Chromatographic fractionation of Aspergillus endotoxins

Summary Crude preparations of the endotoxins extracted from the mycelia ofAspergillus fumigatus andAspergillus flavus, after preliminary concentration by ammonium sulfate, have been fractionated by column chromatography on DEAE-cellulose. Although the biological activity of the chromatographed preparations was not limited to a single fraction, examination of the most active fractions by starch gel electrophoresis showed no bands common to the two nephrotoxins.The highest hemolytic and toxic activities of the fumigatus toxin were found in different fractions of the chromatographed material, and starch gel electrophoresis showed no bands common to these two fractions.The molecular weight of the flavus toxin has been estimated to be in the range of 32,000 to 34,000 as judged by the results of ultracentrifugation and microelectrodialysis in starch gels of the most toxic fractions.Both of the toxins have been shown to contain small amounts of hexosamine and larger amounts of non-amino sugars.Presented in part at the 46th Annual Meeting of the American Society of Biological Chemists, Atlantic City, New Jersey, April, 1962.     

Chromatographic fractionation of histones on Bio-Gel P.

Chromatography of histones on columns of polyacrylamide beads (Bio-Gel P) was studied to determine what factors influence separations. The fractionation achieved on eluting with dilute HCl depends on the HCl concentration, the temperature, and a variable characteristic of the gel which is independent of its pore size and mesh. Somatic histone will be eluted in three, four, or five separate fractions, depending on these variables. Retardations of histones that apparently result from interactions between their charged groups and those of the gel have a strong influence on resolution between different types. Certain histones are retarded more than others when the HCl concentration or temperature is lowered or if the gel has been hydrolyzed. Increases in retardation lead to resolution of more fractions; but if too extreme, coelution of the most retarded fractions will occur.     

Chromatographic fractionation of Escherichia coli transfer RNA on a new support, naphthoyl-Sepharose.

The noncharged naphthoyl-Sepharose CL-6B has been prepared. Escherichia coli tRNA binds to this new adsorbent in 0.75 M ammonium sulphate at neutral pH at room temperature. Using a negative salt gradient, the tRNAs are eluted in a defined order. The chromatographic pattern is clearly different from those of other commonly used tRNA separation techniques.     

Acid-labile amino acid precursors in the Murchison meteorite. 1: Chromatographic fractionation.

The amino acid content of a hot water extract of the Murchison meteorite can be increased by over 100 per cent by subjecting the extract to acid hydrolysis. The acid-labile compounds in the extract that account for this increase were fractionated by column chromatography on a cation exchange resin. Seventy mole per cent behaved as neutral or acidic compounds and were eluted from the column with an initial water wash. The remaining 30 mole per cent (basic precursors) were retained on the column and were eluted with the free amino acids by aqueous NH4OH. The acid-labile amino acid precursors in the water eluate could be retained and further fractionated on an anion exchange column, indicating that they are acidic compounds.     

13C isotope fractionation during rhizosphere respiration of C3 and C4 plants

Stable carbon isotopes are used extensively to partition total soil CO₂ efflux into root-derived rhizosphere respiration or autotrophic respiration and soil-derived heterotrophic respiration. However, it remains unclear whether CO₂ from rhizosphere respiration has the same δ¹³C value as root biomass. Here we investigated the magnitude of ¹³C isotope fractionation during rhizosphere respiration relative to root biomass in six plant species. Plants were grown in a carbon-free sand-perlite medium inoculated with microorganisms from a farm soil for 62 days inside a greenhouse. We measured the δ¹³C value of rhizosphere respiration using a closed-circulation 48-hour CO₂ trapping method during 40~42 and 60~62 days after sowing. We found a consistent depletion in ¹³C (0.9~1.7‰) of CO₂ from rhizosphere respiration relative to root biomass in three C₃ species (Glycine max L. Merr., Helianthus annuus L. and Triticum aestivum L.), but a relatively large depletion in ¹³C (3.7~7.0‰) in three C₄ species (Amaranthus tricolor L., Sorghum bicolor (L.) Moench and Zea mays L. ssp. mays). Overall, our results indicate that CO₂ from rhizosphere respiration is more ¹³C-depleted than root biomass. Therefore, accounting for this ¹³C fractionation is required for accurately partitioning total soil CO₂ efflux into root-derived and soil-derived components using natural abundance stable carbon isotope methods.     

The significance of cytochrome c redistribution during the subcellular fractionation of rat liver

1. The redistribution of mitochondrial cytochrome c during homogenization and subcellular fractionation of the liver was studied. Chromatographically homogeneous (14)C-labelled cytochrome c was added in different amounts to liver suspensions immediately before homogenization and the adsorption of radioactivity was determined in cytochrome c fractions extracted at pH4.0, first with water and then with 0.15m-sodium chloride. 2. The soluble cytochrome c remaining in the cell sap after subcellular fractionation was 7% of the calculated amount of cytochrome c passing through a soluble form during the whole process. The total amount of cytochrome c released in a soluble form and subsequently redistributed was 25-30% of the total liver cytochrome c. 3. In the standard microsomal fraction the cytochrome c extracted with water originated entirely from redistribution whereas that extracted with 0.15m-sodium chloride was 80% endogenous. In the mitochondrial fraction both cytochrome c pools were truly endogenous, so that practically none of the mitochondrial cytochrome c released to the soluble cell sap was readsorbed by the mitochondria. 4. These results support our former hypothesis that the cytochrome c extracted with 0.15m-sodium chloride at pH4.0 from the standard microsomes represents the cytochrome c newly synthesized in situ, since it does not originate from redistribution. However, the microsomal pool extracted with water cannot be an intermediate in the postulated transfer of cytochrome c from the microsomal particles to the mitochondria, since this pool arises from redistribution of mitochondrial cytochrome c.     

Role of the low-affinity binding site in electron transfer from cytochrome C to cytochrome C peroxidase

The interaction of yeast iso-1-cytochrome c (yCc) with the high- and low-affinity binding sites on cytochrome c peroxidase compound I (CMPI) was studied by stopped-flow spectroscopy. When 3 microM reduced yCc(II) was mixed with 0.5 microM CMPI at 10 mM ionic strength, the Trp-191 radical cation was reduced from the high-affinity site with an apparent rate constant >3000 s(-1), followed by slow reduction of the oxyferryl heme with a rate constant of only 10 s(-1). In contrast, mixing 3 microM reduced yCc(II) with 0.5 microM preformed CMPI *yCc(III) complex led to reduction of the radical cation with a rate constant of 10 s(-1), followed by reduction of the oxyferryl heme in compound II with the same rate constant. The rate constants for reduction of the radical cation and the oxyferryl heme both increased with increasing concentrations of yCc(II) and remained equal to each other. These results are consistent with a mechanism in which both the Trp-191 radical cation and the oxyferryl heme are reduced by yCc(II) in the high-affinity binding site, and the reaction is rate-limited by product dissociation of yCc(III) from the high-affinity site with apparent rate constant k(d). Binding yCc(II) to the low-affinity site is proposed to increase the rate constant for dissociation of yCc(III) from the high-affinity site in a substrate-assisted product dissociation mechanism. The value of k(d) is <5 s(-1) for the 1:1 complex and >2000 s(-1) for the 2:1 complex at 10 mM ionic strength. The reaction of horse Cc(II) with CMPI was greatly inhibited by binding 1 equiv of yCc(III) to the high-affinity site, providing evidence that reduction of the oxyferryl heme involves electron transfer from the high-affinity binding site rather than the low-affinity site. The effects of CcP surface mutations on the dissociation rate constant indicate that the high-affinity binding site used for the reaction in solution is the same as the one identified in the yCc*CcP crystal structure.     

Localization of cytochrome C oxidase and cytochrome C peroxidase in mitochondria of Hymenolepis diminuta (Cestoda)

The intramitochondrial localization of cytochrome c oxidase and cytochrome c peroxidase in adult Hymenolepis diminuta was investigated. Mitochondria were fractionated into inner membrane, outer membrane, intermembrane space and matrix and the efficacy of fractionation was monitored employing marker enzymes. Cytochrome c oxidase was associated with the mitochondrial inner membrane. Whereas 55% of the cytochrome c peroxidase activity was in the matrix, 32% of the activity was in the intermembrane space fraction. Based upon the distribution of marker enzymes, a dual compartmentalization of cytochrome c peroxidase is apparent in H. diminuta mitochondria.     

Chromatographic fractionation of yeast extract: A strategy to identify physicochemical properties of compounds promoting CHO cell culture

The study proposes to get a better knowledge of the physicochemical properties of yeast extract (YE) molecules involved in the improvement of CHO cell growth and to reduce YE complexity without losing positive effects. Various chromatographic processes were implemented for fractionation of a nanofiltrated YE (nYE). The nYE was first fractionated by one-step preparative chromatography, either with anion exchange (AEC), hydrophobic interaction (HIC) or size exclusion (SEC) methods. After analysis of its main components, each fraction was added in a control chemically defined medium to assess its impact on CHO cell growth. Results mainly underlined that AEC was the most selective separation process to purify nYE in one step without decreasing cell growth promoting effect. A three-step chromatographic process including successive AEC, HIC, and SEC was then developed to refine the physicochemical properties of nYE compounds. Among fractions that triggered similar cell growth promoting effect than nYE, one also improved IgG specific production. It mainly included cationic and hydrophilic peptides with a great proportion of lysine and arginine, low quantities of polysaccharides and no nucleic acids. Thus, this study allowed us to deepen the YE contribution to animal cell culture as well as to evaluate fractionation strategies to simplify such a complex mixture.