracted twice by suspending it in 0.2 M NaCl and agitating for 12 hrs at 4° C. Ihe extracts were combined, adjusted to pH 8 with NaOH, and centrifuged at 5000 g for 20 minates. (NH)2SO was added to 40% saturation, and after 18 hrs at 4° the precipitate was collected by centrifugation at 12,000 g for 30 minutes. The precipitate was dissolved in 0.2 M NaCl, and the solution was dialyzed against distilled water for 48 hrs at 4°. The soluThis content downloaded from 157.55.39.105 on Fri, 07 Oct 2016 04:31:45 UTC All use subject to http://about.jstor.org/terms 290 Tfiansactions of the Kansas Academy of Ssience tion was centrifuged to remove insoluble material, and the supernatant was concentrated in a dialysis bag suspended in front of an electric fan. The solution was placed on a column of Sephadex G75, and elution was accomplished at room temperature with 0.05 triethanolamine-0.1 M NaCl buffer, pH 7, at a flow rate of 20 ml/hr. The eluate was collected in 5 ml fractions and absorbance of each fraction at 280 nm was used to detect elution of protein components. Inhibitory activity of each fraction was measured, using benzoyl-DL-arginine-p-nitroanilide (BAPA) as trypsin substrate (4). Fractions with inhibitory activity were combined, dialyzed, and placed on a CM-cellulose column (9). Gradient elution was performed using 0.03 hI citrate buffer, pH 4.0, in the mixing chamber and 0.3 M citrate buffer, pH 6.5, in the reservoir. The inhibitory fractions were combined, dialyzed, and lyophilized. Disc electrophoresis was performed on 7% polyacrylamide gels (3). One hundred jug of inhibitor were added to each gel column, and electrophoresis was conducted with Tris-glycine buffer, pH 9.5, at a current of 5 mA per tube for 1.5 hours. Protein bands were located by staining with Coomassie blue (2). Type of inhibition was established by the method of Lineweaver and Burk (7). Thermostability was studied by refluxing water solutionJ containing 100 Jug of inhibitor per ml, withdrawing samples at intenals for up to 8 hrs and determining inhibitor activity. Figure 1. Electrophoretic separation of corn trypsin inhibitor on polyacrylamide gel. Left, normal corn; center, opaque-2; right, floBy-2. This content downloaded from 157.55.39.105 on Fri, 07 Oct 2016 04:31:45 UTC All use subject to http://about.jstor.org/terms Trypsin Inhibitors of Corn 291 Results The three corn sources had similar Sephadex chromatographic patterns, each showing three protein bands with all the inhibitory activity located in the middle band. Further chromatography of the active components on CM-cellulose eliminated a small amount of inactive material from each preparation. After passing through the above columns, the active cornponents were recovered by lyophilization. Yields were 350 and 18 / mg per kilogram for the opaque-2 (HL6) and the normal (H28xK41 ) sources, respentively. Equipment malfunction precIuded accurate yield data for the floury-2 source.

Comparison of polypeptide-chain structure of four mammalian ceruloplasmins by gel filtration in guanidine hydrochloride solutions.

In a recent paper in this journal it was shown that human ceruloplasmin contains a single continuous polypeptide chain composed of about 1050 amino acid residues. Together with the carbohydrate this accounts for the molecular weight of the native protein, 132000. In contrast to this porcine ceruloplasmin has been reported to contain two subunits of molecular weight 70000–80000. This was the reason for performing a direct comparison of polypeptide‐chains from ceruloplasmin from different mammalian sera.Ceruloplasmin was prepared from sera of man, pig, horse and rabbit. The reduced and alkylated proteins were gel filtered on a calibrated column of 6% agarose in 6 M guanidine hydrochloride. The four proteins all contained a single main component eluting in the same position within errors of measurements. It is concluded that the four proteins investigated all contain a single polypeptide chain of very similar size.

Quantitative esterification of lipids on thin-layer adsorbents.

Lipid classes separated by thin-layer chromatography can be determined by gas chromatography of the methyl esters of the component fatty acids with added internal standard. A simplified procedure is described for the complete analysis of all the lipid classes in a given sample in which transesterification is performed on the thin-layer adsorbent itself. The methyl esters are recovered from the reaction mixture with a less polar solvent than would have been required to elute the original component. As a consequence of the elimination of a step that requires preliminary elution of components from the thin-layer plates, there is less contamination of minor components by trace amounts of impurities in large volumes of solvent, and the results obtained are more reproducible.

Biological characterization of structural components of Adenovirus type 12.

SUMMARY Two products with a large mass and five different soluble components of adenovirus type 12 have been identified. The former were virus particles and empty capsids, which both carried group-specific complement-fixing (CF) antigen and were active as complete haemagglutinins (HAs). Their buoyant densities in CsCl were 1·330 to 1·340 and 1·295 to 1·305, respectively. The soluble components were identified as hexons, pentons, isolated fibres, dimers of fibres and isolated vertex capsomeres. Hexons were trypsin-resistant, ther-mostable and carried group-specific CF antigen; ultrastructurally they resembled hexons of other human adenoviruses. Pentons were active as an incomplete HA, causing agglutination in the presence of heterologous antisera against representative members of all three subgroups of adenoviruses. Furthermore, they were trypsin-sensitive and thermolabile. Isolated fibres represented another incomplete HA, exhibiting agglutinating activity only in the presence of antisera against members of subgroup III, e.g. type 2. This component was also identified as the dominating type-specific CF antigen. The length of fibres was estimated as 28 to 32 nm. by electron microscopy. The dimers of fibres represented a complete HA, the activity of which became apparent only after other material was removed. Both isolated fibres and their dimers were trypsin-resistant and thermostable. The assumed pentons and fibre dimers occurred in too low concentration to allow ultrastructural identi-fication. Isolated vertex capsomeres were demonstrated by a haemaggluti- nation enhancement antibody consumption test. They were completely destroyed by trypsin treatment and exhibited a moderate thermostability. Electron microscopy confirmed the presence of capsomere-like structures. These structures occasionally displayed a five-sided contour, thereby differing from hexons. The sequence of elution of different components from an anion exchanger was: isolated vertex capsomeres, hexons + pentons, fibres and dimers of fibres. In zonal centrifugation experiments the components sedimented at decreasing rates in the following order: hexons, pentons+isolated vertex capsomeres, dimers of fibres and isolated fibres. Probably on account of the morphological characteristics of the various components they presented a different pattern of elution in exclusion chromatography. Dimers of fibres, pentons and even isolated fibres were eluted before hexons. The position of the peak activity of type 12 fibres in the elution diagram was similar to that of types 1, 2 and 5 fibres.

Factors Influencing the Elution of High-Boiling Components of Cigarette Smoke from Filters

Factors Influencing the Elution of High-Boiling Components of Cigarette Smoke from Filters

Comparative Studies on the Soluble Components of Adenovirus Types 9 and 15 and the Intermediate Strain 9-15

Five different soluble components of adenovirus types 9, 9-15, and 15 have been identified. These are: (i) a slowly sedimenting, trypsin-resistant, incomplete hemagglutinin (HA). (This component was demonstrable by hemagglutination-enhancement (HE) tests in the presence of heterotypic antisera against members of Rosen's subgroups II and III, but not of subgroup I); (ii) a slowly sedimenting, trypsin-resistant, complete HA, causing only a partial agglutination of cells; (iii) a rapidly sedimenting, incomplete HA, demonstrable by HE tests in the presence of heterotypic antisera against members of all Rosen's subgroups. (Trypsin treatment of this component caused a conversion into slowly sedimenting incomplete HA); (iv) a group-specific complement-fixing (CF) antigen devoid of HA activity; and (v) a rapidly sedimenting, trypsin-sensitive, complete HA, which in the electron microscope was found to represent a dodecahedral aggregate of 12 pentons (a dodecon). On the basis of their biological and physicochemical characteristics, the first four components were interpreted to represent (i) fibers, (ii) a polymer of a few, probably two, fibers, (iii) pentons, and (iv) hexons, respectively. The length of fibers extending from dodecons and virions was estimated to be 11 to 14 nm. A similar value was suggested from exclusion chromatography experiments. Adenovirus types 9 and 15 fibers were recovered in a position intermediate to that of fibers of types 3 and 4, the lengths of which are 10 and 17 nm, respectively. The sequence of elution of different components of types 9 and 9-15 from an anion exchanger was fibers, fiber-aggregate, pentons, hexons, and dodecons. Type 15 components appeared in the same order except for the fact that dodecons eluted before hexons. The molarities of NaCl required to elute the different types 9 and 9-15 components, excluding hexons, were identical. They were distinctly different from those of the corresponding type 15 components. However, hexons of all three serotypes eluted in proximity to each other and there was a slight tendency for type 9-15 hexons to take a position intermediate to those of types 9 and 15.

The resolution of multiple cyanocobalamin-binding components in serum

At least two major cyanocobalamin-binding sites can be demonstrated in human and animal serum by fractionation on CM-cellulose columns when cyanocobalamin is added in vitro. In normal human serum, Component A, representing 50% of the total binding capacity, is associated with serum albumin and does not bind to CM-cellulose at pH 6. Component B, also representing 50% of the total binding capacity is eluted from the column after the serum globulins at 0.3 M NaCl. Sera from cat, dog, rabbit, horse, cow, pig, sheep, rat, and guinea pig are also resolvable into two major components. The two components in different animal sera are similar but vary with respect to concentration and relative elution pattern. In some animal sera, a third minor cyanocobalamin binding complex (Complex C) is present which is eluted at salt concentrations lower than that necessary for elution of Component B. In chicken serum, Component B is entirely absent. Sera of dogs, rabbits and chickens given oral doses of high specific activity [ 57Co]cyanocobalamin, followed by fractionation on CM-cellulose columns, yield cyanocobalamin binding sites essentially similar to that found for cyanocobalamin added to serum in vitro.

Generalization of gas‐liquid partition chromatography to study high pressure vapor‐liquid equilibria of multicomponent systems

AbstractThe use of chromatographic retention volume data to obtain vapor‐liquid equilibrium coefficients has been extended to a ternary system with all components present at finite concentrations in the liquid phase and essentially a light binary gas comprising the vapor phase. The data are obtained by observing the retention time of the solute of interest when that solute is eluted by a flowing gas stream through a column packed with an inert porous firebrick on which is impregnated a fixed, relatively nonvolatile liquid. The retention times are used to compute retention volumes which may be related to the K values of the solute.A previous mathematical description of the elution process is modified to include the case of an N component elution gas, all constituents of which are soluble in the fixed liquid phase. A general solution is derived for the multicomponent elution gas from which K values for each component may be calculated if retention volume data are taken for each, provided molecules of the solute samples used are distinguishable from others of the same species present in the eloution gas. For the case of indistinguishable sample molecules, the rate theory development of Stalkup and Deans is verified for a binary elution gas.Sufficient retention data were taken to completely define the concentration dependence of the K value for propane in the system methane‐propane‐n‐decane at −20°, 0°, 40°, and 70°F. from 20 to 1,000 lb/sq.in.abs. Six methane‐propane binary mixtures were used as elution gases, consisting of 2.08, 4.31, 6.90, 9.44, 13.09, 16.27 mole % propane. Retention data were also taken for methane under the same conditions. Distinguishability of sample molecules was achieved by using radioactive solute samples tagged with carbon‐14.Propane retention data were also obtained for the system methane‐propane‐n‐heptane at −20° and −40°F. from 100 to 1,000 lb./sq.in.abs. for propane at infinite dilution and for binary elution gases composed of 2.08 mole % propane at −40°F. and 4.31 mole % propane at −20°F.All K values determined chromatographically were either compared directly with published static equilibrium values or tested for consistency with such values and found to be in substantial agreement.

The study of gas‐solid equilibrium at high pressures by gas‐chromatography: Part I. Ethane, propane, and n‐butane at essentially infinite dilutions in the methane‐silica gel system

AbstractThe measurement of vapor‐solid distribution coefficients or K values of a solute distributed between a gas phase and an adsorbed phase at essentially infinite dilutions has been investigated. Gas‐solid chromatography is the technique employed in which the solute of interest is eluted through a tube packed with a solid adsorbent by a flowing gas phase which may or may not be appreciably adsorbed. The data taken for the eluted solute samples were then related to the K value and isobaric heat of adsorption for the solutes in the vapor‐solid system.A previous mathematical solution describing the chromatographic process is extended to include the case of an appreciably adsorbed pure component elution gas. This theory allows the calculation of solute K values and heats of adsorption for a solute at essentially infinite dilution in the vapor‐solid system. It is necessary to have adsorption data for the elution gas covering the range of interest. For this reason the necessary elution gas adsorption isotherms were determined gravimetrically for the temperature and pressure range of the study.For comparison purposes a single ethylene adsorption isotherm at 25°C. was obtained along with retention volume data for methane, ethane, and propane at essentially infinite dilution in the ethylene‐silica gel system at 25°C. Agreement of the data of this study and the literature was thought to be sufficiently good to warrant further use of the technique.Methane adsorption isotherms on silica gel were gravimetrically measured at −40°, −20°, 0°, 20°, and 40°C. from 100 to 2,000 lb./sq.in.abs. Retention volume data over the same pressure and temperature ranges were taken for ethane, propane, and n‐butane at essentially infinite dilution in the methane‐silica gel system.

Gas-Liquid Chromatography of Carbohydrate Derivatives

This chapter discusses the general methods and basic principles of gas–liquid chromatography. The development of the method as applied to the various classes of carbohydrate derivatives is surveyed, and this treatment is followed by a discussion of the mobilities of compounds in relation to their structure. Applications of the method to specific problems are described, and the liquid phases used in the gas–liquid chromatography of carbohydrate derivatives are summarized. An attempt is made to assess the relative merits of gas–liquid chromatography and other chromatographic methods as applied to carbohydrate derivatives. The greater resolving power and rapid separations are the distinct advantages of gas–liquid chromatography over other chromatographic techniques. Analysis by gas–liquid chromatography also avoids the errors inherent in paper chromatographic methods involving the elution of components from the paper and their estimation by chemical or colorimetric methods. The results of gas–liquid chromatography are presented as tabulated retention-times, relative to a standard, or by the reproduction of the recorder tracing.

Radioassay by Gas Chromatography of Compounds labelled with Carbon-14

AN efficient device for trapping materials from the effluent of a gas chromatography column has been developed which facilitates collection of components labelled with carbon-14 in close proximity to an efficient scintillator. Collection of components from aliquot s of carrier gas into separate traps for subsequent counting affords a sensitivity of carbon-14 assay limited only by the high sensitivity of liquid scintillation counting technique. Continuous scintillation counting of a single trap through which the column effluent flows permits the high resolution and convenience afforded by simultaneously recording component elution and radioactivity.