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Biomedical subjects

D C Eichler

Publications and source records attributed to D C Eichler.

At least 37 records · Page 2Linked to original sources
PubMed1987

Isolation and characterization of a nucleolar 2'-O-methyltransferase from Ehrlich ascites tumor cells.

A 2'-O-methyltransferase that transfers the methyl group from S-adenosylmethionine to the 2'-hydroxyl group of ribose moieties of RNA has been purified from Ehrlich ascites tumor cell nucleoli. The partially purified enzyme is devoid of other RNA methylase activities and is free of ribonucleases. The enzyme has optimal activity in tris(hydroxymethyl)aminomethane buffer, pH 8.0, in the presence of 0.4 mM ethylenediaminetetraacetic acid, 2 mM dithiothreitol, and 50 mM KCl, and has an apparent Km for S-adenosylmethionine of 0.44 microM. Gel filtration studies of this enzyme gave a Stokes radius of 43 A. Sedimentation velocity measurements in glycerol gradients yield an S20,w of 8.0 S. From these values, a native molecular weight of 145,000 was calculated. The enzyme catalyzes the methylation of synthetic homoribopolymers as well as 18S and 28S rRNA; however, poly(C) is the preferred synthetic substrate, and preference for unmethylated sequences of rRNA was observed. For each RNA substrate examined, only methylation of the 2'-hydroxyl group of the ribose moieties was detected.

Animals↗
PubMed1987

Anti-flavin antibodies.

Antibodies were elicited to FAD by using the hapten N-6-(6-aminohexyl)-FAD conjugated to the immunogenic carrier protein bovine serum albumin. Cross-reactivity was determined by Ouchterlony double-diffusion analysis with N-6-(6-aminohexyl)-FAD coupled to rabbit serum albumin. Anti-FAD IgG was partially purified by (NH4)2SO4 precipitation followed by DEAE-cellulose/CM-cellulose and bovine serum albumin-agarose chromatography. The partially purified anti-FAD IgG fraction failed to inhibit the catalytic activities of the flavin-containing enzymes nitrate reductase, xanthine oxidase and succinate dehydrogenase, whereas enzyme activity could be inhibited by addition of antibodies elicited against the native proteins. However, the partially purified anti-FAD IgG fraction could be used as a highly sensitive and specific probe to detect proteins containing only covalently bound flavin, such as succinate dehydrogenase, p-cresol methylhydroxylase and monoamine oxidase, by immuno-blotting techniques. Detection limits were estimated to be of the order of femtomolar concentrations of FAD with increased sensitivity for the 8 alpha-N(3)-histidyl linkage compared with 8 alpha-O-tyrosyl substitution.

Animals↗
PubMed1986

Characteristics of rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

A procedure for the preparation of rat liver microsomal fractions essentially devoid of contaminating lysosomes is described. When this preparation was examined by immunoblotting with a rabbit antiserum to rat 3-hydroxy-3-methylglutaryl-CoA reductase, a single band corresponding to an Mr of 100000 was observed. No evidence was found for glycosylation of rat liver-3-hydroxy-3-methylglutaryl-CoA reductase. Native rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase differs from the purified proteolytically modified species in that it displays allosteric kinetics towards NADPH.

Acetylglucosaminidase↗
PubMed1985

Spin-labeled erythrocyte membranes: direct identification of nitroxide-conjugated proteins.

The covalent incorporation of a spin-labeled analog of N-ethyl maleimide into erythrocyte membrane proteins has been monitored by electron paramagnetic resonance spectroscopy and the individually labeled proteins detected by immunoblotting techniques, using an anti-nitroxide antibody, following electrophoretic separation of the membrane components. Spin-label was primarily found in the high molecular weight bands (I and II) with no incorporation in proteins with molecular weights less than 35,000. Increasing the reaction time between the spin-label and ghosts altered both the observed labeling pattern and the epr spectra with an increase in the proportion of strongly-immobilized species.

Cyclic N-Oxides↗
PubMed1985

Anti-nitroxide immunoglobulin G: analysis of antibody specificity and their application as probes for spin-labeled proteins.

Antibodies have been elicited to the nitroxide spin-label 4-maleimido-2,2,6,6-tetramethyl-piperidinyl-1-oxy conjugated, via protein sulfhydryl groups, to bovine serum albumin. Antibody-hapten cross-reactivity was demonstrated by double immunodiffusion and by a broadening of the nitroxide electron paramagnetic resonance spectrum. The specificity of the antibodies with respect to hapten structure was examined by means of a simple filter binding assay. Under these conditions, antibodies were shown to distinguish between the nitroxide and hydroxylamine derivatives and between spin-labels comprising either five- or six-membered ring structures. In addition, protein-bound nitroxide spin-labels were detected at the nanogram level by immunoblotting. By use of this method, the specificity of the antibody-hapten reaction predicted by the filter binding assay procedure was utilized to differentially detect various types of bound spin-label. Finally, antibodies were used to identify protein-bound nitroxide spin-label of protein fractionated by gel electrophoresis.

Animals↗
PubMed1985

Purification and properties of a novel nucleolar exoribonuclease capable of degrading both single-stranded and double-stranded RNA.

A ribonuclease that hydrolyzes either linear duplex or single-stranded RNA in an exonucleolytic manner has been partially purified from Ehrlich ascites tumor cell nucleoli and is free from other ribonucleases. The enzyme will also degrade the RNA complement of an RNA X DNA duplex; however, no nuclease activity is observed on linear duplex or single-stranded DNA. The exonuclease acts on RNA nonprocessively from the 3' end releasing 5'-mononucleotides. The enzyme has a broad pH optimum around pH 8.0, requires Mg2+ or Mn2+ (0.06 mM) for optimum activity, and is sensitive to ethylenediaminetetraacetic acid and N-ethylmaleimide inhibition. Monovalent cations including K+, Na+, and NH4+ are inhibitory. Gel filtration studies of this enzyme gave a Stokes radius of 40 A. Sedimentation velocity measurements in glycerol gradients yield a S20,W of 6.0 S. From these values a native molecular weight of 100 000 was calculated. Copurification of the single- and double-stranded activities, identical reaction requirements, and identical heat-inactivation curves strongly suggest that both activities reside with the same enzyme.

Animals↗
PubMed1984

Isolation and properties of a single-strand 5'----3' exoribonuclease from Ehrlich ascites tumor cell nucleoli.

A single-strand-specific, nucleolar exoribonuclease from Ehrlich ascites tumor cells has been isolated and purified free from other nucleases. The exonuclease degraded single-stranded RNA processively from either a 5'-hydroxyl or a 5'-phosphorylated end and released 5'-mononucleotides. The enzyme digested single-strand poly(C), poly(U), and poly(A) equally well but did not degrade duplex poly(C).poly(I) or poly(A).poly(U). Less than 0.2% of duplex DNA or 1.5% of heat-denatured DNA was degraded under the conditions which resulted in greater than 26% degradation of RNA. The ribonuclease required Mg2+ (0.2 mM) for optimum activity and was inhibited by ethylenediaminetetraacetic acid but not by human placental RNase inhibitor. The native enzyme had a Stokes radius of 42 A and a sedimentation coefficient (S20,w) of 4.3 S. From these values, an apparent molecular weight of 76 000 was derived by using the Svedberg equation. The localization and unique mode of degradation suggest a role for the 5'----3' exoribonuclease in ribosomal RNA processing.

Animals↗
PubMed1983

The effect of RNA secondary structure on the action of a nucleolar endoribonuclease.

The effect of the folded macromolecular structure of RNA on the action of a purified single strand specific nucleolar ribonuclease was studied by comparing the limited hydrolysis of defined RNA substrates. The nucleolar RNase was shown to attack only single-stranded regions of the native 5.8 S rRNA, consistent with a computer-derived model for the secondary structure (Nazar, R. N., Stitz, T. O., and Busch, H. (1975) J. Biol. Chem. 250, 8591-8597). The single strand specific nucleolar RNase, unlike S1 nuclease, does not release the end-labeled nucleotide and therefore provides a more useful probe for structural analysis at or near 3'- or 5'-terminal ends of an RNA molecule. Although attacking only single strand regions of the native 5.8 S rRNA, the selectivity of the nucleolar RNase, when compared to S1 nuclease, is distinct and supports the suggestion that other factors besides the proposed secondary structure must also influence nuclease attack. The selective and limited attack of the nucleolar RNase on native RNA was similarly observed using mouse 45 S preribosomal RNA as a substrate and possibly suggests a role for this nucleolar RNase in ribosomal RNA processing.

Animals↗
PubMed1982

Isolation and characterization of a single-stranded specific endoribonuclease from Ehrlich cell nucleoli.

An endoribonuclease which cleaves only single-stranded RNA has been purified from nucleoli of Ehrlich ascites tumor cells. The molecular weight of the ribonuclease is 50,000 to 52,000 as estimated from sedimentation in glycerol density gradients and by gel filtration on Sephadex G-100. The endoribonuclease requires Mg2+ or Mn2+ (0.2 mM) for optimum activity. Monovalent cations including K+, Na+, and NH+4 are inhibitory. The ribonuclease gave an apparent Km for single-stranded RNA of 30 microM. Using ribohomopolymers, we found that the enzyme could digest single-stranded, poly(C), poly(U), and poly(A) equally well, but would not degrade duplex poly(C) . poly(I) or poly(A) . poly(U). The lack of base specificity was further demonstrated using RNA sequence analysis of partial digest products of yeast 5.8 S RNA. The ribonuclease activity is sensitive to EDTA and N-ethylmaleimide, but is not inhibited by human placental RNase inhibitor. The enzyme makes endonucleolytic cleavages which generate 5'-phosphate-terminated oligonucleotides.

Animals↗
PubMed1982

Conjugated dimers of horseradish peroxidase as neuroanatomical tracers.

Horseradish peroxidase dimer (HRP-HRP) and monomer (HRP) were compared in their ability to act as neuroanatomical tracers. Dimers exhibited less diffusion at the site of injection. At 48-72 h survival periods, there were no appreciable differences in the retrograde neuronal labeling produced by these two compounds. However, at 7 days survival, there was little retrograde labeling due to the presence of monomer while dimer labeling was still intense.

Animals↗
PubMed1980

Properties of a purified nucleolar ribonuclease from Ehrlich ascites carcinoma cells.

A nucleolar ribonuclease specific for single-stranded ribonucleic acid (RNA) has been isolated and extensively purified from Ehrlich ascites carcinoma cells. The enzyme is optimally active at neutral pH and degrades RNA via a 2',3'-cyclic intermediate leaving 3'- or 2',3'-cyclic terminated oligonucleotides. The ribonuclease has an apparent molecular weight of 38 500 as judged by sedimentation equilibrium and is a basic protein having an isoelectric point greater than 9.0. The enzyme preferentially cleaves poly(C) over poly (U), poly(A), or poly(C).poly(I). Limit digestion products of poly(C) degratation are on the average tri-, tetra-, and pentanucleotides. In the partial digestion of yeast 5.8S rRNA, the nucleolar ribonuclease cleaves only CpA phosphodiester bonds. Spermidine, spermine, and histone I inhibit the activity of nucleolar ribonuclease. Antibodies directed toward pancreatic RNase do not cross-react with the Ehrlich nucleolar ribonuclease.

Animals↗
PubMed1977

In vitro replication of mitochondrial DNA. Elongation of the endogenous primer sequence in D loop mitochondrial DNA by human DNA polymerase beta.

Whe incubated in the presence of Mn2+ as the divalent metal activator, highly purified human DNA polymerase beta performs a selective and limited replication of KB cell closed circular mtDNA. On the basis of biochemical and electron microscopic analyses of the reaction product, we demonstrate that the polymerase specifically recognizes and elongates the 9 S primer sequence in D loop mtDNA and then proceeds to copy the displaced strand. The point at which the enzyme switches template strands is most likely that at which all negative superhelical turns have been removed and an energetically unfavorable introduction of positive superhelical turns would be required for further synthesis on the initial parental template strand. The product of the reaction is an enlarged D loop that has been converted to a duplex structure. This is the first description of the capacity of a pure eukaryotic DNA polymerase to replicate a naturally occurring, specifically initiated duplex DNA molecule. Our results suggest that this system may be particularly useful in developing an in vitro duplex circular DNA replication system with purified eukaryotic components.

Cell Line↗
PubMed1977

Effect of calcium on the recovery and distribution of DNA polymerase alpha from cultured human cells.

It was recently reported (Lynch, W. E., Surrey, S., and Lieberman, I. (1975) J. Biol. Chem. 250, 8179-8183) that the extraction of regenerating rat liver in solutions of isotonic sucrose containing 4 mM CaCl2 leads to almost quantitative recovery of DNA polymerase alpha (Weissbach, A., Baltimore, D., Bollum, F., Gallo, R., and Korn, D. (1975) Science 190, 401--402) activity in the purified nuclear compartment. Our application of this method to the isolation of the DNA polymerase activities activities from cultured human epithelial and lymphoblastoid cells has led to substantially different results. We have observed that the inclusion of Ca2+ in either isotonic sucrose or hypotonic aqueous extraction media leads to the irreversible inactivation of the majority, cytoplasmic fraction of DNA polymerase alpha activity and is without quantitative effect on the recovery of the nuclear fraction of this activity.

Calcium↗
PubMed1977

On the role of ATP in phosphodiester bond hydrolysis catalyzed by the recBC deoxyribonuclease of Escherichia coli.

The deoxyribonuclease specified by the recB and recC genes of Escherichia coli (recBC DNase; exonuclease V) has been purified to near homogeneity by a new procedure. Although hydrolysis of even a single nucleotide from a duplex DNA molecule by the pure enzyme is absolutely dependent upon ATP, the extent of phosphodiester hydrolysis is strongly inhibited by ATP concentrations of 0.2 mm or greater, and the initial rate is unaffected. Under these conditions, the extent of DNA hydrolysis is proportional to enzyme concentration. In contrast, neither the rate nor the extent of hydrolysis of single-stranded DNA nor ATP is affected by high concentrations of ATP. The amount of large single-stranded polynucleotide generated by the action of the recBC DNase increases as the ATP concentration increases and, at 0.5 mM ATP, becomes equivalent to the amount of acid-soluble nucleotide formed. These findings suggest that high intracellular concentrations of ATP affect the mechanism of the recBC DNase so as to limit the extent of hydrolysis of duplex DNA, while at the same time favoring the formation of single-stranded regions within the duplex. Such regions may be essential intermediates in the recombination process.

Adenosine Triphosphate↗
PubMed1976

Biochemical characterization of mutant forms of DNA polymerase I from Escherichia coli. II. The polAex1 mutation.

DNA polymerase I has been purified to homogeneity from an Escherichia coli K12 strain bearing the temperature-sensitive conditionally lethal mutation, polAex1. The purified enzyme shows no defect in its polymerase or 3' leads to 5'-exonuclease activities; however, its 5' leads to 3'-exonuclease activity is abnormally low at both 30 degrees and 43 degrees. Although the mutant enzyme is able to catalyze the coordinated 5' leads to 3' polymerization and 5' leads to 3' exonucleolytic hydrolysis of nucleotides at a nick in duplex DNA ("nick translation") at a measurable rate at 30 degrees, this reaction is undetectable at 43 degrees. This defect is very likely responsible for the retarded joining of nascent DNA fragments and the consequent loss of viability that occur in the mutant at this temperature.

DNA Nucleotidyltransferases↗