The electrophoresis of histones in polyacrylamide gel and their quantitative determination

1. A new method has been devised for the separation of the histone fractions of calf thymus by electrophoresis in polyacrylamide gel at pH2.4. 2. The fractions have been characterized by their relative mobilities with respect to a marker protein, bovine plasma albumin. 3. A method has been developed for the quantitative determination of the separated histone fractions by measuring the colour yields of dye-histone complexes formed in the gel.     

Chromosomal proteins of Drosophila melanogaster and an approach for their localisation on polytene chromosomes

Chromosomal proteins have been prepared from embryos of Drosophila melanogaster and separated into histone and nonhistone fractions by a procedure which completely avoids exposure to extremes of pH. These fractions have been characterised by amino acid analysis and gel electrophoresis. Antisera have been prepared against whole chromatin and against the two chromosomal protein fractions. — A new method is described for the preparation of Drosophila salivary chromosomes. This method employs microdissection techniques and completely avoids the use of acid fixatives. Preservation of fine structure in these preparations is comparable to, if not better than, that in classical acid-fixed preparations. Antisera against embryo chromatin and chromosomal protein fractions react with the salivary chromosome preparations. These reactions exhibit selectivity with different chromosomal structures. Evidence is presented suggesting a specific distribution of protein antigens along the chromosome.     

Two subforms of eukaryotic topoisomerase I. Purification and structure-function relationships.

A new method for isolation of eukaryotic topoisomerase I from calf thymus and from Jurkat-1 cells using HPLC has been developed. The method allows quantitative purification of high molecular weight topo I and two low molecular weight fractions differing by their isoelectric points. It has been suggested that these fractions be characterized as two subforms of the enzyme possessing structural and functional differences. The differences in their specific activities, sensitivity to camptothecin and in their proteolytic digestion maps have been demonstrated for the two enzymes.     

Countercurrent-distribution studies on histones

1. The possibilities of fractionating histones and histone fractions by means of countercurrent distribution between two phases formed by water and butan-2-ol, in the presence of various concentrations of trichloroacetic acid, have been examined. 2. Although the principal histone fractions differ considerably in their partition ratios, a satisfactory resolution of the principal histone fractions from the whole histone has not been achieved. 3. The histone fractions obtained by other methods can be resolved with suitable concentrations of trichloroacetic acid. Besides the main peak several subsidiary peaks are obtained in most cases, the composition of which corresponds with others of the main fractions. 4. The method is therefore capable of removing from the principal fractions as previously prepared contamination by other fractions. 5. Except in one case, no fraction with composition unlike other fractions has been obtained. In several cases the material isolated from the principal peak behaves as a single component on running again. In two cases fractions with similar compositions were distinguished by countercurrent distribution.     

Use of isoelectric focusing in studying the composition of allergens from mites

The method of the isoelectric separation of allergens isolated from Dermatophagoides ticks with the use of isoelectrofocusing has been improved. 10-12 protein fractions with isoelectric points from 3.50 to 6.55 have been isolated. Allergens obtained from D. farinae and D. pteronyssinus grown on two different nutrient media have been found to contain common protein components. In D. pteronyssinus allergen the presence of a greater number of protein fractions has been noted in the region of pH 5.25-6.00 than in D. farinae allergen.     

Assay of proteins by Lowry's method in samples containing 2-mercaptoethanol

A rapid method of screening chromatographic fractions has been developed for enzymes which metabolize fluorogenic substrates. Samples of the eluted fractions are applied to cellulose acetate gels and then incubated with the specific fluorogenic substrate. Fractions which possess enzymatic activity are visible as fluorescent spots when the gels are examined under long-wave ultraviolet light.     

Isolation of a synaptic junction-enriched fraction from the forebrain of day-old chickens

Synaptosomal plasma membrane (SPM) and other subcellular fractions were isolated from the forebrain of 1-day-old chickens by a procedure based on that of Davis and Bloom (16) and Cotman and Taylor (13). The procedure involves the centrifugation through a discontinuous sucrose gradient of a crude synaptosomal-mitochondrial fraction which has been lysed and weighted with iodonitrotetrazolium. SPM isolated by this method contains only small amounts of lysosomal or mitochondrial membranes and is practically devoid of contaminating microsomal membranes, as estimated by enzyme marker assays. The purity of chick-brain SPM prepared by this method is compared to the purity of chickbrain fractions obtained by two other laboratories, using different methods (4, 59). The SPM were extracted with Triton X-100 and all fractions solubilized in sodium dodecyl sulfate (SDS). The delipidated proteins of all fractions were subjected to SDS-polyacrylamide electrophoresis on slab gels and stained for protein. A distinct difference was observed between the patterns given by the Triton-soluble and-insoluble fractions. Electron microscopy of the synaptic junction fraction showed numerous junctional complexes.     

Separation and identification of ceramides derived from human plasma sphingomyelins

Sphingomyelins from human blood plasma have been converted into ceramides by enzymatic hydrolysis with phospholipase C. After acetylation the ceramides were fractionated by thin-layer chromatography on silica gel containing silver nitrate. Four main fractions obtained by this method were subsequently converted to di-O-trimethylsilyl ether derivatives and separated by gas-liquid chromatography on 1% OV-1. 2-11 components could be distinguished in each of the four fractions. The major fractions emerging from the gas chromatograph were analyzed by mass spectrometry and their main molecular species were identified. Two of the gas chromatographic fractions contained essentially pure molecular species, namely N-tetracosenoyl sphingosine and N-tetracosenoylsphinga-4, 14-dienine.     

A small-scale method for the isolation of insulin-containing secretory granules from islets of Langerhans

A method has been developed which uses small-scale (400 microliter) Percoll gradients and an inexpensive bench-top microcentrifuge for the rapid isolation of insulin-containing secretory granules from islets of Langerhans available from a single rat pancreas. Granule fractions were prepared from homogenates of isolated rat islets by a differential centrifugation step (10 min) to produce a granule-enriched membrane pellet, followed by a further centrifugation (10 min) on a discontinuous Percoll gradient to produce a granule fraction. Measurement of membrane-marker enzyme activities suggested that the yield and purity of granule fractions prepared by this method were comparable to those reported for other methods involving longer centrifugation times in ultracentrifuges. Further purification of the granule fractions by removing lysosomal contamination was achieved by an additional centrifugation (10 min) on another small-scale gradient of higher Percoll concentration. The method proved useful for isolating biosynthetically labeled secretory granule membranes and contents from islets of Langerhans which had been cultured in the presence of 35S-labeled amino acids. The speed and simplicity of this method suggest that it will prove useful in studies requiring the rapid isolation of insulin-containing secretory granules from isolated islets.     

A rapid method for the fractionation of isolated hepatocytes

The fractionation of rat liver hepatocytes using a mechanical disruption technique followed by centrifugation is reported; the whole procedure requires approximately 10 min. Marker enzyme distribution data are in good agreement with distribution data from standard techniques connected with the production of three subcellular fractions—cytoplasmic, mitochondrial, and microsomal. Electrophoretic analysis of the mitochondrial and microsomal fractions show total band correspondence between the fractions produced by the method and traditional techniques. Examination of the fractions by electron microscopy supports the view that the mitochondrial fraction is comprised of both intact mitochondria and mitochondria from which the outer membrane has been removed. The microsomal fraction contains discrete vesicles derived from both rough and smooth endoplasmic reticulum.     

Thin layer gel chromatography of dextrans

Microgram quantities of soluble dextran fractions have been separated by thin layer gel chromatography. The dextrans are first combined with a triazine dye to render them visible during the chromatography and to facilitate the densitometric evaluation of the chromatograms. The method allows the determination of dextran molecular weights up to approximately 100 000 and can also be used in studying the polydispersity of the fractions.     

Fractionation of Neisseria allergenic preparations by a gel chromatographic method on sepharose 4B

The fraction composition of allergens obtained by different methods from the microbial mass of N. meningitidis, N. gonorrhoeae and N. perflava has been studied. Each method produced its characteristic number of fractions, irrespective of the Neisseria species used. Their molecular weights: more than 2,000,000 daltons, 160,000 daltons, 31,000 daltons and 14,000 daltons. All these fractions are biologically active.     

A Labile-Bound Component of Phosphate in the Free Space of Sunflower Plant Roots

An experimental method is described by which it is possible to follow continuously the uptake and release of certain radioactive isotopes and the uptake of water by the roots of young plants. The method has been used in a study of the ion fractions related to the initial uptake of phosphate in the roots of sunflower plants. Two non-bound fractions were identified: one which was leachable in distilled water and another which was released from the roots only by a bathing medium containing inactive phosphate. Experiments using conventional analysis technique corroborated the results obtained by the recording technique.     

Automated determination of DNA using the fluorochrome Hoechst 33258

An automated method for the determination of DNA content in fractions from the alkaline filter elution assay of DNA damage has been developed. DNA-containing fractions are mixed with a fluorochrome (Hoechst 33258) and the DNA concentration is measured fluorometrically in a continuous-flow system. The lower limit of detection is 0.05 micrograms DNA/ml, and the linearity range under the conditions used is 0-8 micrograms DNA/ml. The standard deviation (n = 10) was found to be +/- 0.83%. The results are compared with the manual method.     

The effect of organic solvents on the equilibrium position of enzymatic acylglycerol synthesis

The effect of organic solvents on the equilibrium position of lipase-catalyzed esterification of glycerol and decanoic acid has been investigated. The reaction is carried out in an aqueous-organic two-phase system. In polar solvents, high mole fractions of monoacylglycerol and low mole fractions of triacylglycerol and measured, while in nonpolar solvents, the measured differences in the mole fractions of monodi-, and triacylglycerols are less. There is a good correlation between the ester mole fractions at equilibrium and the log P of the solvent (partition coefficient in n-octanolwater), however, only if the group of tertiary alcohols is excluded. In the plot of the easter mole fractions as a function of the logarithm of hte solubility of water in the organic solvent, the tertiary alcohols can be included; however, in this case other deviations appear.For the prediction of the effect of organic solvents on the ester mole fractions at reaction equilibrium in nondilute reaction systems with a water activity below 1, the program TREP (Two-phase Reaction Equilibrium Prediction) is developed, which is based on the UNIFAC group contribution method. With this model the equilibrium data are essentially predicted from basic thermodynamic data. The required equilibrium constants are estimated from experiments without an organic solvent in the reaction medium. The mole fractions calculated by TREP show the same trends as the experimentally measured mole fractions; however, some variation is observed in the absolute values. These deviations may be due to inaccuracies in the UNIFAC group contribution method. TREP is found to be a correct method to predict within some limits the ester mole fractions at equilibrium for all mixtures of solvents, substrates, and products. The production of monoester can be enhanced in reaction system with a sufficient high concentration of a polar solvent. In experiments with a triglymeto-decanoic acid ratio of 5, almost no di-and triesters can be detected at equilibrium. (c) 1993 John Wiley & Sons, Inc.     

Fractionation of L-cell chromatin into DNA, RNA, and protein fractions on Cs2SO4 equilibruim density gradients

A new procedure is described for fractionation of chromatin into DNA, RNA, and total chromatin protein. By isopycnic gradient centrifugation of chromatin preparations in Cs₂SO₄ solutions containing dimethylsulfoxide and sodium sarcosyl it is possible to obtain highly purified fractions of these components. The method gives a very high yield of these chromatin fractions unlike some other methods, where irreversible binding to columns occurs. Also with this method it is possible to obtain highly concentrated fractions, which after a simple dialysis step, can be conveniently analyzed by polyacrylamide gel electrophoresis.Nuclei from L-929 cells were isolated by a method involving citric acid or by a method using a nonionic detergent. The yields of DNA obtained by both methods was compared. Chromatin was isolated from purified nuclei (prepared in either of the above ways) in two different ways also. In one method, chromatin was extracted from nuclei with 1 m NaCl. A second method involving fractionation of lysed nuclei in sucrose and metrizamide solutions was also used. The yields of DNA obtained by both methods was compared. There appears to be little nuclear membrane contamination of any of these chromatin preparations.A preliminary analysis of L-929 cell chromatin total RNA and protein fractions on polyacrylamide and agarose gels has been made. Both fractions appear to be quite complex with a wide spectrum of subcomponents of differing S values.     

The involvement of the plasma membrane in the development of Dictyostelium discoideum. I. Purification of the plasma membrane

A method for the isolation and purification of plasma membranes of Dictyostelium discoideum by equilibrium centrifugation on sucrose followed by Renografin continuous density gradients has been developed and monitored both with electron microscopy and a number of enzyme assays. On electron microscopy, the final plasma membrane fractions are judged to be freethe basis of of nuclei, rough endoplasmic reticulum, lysosomes and peroxisomes. Some profiles of the mitochondrial inner membranes are found within the plasma membrane fractions, but this contamination has been estimated to be only 5%. On the basis on enzyme assays, the plasma membrane fractions contain all the 5'-nucleotidase activity in the final gradients and are free of catalase, acid phosphatase and malate dehydrogenase activity (markers for peroxisomes, lysosomes, soluble enzymes and the matrix of mitochondria). Their content of glucose-6-phosphatase is reduced by more than 70%. The large majority of RNA and DNA have been removed from the preparation.     

Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure.

Plant cell transformation by Agrobacterium tumefaciens involves the transfer of a single-stranded DNA-protein complex (T-complex) from the bacterium to the plant cell. One of the least understood and important aspects of this process is how the T-complex exits the bacterium. The eleven virB gene products have been proposed to specify the DNA export channel on the basis of their predicted hydrophobicity. To determine the cellular localization of the VirB proteins, two different cell fractionation methods were employed to separate inner and outer membranes. Seven VirB-specific antibodies were used on Western blots (immunoblots) to detect the proteins in the inner and outer membranes and soluble (containing cytoplasm and periplasm) fractions. VirB5 was in both the inner membrane and cytoplasm. Six of the VirB proteins were detected in the membrane fractions only. Three of these, VirB8, VirB9, and VirB10, were present in both inner and outer membrane fractions regardless of the fractionation method used. Three additional VirB proteins, VirB1, VirB4, and VirB11, were found mainly in the inner membrane fraction by one method and were found in both inner and outer membrane fractions by a second method. These results confirm the membrane localization of seven VirB proteins and strengthen the hypothesis that VirB proteins are involved in the formation of a T-DNA export channel or gate. That most of the VirB proteins analyzed are found in both inner and outer membrane fractions suggest that they form a complex pore structure that spans both membranes, and their relative amounts in the two membrane fractions reflect their differential sensitivity to the experimental conditions.     

The involvement of the plasma membrane in the development of Dictyostelium discoideum. I. Purification of the plasma membrane

A method for the isolation and purification of plasma membranes of Dictyostelium discoideum by equilibrium centrifugation on sucrose followed by Renografin continuous density gradients has been developed and monitored both with electron microscopy and a number of enzyme assays. On the basis of electron microscopy, the final plasma membrane fractions are judged to be free of nuclei, rough endoplasmic reticulum, lysosomes and peroxisomes. Some profiles of the mitochondrial inner membranes are found within the plasma membrane fractions, but this contamination has been estimated to be only 5%. On the basis on enzyme assays, the plasma membrane fractions contain all the 5′-nucleotidase activity in the final gradients and are free of catalase, acid phosphatase and malate dehydrogenase activity (markers for peroxisomes, lysosomes, soluble enzymes and the matrix of mitochondria). Their content of glucose-6-phosphatase is reduced by more than 70%. The large majority of RNA and DNA have been removed from the preparation.     

Use of immunological adsorption in heterogeneous immunoenzyme analysis in determining antibodies to the protective determinants of Bacillus anthracis

In a heterogeneous enzyme immunoassay system involving the use of polystyrene assay plates, the method of immunological adsorption has been used for studying the spectrum of specific antibodies to individual chromatographically pure fractions of B. anthracis toxin. The relationship between the characteristics of acquired stability and the level of serum antibodies to individual biologically active and biologically inactive toxin antigens in guinea pigs, immunized with live vaccines in a single injection, has been studied. As revealed in this study, the level of serum antibodies to chromatographically pure toxin fractions does not reflect acquired immunity to anthrax.