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1.
Michael H Woo John R Vance Ana R Otero Marcos Christian Bailly Mary-Ann Bjornsti 《The Journal of biological chemistry》2002,277(6):3813-3822
DNA topoisomerase I (Top1p) catalyzes topological changes in DNA and is the cellular target of the antitumor agent camptothecin (CPT). Non-CPT drugs that target Top1p, such as indolocarbazoles, are under clinical development. However, whether the cytotoxicity of indolocarbazoles derives from Top1p poisoning remains unclear. To further investigate indolocarbazole mechanism, rebeccamycin R-3 activity was examined in vitro and in yeast. Using a series of Top1p mutants, where substitution of residues around the active site tyrosine has well-defined effects on enzyme catalysis, we show that catalytically active, CPT-resistant enzymes remain sensitive to R-3. This indolocarbazole did not inhibit yeast Top1p activity, yet was effective in stabilizing Top1p-DNA complexes. Similar results were obtained with human Top1p, when Ser or His were substituted for Asn-722. The mutations altered enzyme function and sensitivity to CPT, yet R-3 poisoning of Top1p was unaffected. Moreover, top1delta, rad52delta yeast cells expressing human Top1p, but not catalytically inactive Top1Y723Fp, were sensitive to R-3. These data support hTop1p as the cellular target of R-3 and indicate that distinct drug-enzyme interactions at the active site are required for efficient poisoning by R-3 or CPT. Furthermore, resistance to one poison may potentiate cell sensitivity to structurally distinct compounds that also target Top1p. 相似文献
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3.
Comparative study on polypeptide patterns of larvae of Trichinella isolates by two-dimensional electrophoresis 总被引:2,自引:0,他引:2
The two-dimensional patterns (isoelectrofocusing-IEF/polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate-SDS) of S3 fractions of muscle larvae of four Trichinella isolates were compared. The comparative study concerned six groups of polypeptides. It was observed that the Garkavi isolate of Trichinella pseudospiralis was clearly different from the other isolates, and it showed the simplest IEF/SDS polypeptide pattern. The C-76 isolate of T. nelsoni had only four of the six groups, distinguishing it from the GM-1 isolate of T. spiralis and the Boev isolate of T. nativa that showed all the indicated groups. 相似文献
4.
Biochemical dissection of the role of the one-kilodalton carboxyl-terminal moiety of tubulin in its assembly into microtubules 总被引:2,自引:0,他引:2
The 4-kDa C-terminal domain of both tubulin subunits plays a major role in the regulation of microtubule assembly [Serrano et al. (1984) Biochemistry 23, 4675]. Controlled proteolysis of tubulin with subtilisin produces the selective cleavage of this 4-kDa moiety from alpha- and beta-tubulin with a concomitant enhancement of the assembly. Here we show that gradual removal of the last six to eight amino acid residues of the C-terminal region of alpha and beta subunits by an exopeptidase, carboxypeptidase Y, produces a modified protein (C-tubulin) without relieving the modulatory effect of the C-terminal domain and the usual need of MAPs for microtubule assembly. Actually, treatment with this proteolytic enzyme did not change tubulin assembly as promoted by either MAP-2, taxol, MgCl2, dimethyl sulfoxide, or glycerol. The critical concentration for the assembly of C-tubulin remained the same as that for the unmodified tubulin control. Microtubule-associated proteins MAP-2 and tau incorporated into C-tubulin polymers. Clearly, pure C-tubulin did not assemble in the absence of MAPs or without addition of assembly-promoting compounds. However, proteolysis with the exopeptidase induced changes in tubulin conformation as assessed by biophysical methods and double-limited proteolysis. The cleavage with subtilisin after carboxypeptidase digestion did not result in enhancement of the assembly to the levels observed after the treatment of native tubulin with subtilisin. Interestingly, Ca2+ ions affected neither C-tubulin assembly nor depolymerized microtubules assembled from C-tubulin.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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6.
Tryptic digestion of human GPIIIa. Isolation and biochemical characterization of the 23 kDa N-terminal glycopeptide carrying the antigenic determinant for a monoclonal antibody (P37) which inhibits platelet aggregation.
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J J Calvete G Rivas M Maruri M V Alvarez J L McGregor C L Hew J Gonzalez-Rodriguez 《The Biochemical journal》1988,250(3):697-704
Early digestion of pure human platelet glycoprotein IIIa (GPIIIa) leads to a single cleavage of the molecule at 23 kDa far from one of the terminal amino acids. Automated Edman degradation demonstrates that GPIIIa and the smaller (23 kDa) tryptic fragment share the same N-terminal amino acid sequence. A further cleavage occurs in the larger fragment (80 kDa), reducing its apparent molecular mass by 10 kDa. The 23 kDa fragment remains attached to the larger ones in unreduced samples. Stepwise reduction of early digested GPIIIa with dithioerythritol selectively reduces the single disulphide bond joining the smaller (23 kDa) to the larger (80/70 kDa) fragments. Two fractions were obtained by size-exclusion chromatography of early digested GPIIIa after partial or full reduction and alkylation. The larger-size fraction contains the 80/70 kDa fragments, while the 23 kDa fragment is isolated in the smaller. The amino acid compositions of these fractions do not differ very significantly from the composition of GPIIIa; however the 23 kDa fragment contains only 10.2% by weight of sugars and is richer in neuraminic acid. Disulphide bonds are distributed four in the 23 kDa glycopeptide and 20-21 in the 80/70 kDa glycopeptide. The epitope for P37, a monoclonal antibody which inhibits platelet aggregation [Melero & González-Rodríguez (1984) Eur. J. Biochem. 141, 421-427] is situated within the first 17 kDa of the N-terminal region of GPIIIa, which gives a special functional interest to this extracellular region of GPIIIa. On the other hand, the epitopes for GPIIIa-specific monoclonal antibodies, P6, P35, P40 and P97, which do not interfere with platelet aggregation, are located within the larger tryptic fragment (80/70 kDa). Thus, the antigenic areas available in the extracellular surface of GPIIIa for these five monoclonal antibodies are now more precisely delineated. 相似文献
7.
The present study was carried out to evaluate the suitability of the unstable white-zeste system in Drosophila melanogaster by testing 4 organophosphorus insecticides for potential genotoxic activity: dimethoate, fenitrothion, malathion, and methyl parathion. In view of the high sensitivity to insecticides of the unstable zeste strain used in this assay and the negative results obtained in this work, the white-zeste system does not appear to be sufficiently accurate for the evaluation of the mutagenic potential of specifically toxic chemicals, like insecticides. 相似文献
8.
The human platelet integrin GPIIb/IIIa (228 kDa), a Ca-dependent heterodimer formed by the IIb subunit (GPIIb, 136 kDa) and the 3 subunit (GPIIIa, 92 kDa), serves as the fibrinogen receptor at the surface of activated platelets. The degree of dissociation of the GPIIb/IIIa heterodimer (s°20
*, 8.9 S) into its constituent glycoproteins (GPIIb, 5.8 S; and GPIIIa, 3.9 S) has been assessed by analytical ultracentrifugation in Triton X100 buffers, and its Ca2+- and temperature-dependence correlated with Ca2+-binding to GPIIb/IIIa and its temperature dependence. At 21°C half-maximal dissociation of GPIIb/IIIa occurs at 5.5 ± 2.5 × 10–8 M Ca2+, very close to the dissociation constant of the high affinity Ca-binding site of GPIIb/IIIa (Kd1 8 ± 3 × 10–8 M) (Rivas and González-Rodríguez, 1991) and much lower than the Kd of the 3.4 medium affinity Ca-binding sites (Kd2 4 ± 1.5 × 10–5 M), which seems to demonstrate that the stability of the heterodimer in solution at room temperature is regulated by the degree of saturation of the high-affinity Ca-binding site. At 4°C, the stability of the heterodimer is apparently Ca2+-independent, while at room and physiological temperatures (15–37°C) the degree of dissociation of the heterodimer is regulated by the degree of dissociation of the high- and medium-affinity Ca-binding sites, respectively. On increasing the Ca2+ concentration up to 1 × 10–4 M after dissociation in Triton X100 solutions, the reconstitution of the GPIIb/IIIa heterodimer depends on the time and temperature at which the dissociated heterodimer was maintained, being almost complete within the first 5–10 min at 37°C and within the first 1–2 h at 21°C. After this time, a time- and temperature-dependent irreversible autoassociation of GPIIb (covalent) and GPIIIa (non-covalent) occurs, which hinders both the isolation of permanently stable monoamers of GPIIb and GPIIIa and the reconstitution of the GPIIb/IIIa heterodimer in Triton X100 solutions.
Abbreviations: GPIIb, GPIIIa, and GPIIb/IIIa, glycoprotein IIb, IIIa, and the heterodimer formed by them, respectively; s°20
*, the sedimentation coefficient of the glycoprotein-detergent complexes determined at 20°C, after extrapolation to zero-glycoprotein concentration
Offprint requests to: J. González-Rodríguez 相似文献
9.
Proteins blotted on nitrocellulose were stained with either 5-dimethylamino-1-naphthalene-sulfonylchloride (dansyl chloride) or fluorescein isothiocyanate. In both cases the staining procedure can be completed in less than 30 min. The sensitivity for detecting fluorescent-labeled proteins on nitrocellulose was 0.5 ng using a dot test. This was accomplished by transparentizing the nitrocellulose with either immersion oil or toluene. Dansylated proteins were successfully utilized for optimizing the electroblotting procedure. In the presence of 0.2% sodium dodecyl sulfate and 20% methanol the distribution of proteins on the nitrocellulose was an exact replica of the protein pattern seen in the polyacrylamide gel. The fluorescent labeling did not affect the antigenic properties of proteins allowing the subsequent probing with antisera. For this procedure, only one set of samples is needed to obtain accurate photographic records of the gel, the nitrocellulose blot, and the probed blot. 相似文献
10.
F Rivas D García-Cruz H Rivera M L Plascencia R M González J M Cantú 《Annales de génétique》1985,28(2):113-115
A 2 4/12-year-old girl whith a de novo 46,XX,-22 + der(22), t(19;22)(q13.2;p11) karyotype is described. From this and other eight similar cases previously published, a typical phenotype in distal 19q trisomy is concluded. 相似文献