Amberlite IRC-50 Research Articles

Characterization of the ‘very’ lysine-rich histones of active and quiescent anther tissues of Hippeastrum belladonna

Cytochemical studies demonstrated that developmental stages of anther tissue of Hippeastrum belladonna stained differently after exposure to a competitive mixture of eosin and fast green dyes. Nuclei of active tissues stained fast green positive while those of metabolically inactive tissues were eosinophilic. Differences in the polyacrylamide banding patterns of ‘very’ lysine-rich F1 histones occurred in tissues with varying metabolic states. Heterogeneity in the ‘very’ lysine-rich histones was further observed by gradient elution chromatography on Amberlite IRC-50 with a linear gradient of guanidinium chloride solutions. The ‘very’ lysine-rich histones of inactive tissue (tapetal tissue) were resolved into seven components, and five components were observed in the ‘very’ lysine-rich histones of active tissue (pre-meiotic tissue). Distinct mobilities in polyacrylamide-SDS gels of ‘very’ lysine-rich histones components further substantiated biochemical differences in the F1 histones of active and inactive tissues. Analyses of the ‘very’ lysine-rich components of active and inactive tissues showed that their amino-acid compositions, while very similar, revealed consistent differences. The possible role which histones play in gene regulation and, more specifically, the influence of the ‘very’ lysine-rich histones in cell regulation are discussed in light of these findings.

Etude de la fraction F 2b des histones de thymus de veau

Histone fraction F2b isolated from Calf thymus, was fractionated by ion-exchange chromatography on Amberlite IRC-50 columns. Two fractions were obtained. The first fraction corresponds to the serine-rich, lysine-rich histone. The second fraction is an equimolecular complex of serine-rich, lysine-rich histone and of arginine-rich, lysinerich histone. A chemical study of this complex is presented. La fraction F2b des histones de thymus de Veau, soumise à une chromatographie sur Amberlite IRC-50, se sépare en deux sous-fractions: l'une correspond à l'histone riche en sérine et en lysine, l'autre est constituée d'un complexe équimoléculaire d'histone riche en sérine et en lysine et d'histone riche en arginine et en lysine, qui a été caractérisé chimiquement. Die Fraktion F2b der Histone der Kalbsthymusdrüse teilt sich, wenn sie einer Chromatographie über Amberlit IRC-50 unterzogen wird, in zwei Unterfraktionen: die eine entspricht dem an Serin und Lysin reichen Histon, die andere setzt sich aus einem äquimolekularen Komplex des serin — und lysinreichen und des arginin — und lysinreichen Histons zusammen. Eine chemische Untersuchung dieses Komplexes wird vorgelegt.

The microdetermination of calcium by the use of amberlite IRC-50 resin and glyoxal bis(2-hydroxyanil)

Conditions for the use of Amberlite IRC-50 resin and glyoxal bis(2-hydroxyanil) for the microdetermination of calcium in solutions containing phosphate are described.

Purification of Alkaline Proteinase from Aspergillus candidus

An alkaline proteinase of Aspergillus Candidus was purified from wheat bran solid culture by batchwise treatment with Amberlite IRC–50 and sequential chromatography on DEAE-cellulose, hydroxylapatite and Sephadex G–100 gel. This purification results in a 18-fold increase of proteolytic activity and the enzyme preparation was homogeneous in sedimentation analysis of the ultracentrifuge and polyacrylamide gel disc electrophoresis. The molecular weight was estimated to be about 23,000 by gel glltration and 22,000 by calculation from the amino acid composition. The enzyme consisted of Lys14, His4, Arg3, Asp25, Thr15, Ser23, Glu15, Pro7, Gly22, Ala24, Met2, Val16, Ile11, Leu10, Tyr6, Phe7, Trp2 and amide ammonia14 and did not contain cysteine or cystine.

Isolation and Characterization of Human Skin Lysozyme

Lysozyme was isolated from human skin by extraction with 0.1 M acetate buffer, pH 4.5 containing 0.1 M KCl. Crude enzyme was purified by the method of three successive chromatographic processes on Amberlite IRC-50, carboxymethylcellulose and Amberlite IRC-50. The purified human skin lysozyme was determined to be homogenous by poly-acrylamide gel electrophoresis. Molecular weight of the enzyme is estimated to be approximately 15,000 either by dialysis with calibrated membranes or by gel filtration through Sephadex G-50 column. The amino acid composition and several biophysical properties of human skin lysozyme were found to be very similar to those of human milk and leucocyte lysozymes.

Occurrence of a tetrodotoxin-like substance in a goby Gobius criniger

The goby of the Ryukyus which is suspected of being poisonous was identified as Gobius criniger and demonstrated to possess a potent neurotoxin, which appears similar to tetrodotoxin. The toxin was soluble in water, easily dialyzable through a cellophane membrane, and unstable to heat at pH 10. By using activated charcoal and Amberlite IRC-50, a partially purified toxin having a lethality of 0·22 μg per g mouse was obtained. In thin-layer chromatography, this preparation gave a spot coinciding with that of crystalline tetrodotoxin. Symptoms in mice were similar to those induced by tetrodotoxin and saxitoxin. The dosedeath time curve in mice was identical with that for tetrodotoxin. It was also confirmed that both a puffer and G. criniger were neither killed nor paralyzed by an extraordinarily large dose of tetrodotoxin or goby toxin.

Bifidus Factors in Carrot. I. Purification and Some Properties

Procedures for the purification of acidic growth factors for Bifidobacterium bifidum from carrot were developed. These involved adequate solvent extraction and subsequent successive chromatography on Amberlite IR-45, IRC-50, IRA-68, and SE, DEAE, QAE Sephadex which included a proper precipitation procedure for the SE-Sephadex eluate, and preparative paper electrophoresis. Among five kinds of growth factors separated, the major growth factor and the most acidic factor were highly purified. Both factors differed from coenzyme A and its known precursors, while one of the weaker acidic factors coincided with D-pantetheine 4'-phosphate in chromatographic and electrophoretic behaviors.

The influence of the pineal body on the gonadotropic function of the hypophysis.

We conclude that the antigonadotropic activity of the Sephadex G-25 fraction F3 seems to be fairly specific for the pineal body, but that it is unstable under the experimental conditions we have used. The Sephadex G-25 fraction F2 can be partly purified by chromatography on the cation exchanger Amberlite IRC-50, XE-64 and afterwards on the anion exchanger Dowex 1X2. The indoles (melatonin, 5-methoxy-tryptophol and serotonin-creatinine sulphate) show no activity in the bioassay that we always use. Experiments with synthetic arginine-vasotocin indicate that the action of this substance differs in vitro from that of the Sephadex G-25 fraction F3. It is not impossible that the inhibitory activity of the pineal is related to catecholamines.

Isolation and characterization of three phosphoamido-neomycins and their conversion into neomycin by Streptomyces fradiae.

Some amino acids, particularly glycine and serine, favour the accumulation in the fermentation broth of three phosphorylated amino sugar compounds that are intermediates in the pathway of neomycin biosynthesis by Streptomyces fradiae 3535. The compounds were separated and purified further by Amberlite IRC-50 (NH(4) (+) form). The intermediates were characterized by physicochemical methods as neomycin B pyrophosphate (C(23)H(48)N(6)O(19)P(2),3H(2)O), neomycin C pyrophosphate (C(23)H(48)N(6)O(19)P(2),3H(2)O) and neomycin C dipyrophosphate complex (C(24)H(66)N(8)O(33)P(4)).

The specificity of the Vi-phage II bacteriolytic enzyme

Salmonella typhi infected with Vi-phage II produce an enzyme dissolving chloroform-killed, gram-negative bacteria. The enzyme was purified 200 times by chromatography on Amberlite IRC 50. The specificity of its action was investigated using Micrococcus lysodeiktious cells, S. typhi murein and muropeptide C3. It has been shown that the enzyme splits off the cross-linking peptide bond between the amino group of diaminopimelic acid and the carboxyl group of alanine.

EFFECTS OF SURGERY ON PLASMA LEVELS OF CORTISOL, CORTICOSTERONE AND NON-PROTEIN-BOUND-CORTISOL

ABSTRACT The diurnal variation in plasma cortisol, corticosterone and non-protein-bound cortisol was investigated using altogether 991 plasma samples obtained from preoperative chronically ill (control) subjects and patients during and following major surgery. A fluorimetric method using an elution chromatography on Amberlite IRC-50 and the equilibrium dialysis method were used for the determinations. The diurnal variation in plasma cortisol of the preoperative control subjects reached a peak (17.3 μg/100 ml) at 6 a.m. and declined to the lowest level (2.3 μg/100 ml) between 10 p.m. and midnight. The maximum value in the levels of corticosterone (0.6 μg/100 ml) was observed at 6 a.m. Surgery caused a steep rise in plasma cortisol showing a maximum value (30.0 μg/100 ml) 2 to 4 hours after the end of the operation. Although the average of the morning levels of plasma cortisol returned to the control levels within 4 to 5 days, the evening levels returned on the 6th day after the operation. The response of plasma corticosterone in the surgery group was found to parallel the change in plasma cortisol. However, the increase in the corticosterone concentration was significantly higher than that of cortisol. The percentage of non-protein-bound cortisol in the plasma of the preoperative control subjects was found to be 2.5 to 3.3 per cent at 6 and 8 a.m. and 1.5 per cent at midnight, while the percentage remained almost unchanged (about 2%) at 10 a.m., 12 a.m. and 6 p.m. The percentage increased significantly during and for 4 days following the operation, concomitant with the increase in the levels of plasma cortisol. It is suggested that stressful situations are associated with markedly increased plasma levels of biologically active cortisol.

ATP-inhibited ribonuclease of Bacillus subtilis I. Purification and general properties

1. 1. Ribonuclease (ribonucleate nucleotido-2′-transferase (cyclizing), EC 2.7.7.17) activity in the crude extract of Bacillus subtilis K was strongly inhibited by low concentrations of ATP. The inhibitory effect of ATP was thought to be common to all B. subtilis species. 2. 2. Ribonuclease in the crude extract was purified 2100-fold by means of DEAE-Sephadex A-50, Amberlite IRC-50 and Sephadex G-75 column chromatography, and the recovery was 26% efficient. 3. 3. The purified ribonuclease was shown to be almost homogeneous when tested by disc electrophoresis and was inhibited to an extent of 90% in the presence of 10 μM ATP. 4. 4. The ATP-inhibited ribonuclease showed optimum activity at pH 5.5–5.7, was stable between pH 7–8 and almost completely hydrolyzed yeast RNA. 5. 5. The degradation products were identified as Cyd-2′,3′- P, Urd-2′,3′- P, Ado-2′,3′- P and Guo-2′,3′- P.

An attempt to separate a sheep pineal extract fraction showing antigonadotropic activity.

The fraction F3 obtained after gelfiltration of a pineal extract on Sephadex G-25 (Fine) which inhibitsin vitro the secretion of FSH of the anterior hypophysis has been chromatographed on the weak cation-exchanger Amberlite IRC-50, XE-64. This method which was very useful as a purification step of the Sephadex G-25 fraction F2 and has been used with success to isolate the peptide hormones of the neurohypophysis, does appear to be unsuitable for further purification of a pineal fraction with inhibiting activity.

Bioassay of histamine in human urine. An improved method for purification of samples by means of cation exchange chromatography.

Bioassay of histamine on the guinea-pig ileum was performed after puri fication of human urine by means of ion exchange columns prepared from either Dowex-50 or Amberlite IRC-50. The Dowex technique yielded an extract better suited for bioassay than the Amberlite technique. The dif ference was probably due to a lower content of interfering substances in the former extract. Values for the urinary excretion of histamine in healthy subjects are presented.

Trypsin inhibitors from Ascaris lumbricoides var. Suis

The body walls of Ascaris lumbricoides contain two trypsin inhibiting fractions which differ with respect to K i . The K i values are 90 nM for Peak I and 13 nM for Peak II inhibitors. In the presence of 10 mM p- toluenesulfonyl- l-arginine methyl ester, the complex formed between Peak I inhibitor and pork trypsin is dissociated to the extent of 65–75%; the complex with the Peak II inhibitor dissociates to the extent of 19–22%. Peak I inhibitor was obtained in a highly purified form. It chromatographs as a symmetrical peak on an Amberlite IRC-50 column with constant specific activity across the peak. It is homogeneous in the ultracentrifuge with an s 20, w value of 1.00 S. It has a molecular weight of 5520, and is composed of 50 amino acids. Leucine, histidine, methionine and tyrosine are absent. Spectral analysis confirms the presence of one equivalent of tryptophan, two of phenylalanine and four of cystine.

Activation of Factor X in Bovine Prothrombin Preparations

SummaryChromatographic fractionation of factor X (autoprothrombin III) and factor Xa (autoprothrombin C), obtained from bovine prothrombin preparations, on amberlite IRC-50 demonstrates activation of factor X under the conditions of prothrombin activation. The increase in factor Xa protein during prothrombin activation is due to incorporation of a non-active protein into the fraction. Prothrombin Activating Derivative B (PAD-B) is activated by concentrated salt solutions and not by stypven.

A Study of the Multiplicity of Lysine-rich Histones

Fractions of lysine-rich histones have been obtained from rabbit thymus and mammary gland by chromatography on Amberlite IRC-50 with shallow guanidinium chloride gradients. The peaks obtained were rechromatographed using still shallower gradients. Only one of nine peaks was found to be significantly inhomogeneous on rechromatography. Furthermore, each of the other eight peaks was as narrow as a chromatographically homogeneous sample and each was electrophoretically homogeneous on polyacrylamide gel. All fractions from the thymus possessed blocked aminoterminal residues and were terminated at the carboxyl end by 2 adjacent lysine residues. It was concluded that this system of chromatography had been extended nearly to its limit of resolving power and that the complexity of these chromatographic patterns approximates the true multiplicity of lysine-rich histones.

Purification of labeled 203Hg-Neohydrin

An easy method for the purification of labeled Neohydrin was studied by the use of the weak cation exchange resin, Amberlite IRC-50, in a small chromatographic column. The resin shows a high selectivity for the removal of mercury ions from a neutral or alkaline solution. This method allows, in one step, the collection of the pure labeled Neohydrin in a small volume, avoiding evaporation, neutralization, and centrifugation when using the alumina method.

Purification and determination of the antidiuretic substance in human urine.

ABSTRACT This is a contribution to the biological identification and estimation of the antidiuretic substance in human urine. The urine was concentrated and purified by column chromatography on Amberlite IRC-50 and by dialyses. It was possible to estimate the antidiuretic activity of this extract against arginine vasopressin by a fundamentally and statistically valid biological assay using water loaded rats anaesthetized with ethanol. Recovery experiments of vasopressin from urine at different steps of the purification were performed, and the final yield was estimated to be about 60 %. The amount of antidiuretic activity in the urine from four healthy subjects ranged between 11 mU and 24 mU/24 h with a mean of 16 mU/24 h.

Qualitative Species Differences and Quantitative Tissue Differences in the Distribution of Lysine-rich Histones

Abstract Species-specific differences in the distribution of lysine-rich histones were investigated in spleen tissue of three mammalian (calf, cat, and rat) and one avian (chicken) species. Tissue-specific differences in the distribution of the lysine-rich histones were studied in liver, kidney, spleen, and thymus tissues of the rat; liver, spleen, and thymus of the calf; and liver, spleen, and erythrocytes of the chicken. The lysine-rich histone fractions were extracted with aqueous trichloracetic acid from 0.14 m NaCl-washed tissue homogenates and isolated nuclei. The extracts were fractionated by gradient elution chromatography on Amberlite IRC-50 with a linear gradient of guanidinium chloride. Species-specific lysine-rich histone components were observed in the elution profiles for the spleen of calf, rat, cat, and chicken which showed three, five, four, and five distinct components, respectively. Studies on the electrophoretic mobility of the whole lysine-rich histone fractions in polyacrylamide gels showed that the rat spleen fraction contained a component not found in either calf spleen or chicken spleen. In addition, the chicken spleen fraction contained a component not found in the calf spleen fraction. Amino acid analysis of the individual chromatographic components of four different species showed that the over-all compositions were remarkably similar except for a single component found in four tissues of the rat. Treatment of this component with cyanogen bromide indicated that methionine was an integral part of the primary structure. This is the first report of the presence of methionine in a purified lysine-rich histone from any source. Quantitative differences were found between a number of corresponding lysine-rich histones of different tissues of the same species, both mammalian and avian, but the complement of histones for a given species was identical in all tissues studied. Planimetric analysis of duplicate chromatographic profiles of three separate preparations of the rat thymus, spleen, liver, and kidney fractions showed that the thymus fraction contained a 4- to 7-fold greater amount of Component 1 compared to the liver, spleen, and kidney fractions. The kidney fraction contained about one-half the amount of both Components 2 and 5 compared to the thymus, liver, and spleen fractions. All four tissues contained similar relative proportions of Component 3, but the kidney fraction contained approximately twice as much of Component 4 compared to the other three fractions. Absorbance profiles of polyacrylamide gel electrophoresis patterns of the lysine-rich histone fractions of rat thymus, spleen, and liver provided similar results. However, precise quantitative determination of minor components was not possible because of the relatively small differences in mobility between certain components.