Determination of molecular mass of the aroid alternative oxidase by radiation-inactivation analysis.

The functional molecular mass of the cyanide-resistant salicylhydroxamate-sensitive duroquinol oxidase activity from Sympocarpus foetidus (skunk cabbage) and Sauromatum guttatum spadix mitochondria was determined by radiation-inactivation analysis. The functional molecular mass for the oxidase activity was found to be 26,700 Da for skunk cabbage and 29,700 Da for Sauromatum guttatum mitochondria frozen at -70 degrees C. Irradiation of dried mitochondrial samples resulted in a larger target size of 38,000 Da, and in some cases, a stimulation of activity at low dose of radiation. The functional molecular mass of cytochrome c oxidase activity from skunk-cabbage and bovine heart mitochondria was also investigated. Dried and frozen mitochondrial samples from both species yielded similar target sizes, in the range 70,900-73,400 Da. Purified bovine heart cytochrome c oxidase was also irradiated, and yielded a functional molecular mass of 66,400 Da. The target size of cytochrome c oxidase agrees with literature values insofar as the target size is considerably smaller than the molecular mass of the entire complex.

Action mechanism of Escherichia coli DNA photolyase. III. Photolysis of the enzyme-substrate complex and the absolute action spectrum.

The absolute action spectrum of Escherichia coli DNA photolyase was determined in vitro. In vivo the photoreactivation cross-section (epsilon phi) is 2.4 X 10(4) M-1 cm-1 suggesting that the quantum yield (phi) is about 1.0 if one assumes that the enzyme has the same spectral properties (e.g. epsilon 384 = 1.8 X 10(4) M-1 cm-1) in vivo as those of the enzyme purified to homogeneity. The relative action spectrum of the pure enzyme (blue enzyme that contains FAD neutral semiquinone radical) agrees with the relative action spectrum for photoreactivation of E. coli, having lambda max = 384 nm. However, the absolute action spectrum of the blue enzyme yields a photoreactivation cross-section (epsilon phi = 1.2 X 10(3) at 384 nm) that is 20-fold lower than the in vivo values indicative of an apparent lower quantum yield (phi approximately equal to 0.07) in vitro. Reducing the enzyme with dithionite results in reduction of the flavin semiquinone and a concomitant 12-15-fold increase in the quantum yield. These results suggest that the flavin cofactor of the enzyme is fully reduced in vivo and that, upon absorption of a single photon in the 300-500 nm range, the photolyase chromophore (which consists of reduced FAD plus the second chromophore) donates an electron to the pyrimidine dimer causing its reversal to two pyrimidines. The reduced chromophore is regenerated at the end of the photochemical step thus enabling the enzyme to act catalytically.+

The influence of repair systems on the presence of sensitive and resistant fractions in the relation of survival of colony-forming ability in Escherichia coli to UV exposure.

In the experimentally observed relationship between survival of colony-forming ability and the amount of exposure to ultraviolet light, two characteristics are generally found. First, sensitive and resistant components often show. Second, there is often a shouldered character to the survival. We present evidence that the first is largely due to the presence of active replication forks in the genome, and that the second is related to the operation of the recombinational repair system. We are able to describe our data in terms of a superposition of single and multiple-hit fractions and to show that the latter are greatly increased, in excision-repair-competent strains, by prevention of protein synthesis for 1 h prior to irradiation. Applying this analysis and treatment to a number of mutant strains enables us to make suggestions as to the interaction between recombinational and excision repair.

Target size of neurotoxic esterase and acetylcholinesterase as determined by radiation inactivation.

The target size of neurotoxic esterase (NTE), the putative target site for the initiation of organophosphorus-compound-induced delayed neurotoxicity, and acetylcholinesterase (AChE) from hen brain were examined by determining the rate at which the activities of the esterases were destroyed by ionizing irradiation. Samples of hen brain were prepared by slowly drying a microsomal preparation under vacuum. The dried samples were then irradiated with electrons from a 1 MeV Van de Graaff generator. The doses ranged from 0 to 28 Mrad. The radiation doses were calibrated by the rate of inactivation of T1-bacteriophage plaque induction. Following the irradiation procedure, the samples were resuspended in buffer and enzymic activity was measured. The target size of NTE from hen brain was determined to be about 105 kDa, whereas hen brain AChE was found to have a target size of about 53 kDa. The target size of NTE was found to be similar in experiments with rat brain and cat brain. In addition, commercial preparations of electric-eel electric-organ AChE and horse serum butyrylcholinesterase were found to have target sizes that were identical with each other, and also were very similar to that of AChE from hen brain.

Induced radioresistance: an aspect of induced repair.

Irradiation of Escherichia coli cells with UV or X-rays followed by incubation under conditions in which protein synthesis can occur results in a population of cells that is resistant to X-rays; however, this resistance develops only if the cells are recA+ and lexA+, a fact that associates the phenomenon with induced (S.O.S.) repair. By observing separately the component of a culture that is resistant and the component that retains its normal growth, the fraction of induced and uninduced cells for a dose of UV or X-rays can be estimated. Such estimates show that the dose-response for UV induction of resistant cells agrees with that of the recA gene product. Thus induced radioresistance is considered to be due to the changes in the cell occasioned by the derepression of recA and lexA. These changes are expected to be involved with the synapsis of homologous genomes that is necessary for the use of a second genome to repair damage occurring in both strands of a duplex at the same base, as exemplified by a double-strand break or an interstrand crosslink. This consideration is additionally supported by the increased resistance of cells grown to contain multiple genomes in the same envelope, an increased resistance not found in recA- or lexA- cells. The condition of a completed chromosome is also resistant, again not in recA- or lexA- cells. We suggest that cell killing by X-rays is due to the double-strand breaks which are not repaired by molecular synapsis before the arrival of the replication polymerase at the break.