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21.
5-Nitro-2'-deoxyuridylate (NO2dUMP) is a potent mechanism-based inhibitor of dTMP synthetase. After formation of a reversible enzymeìnhibitor complex, there is a rapid first order loss of enzyme activity which can be protected against by the nucleotide substrate dUMP. From studies of model chemical counterparts and the NO2dUMPdTMP synthetase complex, it has been demonstrated that a covalent bond is formed between a nucleophile of the enzyme and carbon 6 of NO2dUMP. The covalent NO2dUMPènzyme complex is sufficiently stable to permit isolation on nitrocellulose membranes, and dissociates to give unchanged NO2-dUMP with a first order rate constant of 8.9 x 10(-3) min-1. Dissociation of the complex formed with [6-3H]NO2dUMP shows a large alpha-secondary isotope effect of 19%, verifying that within the covalent complex, carbon 6 of the heterocycle is sp3-hybridized. The spectral changes which accompany formation of the NO2dUMPènzyme complex support the structural assignment and, when used to tritrate the binding sites, demonstrate that 2 mol of NO2dUMP are bound/mol of dimeric enzyme. The interaction of NO2dUMP with dTMP synthetase is quite different than that of other mechanism-based inhibitors such as 5-fluoro-2'-deoxyuridylate in that it neither requires nor is facilitated by the concomitant interaction of the folate cofactor, 5,10-CH2-H4folate, and that the covalent complex formed is unstable to protein denaturants. 相似文献
22.
S Capitani G Mazzotti S Papa P Santi F A Manzoli 《Biochemical and biophysical research communications》1979,89(4):1206-1211
The terminal transferase activity is modified in the presence of lipid vesicles. A deep inhibitory effect takes place with phosphatidylserine and phosphatidylinositol, while some stimulation is present with sphingomyelin and almost no effect has been detected with phosphatidylethanolamine vesicles. These effects seem to be related to the charge properties of the lipid membranes.A possible involvement of phospholipids in the mechanism of action of the terminal transferase is suggested. 相似文献
23.
Thymidylate synthetase catalyzes an exchange of tritium of [5-3H]dUMP for protons of water in the absence of CH2-H4folate. The turnover number for this reaction is some 45,000-fold lower than that of dTMP formation and Km is 1.2 X 10(-5) M, similar to the dissociation constant of the enzyme-dUMP complex determined by equilibrium dialysis. The presence of 4 mM folate has no effect on Vmax but results in a decrease in the Km of dUMP to a value close to that in the normal enzymic reaction. The exchange reaction provides definitive evidence that the enzymic reaction involves attack of a nucleophile of the enzyme on the 6 position of dUMP to provide a 5,6-dihydro-dUMP intermediate which is covalently bound to the enzyme. Stereochemical considerations of the exchange reaction require proposal of a partial reaction which is not completely sterospecific or a complex reaction in which protons of water are handled with complete stereospecificity in a fashion similar to the one carbon unit of the normal enzymic reaction. 相似文献
24.
Fibronectin: a chromatin-associated protein? 总被引:14,自引:0,他引:14
We have previously reported that chromatin preparations from human cultured fibroblasts contain a single homologous serum protein. In this paper we present evidence, based on immunological identity and physicochemical properties, that this serum protein is fibronectin. Furthermore, using a radioimmunoassay system, we have estimated that fibronectin represents about 0.7% of the total protein in both chromatin preparations and whole fibroblasts. Using a nitrocellulose filter assay system, we also show that fibronectin is a DNA-binding protein having an equilibrium constant of 4.6 x 10(-6) M. Equilibrium competition experiments have demonstrated that fibronectin has the ability to differentiate among nucleotides, indicating that fibronectin-DNA interaction is at least partially specific, and that a minimum polymer length of 12-18 nucleotides is required for effective binding to occur. Fibronectin has been isolated readily from plasma using DNA-affinity chromatography. We do not have direct evidence that fibronectin is an actual nonhistone chromosomal protein, but fibronectin is a DNA-binding protein (at least under in vitro assay conditions) and appears to be a normal constituent of chromatin as chromatin is currently isolated from cell nuclei. 相似文献
25.
26.
Alfonso L. Pogolotti Kathryn M. Ivanetich Hans Sommer Daniel V. Santi 《Biochemical and biophysical research communications》1976,70(3):972-978
Studies are reported on the FdUMP-CH2-H4 folate-peptide obtained upon proteolysis of the complex formed from thymidylate synthetase, FdUMP and 5,10-CH2-H4folate. Contrary to a previous report from this laboratory, the peptide does contain a cysteine residue. The sequence of the largest peptide obtained is Ala-Leu-Pro-Pro-(His,Cys)-Thr. Quantitative modification of the histidine residue with the Pauly reagent indicates that imidazole is not directly linked to the nucleotide. The stability of the peptide indicates the covalent bond to the cofactor involves its 5-nitrogen; from this, it may be concluded that the reactive form of the cofactor is the 5-iminium ion. 相似文献
27.
The interaction of tRNA (m5U54)-methyltransferase (RUMT) with in vitro synthesized unmodified tRNA and a 17-base oligoribonucleotide analog of the T-arm of tRNA in the absence of AdoMet has been investigated. Binary complexes are formed which are isolable on nitrocellulose filters and are composed of noncovalent and covalent complexes in nearly equal amounts. The covalent RUMT-RNA complexes are stable to SDS-PAGE and migrate slower than free enzyme or RNA. Kinetic and thermodynamic constants involved in formation and disruption of noncovalent and covalent binary complexes have been determined and interpreted in the context of steady-state kinetic parameters of the enzyme-catalyzed methylation and 5-H exchange of substrate. The results show that the isolable covalent complex is kinetically incompetent as an intermediate for methylation. Isotope trapping experiments show that when AdoMet is added to preformed binary complex, all bound tRNA is converted to methylated product; thus, the covalent complexes are chemically competent to form products. We have concluded that, after a reversible binary complex is formed, the catalytic thiol adds to the 6-carbon of the U54 of tRNA. The initial adduct leaves the reaction pathway to protonation at carbon 5; the latter can deprotonate and re-enter the pathway to form methylated product. It is speculated that covalent binary RUMT-RNA adducts may serve as depots of enzyme-tRNA complexes primed for methylation, or in unknown roles with RNAs other than tRNA. 相似文献
28.
29.
Julio C. Chávez José L. de la Vega-Beltrán Jessica Escoffier Pablo E. Visconti Claudia L. Trevi?o Alberto Darszon Lawrence Salkoff Celia M. Santi 《PloS one》2013,8(4)
Unlike most cells of the body which function in an ionic environment controlled within narrow limits, spermatozoa must function in a less controlled external environment. In order to better understand how sperm control their membrane potential in different ionic conditions, we measured mouse sperm membrane potentials under a variety of conditions and at different external K+ concentrations, both before and after capacitation. Experiments were undertaken using both wild-type, and mutant mouse sperm from the knock-out strain of the sperm-specific, pH-sensitive, SLO3 K+ channel. Membrane voltage data were fit to the Goldman-Hodgkin-Katz equation. Our study revealed a significant membrane permeability to both K+ and Cl− before capacitation, as well as Na+. The permeability to both K+ and Cl− has the effect of preventing large changes in membrane potential when the extracellular concentration of either ion is changed. Such a mechanism may protect against undesired shifts in membrane potential in changing ionic environments. We found that a significant portion of resting membrane potassium permeability in wild-type sperm was contributed by SLO3 K+ channels. We also found that further activation of SLO3 channels was the essential mechanism producing membrane hyperpolarization under two separate conditions, 1) elevation of external pH prior to capacitation and 2) capacitating conditions. Both conditions produced a significant membrane hyperpolarization in wild-type which was absent in SLO3 mutant sperm. Hyperpolarization in both conditions may result from activation of SLO3 channels by raising intracellular pH; however, demonstrating that SLO3-dependent hyperpolarization is achieved by an alkaline environment alone shows that SLO3 channel activation might occur independently of other events associated with capacitation. For example sperm may undergo stages of membrane hyperpolarization when reaching alkaline regions of the female genital tract. Significantly, other events associated with sperm capacitation, occur in SLO3 mutant sperm and thus proceed independently of hyperpolarization. 相似文献
30.
Jordi Morata Santi Béjar David Talavera Casandra Riera Sergio Lois Gemma Mas de Xaxars Xavier de la Cruz 《PloS one》2013,8(8)
At present we know that phenotypic differences between organisms arise from a variety of sources, like protein sequence divergence, regulatory sequence divergence, alternative splicing, etc. However, we do not have yet a complete view of how these sources are related. Here we address this problem, studying the relationship between protein divergence and the ability of genes to express multiple isoforms. We used three genome-wide datasets of human-mouse orthologs to study the relationship between isoform multiplicity co-occurrence between orthologs (the fact that two orthologs have more than one isoform) and protein divergence. In all cases our results showed that there was a monotonic dependence between these two properties. We could explain this relationship in terms of a more fundamental one, between exon number of the largest isoform and protein divergence. We found that this last relationship was present, although with variations, in other species (chimpanzee, cow, rat, chicken, zebrafish and fruit fly). In summary, we have identified a relationship between protein divergence and isoform multiplicity co-occurrence and explained its origin in terms of a simple gene-level property. Finally, we discuss the biological implications of these findings for our understanding of inter-species phenotypic differences. 相似文献