共查询到20条相似文献,搜索用时 15 毫秒
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Carbamyl phosphate synthetase (EC 2.7.2.9), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3), the first three enzymes in de novo pyrimidine synthesis in Chinese hamster ovary cell strain Kl (CHO-Kl), cose diment through a glycerol gradient. When an extract from Urd- A, a pyrimidine-requiring auxotroph reduced in all three activities, is run on a glycerol gradient, the enzyme activities appear in two peaks higher in the gradient, a peak of aspartate transcarbamylase separated from a peak of carbamyl phosphate synthetase and dihydroorotase. Revertants of Urd- A have increased activity of all three enzymes and give glycerol gradient patterns similar to either CHO-Kl or Urd- A. The gradient pattern for Urd- A and some of its revertants can be mimicked by treating the CHO-Kl cell extract with trypsin. Hybrids made between a CHO-Kl purine-requiring auxotroph (Ade- C) and a Urd- A revertant gave a glycerol gradient pattern which is a composite of the CHO-Kl and revertant patterns. A model is presented for the structure of this multifunctional protein. 相似文献
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Studies of the regulation and reaction mechanism of the carbamyl phosphate synthetase and aspartate transcarbamylase of bakers' yeast. 总被引:1,自引:0,他引:1
Kinetic studies of the carbamyl phosphate synthetase activity (CPSase) of bakers' yeast revealed an absolute requirement for K+ ions ; KM values for two of the substrates, glutamine and bicarbonate, were found to be 5 X 10(-4) M and 3 X 10(-3) M respectively. CPSase activity of the purified enzyme aggregate (M.W. 800,000) was extremely sensitive to UTP with a Ki of 2.4 X 10(-4) M. The purine nucleotide intermediate, XMP, was a strong activator of CPSase, acting at a site different from the regulatory site at which UTP binds ; XMP activation diminished at high concentrations of the substrate Mg-ATP. Studies of the reaction mechanism of CPSase revealed that it involved the sequential addition of the substrates bicarbonate and Mg-ATP, liberation of ADP, addition of glutamine, binding of ATP and then release of ADP and the product carbamyl phosphate. Studies of the reaction mechanism of the aspartate transcarbamylase (ATCase) of the aggregate yielded data which were not compatible with any of the usual models ; whichever reaction mechanism is ultivately found to fit the data, it will probably prove applicable both to the ATCase of the aggregate and to the disaggregated ATCase subunit (MW 138,000). 相似文献
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B P Jarry 《European journal of biochemistry》1978,87(3):533-540
A purification procedure is described by which aspartate transcarbamylase was obtained from cultured cells of Drosophila melanogaster as part of a high-molecular-weight enzyme complex. The complex is shown to contain several polypeptides. An antiserum directed against the complex enzyme inhibited in vitro the activity of aspartate transcarbamylase, carbamylphosphate synthetase and dihydro-orotase which were shown to copurify on a sucrose gradient and by gel electrophoresis. A fast preparation procedure using this antiserum yielded a 220 000-molecular-weight protein in addition to the polypeptides present in the complex. A purification procedure is also described to obtain aspartate transcarbamylase from second instar larvae of Drosophila. At this stage, the enzyme is not complexed with carbamylphosphate synthetase and dihydro-orotase but exhibits the same molecular weight as the aspartate transcarbamylase moiety found in the high-molecular-weight complex of cultured cells. 相似文献
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J Kennedy 《Biochemical and biophysical research communications》1978,80(3):653-658
Activities of aspartate transcarbamylase and dihydroorotase are increased 6 to 8-fold in erythrocytes from individuals with hypoxanthine guanine phosphoribosyltransferase deficiency. The increased enzyme activities do not appear to be due to enzyme activation. 相似文献
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D Pierson V Knight P Hansard E Chan 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1976,152(1):67-70
In mice infected with mouse-adapted influenza A/PR/8/34, hepatic carbamyl phosphate synthetase (CPS) activity was reduced to 88%, and ornithine transcarbamylase (OTC) was reduced to 83% of control values. In mice infected with mouse-adapted B/Lee/40, CPS activity was 98% was 94% of control values. These limited reductions in enzyme activity were attributed to a nonspecific debilitating effect of acute influenzal pneumonia. These findings suggest that the pronounced reduction of CPS and OTC activities reported in Reye's syndrome in man are not a general manifestation of the severity of influenza infection. 相似文献
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An X-ray diffraction study to 2.0 A resolution shows that this enzyme, ATCase, is in the T-state (the c3 to c3 distance is 45.2 A) when ATCase is bound to carbamyl phosphate (CP) and to L-alanosine (an analogue of aspartate). This result strongly supports the kinetic results that alanosine did not inhibit the carbamylation of aspartate in the normal reaction of native ATCase plus CP and aspartate [Baillon, J., Tauc, P., and Hervé, G. (1985) Biochemistry 24, 7182-7187]. The structure further reveals that the phosphate of CP is 4 A away from its known position in the R-state and is in the T-state position of P(i) in a recent study of ATCase complexed with products, phosphate (P(i)) and N-carbamyl-L-aspartate [Huang, J., and Lipscomb, W. N. (2004) Biochemistry 43, 6422-6426]. Moreover, the alanosine position in this T-state is somewhat displaced from that expected for its analogue, aspartate, from the R-state position. The relations of these structural aspects to the kinetics are presented. 相似文献
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J A Ridge F Roberts M H Schaffer G R Stark 《The Journal of biological chemistry》1976,251(19):5966-5975
Some preparations of both native aspartate transcarbamylase from Escherichia coli and catalytic subunit have fewer tight binding sites per oligomer for carbamyl-P than the number of catalytic peptide chains. In contrast, the number of sites for the tight-binding inhibitor N-(phosphonacetyl)-L-aspartate does equal the number of catalytic chains in each case. Binding of the labile carbamyl-P was determined using rapid gel filtration, with conversion to stable carbamyl-L-aspartate during collection. Native enzyme (six catalytic chains) obtained from cells grown under the conditions of J.C. Gerhart and H. Holoubek (J. Biol. Chem. (1967) 242, 2886-2892) has 5.4 tight sites for carbamyl-P at pH 8.0 (KD = 9.9 muM), whereas native enzyme from cells grown with higher concentrations of glucose, uracil, and histidine (to yield more enzyme per unit volume of culture) has only 1.9 tight sites at pH 8.0 (KD = 4.6 muM) and only 2.3 tight sites at pH 7.0 (KD = 2.6 muM). At pH 8.0, catalytic subunit (three catalytic chains) obtained from the former native enzyme has 2.2 tight sites for carbamyl-P (KD = 2.4 muM) and the number of sites is 2.3 in the presence of 35 mM succinate, whereas catalytic subunit obtained from the latter native enzyme has 1.8 tight sites (KD = 3.6 muM) in the absence of succinate and 2.3 tight sites in its presence. The number of tight binding sites is also less than the number of subunit peptide chains in 19F nuclear magnetic resonance experiments performed with catalytic subunit and two fluorinated analogs of carbamyl-P at comparable concentrations of analogs and active sites. A model is proposed in which incomplete removal of formylmethionine from the NH2 termini of the enzyme under conditions of extreme depression affects affinity for ligands. 相似文献
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Aspartate transcarbamylase (carbamoyl-phosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) has been purified from Mycobacterium smegmatis TMC 1546 using streptomycin sulphate precipitation, ammonium sulphate precipitation, DE-52 chromatography, second ammonium sulphate precipitation, Sephadex G-200 gel filtration, and aspartate-linked CNBr-activated Sepharose 4B affinity chromatography in successive order. The enzyme was purified 231.6-fold, and the preparation was found to be homogeneous on column chromatography and polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of 246,000 and was composed of two asymmetrical subunits. The kinetic and regulatory properties of aspartate transcarbamylase from M. smegmatis were also studied. The enzyme was found to be an allosteric in nature with carbamyl phosphate showing positive cooperativity and UMP exhibiting a negative cooperativity. CTP was found to be the most potent inhibitor among nucleotides. Phosphate acted as a non-competitive product inhibitor with respect to aspartate. Succinate and maleate exerted a competitive inhibition when aspartate was the variable substrate. 相似文献