Biomedical subjects
Publications and source records attributed to G C Mueller.
Purification of estrogen receptors by affinity chromatography over diethylstilbestrol-agarose is associated with a major loss of estradiol binding activity. Histone H2B can restore a significant fraction of the binding activity. Cleavage of the H2B molecule into two halves by cyanogen bromide reveals that the carboxyl terminus is responsible for the major reactivating effects.
12-O-Tetradecanoylphorbol 13-acetate (TPA) has been shown to induce the formation of an unusual acidic phospholipid, phosphatidylethanol, in HL-60 cells. The synthesis of this lipid is dependent upon the presence of ethanol in the culture medium of TPA-treated cells; however, other exogenous alcohols can substitute as headgroup precursors with the formation of the corresponding phosphatidyl alcohol. The activation of the pathway for phosphatidyl alcohol synthesis appears to be mediated through protein kinase C. Studies of the time-course for the synthesis and accumulation of phosphatidylethanol suggest a possible involvement of the pathway for phosphatidyl alcohol synthesis in the TPA-induced differentiation of HL-60 cells.
Two versions of an approach to identify DNA-protein interactions at sites of DNA replication in HeLa cell nuclei are described. In this procedure, newly replicated DNA chains are first labeled and photosensitized in vitro by the incorporation of [alpha-32P] dCTP and bromodeoxyuridine triphosphate, respectively. Irradiation with ultraviolet light is then used to covalently crosslink the proteins that are adjacent to the photosensitized and isotopically labeled strands of newly replicated DNA. After the bulk of the DNA is digested with nucleases, the crosslinked proteins--marked by short covalently linked radioactive DNA tags--are fractionated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and detected by autoradiography. With this technology, certain proteins have been shown to associate selectively with newly replicated DNA. The method appears adaptable for application to a variety of problems involving DNA-protein association.
Tumor-promoting phorbol esters acutely activate a pathway in lymphocytes leading to the synthesis and accumulation of phosphatidylethanol, using exogenous ethanol as a precursor. This product is a representative of a unique class of acidic glycerophospholipids in which the head group is a primary alcohol. The formation of this lipid, in response to different phorbol ester derivatives, correlates with their activity as tumor promoters and inducers of growth changes in a variety of animal cells. Since phosphatidylethanol represents an unusual metabolite of ethanol, it is proposed that studies of its synthesis and biological functions may also provide new perspectives on the biology of alcohol addiction as well as the role of this biological pathway in tumor promotion.
Two estrogen binding proteins (Mr = 50,000 and 65,000) were purified from rabbit uterine cytosol using an improved procedure for affinity chromatography on diethylstilbestrol-agarose. The estrogen receptors were radioiodinated while adsorbed to the resin using the lactoperoxidase or Bolton-Hunter techniques. After elution, the labeled receptors were utilized for peptide mapping studies and investigations of receptor function. Partial peptide mapping revealed strong homology between the Mr 50,000 and 65,000 proteins suggesting common structural features. Estrogen receptors labeled by the lactoperoxidase procedure were rendered unable to bind immobilized heparin or hormone; in contrast, the Bolton-Hunter labeling technique yields proteins that retain both their ability to bind hormone and to absorb on heparin-agarose. The development of these iodination methodologies appears useful for the investigation of both the structure and functional properties of the receptor proteins.
Fatty acids rapidly accelerate (1.5-7.0-fold) the incorporation of [methyl-3H]choline chloride into the phosphatidylcholine fraction of bovine lymphocyte lipids. This ability of fatty acids to activate choline phospholipid synthesis has been correlated with certain structural features of fatty acids. Mono- and polyenoic unsaturated fatty acids of 18 and 20 carbons in length are highly active, whereas their saturated analogues are nearly inactive. Among the unsaturated fatty acids, the cis-isomers are active, while the trans-isomers are relatively ineffective. The delayed addition of bovine serum albumin (5 mg/ml) and other lipid-binding proteins to activated cells rapidly counteracts the lipid effects. The activated state of the cell membrane thus appears to be a dynamic one, requiring the continued interaction of the fatty acid with a lipid-sensitive target molecule of the cell surface that in turn appears to coordinate the enzymatic components of this pathway.
Phosphatidylcholine synthesis in cultured bovine lymphocytes is stimulated by cis-unsaturated fatty acids. This stimulation is correlated with an activation of the enzyme cytidyltransferase (EC 2.7.7.15) and its apparent translocation from the cytosol to the membrane/particulate of cells. In addition, these agents increase the levels of cytidine diphosphocholine - a product of the cytidyltransferase reaction and a precursor to phosphatidylcholine. Retinoic acid and 5,8,11,14-eicosatetraynoic acid both activate cytidyltransferase activity and raise cytidine diphosphocholine levels, yet they are ineffective as stimulators of overall phosphatidylcholine synthesis. The effects of all of these lipids are reversed by the delayed addition of bovine serum albumin. The data point to the view that cytidyltransferase activation is required but is not sufficient for stimulation of phosphatidylcholine synthesis: regulation at another step is suggested.
Hemin (ferriprotoporphyrin IX-chloride) can mediate the covalent cross-linking and degradation of yeast glutathione reductase. This reaction requires both NADPH and oxygen suggesting the involvement of a reduced oxygen species in the cross-linking and degradation process. During the course of the reaction the enzymatic activity of glutathione reductase is rapidly destroyed. Implications of these findings for a regulatory role of hemin in cell biology are discussed.
Hemin, in the presence of 2-mercaptoethanol and oxygen, catalyzes the selective degradation of heme-binding proteins to small peptide fragments. Among the proteins examined, the heme-binding protein of rabbit serum (HBP-93) proved to be unusually sensitive. Myoglobin also exhibited considerable sensitivity whereas hemopexin and bovine serum albumin were only slightly susceptible to this degradative action of hemin. The reaction with HBP-93 depended upon coordination of the protein with hemin, was optimal at pH 6.5 and increased 4-fold as the temperature was elevated from 10 to 60 degrees C. The requirement for both oxygen and the reducing agent, 2-mercaptoethanol, and the partial protection of HBP-93 to degradation by catalase, superoxide dismutase, mannitol, and thiourea suggest the involvement of reduced oxygen species in the reaction. A possible role for the heme-mediated degradation of proteins in cell differentiation and other biological responses is discussed.
Diethylstilbestrol was coupled to epoxy-activated agarose yielding an affinity resin which is highly efficient for the isolation of estrogen receptors. This resin, diethylstilbestrol-agarose (DES-agarose), bound two proteins (Mr = 50,000 and 65,000) from rabbit uterine cytosol that show a specific interaction with estradiol. A two step procedure--adsorption on DES-agarose followed by a selective elution with p-sec-amylphenol and NaSCN, yielded highly purified estrogen receptors which can be used in the studies of estradiol-receptor interactions with other cell constituents.
An antiestrogen affinity resin was synthesized by conjugating LY117018, a benzothiophene -derived antiestrogen, to epoxy-activated agarose. This affinity resin bound the Mr = 50 000 and 65 000 estrogen receptor proteins of rabbit uterine cytosol; in addition, it retained a protein from the cytosols of both rat and rabbit uteri that exhibited an ability to interact specifically with LY117018. The possibility that the LY117018 binding protein, which is distinct from estrogen receptors, may play a role in the antiestrogenic actions of LY117018 is discussed.
3H-Estradiol-estrogen receptor complexes were adsorbed on a column of heparin-agarose and subjected to a gradient of increasing concentrations of p-sec-amylphenol. At least five peaks of released 3H-estradiol were observed--demonstrating the existence of subsets of heparin-immobilized receptors with different affinities for estradiol. This finding is presented as further evidence for a functional micro-heterogeneity among estrogen receptors. The origin of the observed differences in estrogen receptors and possible relevance of the findings to receptor-mediated responses are discussed.
Hemin (ferric protoporphyrin IX chloride) has been shown to cause strand scission in DNA in a reaction which requires the presence of oxygen and the reducing agent, 2-mercaptoethanol. In model studies, circular supercoiled plasmid DNA is converted within 30 min to the open circle and linear forms. With longer incubation times the DNA is degraded to small pieces. The reaction is markedly influenced by the addition of divalent cations; Mg2+ and Ca2+ inhibit the reaction while the transition metals Co2+, Zn2+, Ni2+, and Cu2+ promote the degradation. These observations are discussed in relation to the role of hemin in the modulation of gene expression during cell differentiation.
Upon treatment of HeLa nucleoids with EcoR1 restriction enzyme, a minute fraction of the DNA remains attached to the nuclear matrix. This matrix-DNA complex appears to be composed of a unique set of proteins. Electron microscopic analysis of the DNA fragments present shows an enrichment for HeLa DNA replicating forks.Images
Treatment of bovine lymphocytes with phorbol esters activates an oxygen-dependent system for the formation of a class of arachidonic acid metabolites which appears distinct from prostaglandins, prostacyclins, thromboxanes and leukotrienes. A full activation of the system is achieved within minutes by 10(-7) M 12-O-tetradecanoylphorbol-13-acetate. This activation can be largely prevented by the lipoxygenase inhibitors, 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid.
Treatment of bovine lymphocytes with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (10(-8) M) for as little as 5 min significantly alters the ability of membrane-particulate fractions to phosphorylate proteins using in situ protein kinases and exogenous [gamma-32P]ATP. After 20 min of treatment, the phosphorylation of two proteins with mol. wts. of 65K and 74K in the isolated membrane-particulate fraction is increased approximately 30-35%, whereas the phosphorylation of a 130K protein is almost completely suppressed. The ability of different phorbol esters to alter membrane protein phosphorylation correlates well with their potency as tumor promoters in mouse epidermis and as comitogens in phytohemagglutinin-treated lymphocytes. This phorbol ester response appears to have a low temperature coefficient since cells treated with TPA at 4 degrees C also responded, although at a slower rate. This action of TPA is neither mimicked nor antagonized by dibutyryl cAMP (1 mM); moreover, it is not affected by retinoic acid, an agent which blocks several other phorbol ester effects in these cells. Inhibition of protein synthesis with cycloheximide also fails to influence this response. In contrast, trifluoperazine, an inhibitor of calmodulin function and certain lipid-dependent protein kinases, depressed the phosphorylation of the 65K and 74K proteins to below the control level.
Plasmid subclones of the 5' and 3' regions of the beta major-globin gene have been used as probes to assay the level and character of the transcripts of this gene in differentiating Friend erythroleukemia cells treated with dexamethasone. Using an S1 nuclease protection protocol, it is shown that dexamethasone reduces the amount of precursor-specific sequences in differentiating Friend cells within 30 min without significantly affecting the nuclear turnover of these molecules. The sites of initiation and termination of transcription remained the same during the shutdown of transcription by the hormone. In contrast to the situation in Friend cells, dexamethasone failed to inhibit globin gene transcription in three normal erythroid tissues. The manner in which the Friend virus complex might render the globin genes sensitive to glucocorticoids is discussed.
A 93,000 molecular weight protein (HBP.93) which binds hemin and protoporphyrin IX with high affinity has been isolated from rabbit serum using affinity chromatography on hemin-conjugated agarose. The amino acid composition of this protein is unique in that the proline and histidine contents are remarkably high (16.6 and 9.9 mol %, respectively). A large increase in the absorbance of the Soret region arises from the heme-protein interaction. The spectrophotometric titration showed that the protein can bind 25-35 mol of hemin/mol of protein. The apparent dissociation constant was estimated to be 1-4 X 10(-7) M for hemin at pH 7.4 and approximately 10(-6) M for protoporphyrin IX at pH 9.2. The similarity of the difference spectrum of heme-HBP.93 complex to that of heme-hemopexin complex suggests that a bisimidazol-type coordination of heme iron is involved in the binding. The extremely high capacity of HBP.93 to bind heme is also demonstrated by a large increase in the sedimentation velocity of the protein upon heme binding. The native heme-protein complex migrates faster than the heme-free protein in a polyacrylamide gel at pH 8.8; the increased mobility appears to be due to the charge on the carboxyl groups of the bound heme. Although the use of a hemin-agarose column has failed to reveal a protein of similar size and heme affinity in the sera of a number of other species, including man, the heme-binding properties and high histidine level of the human alpha 2-histidine-rich glycoprotein raise the possibility that the two proteins are related.