Biochemistry
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The release of fibrinopeptides A and B by the slow and fast forms of thrombin was studied over the temperature range from 5 to 45 degrees C and the salt concentration range from 100 to 800 mM. The sequential mechanism for the release of fibrinopeptides originally proposed by Shafer was found to be obeyed under all conditions examined. The origin of preferential binding of fibrinogen and fibrin I to the fast form of thrombin in the transition state is in the second-order rate constant for association, k(l). ⋯ This anion drastically and specifically reduces the thickness of fibrin fibers, as judged by the 10-fold decrease in the equilibrium turbidity of clots developed in NaCl as compared to the turbidity of clots developed in NaF. Hence, the transition from a "coarse" to a "fine" clot induced by an increase in ionic strength as first described by Ferry is, instead, due to the specific binding of Cl- to intermediates in the ensuing polymerization. In fact, no change in the clotting curve is observed when the ionic strength is changed with NaF.
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Comparative Study
Discodermolide, a cytotoxic marine agent that stabilizes microtubules more potently than taxol.
Computer-assisted structure analysis indicated (+)-discodermolide, a polyhydroxylated alkatetraene lactone marine natural product, was an antimitotic compound, and we confirmed this prediction. Previous work had shown an accumulation of discodermolide-treated cells in the G2/M portion of the cell cycle, and we have now found that discodermolide arrests Burkitt lymphoma cells in mitosis. Discodermolide-treated breast carcinoma cells displayed spectacular rearrangement of the microtubule cytoskeleton, including extensive microtubule bundling. ⋯ Without MAPs and/or without GTP, tubulin assembly was also more vigorous with discodermolide than with taxol under every reaction condition examined. Discodermolide-induced polymer differed from taxol-induced polymer in that it was completely stable at 0 degree C in the presence of high concentrations of Ca2+. In a quantitative assay designed to select for agents more effective than taxol in inducing assembly, discodermolide had an EC50 value of 3.2 microM versus 23 microM for taxol.
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Bizelesin is a bifunctional covalent minor groove binding agent which forms adducts with 3'-adenines on opposite DNA strands. DNA lesions induced by bizelesin in genomic DNA of BSC-1 cells, as well as intracellular and purified simian virus 40 (SV40) DNA, were examined. Alkaline sucrose sedimentation analysis indicated a nonrandom distribution of heat-labile damage in BSC-1 cell genomic DNA with frequencies of 1-60 lesions/10(6) base pairs (bp) for bizelesin concentrations from 10 to 400 nM, respectively. ⋯ Bizelesin adduction sites (mapped on the SV40 genome as thermally-induced strand breaks at 50-100 bp resolution) are found in regions centered at 4200, 3900, 4700, and approximately 5200. The location of these regions of intense bizelesin bonding coincides with the sites of potential cross-links predicted using the 5'-T-(A/T)4-A-3' sequence. The analysis of bizelesin adducts at the sequence level in the 3943-4451 SV40 DNA fragment indicated that 40% of total damage was in potential cross-linking sites and an additional 35% in the 5'-A-(A/T)4-A-3' monoalkylating sites.(ABSTRACT TRUNCATED AT 250 WORDS)
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Comparative Study
The spongistatins, potently cytotoxic inhibitors of tubulin polymerization, bind in a distinct region of the vinca domain.
The highly cytotoxic, sponge-derived, antimitotic macrolide polyether spongistatin 1 has been previously shown to inhibit microtubule assembly, the binding of vinblastine and GTP to tubulin, and displacement of GDP bound in the exchangeable site of tubulin. We have now examined in detail inhibition by spongistatin 1 of both [3H]vinblastine and [3H]dolastatin 10 binding to tubulin. We found spongistatin 1 to be a noncompetitive inhibitor of the binding of both radiolabeled drugs to tubulin, in contrast to competitive patterns obtained with vincristine versus [3H]vinblastine and with a chiral isomer of dolastatin 10 versus [3H]dolastatin 10. ⋯ These two compounds, despite activity comparable to spongistatin 1 as inhibitors of tubulin polymerization and [3H]vinblastine binding, had much reduced activity as inhibitors of nucleotide exchange and [3H]dolastatin 10 binding. Spongistatins 1 and 6 were compared for effects on dolastatin 10-induced aggregate formation in conjunction with effects on [3H]dolastatin 10 binding. Spongistatin 6 was about 4-fold less active than spongistatin 1 as an inhibitor of aggregation and over 20-fold less active as an inhibitor of dolastatin 10 binding.
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Vinca site agents are antimicrotubule compounds that bind to the same site on tubulin as do the vinca alkaloids. These include agents that induce the formation of nonmicrotubule oligomers of tubulin (vinblastine and vincristine) and agents that do not (maytansine and rhizoxin). All of these quench the fluorescence of tubulin upon binding. ⋯ The vinca alkaloids differ from maytansine and rhizoxin by causing a large enhancement of chymotryptic cleavage of beta and a large inhibition of typtic cleavage of alpha, after Arg-339. These effects are interpreted as due to vinca induced oligomerization of tubulin. It is argued that the common binding site for the vinca site agents is located on beta-tubulin, close to the helix that is disrupted by colchicine.