Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.1.6 (CAD)
 4,420 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The multifunctional protein CAD catalyzes the first three steps in pyrimidine biosynthesis in mammalian cells, including the synthesis of carbamyl phosphate from bicarbonate, MgATP and glutamine. The Syrian hamster CAD glutaminase (GLNase) domain, a trpG-type amidotransferase, catalyzes glutamine hydrolysis in the absence of MgATP and bicarbonate (Km = 95 microM and kcat = 0.14 s-1). Unlike E. coli carbamyl phosphate synthetase (Wellner, V.P., Anderson, P.M., and Meister, A. (1973) Biochemistry 12, 2061-2066), a stable thioester intermediate did not accumulate when the mammalian enzyme was incubated with glutamine. However, a covalent adduct could be isolated when the protein was denatured in acid. The steady state concentration of the intermediate increased with increasing glutamine concentration to nearly one mole per mole of enzyme with half saturation at 105 microM, close to the Km value for glutamine. The adduct formed at the active site of the glutaminase domain. The rate of breakdown of the intermediate (k4), determined directly, was 0.17 s-1 and the rate of formation (k3) was estimated as 0.52 s-1. In the absence of MgATP and bicarbonate, k4 = kcat indicating that the decomposition of the intermediate is the rate-limiting step. The intermediate was chemically and kinetically competent, and the glutamine dissociation constant (330 microM) and rate constants were consistent with steady state kinetics and accurately predicted the steady state concentration of the intermediate. These studies suggest a mechanism similar to the cysteine proteases such as recently proposed by Mei and Zalkin (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619) who identified a catalytic triad in glutamine phosphoribosyl-5'-pyrophosphate amidotransferase, a purF-type enzyme. MgATP and bicarbonate increased kcat of the glutaminase reaction 14-fold by accelerating both the rate of formation and the rate of breakdown of the intermediate, and prevented the accumulation of the intermediate; however, the Km value for glutamine was not significantly altered. The instability of the thioester intermediate leads to appreciable hydrolysis of glutamine in the absence of the other substrates. However, bicarbonate alone spares glutamine by increasing the Km and Ks of glutamine to 600 and 8960 microM, respectively, thus reducing kcat/Km 3-fold when MgATP is limiting. In the absence of MgATP and bicarbonate, ammonia decreased the rate of hydrolysis and the accumulation of the thioester intermediate indicating that ammonia had direct access to the thioester at the GLNase domain active site.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The catalytic mechanism of the amidotransferase domain of the Syrian hamster multifunctional protein CAD. Evidence for a CAD-glutamyl covalent intermediate in the formation of carbamyl phosphate. 167 73

CAD is a 243-kDa multidomain polypeptide which catalyzes the first three steps in mammalian de novo pyrimidine biosynthesis. The largest cDNA clone obtained thus far, pCAD142 (Shigesada, K., Stark, G.R., Maley, J. A., Niswander, L. A., and Davidson, J. N. (1985) Mol. Cell. Biol. 5, 1735), lacks the 5' end of the mRNA which encodes the amino terminus of CAD. To clone this missing segment, a synthetic oligonucleotide complementary to pCAD142 and poly(A)+ RNA template, isolated from a Syrian hamster cell line which overproduces the CAD mRNA, were used for cDNA synthesis. The resulting clone pKB11, which has a 1369-base pair (bp) cDNA insert, overlapping pCAD142 by 781 bp, was identified by hybridization methods and sequence analysis and found to contain the entire cDNA sequence for the amino end of the CAD polypeptide. The deduced amino acid sequence is homologous to seven carbamyl phosphate synthetases. Primer extension, oligonucleotide-directed RNase H digestion, and RNA sequencing indicated that pKB11 extends to within 68 bases of the 5' end of the CAD mRNA. This conclusion was confirmed by Northern blotting analysis of the 5'-flanking region of CAD gene. The probable 3' end of an unidentified gene which codes for a 1-kilobase (kb) transcript was identified immediately upstream of the CAD gene. Northern analysis using probes complementary to the region between the CAD and the 1-kb genes detected the presence of a small transcript of less than 300 nucleotides. The sequence revealed three potential translation initiation sites raising the possibility of more than one CAD translation product. The major translation start codon was identified as the first ATG in pKB11 by sequence homology, in vitro transcription and translation, and protein studies. Starting from this ATG within pKB11, the clone encodes a 143-residue domain of unknown function. This study completes the determination of the primary structure of the CAD polypeptide. The CAD mRNA is 7.5 kb in length and has 6675 bp of coding sequence and about 200 bp and 600 bp of untranslated sequence at the 5' and 3' ends, respectively.
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PMID:Molecular cloning of a cDNA encoding the amino end of the mammalian multifunctional protein CAD and analysis of the 5'-flanking region of the CAD gene. 167 75

There are several hundred thousand members of the Alu repeat family in the human genome. Those Alu elements sequenced to date appear to fit into subfamilies. A novel Alu has been found in an intron of the human CAD gene: it appears to be due to rearrangement between Alu repeats belonging to two different subfamilies. Further sequence data from this intron suggest that the Alu element may have rearranged prior to its entry into the CAD gene. Such findings indicate that, in addition to single nucleotide substitutions and deletions, DNA rearrangements may be a factor in generating the diversity of Alu repeats found in primate genomes.
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PMID:An unusual Alu repeat sequence within the CAD gene. 167 52

The CAD multidomain protein, which includes active sites of carbamyl phosphate synthetase II (CPS II, glutamine-dependent), aspartate transcarbamylase, and dihydroorotase, was immunostained in normal rat brains, the gliotic brains of myelin-deficient mutant rats, and brains from normal weanling hamsters. In each of these tissues CAD was observed in cells resembling astrocytes. In hamster brain, CAD immunofluorescence was also found in cells closely related to astrocytes, i.e., the Bergmann glia in cerebellum and the tanycytes surrounding the third ventricle. The astrocytic identity of the CAD-positive cells in rat brain was confirmed by double immunofluorescence staining with antibodies against glial fibrillary acidic protein (GFAP). The two enzymes carbonic anhydrase and glutamine synthetase occur in the cytoplasm of normal astrocytes in gray matter and of reactive astrocytes during gliosis. Products of each enzyme, i.e., bicarbonate and glutamine, are required for the CPS II reaction, which is the first step in the biosynthesis of pyrimidines. Therefore, the present results suggest roles for carbonic anhydrase and glutamine synthetase, as well as CAD, in pyrimidine biosynthesis in brain and a role for the astrocytes in the de novo synthesis of pyrimidines.
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PMID:Localization of the multifunctional protein CAD in astrocytes of rodent brain. 167 39

CAD is the multifunctional protein of higher eukaryotes which catalyzes the first three steps of pyrimidine biosynthesis. Its enzymatic activities exist as independent domains in the order: N terminus-carbamylphosphate synthetase II(CPSase)-dihydroorotase(DHOase)-aspartate transcarbamylase(ATCase)-C terminus. To functionally define the minimum hamster cDNA region required to encode an active DHOase, expression constructs were generated. Many such constructs complement Escherichia coli mutants defective not only in DHOase but also in ATCase. Constructs deleted for most of the sequence encoding the ATCase domain continue to complement E. coli mutants defective in DHOase. All of these smaller constructs also lack the region encoding CPSase. Therefore, a 'genetic cassette', containing information for neither the CPSase nor the ATCase domain, can direct the synthesis of a polypeptide with DHOase activity. Interestingly, inclusion of a portion of the DHOase-ATCase interdomain bridge appears to be required for optimum activity.
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PMID:Synthesis of the nonconserved dihydroorotase domain of the multifunctional hamster CAD protein in Escherichia coli. 167 66

Extrachromosomal elements are common early intermediates of gene amplification in vivo and in cell culture. The time at which several extrachromosomal elements replicate was compared with that of the corresponding amplified or unamplified chromosomal sequences. The replication timing analysis employed a retroactive synchrony method in which fluorescence-activated cell sorting was used to obtain cells at different stages of the cell cycle. Extrachromosomally amplified Syrian hamster CAD genes (CAD is an acronym for the single gene which encodes the trifunctional protein which catalyzes the first three steps of uridine biosynthesis) replicated in a narrow window of early S-phase which was approximately the same as that of chromosomally amplified CAD genes. Similarly, extrachromosomally amplified mouse adenosine deaminase genes replicated at a discrete time in early S-phase which approximated the replication time of the unamplified adenosine deaminase gene. In contrast, the multicopy extrachromosomal Epstein-Barr virus genome replicated within a narrow window in late S-phase in latently infected human Rajii cells. The data indicate that localization within a chromosome is not required for the maintenance of replication timing control.
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PMID:Replication timing control can be maintained in extrachromosomally amplified genes. 167 57

We conducted a series of studies aimed at investigating the effect of beta-blockers on exercise physiology. On the basis of these and other existing studies, it is possible to draw the following conclusions and to make the following tentative recommendations for patients engaged in exercise training who receive beta-blocker therapy: i) CAD patients treated with beta-blockers are capable of deriving the expected enhancement of cardiorespiratory fitness during training, irrespective of the type of drug used; ii) beta1-selective blockers are preferable to nonselective agents for hypertensive patients engaged in exercise training; iii) because beta1-selective blockers impair exercise tolerance in some hypertensive patients, physicians should look out for this adverse reaction and, if present, consider alternative antihypertensive therapy; iv) intrinsic sympathomimetic activity confers no advantage during exercise training; v) exercise intensity prescription for patients receiving beta-blockers should be in accordance with traditional guidelines and based on results of individualized exercise testing performed on medication; vi) exercise training is desirable during beta-blocker therapy in that it appears to offset adverse alterations in lipoprotein metabolism; and vii) nonselective beta-blockers may increase predisposition to exertional hyperthermia, and patients must therefore be encouraged to adhere strictly to accepted guidelines for heat injury prevention.
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PMID:Effect of beta-blockers on exercise physiology: implications for exercise training. 167 17

The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA) was used to chemically modify the ATP binding sites of the carbamyl phosphate synthetase domain of CAD, the multifunctional protein that catalyzes the first steps in mammalian pyrimidine biosynthesis. Reaction of CAD with FSBA resulted in the inactivation of the ammonia- and glutamine-dependent CPSase activities but had no effect on its glutaminase, aspartate transcarbamylase, or dihydroorotase activities. ATP protected CAD against inactivation by FSBA whereas the presence of the allosteric effectors UTP and PRPP afforded little protection, which suggests that the ATP binding sites were specifically labeled. The inactivation exhibited saturation behavior with respect to FSBA with a K1 of 0.93 mM. Of the two ATP-dependent partial activities of carbamyl phosphate synthetase, bicarbonate-dependent ATPase was inactivated more rapidly than the carbamyl phosphate dependent ATP synthetase, which indicates that these partial reactions occur at distinct ATP binding sites. The stoichiometry of [14C]FSBA labeling showed that only 0.4-0.5 mol of FSBA/mol of protein was required for complete inactivation. Incorporation of radiolabeled FSBA into CAD and subsequent proteolysis, gel electrophoresis, and fluorography demonstrated that only the carbamyl phosphate synthetase domain of CAD is labeled. Amino acid sequencing of the principal peaks resulting from tryptic digests of FSBA-modified CAD located the sites of FSBA modification in regions that exhibit high homology to ATP binding sites of other known proteins. Thus CAD has two ATP binding sites, one in each of the two highly homologous halves of the carbamyl phosphate domain which catalyze distinct ATP-dependent partial reactions in carbamyl phosphate synthesis.
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PMID:Identification of the ATP binding sites of the carbamyl phosphate synthetase domain of the Syrian hamster multifunctional protein CAD by affinity labeling with 5'-[p-(fluorosulfonyl)benzoyl]adenosine. 168

Lipoprotein (Lp) cholesterol and apolipoproteins (apo) A-I and B levels have been shown to be better markers for the presence of coronary artery disease than total cholesterol. In this study, we determined the plasma levels of lipoprotein particles containing apo A-I only (LpA-I), apo A-I and A-II (LpA-I:A-II), apo B and C-III (LpB:C-III) and apo B and E (LpB:E) in 145 patients with coronary artery disease (mean age +/- SD, 51 +/- 7 years) and 135 healthy control men (mean age 49 +/- 11 years). Patients with CAD had lower high density lipoprotein (HDL) cholesterol and apo A-I levels and higher triglycerides and apo had lower high density lipoprotein (HDL) cholesterol and apo A-I levels and higher triglycerides and apo B levels than controls. In patients with CAD, LpA-I (0.341 +/- 0.093 vs. 0.461 +/- 148 g/l) and LpA-I:A-II (0.694 +/- 0.171 vs. 0.899 +/- 0.148 g/l) were lower, whereas LpB:E (0.372 +/- 0.204 vs. 0.235 +/- 0.184 g/l) were higher than in controls (cases vs. controls, all P less than 0.005). No significant differences were observed for LpB:C-III (0.098 +/- 0.057 vs. 0.107 +/- 0.061 g/l, p = 0.235) particles. Discriminant analysis indicates that LpA-II:A-I, LpE:B, LpA-I, and triglycerides best differentiate between cases and controls. Plasma apo C-III (0.027 +/- 0.008 vs. 0.036 +/- 0.020 g/l) and E (0.040 +/- 0.015 vs. 0.055 +/- 0.029 g/l) were lower in the CAD group (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Plasma apolipoprotein A-I, A-II, B, E and C-III containing particles in men with premature coronary artery disease. 168 7

The initial promising results with alternating chemotherapy regimens (mechlorethamine, vincristine, procarbazine, and prednisone/doxorubicin, bleomycin, vinblastine, and dacarbazine [MOPP/ABVD]; lomustine, melphalan, and vindesine [CAD] plus MOPP plus ABV) combined with intermediate-dose radiation therapy (RT) have been sustained with further follow-up; 82.2% of patients (152 of 185) achieved a complete remission (CR), and overall survival is 71.7% +/- 4.4% at 8 years (median follow-up is 55 months among the survivors). No statistically significant differences were found in CR percentage, CR duration, or survival between stages IIB, IIIB, and IV patients. For that reason, stepwise Cox regression analyses to identify the important prognostic factors were performed on overall survival, tumor mortality, freedom from disease progression, and survival following disease progression. Pretreatment characteristics were also tested for association with the probability of achieving CR, CR duration, and death due to other causes. Characteristics that were consistently associated with an independently unfavorable prognosis were low hematocrit, high serum lactic acid dehydrogenase (LDH), age more than 45 years, inguinal node involvement, mediastinal mass greater than .45 of the thoracic diameter, and bone marrow involvement. Patients with two or more unfavorable characteristics were much more likely to fail treatment (median survival, 62.4 months) than those with none or only one unfavorable factor (greater than 95% survival). This striking difference between the low- and high-risk groups remained even if the comparison was restricted to patients less than or equal to 45 years of age. These results provide a basis for selecting the young patients at high risk of failure for more intensive initial treatment with either autologous bone marrow rescue or hematopoietic growth factors.
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PMID:Prognostic factors among 185 adults with newly diagnosed advanced Hodgkin's disease treated with alternating potentially noncross-resistant chemotherapy and intermediate-dose radiation therapy. 169 35


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