Inactivation of secretory phospholipase A2 by ionizing radiation.

The extracellular phospholipase A2s (PLA2) from cobra venom, rattlesnake venom, and porcine pancreas were analyzed by radiation inactivation to determine their functional aggregation states. The analysis was performed in the presence of the protein transferrin at two different concentrations of PLA2: 5 micrograms/ml. The small size of these proteins necessitated the use of high radiation dosages. The catalytic activity of all samples decreased as a single exponential as a function of radiation dosage, to > 97% inactivation. Target size analysis of these curves yielded sizes corresponding to dimers for all three PLA2s, indicating that all three enzymes exist as dimers or larger aggregates under the conditions studied. An analysis of the amount of intact protein remaining by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the loss of protein also followed a dimeric size for all three PLA2s. The loss of protein as a dimer indicates that transfer of radiation energy is occurring between polypeptides.     

Inactivation of macromolecules by ionizing radiation. Deterministic single-hit or stochastic multievent process?

A stochastic theory concerning the radiation inactivation of macromolecules such as enzymes or receptors is elaborated. In contrast with the single-hit theory, which assumes a complete inactivation of the target as the result of one hit, the stochastic theory postulates that the degree of inactivation by one hit is a random variable. This distinguishing feature has been considered in order to give a possible interpretation to the observed effect of temperature on the radiation-sensitivity of enzymes. As a consequence of the progressive inactivation during irradiation, the binding affinity of a ligand for the macromolecule is impaired by irradiation. Although this property might discriminate the stochastic theory from the classical single-hit theory on the basis of a statistical analysis of experimentally obtained data, it is shown that the commonly obtained degree of inaccuracy may render the statistical test non-conclusive.     

Inactivation of ribonuclease inhibitor by thiol-disulfide exchange.

Porcine ribonuclease inhibitor (RI) contains 30 1/2-cystinyl residues, all of which occur in the reduced form. Reaction of the native protein with 5,5'-dithiobis (2-nitrobenzoic acid) resulted in the release of 30 mol of the product 5-mercapto-2-nitrobenzoate, and the loss of the RNase inhibitory activity. A linear relationship between the degree of modification and inactivation was observed. The rate of modification was greatly increased in the presence of 6 M guanidinium HCl. Reaction with substoichiometric amounts of 5,5'-dithiobis(2-nitrobenzoic acid) was found to yield a mixture of fully reduced active molecules, and fully oxidized inactive ones, but no partially oxidized forms were detected. This suggests that an "all-or-none" type of modification and inactivation took place. All 1/2-cystinyl residues in the inactive, monomeric inhibitor had formed disulfide bridges, judged by the absence of either free thiol groups or mixed disulfides with 5-mercapto-2-nitrobenzoate. This fully disulfide-cross-linked molecule had an open conformation compared to the native one, as shown by gel filtration and limited proteolysis. Reaction of phenylarsinoxide with vicinal dithiols yields products that are much more stable than those with monothiols. Titration of RI with this reagent yielded complete inactivation at a reagent/thiol ratio of 0.5. Taken together, these observations suggest that the thiol groups in RI have a diminished reactivity due to three-dimensional constraints. After the initial modification of a small number of thiol groups, a conformational change occurs which causes an increase in reactivity of the remaining thiols. The thiol groups are situated close enough together to permit the formation of 15 disulfide bridges in the inactive molecule.     

Inactivation by ionizing radiation of Salmonella enteritidis serotype montevideo grown in composed sewage sludge.

S. enteritidis ser. montevideo were grown in composted sewage sludge to levels of approximately 10(9)/g. These bacteria were found to be inactivated by ionizing radiation at approximately the same rate (30 krads/log) as Salmonella species in liquid digested sludge.     

Inactivation and reactivation of ribonuclease A studied by computer simulation

The year 2011 marked the half-centenary of the publication of what came to be known as the Anfinsen postulate, that the tertiary structure of a folded protein is prescribed fully by the sequence of its constituent amino acid residues. This postulate has become established as a credo, and, indeed, no contradictions seem to have been found to date. However, the experiments that led to this postulate were conducted on only a single protein, bovine ribonuclease A (RNAse). We conduct molecular dynamics (MD) simulations on this protein with the aim of mimicking this experiment as well as making the methodology available for use with basically any protein. There have been many attempts to model denaturation and refolding processes of globular proteins in silico using MD, but only a few examples where disulphide-bond containing proteins were studied. We took the view that if the reductive deactivation and oxidative reactivation processes of RNAse could be modelled in silico, this would provide valuable insights into the workings of the classical Anfinsen experiment.     

DNA damage caused by ionizing radiation.

A survey is given of continuous-time Markov chain models for ionizing radiation damage to the genome of mammalian cells. In such models, immediate damage induced by the radiation is regarded as a batch-Poisson arrival process of DNA double-strand breaks (DSBs). Enzymatic modification of the immediate damage is modeled as a Markov process similar to those described by the master equation of stochastic chemical kinetics. An illustrative example is the restitution/complete-exchange model. The model postulates that, after being induced by radiation, DSBs subsequently either undergo enzymatically mediated restitution (repair) or participate pairwise in chromosome exchanges. Some of the exchanges make irremediable lesions such as dicentric chromosome aberrations. One may have rapid irradiation followed by enzymatic DSB processing or have prolonged irradiation with both DSB arrival and enzymatic DSB processing continuing throughout the irradiation period. Methods for analyzing the Markov chains include using an approximate model for expected values, the discrete-time Markov chain embedded at transitions, partial differential equations for generating functions, normal perturbation theory, singular perturbation theory with scaling, numerical computations, and certain matrix methods that combine Perron-Frobenius theory with variational estimates. Applications to experimental results on expected values, variances, and statistical distributions of DNA lesions are briefly outlined. Continuous-time Markov chains are the most systematic of those radiation damage models that treat DSB-DSB interactions within the cell nucleus as homogeneous (e.g., ignore diffusion limitations). They contain virtually all other relevant homogeneous models and semiempirical summaries as special cases, limiting cases, or approximations. However, the Markov models do not seem to be well suited for studying spatial dependence of DSB interactions, which is known to be important in some situations.     

Inactivation of thrombomodulin by ionizing radiation in a cell-free system: possible implications for radiation responses in vascular endothelium

Normal tissue radiation injury is associated with loss of vascular thromboresistance, notably because of deficient levels of endothelial thrombomodulin (TM). TM is located on the luminal surface of most endothelial cells and has critical anticoagulant and anti-inflammatory functions. Chemical oxidation of a specific methionine residue (Met388) at the thrombin-binding site in TM reduces its main functional activity, i.e., the ability to activate protein C. We examined whether exposure to ionizing radiation affects TM in a similar manner. Full-length recombinant human TM, a construct of epidermal growth factor-like domains 4-6, which are involved in protein C activation, and a synthetic peptide containing the methionine of interest were exposed to gamma radiation in a cell-free system, i.e., a system not confounded by TM turnover or ectodomain shedding. The influence of radiation on functional activity was assessed with the protein C activation assay; formation of a TM-thrombin complex was assessed with surface plasmon resonance (Biacore), and oxidation of Met388 was assessed by HPLC and confirmed by mass spectroscopy. Exposure to radiation caused a dose-dependent reduction in protein C activation, impaired TM-thrombin complex formation, and oxidation of Met388. These results demonstrate that ionizing radiation adversely affects the TM molecule. Our findings may have relevance to normal tissue toxicity in clinical radiation therapy as well as to the development of radiation syndromes in the non-therapeutic radiation exposure setting.     

Exposure of human lymphocytes to ionizing radiation reduces mutagenesis by subsequent ionizing radiation

The effect of prior incubation with [3H]thymidine on survival and mutagenesis after X-irradiation of human lymphocytes was studied by incubating lymphocytes with 0.001-1.0 mu Ci/ml [3H]thymidine for 6 h at 37 degrees C and then irradiating with 150 or 300 rad. Survival was measured using lymphocyte cloning and mutagenesis was measured using 6-thioguanine selection to detect clones mutated at the hypoxanthine phosphoribosyltransferase locus. [3H]Thymidine alone had no effect on survival or mutagenesis and X-radiation alone produced the expected decrease in survival and increase in mutations. [3H]Thymidine prior to X-radiation had no effect on lethality of X-radiation but at concentrations of 0.1 and 1.0 mu Ci/ml produced a significant decrease in the number of mutations induced after both 150 and 300 rad. The results suggest that ionizing radiation, produced by disintegration of 3H, reduces the mutagenic effect of a subsequent exposure to ionizing radiation by induction of a system which prevents or repairs a restricted class of radiation damage.