Trichloroacetic Acid-soluble Fraction Research Articles

Phosphorus and the growth of euglena gracilis.

SYNOPSIS. Growth of streptomycin‐bleached Euglena gracilis depends in part on the availability of phosphorus. Maximum cell density on a defined medium is reached at a phosphorus (supplied as inorganic phosphate) concentration of 4–5 μ/ml. At lower concentrations, the cells apparently deplete the medium of phosphorus. Inorganic phosphorus at > 1 mg/ml inhibits growth in terms of cell density per ml and generation time. Phosphorus‐limited cells survive for at least 6–7 days and are able to undergo mitosis following a lag period when returned to phosphorus‐containing medium. The majority of inorganic 32P incorporated by these Euglena ends up in the hot trichloroacetic acid soluble fraction and in the ethanol‐ether soluble fraction.

Ultraviolet-induced pyrimidine dimers in tetrahymena: I. Removal in the dark

Thymine-containing pyrimidine dimers have been isolated and identified chromatographically from DNA hydrolysates of ultraviolet-irradiated Tetrahymena. Removal of these photoproducts has been observed during post-irradiation growth of cells in the dark. After 47 h, about 83 % of the pyrimidine dimers disappear from the trichloroacetic acid-insoluble fraction. Further analysis of both the trichloroacetic acid-insoluble and trichloroacetic acid-soluble fractions indicated that photoproducts accumulating in the soluble fraction are eventually lost from the cytoplasm of irradiated cells.

麹酵素による蛋白質の分解(第1報)

Oryzenin, the principal protein of milled rice, was fractionated into 3 fractions by Sepharose 4B gel filtration. The hydrolysis of oryzenin by the acid protease prepared from Taka-diastase was examined at several pH; 3.0, 4.0 and 6.0. (1) Optimum pH of the acid protease for the hydrolysis of oryzenin was found to be near 3.0. (2) Ratios of lower molecular peptides (phosphotungstic acid-soluble fraction) to higher molecular peptides (trichloroacetic acid-soluble fraction) liberated from oryzenin by the enzyme were determined occasionally during the long time reactions and it was found that the ratios were higher when reacted at pH 4.0 or 6.0 than at pH 3.0. (3) Elution patterns of the protein fractions of the reactants from Sepharose 4B column were obtained at times during the reactions. The gel filtration patterns were somewhat different depending on the reaction pH; Fraction I (eluted at void volume and considered to be the largest molecular fraction) disappeared rapidly and completely when reacted at pH 4.0 and the disappearance of Fraction II (eluted succesive to Fraction I) was comparatively rapid at pH 3.0. (4) Lower molecular fractions of the reactants were occasionally fractionated by Sephadex G-15 gel filtration during the reactions. It was also found that the gel filtration patterns were somewhat different depending on the reaction pH; When the protein was reacted at pH 3.0, there appeared several peaks in the gel filtration pattern between the high molecular fraction (void volume) and the small molecular fraction (eluting out volume)mainly consisted of free amino acids presumably, while these peaks were not appeared at all when the protein was reacted at pH 4.0 or 6.0. (3) Amino acid liberation patterns were again different depending on the reaction pH; Amounts of the liberated amino acids were larger when reacted at pH 4.0 or 6.0 than at pH 3.0.

Adaptation de la vitesse de biosynthèse de l'amylase pancréatique et du chymotrypsinogène a des régimes riches en amidon ou en protéines

1. 1. Previous investigations in this laboratory have shown that the amounts of amylase in rat pancreas and pancreatic juice are 3–4 time higher, and the amounts of chymotrypsinogen 3 times lower, when the animals receive a starch-rich diet (diet G) rather than a protein-rich diet (diet P). With these variations are associated similar variations in the relative rates of biosynthesis of the enzymes (radioactivity incorporated into the enzymes versu radioactivity incorporated under the same conditions into the total proteins of pancreas). The specific radioactivity of the precursor (labelled valine) has now been evaluated as accurately as possible in order to calculate and to compare the biosynthetic rates themselves in pancreas G and P. 2. 2. Two series of experiments have been carried out, the first using intravenous injection of labelled precursor into rats G and P in vivo, and the second using incubation of slices of pancreas G and P in the presence of precursor. In both cases, the specific radioactivity of the precursor between 2–15 min in the trichloroacetic acid-soluble fraction of the homogenates and the radioactivity incorporated after 5, 10 and 15 min into pure amylase and chymotrypsinogen have been determined. The evaluation of the magnitude of the adaptation effect is greatly facilitated by the fact that the specific radioactivity of the precursor is always about the same in pancreas G and P. 3. 3. Radioactivity measurements on pancreatic juice samples collected from injected animals, and chase experiments with pancreas slices, have shown that no loss of labelled enzymes by secretion and degradation is likely to occur during the assays. 4. 4. As a result, it may be concluded that the ingestion of diet G, as compared with diet P, induces an 8-fold increase in the rate of biosynthesis of amylase and a 2-fold decrease in that of chymotrypsinogen. The fact that the results obtained with pancreas slices are similar to those obtained in vivo suggests that the alimentary stimulus determines a specific modification in the biosynthetic system of pancreas acinar cells. 5. 5. Finally, the experimental values may be used for calculating the number of amylase and chymotrypsinogen molecules synthesized per minute by one acinar cell of rat pancreas.

Role of Acetate in Sporogenesis of Bacillus cereus

Nakata, H. M. (Washington State University, Pullman). Role of acetate in sporogenesis of Bacillus cereus. J. Bacteriol. 91:784-788. 1966.-The distribution of radioactivity associated initially with acetate-2-C(14) was followed during sporogenesis of Bacillus cereus strain T. This was accomplished by replacing cells committed to sporulation into a chemically defined sporulation medium. It was observed that 65 to 70% of the initial radioactivity was incorporated into poly-beta-hydroxybutyrate, whereas 20 to 25% was found in other cellular constituents. Virtually no radioactivity was lost as C(14)O(2) during the first 5 to 6 hr after replacement. Then, a gradual evolution of C(14)O(2) coincident with poly-beta-hydroxybutyrate degradation, was observed until about the ninth hour. By this time, the polymer was essentially depleted, and the first spore structures were observed in stained preparations. The total amount of radioactivity lost as C(14)O(2) was 20 to 25%. The major portion of products derived from poly-beta-hydroxybutyrate was incorporated into the spores. As much as 17% of the radioactivity associated with the spores was found in dipicolinic acid. More than 50% was located in spore proteins, 20 to 25% in the hot 5% trichloroacetic acid-soluble fraction, 4 to 5% in the lipid fraction, and 15 to 20% in the cold 5% trichloroacetic acid-soluble fraction. These data, accounting for 70 to 75% of the initial radioactivity, confirmed the hypothesis that the major role of acetate, and subsequently of poly-beta-hydroxybutyrate, in sporulation of B. cereus T is to provide carbon precursors and energy for sporogenesis.

Correlation of Carbohydrate Catabolism and Synthesis of Macromolecules During Enzyme Synthesis in Pseudomonas fluorescens

Kirkland, Jerry J. (Oklahoma State University, Stillwater), and Norman N. Durham. Correlation of carbohydrate catabolism and synthesis of macromolecules during enzyme synthesis in Pseudomonas fluorescens. J. Bacteriol. 90: 23-28. 1965.-Glucose, ribose, and fructose shorten the lag period required for synthesis of protocatechuate oxygenase. Radioactivity from uracil-2-C(14) is incorporated into the hot trichloroacetic acid-soluble fraction after a lag period of approximately 20 min after addition of protocatechuic acid. Addition of glucose or ribose simultaneously with the inducer shortens the lag period to approximately 5 min and increases the rate of uracil incorporation. The inducer must be present to initiate incorporation of radioactivity, and the exogenous carbon source accelerates incorporation but is not sufficient to initiate synthesis by itself. The addition of protocatechuic acid increases the rate and total incorporation of radioactivity from uniformly labeled glucose or ribose-1-C(14) into the hot trichloroacetic acid-soluble fraction. Ribose decreases the incorporation of radioactivity from uniformly labeled glucose into the hot trichloroacetic acid-soluble fraction, and glucose shows a similar effect on incorporation of radioactivity from ribose-1-C(14), indicating the two sugars are serving in the same capacity to enhance enzyme synthesis. Radioactivity from glucose-1-C(14) is not incorporated into the hot trichloroacetic acid-soluble fraction. The results suggest that glucose and ribose shorten the lag period for inducible enzyme formation by serving as a "specific" carbon source for synthesis of macromolecules such as ribonucleic acid.

EFFECT OF THYROTROPIN ON THE WEIGHT, PROTEIN, RIBONUCLEIC ACID, AND THE RADIOACTIVE PHOSPHORUS UPTAKE OF CHICK THYROIDS.

A single injection of 0.020 U of thyrotropin (TSH) was given 2, 6, 12, 18 or 24 hr before autopsy to day-old chicks. The dry weight of the thyroids and the total thyroidal protein were significantly decreased by 2 hr after TSH stimulation. The total ribonucleic acid (RNA) in the thyroids was significantly increased by 12 hr after TSH administration. The 32P incorporation into the whole thyroid gland, the trichloroacetic acid (TCA) soluble, the ethyl alcohol soluble, the RNA and the TCA precipitate fractions of thyroids was increased by TSH treatment. The greatest and earliest increase was in the TCA soluble fraction, and, as 32P was lost from this fraction, it increased in the other fractions of stimulated glands. The 32P uptake into subcellular components such as nuclei, mitochondria and microsomes was significantly increased by at least 12 hr after TSH injection, but the 100,000 g supernatant of thyroids showed a significantly increased uptake by 2 hr after TSH. This was the most heavily labeled cellula...

Immunologically induced aspermatogenesis in guinea pigs.

The general background of the aspermatogenic syndrome and its immunological basis is summarized as well as recent additional findings on the question of specificity of tissue antigen and of injection route along with the immunochemical characterization of the aspermatogenic factor in the guinea pig. There was no significant difference in the germinal lesions induced by intramuscular and intracutaneous routes of injection of testicular antigen. 100 mg of antigen were less effective in sensitization than 10 mg and humoral antibody titers also decreased rather than increased at higher levels of antigenic stimulation. Acid-extracted brain was capable of inducing lesions similar to that from sensitization by testis. It is concluded that there may be an autoimmune factor common to these 2 tissues.

Amitrole Interference with Adenine-2-[superscript fourteen]C Metabolism by Saccharomyces cerevisiae

Metabolic incorporation of adenine-2-14C by Saccharomyces cerevisiae was examined for alterations produced by 3-amino-1,2,4-triazole (amitrole) and histidine. The radioactivity recovered from cells after a 4-hour incubation was found predominantly in ethanol-insoluble components (nucleic acids and proteins) of untreated cultures. Amitrole inhibited incorporation of 14C into insoluble components. Carbon-labeled imidazoleglycerol phosphate, a precursor of histidine, and labeled imidazoleglycerol accumulated in the ethanol-soluble fraction of amitrole treated cells. Imidazoleglycerol accumulated in the external medium. Histidine prevented 14C incorporation into the hot trichloroacetic acid-insoluble residue (protein), prevented amitrole-induced imidazoleglycerol and imidazoleglycerol phosphate accumulation, and circumvented amitrole inhibition of 14C incorporation into the hot trichloroacetic acid-soluble (nucleic acids) fraction.

Chemical forms of plant and soil iron as influenced by soil moisture

Experiments were conducted to determine whether soil moisture content has an effect on the chemical forms of plant and soil iron. Soybean plants, variety Lee, were grown on Adelanto loam soil under greenhouse conditions. Two different moisture levels, 75 per cent and 120 per cent of the moisture equivalent, were maintained in soil samples placed in individual containers. The same moisture treatments were used for separate soil samples on which the test plants were grown. Soil iron forms were determined in the moisture-treated soil by using different extracting agents. A significant decrease in soil iron extracted with 10−4 M EDTA from soil at the high moisture level was attributed to a relative increase in the free calcium ion. Soybean plants grown under the high moisture level were chlorotic while those under the low moisture level were green in appearance. Plant samples were taken at two stages of growth for subsequent analysis. The chemical analysis of the leaf tissues have shown the presence of equal amounts of total iron and less amounts of water-soluble and “active iron” in chlorotic tissues as compared to non-chlorotic tissues. The difference found between chlorotic and non-chlorotic plants in the amount of iron in the water extract was in the trichloroacetic acid-soluble fraction. The water- and salt-soluble protein nitrogen was approximately the same in chlorotic and non-chlorotic leaves.

Tryptic hydrolysis of paramyosin from invertebrates: Rate and extent of proteolysis

The rate extent of the action of trypsin (EC 3.4.4.4) on Pinna nobilis and Pecten maximum paramyosin were followed by different methods, namely total nitrogen, α-amino nitrogen and arginine content in the trichloroacetic acid-soluble fraction, alkali uptake and N-terminal residues in the digest mixture. The results obtained permi one to say that the number of bonds split typrsin in both cases is approximately half of the susceptible bonds present in the whole molecule. About 80% of the total arginine liberated by the enzyme is free or in an N-terminal position. The kinetics of proteolysis were studied by two different methods and they clearly indicate the existence of at least two and possibly three simulataneous reactions proceedings at different rates. The rate contants suggest that one class of peptide bonds is much more susceptible to attack than others. The results are discussed in the light of the proposed secondary and tertiary structure of paramyosin.

COMPOSITION OF THE SHEATH OF SPHAEROTILUS NATANS.

Romano, Antonio H. (University of Cincinnati, Cincinnati, Ohio) and Joyce P. Peloquin. Composition of the sheath of Sphaerotilus natans. J. Bacteriol. 86:252-258. 1963.-The sheath of Sphaerotilus natans was isolated and subjected to chemical analysis. Isolation of the sheaths was accomplished by incubating cells in the presence of lysozyme and ethylenediaminetetraacetic acid in tris(hydroxymethyl)aminomethane buffer and adding sodium dodecyl sulfate subsequently. Under these conditions, there was complete dissolution of cells. The sheaths, which were left intact by this treatment, were recovered by centrifugation, washed exhaustively, lyophilized, and subjected to analysis. Hydrolysis of the sheath material with 2 n HCl at 100 C resulted in the liberation of reducing sugars amounting to 36% of the dry weight. Amino sugar accounted for 11% of the dry weight. Paper chromatography of hydrolysates showed the presence of glucose and hexosamine. Tests for muramic acid were negative. In addition to carbohydrate, 27% protein and 5.2% lipid were found to be present. Fractionation studies indicated that essentially all of the polysaccharide was associated with a trichloroacetic acid-soluble fraction. The sheath is therefore considered to be a protein-polysaccharide-lipid complex, which is chemically and anatomically distinct from the cell wall and the slime layer. It is hypothesized that this unique structure may be related to the microcapsule found in many gram-negative bacteria, and may represent a structural specialization of this more common structure.

Intracellular distribution of sulfur during the synchronous growth of chlorella pyrenoisoda

Intermittent illumination was used to synchronize cells of Chlorella pyrenoidosa. The degree of synchrony of the cultures was evaluated during three consecutive generations of growth in continuous light following the synchronization procedure. The loss in synchrony, as measured by the coefficient of variation, was only 8.53% for the three generations.The uptake and intracellular distribution of [35S]sulfate was followed during synchronous growth. The total cellular-35S (percentage of cellular dry wt.) remained essentially constant throughout cellular development. However, the percentage 35S (percentage of total cellular-35S) in the total cellular protein, cold trichloroacetic acid extract, and sulfolipid fractions of the cells showed dennite periodism.Between the third and fifth hours of cellular development, during a 14-h synchronous growth cycle, the cold trichloroacetic acid-soluble fraction showed a marked increase in percent 35S while the protein fraction showed a decrease of equal magnitude. The decrease in protein-35S was traced to a decrease in the percentage of protein cystine-cysteine-35S. The percentage of protein methionine-35S increased during this period but not enough to compensate for the decrease in protein cystine-cysteine-35S. Thus, the percentage of protein-35S decreased. Between the fifth and seventh hours of cellular maturation, the percentage of 35S in the protein and trichloroacetic acid-soluble fractions returned to their original levels. The protein-sulfur-amino acid composition likewise returned to its original value. The change in the 35S content of the cold trichloroacetic acid-soluble fraction was attributed to a single spot on paper chromatograms which absorbed ultraviolet light and was ninhydrin negative. Acid hydrolysis of the compound(s) in this spot released six or seven ninhydrin-positive spots and two ultraviolet-absorbing spots. After acid hydrolysis, most of the radioactivity was associated with a ninhydrin-positive spot which had the characteristics of cysteic acid.Immediately after the 35S content of the protein and trichloroacetic acid-soluble fractions returned to their original values, nuclear division was initiated. A close relationship between these shifts in sulfur metabolism and the initiation of nuclear division seems possible.

Propylthiouracil and hepatic clearance of thyroid hormones.

Biliary radioactivity derived from injected tracer amounts of either radioactive thyroxin or triiodothyronine was increased following injection of propylthiouracil (PTU). In one experiment PTU was administered 18 hr and again 1 hr before the measurement of biliary radioactivity. The tracer hormones were injected just prior to the first of four hourly periods of bile collection. Total radioactivity and trichloroacetic acid-soluble radioactivity were determined in hourly plasma samples. In another experiment, thyroxin was injected 18 hr prior to bile collection. Radioactivity in bile collected from each animal for 1 hr was measured and then PTU was infused at a rate of 5 mg/hr intravenously by constant infusion pump for the next 2 hr. While large alterations were occurring in radioactivity of bile, no discernible alterations were seen in either total plasma counts or counts in the trichloroacetic acid-soluble fraction of the plasma. These results, considered along with those of other investigators, were interpreted to mean that PTU probably has more than one extrathyroidal effect on the metabolism of thyroid hormones.

Influence of radiation on the incorporation of radioactivity in different cell constituents.

The effect of radiation on the incorporation of 32 P into cold trichloroacetic acid-soluble fraction, lipid and nucleic acid has been studied in Escherichia coli . The fraction of the total cells capable of giving rise to visible colonies after a dose of X-rays was determined from the agar plating. It was observed that the process of colony formation was about four times as sensitive as the nucleic acid synthesis in E. coli . Incorporation of radioactivity in lipid and acid-soluble phosphate was even less sensitive to radiation than nucleic acid synthesis. It has been suggested that radiation possibly breaks the DNA-enzyme complex, one such break resulting in the inhibition of cell division but in only a slight decrease in nucleicacid synthesis. This was considered to be the reason for the observed greater radiation sensitivity of colony formation than that of nucleic acid synthesis.

STUDIES OF ASPOROGENIC MUTANTS OF BACILLUS CEREUS: PRELIMINARY INVESTIGATIONS WITH CALCIUM AND ZINC

The physiology of spore formation was studied in Bacillus cereus and a temperature-sensitive asporogenic mutant. The parent organism sporulates when cultured in a minimal medium at either 28 °C or 37 °C while the mutant sporulates only at 28 °C. The blocking of sporulation at 37 °C has been referred to as "abortive" sporulation. Uptake of calcium and zinc was followed during growth and sporulation or "abortive" sporulation. Calcium and dipicolinic acid (DPA) levels in sporogenic cultures increased as the medium calcium was increased. The asporogenic mutant took up less calcium and synthesized little DPA. Heat resistance of spores increased as the calcium and DPA increased. Over 99% of Ca45or Zn65were released from labelled spores when autoclaved to release DPA. Chemical fractionations were made of cells labelled with Zn65and Ca45and harvested at different times during the culture cycle. Smaller percentages of calcium than of zinc were located in the cold trichloroacetic acid soluble fraction. The alcohol-soluble, ether-insoluble fraction of spores contained a greater percentage of calcium than was found in vegetative cells. Cells which had undergone "abortive" sporulation contained the same percentage of calcium in this fraction as homologous vegetative cells.

OXIDATIVE ASSIMILATION OF GLUCOSE BY PSEUDOMONAS AERUGINOSA

Duncan, Margaret G. (The University of British Columbia, Vancouver, British Columbia, Canada) and J. J. R. Campbell. Oxidative assimilation of glucose by Pseudomonas aeruginosa. J. Bacteriol. 84:784-792. 1962-Oxidative assimilation of glucose by washed-cell suspensions of Pseudomonas aeruginosa was studied using C(14)-labeled substrate. At the time of glucose disappearance, only small amounts of radioactivity were present in the cells, and alpha-ketoglutaric acid accumulated in the supernatant fluid. Most of the material synthesized by the cells during oxidative assimilation was nitrogenous, the ammonia being supplied by the endogenous respiration. The cold trichloroacetic acid-soluble fraction and the lipid fraction appeared to be important during the early stages of oxidative assimilation, and the largest percentage of the incorporated radioactivity was found in the protein fraction. In the presence of added ammonia, assimilation was greatly increased and no alpha-ketoglutaric acid was found in the supernatant fluid. Sodium azide partially inhibited incorporation into all major cell fractions, and at higher concentrations depressed the rate of glucose oxidation. During oxidative assimilation, chloramphenicol specifically inhibited the synthesis of protein. Oxidative assimilation of glucose by this organism did not appear to involve the synthesis of a primary product such as is found in the majority of bacteria.

Inhibition of deoxyribonuclease I of human serum in vitro by nitrogen mustard or leucocyte extracts

The temperature, time of incubation, concentration of MgCl 2 · 6H 2O and concentration of serum required for an optimum activity of deoxyribonuclease I (DNase I) of human serum were studied. The final incubation mixture consisted of 2 ml. of serum, 2 mg. deoxyribonucleic acid (DNA), 3 mg. MgCl 2 · 6H 2O, 0.5 ml. of 0.1 tM tris(hydroxymethyl) aminomethane buffer pH 7.3, and 0.1 ml. toluene in a total volume of 3 ml. After 20 hr. of incubation at 45 °C., determinations of DNase I were made by measuring deoxyribose-purine-containing fragments in the trichloroacetic acid-soluble fraction. Addition of 2 mg. nitrogen mustard or 0.5 ml. leucocyte extract to the incubation mixture inhibited the DNase I activity of the human serum. Enzyme kinetic studies indicate a noncompetitive relationship between DNA and nitrogen mustard [methylbis(β-chloroethyl)amine hydrochloride]for the DNase I of human serum; however, the inhibitor in the leucocyte extracts lowered the human serum DNase I activity in a competitive manner.

Glycogen fractions and their role in the histochemical detection of glycogen.

Glycogen fractions have been investigated in guinea pig liver by the anthrone technique and histochemically by Gomori's hexamine silver method. It is concluded that it is only the trichloroacetic acid soluble fraction of tissue glycogen that is demonstrated by this histochemical technique.

Acidic peptide conjugates in mammalian liver

Examination of the trichloroacetic acid-soluble fraction of several mammalian tissues has revealed the presence of a variety of peptides that have acidic properties and that are closely associated with material showing maximum absorbance at about 268 mμ. These conjugates contained from two to seven different amino acids. Although the peptides and the materials absorbing u.v. were closely associated during the early stages of the purification, they disassociated in the course of further fractionation. The nature of the linkage between these components remains to be established.