ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1992.tb08543.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract—The acrylamide quenching of the tryptophan fluorescence of apo and holo glyceraldehyde‐ 3‐phosphate dehydrogenase (GAPDH) was studied. In the case of apo‐GAPDH, the steady state fluorescence quenching cannot be described by the classical Stern‐Volmer equation: strong cooperative quenching is observed. In the presence of Pi and/or cofactor NAD+an inaccessible fraction appears. Cooperative quenching is partially suppressed in the presence of Pi and fully absent in the presence of NAD+.The measurements of the fluorescence lifetimes of the holo‐enzyme by phasefluorometry allow the resolution of two lifetimes. The long‐lived component is quenched by acrylamide, the short‐lived component is not. Quenching induces a red shift of the steady state emission peak. The quenching parameters from the lifetime measurements allow the quantitative description of the steady state fluorescence quenching data.In agreement with the observations of orstan and Gafni (Photochemistry and Phorobiology, (1990) 31, 725–731), we find that acrylamide causes a slow, irreversible loss of activity and a reduction of titratable thiol groups when it acts on the apo‐enzyme. This inactivation is strongly reduced in the presence of NAD+. We show that this inactivation is also slowed down by the presence of Pi, and that it is accompanied by a loss of the NAD+binding site.Blocking the thiol groups with 5,5'‐dithio‐bis‐(2‐nitrobenzoic acid) does not lead to a protection against the irreversible inactivation by acrylamide, showing that reactions other than thiol modifications are involved in the irreversible effect. A fraction of the inactivation can be reversed by trea

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1992.tb04203.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract—Derivatives of the tubulin polymerization inhibitors colchicine and podophyllotoxin bearing the photoreactive 2‐diazo‐3,3,3‐trifluoropropanoyl (DTFP) group were synthesized for evaluation as potential photoaffinity labels of the tubulin binding site. All labels were assayed for their ability to inhibit tubulin polymerization, andN‐DTFP‐deacetylthiocolchicine was shown to competitively inhibit tubulin‐colchicine binding with aKiof 4–5 μM.The tubulin off‐rate of this analog was similar to that of podophyllotoxin, rather than to the relatively irreversibly bound colchicine. Photochemical solvent insertion reactions of the labels were investigated. Radioactive samples of the two most active labels were prepared and used in initial protein‐labeling experiments, during which the fractional occupancy of tubulin and extent of covalent incorporation were determined. A rearrangement of DTFP amides was encountered which is relevant to the utility of this moiety for use in synthesis of

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1992.tb04204.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract—Steady‐state and time‐resolved fluorescence studies have been performed with human epidermal growth factor, a small globular protein having two adjacent tryptophan residues near its C‐terminus. Based on the relatively red fluorescence and accessibility to solute quenchers, the two tryptophan residues are found to be exposed to solvent. Anisotropy decay measurements show the dominant depolarizing process to have a sub‐nanosecond rotational correlation time indicating the existence of rapid segmental motion of the fluorescing tryptophan residues. From an analysis of the low‐temperature excitation anisotropy spectrum of the protein (and in comparison with that of tryptophan, the peptide melittin, and the dipeptide trp‐trp), it is concluded that homo‐energy transfer and/or exciton interaction occurs between the adjacent tryptophan residues. A thermal transition in the structure of the protein, which is observed by circular dichroism measurements, is not sensed by the steady‐state fluorescence of the protein. This result, in conjunction with the anisotropy decay results, indicates that the two tryptophan residues are in a highly flexible C‐terminus segment, which is not an integral part of the three‐dimensional structure of the protein. Fluorescence measurements with three site‐directed mutants also sho

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1992.tb04205.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract—Sanguinarine, a commercial drug exhibiting antimicrobial and antitumor properties, was studied with respect to its basic photochemical characteristics and also with regard to its phototoxicity to mosquito larvae (Aedes atropalpus). Sanguinarine proved to be clearly phototoxic to larvae, with an LD50of 0.096 mg/mL with near UV exposure as compared with 23.3 mg/mL without. Flash photolysis experiments enabled the study of the triplet state of sanguinarine to be undertaken. Quenching by oxygen occurs with a rate constant of 6 × 109M‐1s‐1time‐resolved emission studies indicate that sanguinarine produces a significant amount of singlet oxygen (ΦΔ= 0.16) as does the isoquinoline alkaloid, berberine (ΦΔ= 0.25). These values represent the first direct quantitative measurements of photosensitization parameters of these compounds. Additionally, sanguinarine exhibits efficient electron donation properties, undergoing reaction with methyl viologen with a rate constant>1010M‐1s‐1but is a poor electron acceptor. Phototoxicity of sanguinarine can thus be explained in terms of its photosensitiz

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1992.tb04206.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract—The effects of singlet oxygen (1O2), generated by the thermal decomposition of water soluble NDPO2(endoperoxide of the disodium 3,3'‐(1,4‐naphthylidene) dipropionate), on a single‐stranded shuttle vector were analysed.1O2induces a much higher level of breaks in the phosphodiester backbone of single‐stranded than double‐stranded DNA. This may be due to a higher accessibility of guanine residue, primarily damaged by1O2. The damaged vector was transfected into monkey COS7 cells where single‐stranded DNA was converted to the double‐stranded replicative form DNA. After 3 days, extrachromosomal DNA was extracted and the plasmids rescued inE. colito study mutagenesis. There is a significant increase in mutation frequency of damaged single‐stranded DNA in comparison to untreated DNA. It is concluded that1O2induces breaks in the backbone of single‐stranded DNA and that the1O2‐damaged molecules are mutated after passage th

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1992.tb04207.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract—Photosensitized inhibition of mitochondrial succinoxidase by hypericin was measuredin vitroand found to be drug‐dose, light‐dose, and wavelength dependent. Singlet oxygen generation, monitored using the singlet oxygen trap tetramethylethylene, and oxygen consumption in isolated mitochondria sensitized by hypericin were also light‐dose and wavelength dependent. Unequivocal evidence for the generation of singlet oxygen was obtained using kinetic isotope ratios of products from the reaction between singlet oxygen and geminally deuterated tetramethylethylene. An action spectrum for the inhibition of succinoxidase was measured at wavelengths between 400 and 700 nm and found to parallel the recorded visible absorption spectrum of hypericin in isolated mitochondria. The greatest singlet oxygen generation, oxygen consumption, and succinoxidase inhibition occurred with white light or 600 nm irradiation. These data are consistent with a type II singlet‐oxygen‐mediated mechanism for hypericin induced photosensitized inhibition of mitochondrial su

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1992.tb04208.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract—Irradiation with 365 nm light of Wi26VA4 SV40‐transformed human fibroblasts cultured for 24 h in the presence of low density lipoproteins loaded with the anticancer porphyrin mixture Photofrin II resulted in a near complete inhibition of [14C]oleic acid incorporation into triacylglycerols, cholesteryl esters and phospholipids. More than 80% reduction of the fatty acid incorporation in all lipid classes was observed following an irradiation dose of 1 J/cm2. The activities of the respective acyltransferases, measuredin vitroon cell homogenates, were also markedly diminished, but to a lesser extent than lipid synthesis from oleic acid. Moreover, oleic acid uptake by cells was strongly and rapidly reduced. It is suggested that the rapid inhibition of membrane phospholipid synthesis upon cell photosensitization, due to both a direct inactivation of acyltransferases and to a reduction of fatty acid utilization, could play an important role in the photocytotoxic effect of Photofrin

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1992.tb04209.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract—The natural product 2‐chloro‐3, 11‐tridecadiene‐5, 7, 9‐triyn‐l‐ol (l) photosensitized the inactivation ofEscherichia coliin the presence of near‐ultraviolet light (320–400 nm; NUV) under both aerobic and anaerobic conditions. A series ofE. colistrains differing in DNA repair capabilities and catalase proficiency exhibited indistinguishable inactivation kinetics following treatment with the chemical plus NUV. The presence of carotenoids did afford some protection toE. coliagainst inactivation under aerobic conditions, consistent with the involvement of singlet oxygen. The photosensitized hemolysis of human erythrocytes occurred more rapidly in the absence than in the presence of oxygen. Aerobically, the onset of hemolysis was partially inhibited by NaN3and by 2,6‐di‐t‐butyl 4‐methylphenol (BHT) but not by superoxide dismutase (SOD). The aerobic lipid peroxidation observed in the membranes of erythrocyte ghosts was completely inhibited by BHT, and partially by NaN3, but not by SOD. These results suggest that either lipid peroxidation of the membrane is not the main cause of photohemolysis or that BHT has insufficient access to intact erythrocyte lipids to protect them. Aerobically, crosslinking of membrane proteins was also observed; it was not affected by SOD, but was partially inhibited by BHT and NaN3. The anaerobic photosensitized hemolysis of erythrocytes was more rapid; a radical mechanism was suggested since BHT inhibited the hemolysis to a greater extent than under aerobic conditions. Neither lipid peroxidation nor protein crosslinking was observed under conditions believed to be anaerobic. A light‐dependent electron transfer to cytochrome c was obtained under argon but not under oxygen. Although induced mutations were not observed in the experiments withE. coli, I was capable of damaging both supercoiled pBR322 andHaemophilus influenzaetransforming DNA in a manner that seemed to be equivalent under aerobic and anaerobic conditions. In conclusion, I can behave as typical photodynamic molecule under aerobic conditions but, in contrast to most photodynamic molecules, it is also phototoxic under anaerobic conditions. The extent to which the radical reactions detected under anaerobic reactions compete with the photodynamic processes when o

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1992.tb04210.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Abstract—Photosensitization mediated by Photofrin II (PFII) was found to be mutagenic at the heterozygous thymidine kinase (tk) locus in mouse L5178Y lymphoma strains LY‐S1 and LY‐R16 but not in strain LY‐R83 which is hemizygous at thetklocus. After treatments yielding 37% survival, the mutagenicity of photosensitization with PFII in strain LY‐S1 was similar to that of other mutagenic agents including x‐radiation, ethyl methanesulfonate, and photosensitization with chloroaluminum phthalocyanine (AIPcCI). Although both strain LY‐S1 and strain LY‐R16 were mutagenized by photosensitization with PFII, only strain LY‐S1 was mutagenized by photosensitization with AIPcCI. The non‐mutability of strain LY‐R83 following photodynamic treatment with either sensitizer may be because of the poor recovery of mutants with intergenic mutations in this TK+/0hemizygous strain, whereas the non‐mutability of strain LY‐R16 subjected to photodynamic treatment with AIPcCI may be because LY‐R16 cells sustaining mutagenic damage do not survive for reasons other than th

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1992.tb04211.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY