S fractions

Many enzymes are soluble and will be recovered in the S-100 fraction. In addition, their activity can be measured rather conveniently by adding a sample of the S-100 fraction directly to a reaction mixture. Of course, since the S-100 fraction will contain an excessive amount of extraneous protein, it will be necessary to terminate the reaction and filter the sample prior to injecting it onto the HPLC for analysis. 

Note that while the S-100 fraction can be used in this form, it would be best to have it dialyzed to remove unwanted low molecular weight compounds before using it in an assay. In addition to dialysis, a simple salting out can be performed to remove some of the extraneous protein material. Ammonium sulfate is often added for this purpose to remove unwanted proteins or to precipitate the enzyme in question. Any ammonium sulfate should be removed before the sample is used in an assay, because the salt might affect activity. Again, dialysis can be used, or alternatively the sample can be passed through a gel filtration (G-25) column. 

Finally, HPLC can be used as an analytical method to monitor the efficiency of different purification methods. For example, imagine that gel filtration chromatography has just been carried out on a sample of an S-100 fraction. Several peaks are observed. A question usually asked at this point relates to the homogeneity of each of the peaks. HPLC is ideal to answer this question, since it allows each peak to be analyzed in just a few minutes. Also, different columns can be used for the analysis, and therefore the homogeneity of any peak can be verified under a variety of conditions. 

As another example, imagine that an S-100 fraction has been prepared, ammonium sulfate has been added, and the fraction that precipitates between 30 and 50% has been obtained. This sample is subjected to affinity and ion-exchange chromatography. Have these procedures been successful in removing extraneous proteins While it is possible to answer this question by a determination of total protein content, it may be more informative to analyze each of the samples for its constituent proteins. 

Figure 9.95 HPLC elution profiles of an adenylate deaminase incubation mixture. The reaction mixture contained 15 /xmol of imidazole-HCl (pH 6.8), 250 nmol of FoMP, 250 nmol of ATP, and 5 /u,mol of KCl in a final volume of 250 fiL. The reaction was initiated by the addition of activity obtained from the S-100 fraction and incubated at 37°C. At 10-minute intervals, 25 fiL samples were injected onto the HPLC column. There is a decrease in the FoMP peak (retention time, 1.7 min) and a significant rise in the peak corresponding to FoIMP (retention time, 3.1 min) up to 30 minutes. Inset Graphical representation of the first 30 minutes of the reaction. (From Jahngen and Rossomando, 1984.)...

The activity was prepared from the microorganism Dictyostelium dis-coideunt. Cells were lysed, and an S-100 fraction was prepared and used as the source of the deaminase activity. 

Escherichia coli K12 ML1629, . coU K12 ML1410 R81, and E. coli K12 J5 Rll-2 showed similar resistant patterns to aminoglycosidic antibiotics, and the S-100 fraction contained kanamycin-neomycin phosphate transferase I. This enzyme solution also inactivated lividomycins A and B (6 and 7) in the presence of adenosine 5 -triphosphate. A crude powder of the inactivated lividomydn A extracted by column chroma-... 

Although it only stimulates the reaction in a fresh extract, creatine phosphate is essential for activity after frozen storage. Reticulocyte lysate stimulates both fresh and frozen extracts but, alone, will not rescue activity in a frozen extract. An equivalent effect on activity is seen whether whole reticulocyte lysate or an S-100 fraction (which is depleted of ribosomes) is used, indicating that the reticulocyte ribosomes contribute little if anything to translation. 

For labelled S-phase fraction, both compounds and all treatments of cell number was reduced, comparing to control, as expected to Topi mechanism of action. Cytostatic effects were found for unlabeled cells as show in normalized curves (Figure 8) with a progressive cytotoxicity at a longer time (48-72h) starting to 100 nM for edotecarin and 300 nM for SN-38. Cytotoxic effects... 

The di-, tri-, and sulfallates were classified as potent mutagens in the Ames assay with TA 100 and TA 1538 (base-pair substitution mutants), with metabolic activation required (1978). More recent in vitro testing (1981) of di- and triallate with S. typhimurium (strains TA1535.-100, and -98) showed dose-related increases without metabolic activation, but greatly enhanced mutagenicities in the presence of Aroclor 1254-induced rat liver S-9 fraction. These tests indicated that both the di- and triallates can induce base pair substitution and frame shift mutations. 

Sulfur, carbon and hydrogen stable isotope ratios of pyrite, kerogens, and bitumens of two high-sulfur Monterey formation samples from the onshore Santa Maria Basin in California were determined. Kerogens from these were pyrolyzed at 300°C for periods of 2, 10 and 100 hours in closed systems and the yields and isotopic compositions of S-containing fractions (residual kerogens, bitumens and hydrogen sulfide) were determined. 

DNA was isolated from mouse skin (9,10) and cells in culture (11-13) exposed to [%]- or [ C]-DMBA, or recovered from mixtures containing calf thymus DNA, [3h]-DMBA, cofactors and either Aroclor-induced rodent liver microsomes (14-17), S-9 fraction (16-18) or 12 h DMBA-induced (100 nmoles in 0.1 ml benzene) mouse... 

The quality of enantioselective reactions is numerically expressed as the so-called enantiomeric excess (ee). It is equal to the yield of the major enantiomer minus the yield of the minor enantiomer in the product whose total yield is normalized to 100%. For example, in the Sharpless epoxidation of allyl alcohol (see Figure 3.7). S - and R-glycidol are formed in a ratio of 19 1. For a total glycidol yield standardized to 100%, the S-glycidol fraction (95% yield) thus exceeds the /f-glycidol fraction (5% yield) by 90%. Consequently, S-glycidol is produced with an ee of 90%. 

S-30 fraction from wheat germ—commercial (Fisher catalog L-4440) or freshly prepared (see Appendix) supplemented with 125 mM ATP (potassium salt), 2.5 mM GTP (potassium salt), 100 mM creatine phosphate, and 0.5 mg/ml creatine Reaction buffer (300 mM HEPES, 30 mM dithiothreitol [pH 7.1])... 

To the dialyzed, cloudy enzyme solution an equal volume of 1 M KPi 7.75) was added to further precipitate the urea-soluble proteins. The suspension was centrifuged and the pellet was collected and redissolved in ca. 130 ml of 6 M urea, 0.03 M NaCl, 16 mM KPi (pH 7.8) buffer prior to being loaded on a Sephacryl S-2(X) column previously equilibrated with 6 M urea, 0.03 M NaCl, 16 mM KPi (pH 7.8). The column was eluted with the same buffer and fractions were collected. TTiose containing enzyme activity or having an enriched protein species of 32 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (16) were pooled and concentrated. The concentrated eluant (20 ml) was applied to a Sephacryl S-100 column previously equilibrated with 6 M urea, 0.03 M NaCl, 16 mM KPi, pH 7.8. The column was eluted with the same buffer, fractions were collected and those containing only 32 kDa (LO) and 24 kDa species were pooled and concentrated. This is referred to as the "two-banded" protein, and was used without further purification (to reduce protein loss) for phenylhydrazine inactivation (see below). 

Suitability of reference elements have been tested by Ackermann (1980) on sediment samples from the Ems River estuary in Northern Germany. Table 3-3 summarizes the correlation coefficient "r between the contents of conservative elements and the percentage of grain size fraction <20 um and <63 um, respectively, and the slope of the regression line, the quotient s(100%)/s(0%) from the ordinate values extrapolated... 

The antimutagenic effects of ajoene were investigated by the Ames test. Ajoene inhibited mutagenesis induced by both benzo[a]pyrene (B[a]P) and 4-nitro-l,2-phenylenediamine (NPD) in a dose-dependent manner. In particular, NPD-induced mutagenesis was more effectively suppressed by ajoene than the B[a]P-induced type. Furthermore, the inhibition of mutagenesis by ajoene was more effective for transition-type mutations than for the frame shift type. HPLC analysis of B[a]P metabolism in the presence of the rat liver microsomal fraction (S-9) showed that ajoene dose-dependently inhibited the metabolic activation of B[a]P which suggests that ajoene affected the metabolic enzymes in the S-9 fraction [100]. 

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