Sequential Ion Exchange Research Articles

A superoxide dismutase of metacestodes of Taenia taeniaeformis

Superoxide dismutase was purified from Taenia taeniaeformis metacestodes by sequential ion exchange chromatography on quaternary-amino-ethyl-cellulose, gel filtration chromatography on ACA 44 and ion exchange chromatography on DEAE-cellulose, followed by chromatofocusing on polybuffer exchanger 94. This isolation procedure resulted in the detection of a single protein-staining band on alkaline gels, coincident with enzyme activity. We have, however, detected what appear to be two peaks of enzyme activity within this single protein-staining band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis using gradient slab gels and analysis under reducing conditions, resulted in the detection of only one protein at an apparent M r of 16 600, while analysis under non-reducing conditions, gave a single protein of an apparent M r of 64000. The isoelectric point of the purified protein is 6.6. Boiling for 3 min completely destroyed the enzyme, whereas incubation for 2 h at 37°C resulted in the loss of 56% of the enzymic activity. Incubation with 10 mM KCN resulted in 83% inhibition of the enzyme. We have detected up to 168 U ml −1 of enzyme activity in the cyst fluid surrounding the parasite in situ. This is the first instance in which any parasite superoxide dismutase has been purified to apparent homogeneity. Parasite-mediated enzymic destruction of superoxide anion can not only protect against oxygen toxicity as a result of normal parasite respiratory processes but also may serve as yet another mechanism used by tissue-dwelling parasites to evade host immunologic attack.

Overproduction of 5-enolpyruvylshikimate-3-phosphate synthase in a glyphosate-tolerant Petunia hybrida cell line

Analysis of a Petunia hybrida cell culture (MP4-G) resistant to 1 m m glyphosate revealed a 15- to 20-fold increased level of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in the herbicide-tolerant strain. Immunoblotting and enzyme kinetic measurements established that the increased EPSP synthase activity resulted from overproduction of a herbicide-sensitive form of the enzyme. Homogenous enzyme preparations were obtained from the herbicide-tolerant cell line by sequential ion-exchange, hydroxyapatite, hydrophobic-interaction, and molecular sieve chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve chromatography established the Petunia enzyme to be a monomeric protein with M r 49,000–55,800. K m values for phosphoenolpyruvate and shikimate 3-phosphate were about 14 and 8 μ m, respectively. Glyphosate inhibited the enzyme competitively with phosphoenolpyruvate ( K i = 0.17 μ m). These experiments provide further evidence that EPSP synthase is a major site of glyphosate action in plant cells.

A protein of the Z class of liver cytosolic proteins in the rat that preferentially binds heme.

A low molecular weight protein purified from rat liver cytosol was observed to bind heme with an affinity higher than that for other organic anions. Purification was achieved by two procedures, one employing affinity chromatography on oleic acid-agarose, and the other using sequential ion-exchange and gel filtration chromatography after initial removal of aprotinin-sensitive proteases. Removal rather than inhibition of proteases improved the yield four times. Both procedures produced a stable protein. The purified protein binds heme with a higher affinity (Kd 0.15 microM) than any other organic anion tested including other (metallo)porphyrins, bilirubin, and oleic acid. Based on its molecular weight, amino acid composition, immunological properties, and the increase of its tissue levels in response to the administration of hypolipidemic agents, the protein was identified as being related to proteins of the Z class, whose members include fatty acid binding protein and sterol carrier protein. Like other Z proteins, our protein exhibits several forms on electrofocusing, but differs from fatty acid-binding protein and sterol carrier protein in that its major form exhibits a pI of 7.4. In view of its distinct isoelectric focusing pattern, its higher affinity for heme than for oleic acid, and its apparent inability to bind cholesterol and steroids, we cannot identify this protein as any of the above-mentioned proteins of the Z class. Consequently we have provisionally designated it heme-binding protein.

Zeolite photochemistry: energy transfer between rare-earth and actinide ions in zeolites

Energy transfer is an important process in various display and luminescence devices. This paper shows that energy transfer efficiencies can be controlled by selectively placing certain inorganic ions in zeolite molecular sieves. The energy transfer in these zeolites occurs between uranyl ions and europium(III) ions in various zeolites. Three-dimensional channels in crystalline materials provide better interactions for energy transfer than two-dimensional or zigzag channels such as in mordenite and ZSM-5. Different synthetic procedures involving simultaneous and sequential ion exchange yield different efficiencies of energy transfer. The mechanism is believed to be of the short-range electron-exchange type for crystalline ZSM-5, modenite, and Y zeolite and of the long-range type for amorphous materials derived from zeolite A. 26 references, 2 figures, 2 tables.

Purification and characterization of mitochondrial thioesterase from rabbit myocardium and its inhibition by palmitoyl carnitine.

Mitochondrial thioesterase from rabbit myocardium was purified to homogeneity by sequential ion exchange, hydroxylapatite, chromatofocusing and gel filtration chromatographies. The purified protein had an apparent molecular mass of 170 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent silver staining revealed a single band (Mr 43 000) demonstrating that the protein is a tetramer. The specific activity of the purified thioesterase for palmitoyl-CoA hydrolysis was 1.8 mumol/mg per min. Thioesterase activity was maximal at pH 8 and was activated by Mg2+ but inhibited by Ca2+. Pathophysiological concentrations of L-palmitoyl carnitine (20-400 microM) competitively inhibited enzymic activity. The purified enzyme was also inhibited by high concentrations of substrate (over 20 microM palmitoyl-CoA).

Müllerian inhibiting substance inhibition of a human endometrial carcinoma cell line xenografted in nude mice

Müllerian inhibiting substance (MIS) is a fetal testicular product which causes regression of the müllerian duct in the male mammalian embryo. This material has been partially purified from a neonatal bovine source and in cruder fractions has shown antitumor effects when tested against the HOC-21 ovarian carcinoma line in monolayer cytotoxicity, in soft agar colony inhibition assay, and in nude mouse xenografts. The glycoprotein used for the present studies was more highly purified by sequential ion exchange, carbohydrate affinity, and dye affinity chromatography. After a 1-hr exposure with 1.0 × 10 6 tumor cells prior to heterotransplantation, this more purified preparation with MIS biological activity as determined by organ culture assay of embryonic urogenital ridges delayed the appearance of palpable tumor nodules. That this response may be specific for tumors derived from the coelomic epithelium of the embryo is further supported by the absence of any antitumor effect when this substance was tested against the SW-48 colon carcinoma line. It is of interest that the antitumor activity followed the biological activity as the preparation was further purified to 30,000-fold.

Lymphocyte recognition of lymph node high endothelium. V. Isolation of adhesion molecules from lysates of rat lymphocytes.

Lymphocytes migrate from blood into lymph nodes (LN) of rats specifically at segments of venules lined by high endothelium (HEV). Investigation of lymphocyte surface molecules mediating this interaction has been carried out using an in vitro assay in which lymphocytes adhere selectively to HEV when overlaid onto sections of peripheral LN. Using this assay we have previously isolated a thoracic duct lymph component termed High Endothelial Binding Factor (HEBF), which is detected by its ability to block HEV sites where lymphocytes attach. In the current study, we present evidence that anti-HEBF antibody recognizes surface molecules of lymphocytes involved in adhesion to high endothelium. Rabbit antibody was produced against HEBF isolated from lymph by sequential ion exchange [diethylaminoethyl (DEAE)-cellulose and carboxymethyl (CM)-Sepharose] and Sephacryl S-200 gel filtration. Anti-HEBF F(ab')2 bound to 60 to 70% thoracic duct lymphocytes (TDL), spleen, and LN cells but to only 2 to 9% thymus and bone marrow cells (indirect immunofluorescence). In addition, biologically active HEBF was isolated from lymph and detergent lysates of lymphocytes by antibody affinity chromatography. Comparable amounts of the factor were isolated from lysates of TDL, LN, and spleen cells whereas little or no HEBF was detected in lysates of thymus or bone marrow cells or in serum. HEBF obtained from TDL appeared to be a glycoprotein because it was trypsin sensitive, bound to lentil lectin, and was eluted with alpha-methyl-D-mannoside. The finding that HEBF was not recovered from lysates of trypsinized TDL indicates that the activity was mediated by components expressed on cell surface membranes. Immunoprecipitation studies revealed that anti-HEBF antibody recognized radioiodinated surface membrane proteins of TDL and TDL-derived T cells and B cells that resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography into three major bands with m.v. of 230,000, 210,000, and 92,000 (nonreduced) and one major band with m.w. of 70,000 and two minor bands with m.w. of 92,000 and 45,000 (reduced). These observations indicate that HEBF is a surface membrane component that is not class-specific. It is suggested that lymphocyte surface HEBF is composed of high endothelial adhesion molecules that mediate cell-cell interactions involved in entry of lymphocytes from blood into peripheral lymph nodes.

A new radioenzymatic assay for histamine using purified histamine N-methyltransferase

Radioenzymatic assays for histamine (Hm) have found wide application. However, these procedures may lack the sensitivity necessary to quantify Hm in certain biological samples, such as human plasma. Purification of histamine N-methyltransferase (HNMT) has permitted the development of a new and highly sensitive radioenzymatic assay for Hm. HNMT was purified by sequential ion exchange, hydrophobic and molecular exclusion chromatography. The use of purified HNMT in the Hm assay has allowed the inclusion of high specific activity tritiated S-adenosyl-L- methionine ([ 3H]SAME) and the development of a simplified solvent extraction product isolation procedure. This assay has a sensitivity of approximately 2 picograms and is specific for Hm. Hm was easily quantified in human plasma and was found to be 303 ± 81 pg/ml (mean ±SD) in 8 male subjects. Substantial blank reduction and increased product conversion occur when purified HNMT is utilized in the Hm radioenzymatic assay, thus, increasing the sensitivity and possibly improving the specificity of this procedure.

Mullerian Inhibiting Substance Inhibits Growth of a Human Ovarian Cancer in Nude Mice

DONAHOE, PATRICIA K.; FULLER, ARLAN F. JR.; SCULLY, ROBERT E.; GUY, STEPHEN R.; BUDZIK, GERALD P. Author Information

Mullerian Inhibiting Substance Inhibits Growth of a Human Ovarian Cancer in Nude Mice

Mullerian inhibiting substance (MIS) was investigated for its ability to inhibit growth of a human ovarian cancer in nude mice. Biologically active preparations from newborn calf testes, obtained after sequential ion exchange chromatography, delayed or prevented growth of a human ovarian cancer (HOC-21) when 2 X 10(6) cells were preincubated with them prior to subcutaneous injection of the tumor cells into Balb/C homozygous nude mice. Preincubation of a human colon carcinoma cells (SW-48) with similar preparations of MIS failed to inhibit growth of the tumor cells in nude mice. Human serous carcinomas are thought to arise from the ovarian surface epithelium, a derivative of the coelomic epithelium of the urogenital ridge, which invaginates to form the mullerian duct early in embryonic life. The neoplastic cells of serous tumors simulate morphologically the lining cells of the fallopian tube, which are derivatives of mullerian duct epithelium. This study provides physiologic confirmation of the mullerian nature of this type of tumor and suggests that MIS may ultimately prove to be effective in its therapy.

Characterization of Chrysaora quinquecirrha (sea nettle) nematocyst venom collagenase

1. 1. The collagenase isolated from Chrysaora quinquecirrha (sea nettle) nematocyst venom was purified 237 fold using three sequential ion exchange chromatographic steps. 2. 2. The collagenase had optimal activity at physiological pH and temperature ranges. 3. 3. The enzyme was activated by Ca 2+ and Na + and reversibly inhibited by EDTA. 4. 4. The enzyme was not inhibited by phenylmethyl-sulfonyl fluoride but a number of chelating agents, cysteine, and many metal ions inhibited the collagenase.

Solubilization and isolation of the human placenta receptor for epidermal growth factor-urogastrone.

We describe the solubilization and purification of both the photoaffinity-labeled and unlabeled human placenta receptor for epidermal growth factor-urogastrone (EGF-URO). The photolabeled receptor can be purified 300- to 500-fold from membrane extracts by combined immunoaffinity and lectin-agarose affinity chromatography. This isolation approaches theoretical purity. Upon gel filtration and wheat germ agglutinin-Sepharose chromatography, the photolabeled receptor and the EGF-URO binding activity co-migrate, as measured by a newly developed lectin-agarose immobilization assay of receptor binding. Sequential ion exchange, Cibacron blue-Sepharose, wheat germ agglutinin-Sepharose, and gel filtration chromatography yield a 110-fold purification of the EGF-URO binding activity; this purified fraction contains protein constituents that exhibit electrophoretic mobilities parallel to those of the photolabeled receptor constituents, that have apparent molecular weights of 180,000 and 160,000. This molecular weight range is consistent with a measured apparent Stokes radius for both the photolabeled and unlabeled receptor of 5.1 nm (Sephacryl S-200 gel filtration), although an apparent radius of 4.3 nm is estimated by gel filtration on Sepharose-6B. The apparent molecular weight of the photolabeled receptor is unaffected by 2-mercaptoethanol. This work provides a basis for further detailed studies of this growth factor receptor.

Purification and characterization of androgen binding protein from the rat epididymis.

Androgen binding protein (ABP) was purified from rat epididymides by sequential ammonium sulfate precipitation, affinity chromatography, gel filtration, and DEAE ion exchange chromatography. The column matrix formed by coupling dihydrotestosterone-17 alpha-(hexanoic acidY to agarose via diisopropylamine was stable during the extensive washing required following application of crude tissue extracts to the affinity matrix. In addition, when used under the optimal conditions, the column produced a 1600-fold purified in a single step. Apparent homogeneity of the final product was shown by polyacrylamide gel electrophoresis, sedimentation equilibrium, and constant specific activity across the peak of the final chromatograph. The molecular weight determined by sedimentation equilibrium at pH 7.4 was 85 000. By contrast, the molecular weight determined by sedimentation equilibrium in guanidine.HCl and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately one-half that of the native protein, suggesting that suggesting that ABP is comprised of subunits.

Biochemical characterization of guanidinium chloride-soluble dentine collagen from lathyritic-rat incisors.

alpha- and beta-Chains were isolated by sequential ion-exchange and gel-filtration chromatography of guanidinium chloride-soluble dentine collagen obtained from Tris/NaCl-extracted EDTA-demineralized lathyritic-rat incisors. The alpha-chains were identified as alpha 1 I and alpha 2 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and amino acid analysis of the intact chains and their CNBr peptides. The dentine alpha-chains exhibited higher lysine hydroxylation and phosphate content, but lower hydroxylysine glycosylation, than alpha-chains from skin. Increased lysine hydroxylation was observed in the helical sequences. The alpha 1 I/alpha 2 ratio was approx. 3:1, and was presumably due to the presence of (alpha 1 I)3 molecules along with (alpha 1 I)2 alpha 2 molecules as shown recently for neutral-salt-soluble dentine collagen [Wohllebe & Carmichael (1978) Eur. J. Biochem. 92, 183--188]. In the borohydride-reduced beta 11- and beta 12-chains from guanidinium chloride-soluble dentine collagen, the reduced cross-links hydroxylysinohydroxynorleucine and hydroxylysinonorleucine were present. A higher proportion of hydroxylysinonorleucine in the reduced beta 12-chain probably reflects differences in extent of hydroxylation of specific lysine residues of the alpha 1 I- and alpha 2-chains.

Type-I trimer and type-I collagen in neutral-salt-soluble lathyritic-rat dentine.

Triple-helical collagen molecules have been obtained from EDTA-demineralized lathyritic rat incisors by neutral buffer extraction. Component alpha chains, isolated by sequential ion-exchange and gel-filtration chromatography, were shown to be alpha1 I and alpha2 chains by cyanogen bromide peptide analysis. The alpha1 I:alpha2 chain ratio was approximately 3:1, which is greater than expected for type I collagen. The excess of alpha1 I chains over that required for type I collagen was due to the presence of type I trimer molecules. Fractional salt precipitation separated type I collagen from type I trimer. It is not known at present if type I trimer synthesis also occurs in normal rat tissues.

Acidic peptide and polyribonucleotide crystal growth inhibitors in human urine.

Urine contains nondialyzable inhibitors of calcium oxalate crystal growth. We have pursued the hypothesis that these inhibitors may, in part, be acidic peptides and polyribonucleotide fragments. Homopolyribonucleotides and RNA inhibit calcium oxalate crystal growth at 5 x 10(-6) M of constituent ribonucleotide, whereas the monomer nucleotides are inactive at 10(-4) M. Poly-L-aspartic or glutamic acid are also inhibitory at 5 X 10(-6) M of amino acid, whereas the monomeric amino acids are inert. Gastric pepsin, a naturally occurring acidic peptide, is inhibitory. Incubation with nonspecific protease reduced the inhibitory effectiveness of normal human urine consistently and significantly, a fact compatible with an important contribution of peptides. A variable additional reduction was produced by subsequent treatment with ribonuclease, suggesting only a small role for polyribonucleotide. Sequential ion exchange and gel filtration chromatography and preparative disc gel electrophoresis yielded inhibitory material enriched with peptides that were strongly acidic and high in proline. Peptides and ribonucleotides seem to contribute to urinary nondialyzable crystal growth inhibitory activity.

Structural polypeptides of mammalian type C RNA viruses. Isolation and immunologic characterization of a low molecular weight polypeptide, p10.

Low molecular weight polypeptides of several mammalian type C RNA tumor viruses were purified by sequential ion exchange chromatography and molecular sizing techniques. These included a polypeptide with a molecular weight of 10,000 to 11,000, p 10, from two type C viruses of mouse origin. Rauscher- and Moloney-murine leukemia virus (MuL virus), and from an infectious type C virus isolate of the woolly monkey. The p12 structural polypeptides of these viruses as well as Rauscher-MuL virus p15 were also purified. By using radioimmunoassays developed for each polypeptide, it was possible to demonstrate that all three low molecular weight polypeptides, p15, p12, and p10, were immunologically unique. Among type C viral structural polypeptides, p10 has been least well characterized immunologically. The results of the present study indicate that p10 is virus-coded and possesses strong group-specific antigenic determinants. By use of appropriate immunoassays, broadly reactive interspecies determinants shared by mammalian type C virus isolates of murine, feline, and primate origin, were also demonstrated. The interspecies antigenic determinants of p10 were shown to be as broadly cross-reactive as those exhibited by the major type C virus structural polypeptide, p30.

Immunochemical characterization of two major polypeptides from murine mammary tumor virus.

A major murine mammary tumor viral (MMTV) antigen, sl, originally described by Nowinski et al. (1967, 1968, 1971), has been purified from RIII mouse milk MMTV by sequential ion-exchange and gel chromatography. The purified protein with sl antigenic reactivity contains carbohydrate, and has an apparent minimal molecular weight of 52,000. It can be designated as gp52 (sl). Another major MMTV viral protein with a molecular weight of 27,000 has also been isolated, and antisera have been prepared against it. Both MMTV gp52 (sl) and p27 viral polypeptides have been iodinated with (125)I and used in immunoprecipitation and competition assays. The two MMTV proteins differ absolutely from each other and from major mouse type C viral polypeptides in molecular weight, immunological reactivity, and amino acid composition. Purified gp52 (sl) in radioimmunoprecipitation inhibition assays reacted in two distinct patterns. One pattern showed partial displacement of antibody which could be converted to the second, a complete displacement, by heating the antigen, presumably by exposing additional reactive determinants. Biologically, the patterns of major MMTV polypeptide expression in milk correlated with spontaneous mammary tumor incidence in different strains of mice, indicating that the sl antigen is group specific for MMTV or that several mouse strains contain the same virus type.

Preparation and isolation of immunologically active glycopeptides from carcinoembryonic antigen (CEA).

AbstractIn order to determine the structure of the tumor‐specific immunodominant group of the carcinoembryonic antigen (CEA) of human gastro‐intestinal tumors, immunologically active fragments from this glycoprotein were prepared by partial enzymatic hydrolysis using the proteolytic enzyme nagase. The fragments were purified by sequential molecular sieve, adsorption and ion exchange chromatography, and were analysed for carbohydrate and amino‐acid composition by gas‐liquid chromatography. The antigenic activity of each fragment was tested using both the Z‐gel and Farr radioimmunoassay techniques for CEA. Immunologically active glycopeptides with molecular weights of 1,000–5,000 Daltons, which contained the tumor antigenic determinant of CEA, had relatively high contents of N‐acetyl‐D‐glucosamine, aspartic acid or asparagine, and glutamic acid or glutamine.

Sequential Ion-Exchange Separation of Heavy Elements and Selected Fission Products for Burnup Measurement

Sequential Ion-Exchange Separation of Heavy Elements and Selected Fission Products for Burnup Measurement