Distinct Peaks Of Radioactivity Research Articles

Covalent binding of polycyclic aromatic hydrocarbon components of manufactured gas plant residue to mouse lung and forestomach DNA.

The present study characterized the DNA adducts induced by manufactured gas plant residue (MGP) and benzo[alpha]pyrene (B[alpha]P) in mouse lung and forestomach. The dose levels used in the present study were comparable to the levels used in a previous animal bioassay. Adduct formation was evaluated in female A/J mice (7 weeks old) fed MGP (0.25%) or B[alpha]P (16 and 98 ppm) for 14 days. In addition, adduct formation was also evaluated in mice 24 h after the ip administration of 1.8 mg of B[alpha]P in 0.5 mL of tricaprylin. 32P-Postlabeling combined with multidimensional TLC and reverse phase HPLC was used to evaluate hydrocarbon-DNA adducts. HPLC separation of chemical-DNA adducts formed in lung following MGP ingestion resulted in three distinct peaks of radioactivity eluting at 22, 32.4, and 33.5 min. These peaks accounted for 13, 10, and 41% of the total adducts detected. The adducts isolated from forestomach eluted as a series of minor peaks with two more distinct peaks of radioactivity at 32.4 and 33.5 min. These peaks accounted for 47 and 32% of the total adducts detected in forestomach, respectively. Ingestion of B[alpha]P (16 or 98 ppm) and the ip administration of B[alpha]P resulted in a single major adduct with a retention time of 32.4 min. The DNA adducts formed from MGP administration were further characterized by comparison with adducts formed following the administration of individual hydrocarbons and a mixture of hydrocarbons.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of human progesterone receptor by high performance hydrophobic interaction chromatography

High performance hydrophobic interaction chromatography has been used to separate progestin receptors (PRs) from human uterus and from the T47D human breast cancer cell line. Reproducible separations of high resolution were achieved using a TSK Phenyl-5PW column and a reverse salt gradient of 400 mM to 0 mM sodium sulfate in phosphate buffer, pH 7.4. Peaks of radioactivity exhibiting hydrophobic behaviour were isolated, as well as a smaller proportion of specific bound receptors located in the void volume fraction. No differences in retention times were observed between uterine and breast cell line samples. When the technique was used in conjunction with rapid vertical tube sucrose density gradient centrifugation, the 8S sedimenting PR from fresh, low-salt cytosol always eluted with a retention time of 24 min. The natural 4S receptor chromatographed as a single peak at 29 min while the 4S receptor species from high-salt cytosol appeared as two distinct peaks of radioactivity with retention times of 29 and 33 min. While specific binding was shown to occur in the void volume of the column, the origin of these receptors were indeterminate. These results would suggest that under these conditions the 8S receptor occurs as a single hydrophobic class of protein, whereas the data provides evidence that transformed 4S receptor may be proportioned into two unequal entities as a function of exposure to salt.

Amino acid mdification of proteins in regenerating sciatic nerves of rats

Recent experiments have shown that Arg, Lys, and Leu can be incorporated posttranslationally into proteins of regenerating sciatic nerves of rats. The present experiments investigate a mixture of 15 radioactive amino acids to determine if additional amino acids can be conjugated posttranslationally to proteins of regenerating nerves. Proteins of regenerating sciatic nerves of rats were able to incorporate Arg, Lys, Leu, Pro, Val, Ala, Phe, and Ser in relatively large amounts and Asp, Glu, Thr, Gly, Ile, His, and Tyr in relatively low or undetectable amounts, in the most advanced portion of the regenerating nerves. Two-dimensional SDS PAGE showed incorporation of the amino acid mixture into distinct radioactive peaks with molecular weights in the 80-90 kD, 53-66 kD, 22-46 kD, and 17 kD ranges with isoelectric points between 5.0 and 7.9. Most of the amino acids were incorporated into proteins in all of the molecular weight ranges. But Ser was incorporated in highest amounts in the 17 kD range, and Val was most abundant in the 22-46 kD range. In some cases results indicated that single proteins were modified by several amino acids. While we do not yet know which amino acids modify specific nerve proteins or the function of the modifications in nerve regeneration, these studies demonstrate the participation of some but not all amino acids in posttranslational modification reactions and the selective modification of specific groups of nerve proteins by these amino acids.

Microheterogeneity of agonist and antagonist glucocorticoid receptor complexes detected by isoelectric focusing and modifications induced by receptor activation

Rat-liver glucocorticoid receptor was incubated with either [ 3H]triamcinolone acetonide or [ 3H]RU 486, a well known antiglucocorticoid. Once formed, the steroid-receptor complexes were analyzed by isoelectric focusing in agarose gel slabs. A careful slicing of the receptor tracks revealed the presence of three distinct radioactive peaks focused at the following p I values: 5.3 ± 0.2 ( n = 17), 4.83 ± 0.04 ( n = 17) and 4.4 ± 0.1 ( n = 17). All these peaks correspond with receptor isoforms as suggested by control experiments. The receptor state was analyzed after focusing by a chromatographic assay on DNA-cellulose, DEAE-trisacryl TM and hydroxyapatite minicolumns. The peak of p I 4.4 apparently corresponded to the non-transformed receptor and was greatly stabilized in the presence of RU 486, whereas the peaks of p I 4.8 and 5.3 were probably made of transformed receptor and meroreceptor. These results were confirmed by autoradiographic studies after isoelectric focusing of receptor molecules covalently labelled with [ 3H]dexamethasone mesylate. Thus, the rat-liver glucocorticoid receptor appeared to be a rather acidic protein which became less acidic after transformation by heat, displaying a p I shift which was strongly reduced in case of steroid-receptor complexes formed with the antiglucocorticoid RU 486.

The formation of inositol 1,2-cyclic 4,5-trisphosphate and inositol 1,2-cyclic 4-bisphosphate on stimulation of mouse pancreatic minilobules with carbamylcholine.

When [3H]myoinositol-prelabeled pancreatic minilobules were incubated with carbamylcholine (CCh) for 30 min, followed by ionophoresis on paper of the aqueous extracts, there were distinct peaks of radioactivity immediately preceding inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3), which, based on earlier studies with inositol 1,2-cyclic phosphate (IcP), are the expected positions for inositol 1,2-cyclic 4-bisphosphate (IcP2) and inositol 1,2-cylic 4,5-trisphosphate (IcP3). These peaks were essentially absent on ionopherograms of extracts from minilobules not incubated with CCh. Similar results were obtained with high performance liquid chromatography (HPLC), except that the putative inositol cyclic phosphate peaks eluted immediately before the non-cyclic inositol polyphosphates, as to be expected. Taking advantage of the unique acid lability of the inositol cyclic phosphates, we demonstrate that the putative inositol cyclic polyphosphate peaks were specifically eliminated by prior hydrolysis of the aqueous extracts, as shown by either ionophoresis or HPLC. After preparative isolation of putative IcP2 and IcP3 by ionophoresis, acid hydrolysis shifted the positions of putative IcP2 and IcP3 peaks to the positions of standard IP2 and IP3, respectively, as shown by either ionophoresis or HPLC. The amounts of IcP, IcP2, and IcP3 formed on CCh stimulation, as measured by ionophoresis, were 0.7, 6.8, and 29.8% of that of, IP, IP2, and IP3, respectively (average of two experiments which agreed within 10%).

Intracellular modification of 125I-labeled epidermal growth factor by normal human foreskin fibroblasts.

Intracellular processing of 125I-labeled epidermal growth factor (EGF) in normal human foreskin fibroblasts was examined after incubation with saturating concentrations of [125I]EGF. This report describes the column chromatographic separation of multiple processed forms of EGF generated by human foreskin fibroblasts and their structural characterization. More than 95% of the cell-bound [125I]EGF was converted into multiple forms, which were separated into four distinct peaks of radioactivity using columns of Bio-Gel P-150 equilibrated with 0.2% sodium dodecyl sulfate. These were designated peaks 1-4. Cellular generation of these four peaks was dependent on culture conditions. Differences in absolute and relative amounts of peaks 1-4 were observed as a function of time of incubation at 37 C. In addition, chromatographic profiles of cell-associated 125I varied in relation to cell density. The radioactivity in peak 1 comigrated with 125I-labeled native EGF on nondenaturing polyacrylamide gels (pH 9.5), whereas peaks 2 and 3 exhibited more rapid electrophoretic mobilities. Electrophoretic mobilities of the radioactivity in peaks 2 and 3 were indistinguishable from those of chemically prepared derivatives of [125I]EGF which were lacking either one or six amino acid residues from the carboxyterminus, respectively. The EGF receptor bound the radioactive material in peak 2 with an affinity equal to or greater than that of EGF; however, the radioactivity in peak 3 was bound to a much lesser extent. The radiolabel in both peaks 2 and 3 was greater than 95% precipitable by antiserum to native EGF. The labeled material in peak 4 was composed of [125I]monoiodotyrosine, 125I-, and an unidentified peptide. None of the radiolabeled compounds in peak 4 interacted with the EGF receptor or with antiserum to native EGF.

Synthesis of adult-type hemoglobin in human erythremia cell line

KMOE -2/05 cells, derived from a patient with acute erythremia, became benzidine-positive after the addition of cytosine arabinoside (CA). Radioimmunoassays using antihuman hemoglobin antibodies revealed an elevated amount of hemoglobin in the CA-exposed cells over that in the control cells (without CA). Isoelectric focusing of the CA-exposed cell lysate formed benzidine-positive foci in the positions of human adult Hb (HbA) and human fetal Hb (HbF). To determine the types of globin synthesized in the CA-exposed cells, globin chains internally labeled with [3H] leucine were purified by carboxy-methyl (CM)-Sephadex column chromatography, immunoadsorption by Sepharose-coupled antihuman Hb antibodies and Sephadex G-100. The labeled globin chains were finally separated by CM-cellulose chromatography in urea. Two distinct peaks of radioactivity were shown in exactly the same fractions as carrier human globin alpha- and beta-chains. These observations indicate that these KMOE -2/05 cells synthesize HbA.

Synthesis of Adult-Type Hemoglobin in Human Erythremia Cell Line

KMOE -2/05 cells, derived from a patient with acute erythremia, became benzidine-positive after the addition of cytosine arabinoside (CA). Radioimmunoassays using antihuman hemoglobin antibodies revealed an elevated amount of hemoglobin in the CA-exposed cells over that in the control cells (without CA). Isoelectric focusing of the CA-exposed cell lysate formed benzidine-positive foci in the positions of human adult Hb (HbA) and human fetal Hb (HbF). To determine the types of globin synthesized in the CA-exposed cells, globin chains internally labeled with [3H] leucine were purified by carboxy-methyl (CM)-Sephadex column chromatography, immunoadsorption by Sepharose-coupled antihuman Hb antibodies and Sephadex G-100. The labeled globin chains were finally separated by CM-cellulose chromatography in urea. Two distinct peaks of radioactivity were shown in exactly the same fractions as carrier human globin alpha- and beta-chains. These observations indicate that these KMOE -2/05 cells synthesize HbA.

Characterization of testosterone binding protein of rat liver cytosol.

Testosterone binding protein from rat liver cytosol, which had been incubated with [3H]testosterone followed by treatment with dextran-coated charcoal, was analyzed by DEAE-cellulose and phosphocellulose chromatography. On DEAE-cellulose chromatography, two distinct peaks of radioactivity were eluted at 0.07 M and 0.19 M KCl, both sedimented in 4 S regions. Phosphocellulose chromatography resulted in a broad peak at 0.08 M KCl, with a shoulder at 0.04 M KCl, both sedimented at 4 S. These findings indicated that testosterone binding protein consists of two types of components each with 4 S.

Identification of human DNA sequences complementary to chromosomal RNA with an improved RNA/DNA gradient hybridization technique

Native DNA components in the human genome were identified with an improved RNA gradient hybridization technique. 3H-Labelled complementary RNA transcribed from total human DNA was hybridized to homologous DNA which had been mildly sheared to generate single-stranded ends. Unhybridized RNA was eliminated from the reaction mixture by column chromatography before the hybrids were subjected to CsCl gradient centrifugation. Nine radioactive peaks representing RNA hybrids were reproducibly found at buoyant densities of 1.687, 1.693, 1.696, 1.699, 1.702, 1.705, 1.708, 1.711 and 1.715 g/cm 3. The technique was also utilized to hybridize 125I-labelled human chromosomal RNA to DNA. Three distinct radioactive peaks were found at the heavy buoyant densities of 1.702, 1.705, and 1.708 g/cm 3, suggesting a differential enrichment of DNA components with sequences complementary to chromosomal RNA.

Human urinary and liver conjugates of 17α-ethynylestradiol

Chromatographic profiles of the conjugates of 17α-ethynylestradiol (EE 2) were obtained from the urine of castrate and normal women given tritium labeled EE 2 orally. Five distinct radioactive peaks were observed, with considerable quantitative variation between individuals. Glucosiduronate fractions comprised the dominant excreted radioactivity. In vitro incubation of normal human liver also produced five conjugate types. Simultaneous intravenous/oral ( 14C/ 3H) administration of EE 2 demonstrated conversion to identical urinary conjugate and free steroid products, with a greater excretion of the intravenous material. Authentic 3-glucuronide of EE 2 was synthesized and its position in the chromatographic system relative to the urinary conjugate peaks demonstrated.

Glycoprotein biosynthesis in plants. Demonstration of lipid-linked oligosaccharides of mannose and N-acetylglucosamine.

Previous studies from this laboratory have shown that particulate preparations from maturing cotton fibers catalyze the transfer of mannose from GDP-[14C]mannose into mannosylphosphorylpolyisoprenol (Forsee, W. T., and Elbein,A. D. (1973) J. Biol. Chem. 248, 2858-2867). In this report, we show that these particulate preparations also catalyze the inocoporation of mannose from GDP-[14C]mannose into lipid-linked oligosaccharides and into glycoprotein. The oligosaccharide-lipids were treated with dilute acid to liberate the water-soluble oligosaccharides and these oligosaccharides could then be separated into seven or eight distinct radioactive peaks by paper chromatography in isobutyric acid/NH4OH/H2betaO (57/4/39). The smallest of the oligosaccharides appears to be a trisaccharide with the structure Man leads to GlcNAc-GlcNAc. Thus the oligosaccharides attached to the lipids apparently range in size from those having 3 glycose units to those having approximately 8 to 10 glycose units. The radioactivity in the smaller-sized oligosaccharide-lipids could be chased into the larger oligosaccharide-lipids by a second incubation in the presence of unlabeled GDP-mannose. The sugar at the reducing ends of the oligosaccharides was identified as GlcNAc while some mannose (20 to 30%) was present in alpha linkages at the nonreducing ends...

Formation, Size, and Solubility in Chloroform/Methanol of Products of Protein Synthesis in Isolated Mitochondria of Rat Liver and Zajdela Hepatoma

The number, size, solubility in chloroform/methanol and some aspects of the formation of the components labeled by radioactive amino acids in isolated mitochondria of rat liver and Zajdela hepatoma were studied. Isolated mitochondria were labeled with radioactive amino acids under various conditions, and the distribution of radioactivity in sodium dodecylsulfate-polyacrylamide gels after electrophoresis of mitochondrial membrane fraction was analysed. 1. Isolated mitochondria of rat liver and Zajdela hepatoma incroporated radioactive amino acids almost exclusively into the membrane fraction. Electrophoretic analysis of this fraction revealed the presence of 15 distinct peaks of radioactivity with corresponding apparent molecular weights of 10 000 to 58 000. The electrophoretic mobility of the labeled components was identical and the general pattern of the radioactivity distribution in the gel for the rat liver and the tumour mitochondria was very similar. 2. Components of the membrane fraction of rat liver mitochondria labeled in vitro displayed an unequal solubility in acidic (2 mM HC1) chloroform/methanol (2/1) mixture; as detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis a single labeled component with apparent molecular weight of 10 000 was soluble in neutral chloroform/methanol. 3. Inverse relation was observed between amino acid incorporation activity of isolated mitochondria and the portion of the label incorporated into the component with apparent molecular weight 10 000. The identity of this component with that soluble in neutral chloroform/methanol mixture has been indicated. 4. The rate of incorporation of [3H]leucine by isolated Zajdela hepatoma mitochondria into the components with lower (10 000-25 000) apparent molecular weights decreased with time, whereas that into components with higher (above 25 000) apparent molecular weight remained approximately constant within the time interval tested (30 min). 5. From the total radioactivity incorporated into the membrane fraction during 5-min pulse labeling of isolated Zajdela hepatoma mitochondria by [3H]leucine up to 25% was recovered in the region of the gel corresponding to a component with apparent molecular weight 10 000. After 25 min chase the radioactivity in this region decreased about 3.5 times while the specific radioactivity of the total membrane fraction did not change significantly. The pattern of radioactivity distribution observed after the pulse was preserved by chloramphenicol. 6. Unlabeled sonicated mitochondria or postribosomal supernatant from rat liver regenerating in the presence of chloramphenicol were incubated with neutral chloroform/methanol extract of in vitro with [14C]leucine labeled rat liver mitochondria. After this incubation several labeled components with apparent molecular weights above 10 000 were recovered in the electrophoreograms of the originally unlabeled fractions.

Separation of conjugated urinary estrogens on columns of sephadex® : II. Sequential separations

1 1 See preceding paper (ref. 1) for abbreviations used here. Columns of DEAE-Sephadex were used to separate conjugated estrogens in urine (metabolites of [4- 14C]estradiol, administered intravenously) and reference standards. A scheme has been devised whereby many of the conjugated estrogens in urine may be separately by means of a sequence of three chromatographic procedures on columns of Sephadex G-25, DEAE-Sephadex and Sephadex G-15. The composite elution profile contains at least eighteen distinct peaks of radioactivity.