ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1977.tb09163.x

出版商:Blackwell Publishing Ltd    

年代:1977

数据来源: WILEY

摘要:

Abstract—Escherichia coliphotoreactivating enzyme (PRE) has been purified in large amounts from anE. colistrain lysogenic for a defective Λ bacteriophage carrying thephrgene. The resulting enzyme has a pH optimum of 7.2 and an ionic strength optimum of 0.18. It consists of an apoprotein and cofactor, both of which are necessary for catalytic activity. The apoprotein has a monomer molecular weight of35,200 and shows stable aggregates under denaturing conditions. The amino acid analysis of theE. colienzyme is very similar to that of the photoreactivating enzyme from orchid seedlings (Cattelya aurantiuca). Both have arginine at the amino terminus. The cofactor, like the holoenzyme, shows absorption, magnetic circular dichroism, and emission properties indicative of an adenine moiety. Although the isolated enzyme has an action spectrum which peaks at about 360 nm, neither the cofactor, apoenzyme nor holoenzyme shows any detectable absorption between 300 and 400

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1977.tb09164.x

出版商:Blackwell Publishing Ltd    

年代:1977

数据来源: WILEY

摘要:

Abstract—Yeast DNA photolyase purified twice by affinity chromatography was analyzed by electrophoresis on polyacrylamide gradient gels or by sedimentation velocity through 5–200/, sucrose gradients containing 0.4MKC1. Its molecular weight estimated by both these methods was130,000 and136,000, respectively. However, the enzyme dissociated into two bands having molecular weights of60,000 and85,000 when it was examined by electrophoresis on SDS polyacrylamide gradient gels. The subunit structure of the enzyme was confirmed when two absorption maxima corresponding to polypeptides of54,000 and82,500 daltons were observed in sucrose gradients run in 1.0 M KCI. Upon mixing these two fractions, a time‐dependent increase in activity occurred, demonstrating that active enzyme could be reconstituted from these subunits.The activity of photolyase purified by affinity chromatography is enhanced by a compound (activator III) obtained from yeast by acidification, neutralization, ion exchange chromatography and gel filtration. Activator III emits at 350 and 440 nm when excited at 290 nm, and emits at 440 nm when excited at 358 nm. After acid hydrolysis, emission at 440 nm is produced only by excitation at 358 nm, indicating that it contains two separate chromophoric moieties. The chromophore excited by 358 nm light has a pK of 9–11, while the other has a pK of 4–5. Enhancement of photolyase activity by activator III at a concentration equimolar with that of the enzyme and the similarity of the fluorescent spectra of the activator and heat‐denatured photolyase suggest that the activator may be the chromophore associated with

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1977.tb09165.x

出版商:Blackwell Publishing Ltd    

年代:1977

数据来源: WILEY

摘要:

Abstract—Indole derivatives including tryptophan can be used as photosensitizers of the splitting of pyrimidine dimers. The reaction can take place in frozen aqueous solutions as well as in fluid medium. Electron transfer from the indole ring to the dimer appears to be involved in the photosensitized reaction. Solvated electrons produced by flash photolysis in the presence of indoles or by pulse radiolysis are also able to split thymine dimers.The splitting of pyrimidine dimers in DNA can be photosensitized by indole derivatives such as serotonin and by tryptophan‐containing oligopeptides. Several methods including fluorescence and nuclear magnetic resonance have been used to show that the indole ring of these oligopeptides is able to stack with bases in nucleic acids. These stacked complexes are involved in the photosensitized reaction.The splitting of pyrimidine dimers in DNA has also been photosensitized by the protein coded by gene 32 of phage T4 which binds strongly and cooperatively to single‐stranded DNA. The mechanism of the splitting reaction as well as the possible use of this reaction to investigate the role of tryptophan residues in the binding of proteins to nucleic acids are disc

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1977.tb09166.x

出版商:Blackwell Publishing Ltd    

年代:1977

数据来源: WILEY

摘要:

Abstract—Photoreactivating enzyme (PRE) monomerizes cyclobutyl pyrimidine dimers formed in DNA by UV light (Λ<300 nm). The enzyme requires near UV and visible wavelengths (300<Λ<600 nm) for activity. Possible mechanisms of action of the PRE are suggested by non‐enzymatic processes in which pyrimidine dimers are monomerized by UV and visible light. Two such non‐enzymatic processes are (a) photolysis of dimers resulting from direct absorption of UV, and (b) sensitized monomerization involving charge transfer complexes. Several lines of evidence suggest that the mechanism of action of the PRE more closely resembles (b) than (a). Recent experiments on the PRE fromE. colireveal the presence of new long wavelength absorption which may indicate the presence of a ground state complex. The known ability of PRE to monomerize dimers of thymine, cytosine and uracil suggests that the carbonyl groups at 2 position of the pyrimidine ring may be important in the interaction between enzyme an

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1977.tb09167.x

出版商:Blackwell Publishing Ltd    

年代:1977

数据来源: WILEY

摘要:

Abstract—The fluorescence excitation spectrum and the excitation polarization spectrum of indole in propylene glycol were measured at — 58°C, after selecting by optical filters the emission originating from the1Laelectronic level. From the analysis of these spectra, the excitation spectrum was resolved into the1Laand1Laexcitation bands. A similar resolution of the excitation spectrum of tryptophan is given. This method can also be applied to the resolution of the emission spectrum in cases of dual emis

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1977.tb09168.x

出版商:Blackwell Publishing Ltd    

年代:1977

数据来源: WILEY

摘要:

Abstract—Flash photolysis techniques have been used to study the effect of 8α‐substitution on flavin triplet state formation and decay and on the properties of neutral and anionic serniquinones. Compared with riboflavin, the N(1) and N(3) isomers of 8α‐histidylriboflavin show a lower triplet yield (˜10%) and a faster rate of decay (˜ 4‐Cfold). Acetylation of the histidyl a‐amino groups and of the flavin ribityl side chain results in a 2‐fold increase in triplet yield and a 2‐fold slower rate of decay.The yield of neutral 8α‐substituted flavin semiquinones upon flash photolysis in the presence of EDTA was approximately 50% that given by riboflavin. These substituted flavin neutral semiquinones dismutated at a rate 2–3 times slower than the corresponding unsubstituted form, although the anionic semiquinones dismutated at approximately the same rate.In the presence of oxygen, the kinetics of semiquinone decay changed from second order to pseudo‐first order upon raising the pH, thus showing anionic semiquinone oxidation as seen previously with unsnbstituted flavins. The pK values for the ionization of the neutral 8α‐substituted Aavin semiquinones are 1–1.5 units lower than the unsubstituted form. The anionic 8α‐substituted flavin semiquinones react with oxygen at a rate 2–10 times more slowly than does the riboflavin form. Such alterations in properties probably reflect the electron‐withdrawing effect of the 8α

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1977.tb09169.x

出版商:Blackwell Publishing Ltd    

年代:1977

数据来源: WILEY

摘要:

Abstract—In order to investigate the possibility of the tautomerization of alloxazine to isolloxazine in its ground state, the parameters affecting the redistribution of charges in the lumichrome molecule were studied. The absorption and emission spectra of lumichrome as a function of pH in the rangeH0= ‐ 6 to pH = 12 were recorded. At extreme pH conditions the spectra of lumichrome are similar to those of the isoalloxazine system. At high acid concentration (H0<‐ 3.0) the absorption spectrum of lumichrome protonated at N10, is practically identical to that of lumiflavin. The fluorescence quantum yield of the two cations is negligible at room temperature.At pH = 10.5 lumichrome is deprotonated at positions N1or N3, The two monoanions have different excitation spectra. Except for slight differences in the extinction coefficients, the absorption of the anion deprotonated at N3is very similar to the lumichrome spectrum. The absorption spectra of the N1monoanion and of the di‐anion are similar to the spectrum of lumiflavin except for a blue shift of about 20 nm. Furthermore, the emission spectrum of the N1‐monoanion is identical to that of the isoalloxazine system. These results indicate that the charge distribution in the lumichrome molecule depends on the protonation and deprotonation of the nitrogen atoms at positions 10 and l. Both processes cause a redistribution of charges so that an isoalloxazine ring system

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1977.tb09170.x

出版商:Blackwell Publishing Ltd    

年代:1977

数据来源: WILEY

摘要:

Abstract—UV‐induced alkaline labile viral DNA damage was detected following irradiation of adenovirus type 2 and found to be repaired following the infection of human KB cells. Human adenovirus type 2 was irradiated with various doses of UV and subsequently used to infect human KB cells in tissue culture at approximately 2 × 103particles per cell. Before, and at various times after infection, the viral DNA was examined on alkaline sucrose gradients. Irradiated free virus DNA showed a dose dependent decrease in molecular weight compared to unirradiated virus DNA, indicating the presence of UV‐induced alkaline labile lesions. Furthermore, an increase in the molecular weight of the irradiated virus DNA was found after infection indicating that alkaline labile lesions were removed from the viral DNA by a host mediated repair mechanism. After infection, the molecular weight of the irradiated virus DNA reached a value similar to that of unirradiated virus DNA for all the UV doses s

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1977.tb09171.x

出版商:Blackwell Publishing Ltd    

年代:1977

数据来源: WILEY

摘要:

Abstract—The eosin‐sensitized photooxidation of tyrosine and a number of compounds related to tyrosine (substituted phenylalanines) was studied by steady‐state kinetic and flash photolysis techniques. In particular, the role of the phenolic group and the amino and carboxyl groups of the alanyl side chain in the photooxidation mechanism was investigated in detail. Several relationships between substrate structure and susceptibility to photooxidation as well as effects of substrate structure on photooxidation mechanisms were found.For example, phenylalanine is not photooxidizable, hut substitution of electron‐donating (activating) groups such as‐OH (as in tyrosine) or‐NH2(as in p‐aminophenylalanine) results in rapidly photooxidized derivatives. However, substituting deactivating groups such as—C1 (as inp‐chlorophenylalanine) or weakly activating groups such as ‐ OCH3(as in 4‐methoxyphenylalanine) result in non‐photooxidiz‐able derivatives. Substitution of additional activating groups to the ring of hydroxy‐substituted phenylalanines results in increased rates of photooxidation, whereas additional deactivating groups result in decreased photooxidation rates.The rate‐determining step in the photooxidation mechanism is shown to be dependent on the presence and position of an electron‐donating substituent on the benzenoid ring. Only minor involvement of the side chain amino and carboxyl groups was found. Both singlet oxygen and hydrogen abstraction mechanisms are involved in the eosin‐sensitized photooxidation of hydroxy‐substituted phenylalanines (e.g. tyrosine). The hydrogen abstraction mechanism probab

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1977.tb09172.x

出版商:Blackwell Publishing Ltd    

年代:1977

数据来源: WILEY