Role of superoxide in radiation-killing of Escherichia coli and in thymine release from thymidine

The role of superoxide and hydroxyl radicals in gamma-radiation-killing of Escherichia coli K12 was studied in aerated suspensions supplemented with formate, phosphate, superoxide dismutase, catalase and saturated with nitrous oxide. Nitrous oxide, which converts e-aq to .OH, caused decreased radiosensitivity. On the other hand, formate, which results in conversion of .OH to .O2-, resulted in an increased radiosensitivity. The results implicated .O2- as a major cause of radiation-mediated cell-killing. The addition of the enzymes, superoxide dismutase or catalase to the E. coli suspensions prior to and during irradiation had no effect on cell survival, indicating that the biologically significant site of generation and action of .O2- is an intracellular one. Further studies were undertaken to examine the role of superoxide in DNA damage. The release of thymine from the DNA base, thymidine was studied as a result of gamma-irradiation and of chemically generated superoxide (using KO2 in dimethyl sulfoxide). Thymine was identified by HPLC and mass spectrometry. C-13 NMR analysis of the reaction mixture of thymidine with KO2 in dimethyl sulfoxide provided evidence for attack of .O2 at the ribosyl Cl' atom.     

Flash spectroscopy of purple membrane.

Flash spectroscopy data were obtained for purple membrane fragments at pH 5, 7, and 9 for seven temperatures from 5 degrees to 35 degrees C, at the magic angle for actinic versus measuring beam polarizations, at fifteen wavelengths from 380 to 700 nm, and for about five decades of time from 1 microsecond to completion of the photocycle. Signal-to-noise ratios are as high as 500. Systematic errors involving beam geometries, light scattering, absorption flattening, photoselection, temperature fluctuations, partial dark adaptation of the sample, unwanted actinic effects, and cooperativity were eliminated, compensated for, or are shown to be irrelevant for the conclusions. Using nonlinear least squares techniques, all data at one temperature and one pH were fitted to sums of exponential decays, which is the form required if the system obeys conventional first-order kinetics. The rate constants obtained have well behaved Arrhenius plots. Analysis of the residual errors of the fitting shows that seven exponentials are required to fit the data to the accuracy of the noise level.     

Synthesis of cytochrome c oxidase in the liver of Rana catesbeiana tadpole treated with griseofulvin

The effect of griseofulvin treatment on the synthesis of cytochrome c oxidase was studied with the liver of the tadpole, Rana catesbeiana. (1) In the liver of tadpole treated with griseofulvin, a ferrochelatase inhibitor, the synthesis of heme a, but not cytochrome c oxidase protein, is inhibited. (2) The apocytochrome c oxidase which is formed in the liver of tadpole treated with griseofulvin is converted to the active holoenzyme by exogenously added heme a.     

The reaction of arsenite-complexed xanthine oxidase with oxygen. Evidence for an oxygen-reactive molybdenum center

The effects of arsenite on the reaction of reduced xanthine oxidase with oxygen are determined. The kinetics of the reaction monitoring the return of enzyme absorbance are investigated as are the kinetics and stoichiometries of peroxide and superoxide formation. Although some of the effects of arsenite are qualitatively consistent with expectations based on the known perturbation of the molybdenum midpoint potentials by arsenite, several results cannot be so easily explained. Specifically, arsenite introduces a very rapid phase (kobs = 110 s-1 at 125 microM oxygen) to the oxidative half-reaction which is not observed with the native enzyme. Arsenite also diminishes the amount of superoxide produced and eliminates one-electron reduced enzyme as a detectable kinetic intermediate in the reoxidation pathway. These differences appear to result from the ability of arsenite to greatly enhance the oxygen- and/or superoxide-reactivity of the reduced molybdenum center. This is reflected in the observation that reduced forms of arsenite-complexed xanthine oxidase lacking functional FAD (iodoacetamide-alkylated enzyme and deflavo enzyme) react relatively rapidly with oxygen whereas these reactions are quite slow in the absence of arsenite.     

Cascade of autophosphorylation in the beta-subunit of the insulin receptor

Insulin stimulated autophosphorylation of the beta-subunit of the insulin receptor purified from Fao hepatoma cells or purified from Chinese hamster ovary (CHO/HIRC) or Swiss 3T3 (3T3/HIRC) cells transfected with the wild-type human insulin receptor cDNA. Autophosphorylation of the purified receptor occurred in at least two regions of the beta-subunit: the regulatory region containing Tyr-1146, Tyr-1150, and Tyr-1151, and the C-terminus containing Tyr-1316 and Tyr-1322. In the presence of antiphosphotyrosine antibody (alpha-PY), autophosphorylation of the purified receptor was inhibited nearly 80% during insulin stimulation. Tryptic peptide mapping showed that alpha-PY inhibited autophosphorylation of both tyrosyl residues in the C-terminus and one tyrosyl residue in the regulatory region, either Tyr-1150 or Tyr-1151. Thus, a bis-phosphorylated form of the regulatory region accumulated in the presence of alpha-PY, which contained Tyr(P)-1146 and either Tyr(P)-1150 or 1151. In intact Fao, CHO/HIRC, and 3T3/HIRC cells, insulin stimulated tyrosyl phosphorylation of the beta-subunit of the insulin receptor. Tryptic peptide mapping indicated that the regulatory region of the beta-subunit was mainly (greater than 80%) bis-phosphorylated; however, all three tyrosyl residues of the regulatory region were phosphorylated in about 20% of the receptors. As the phosphotransferase was activated by tris-phosphorylation but not bis-phosphorylation of the regulatory region of the beta-subunit (White et al.: Journal of Biological Chemistry 263:2969-2980, 1988), the extent of autophosphorylation in the regulatory region may play an important regulatory role during signal transmission in the intact cell.     

Calcium ion stimulated endogenous protein kinase catalyzed phosphorylation of basic proteins in myelin subfractions and myelin-like membrane fraction from rat brain

Polypeptide composition and endogenous phosphorylation were investigated in the subfractions of rat brain myelin isolated by either discontinuous or continuous sucrose density gradient centrifugation of myelin. Similarly, a myelin-like membrane fraction (SN4) was also studied. Observations were made that strongly indicated the presence of a calcium-stimulated protein kinase in a highly purified myelin preparation and which exclusively phosphorylated myelin basic proteins of the membrane preparation. Adenosine cyclic 3',5'-phosphate (cAMP) stimulated kinase on the other hand was found to be considerably enriched in the myelin-like membrane fraction. Although this latter enzyme is also capable of phosphorylating the basic proteins, its effect was at least 5 times weaker compared to the calcium-stimulated myelin protein kinase. Within the gradient subfractions there appeared a close relation between the amount of basic proteins and their calcium-stimulated phosphorylation; a similar relationship, however, was not obtained in the case of cAMP-dependent phosphorylation of myelin basic proteins. The former (i.e., Ca2+-stimulated phosphorylation) was found to require a protein factor that functionally resembled calmodulin. The results thus raises an interesting possibility of the presence of calmodulin-like proteins and a calcium-stimulated protein kinase in adult myelin membrane from mammalian brain, both of which have been hitherto unrecognized constituents of myelin membranes.     

A resonance Raman study of Ambystoma tigrinum hemoglobins: evidence for intraspecies hemepocket variations

Using resonance Raman difference spectroscopy, the Raman-active vibrational modes of hemoglobins from adult, neotenic, and larval forms of the salamander, Ambystoma tigrinum have been compared to each other and to human hemoglobin. The local heme environment of the adult and neotenic proteins were identical and differed from that of the larval protein. Differences were observed in modes sensitive to porphyrin pi electron density and axial ligation. Systematic differences were also observed between human and adult salamander hemoglobins particularly in modes sensitive to the heme vinyl environment. The relationship between these environmental differences, oxygen binding affinity, and the effects of allosteric modulators are discussed.     

Depurination-induced infidelity of deoxyribonucleic acid synthesis with purified deoxyribonucleic acid replication proteins in vitro

Removal of purine bases from phi X174 single-stranded DNA leads to increased reversion frequency of amber mutations when this DNA is copied in vitro with purified DNA polymerases. This depurination-induced mutagenesis is observed at three different genetic loci and with several different purified enzymes, including Escherichia coli DNA polymerases I and III, avian myeloblastosis virus DNA polymerase, and eukaryotic DNA polymerases alpha, beta, and gamma. The extent of mutagenesis correlates with the estimated frequency of bypass of the lesion and is greatest with inherently inaccurate DNA polymerases which lack proofreading capacity. With E. coli DNA polymerase I, conditions which diminish proofreading result in a 3-5-fold increase in depurination-induced mutagenesis, suggesting a role for proofreading in determining the frequency of bypass of apurinic sites. The addition of E. coli single-stranded DNA-binding protein to polymerase I catalyzed reactions with depurinated DNA had no effect on the extent of mutagenesis. Analysis of wild-type revertants produced during in vitro DNA synthesis by polymerase I or avian myeloblastosis virus DNA polymerase on depurinated phi X174 amber 3 DNA indicates a preference for insertion of dAMP opposite the putative apurinic site at position 587. These results are discussed in relation both to the mutagenic potential of apurinic sites in higher organisms and to studies on error-prone DNA synthesis.