Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:4.1.1.6 (CAD)
4,420 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dihydroorotase (DHOase) catalyzes the third step in eukaryotic de novo pyrimidine biosynthesis. In mammalian cells, this enzyme activity is carried by a large chimeric protein, CAD, that also catalyzes the first two steps in the pathway: glutamine-dependent carbamyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). Controlled elastase cleavage of CAD released a 44,000 +/- 2,000-dalton proteolytic fragment which catalyzed only the dihydroorotase reaction. We have devised a rapid and simple method for the isolation of the DHO domain from elastase digests. The domain, which was obtained in 36% yield, was found to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The domain was also characterized by amino acid analysis and analytical high pressure liquid chromatography peptide mapping. The amino terminus of both the DHO domain and intact CAD was blocked suggesting that this domain is located at the extreme amino terminus of the CAD polypeptide, a result consistent with the suspected juxtaposition of domains as DHO-CPS-ATC. The isoelectric point of the DHO domain was 5.1, while that of the ATC domain was 9.4, so that the ends of the CAD polypeptide are oppositely charged at physiological pH. Immunoblotting with DHO domain-specific antibodies showed that a 47-kDa species was generated in the early stages of controlled proteolysis of CAD. Thus there are two elastase cleavage sites within a 3-kDa connecting region that links the DHO and CPS domains. The domain was shown by atomic absorption spectrophotometry and by isolating a 65Zn-containing DHO domain from mammalian cells grown in the presence of the radionuclide to contain 1 g eq of tightly bound zinc in each polypeptide chain. Zinc was not found in any other CAD domain. Chelating agents inhibit dihydroorotase activity of the isolated domain supporting the conclusion, based on studies of intact CAD by others, that zinc participates in catalysis. At moderate protein concentrations the DHO domain was a 88,000 dimer with a Stokes radius of 37.6 A, a S20,w = 5.1 X 10(-13) s, a diffusion coefficient of 3.17 X 10(-7) cm2 s-1, and a frictional ratio of 1.26. On dilution the dimer dissociated and was in rapid concentration-dependent equilibrium with a 43,500 monomer. The hydrodynamic parameters of the monomer have also been estimated (Stokes radius of 29.8 A, D20,w = 4.11 X 10(-7) cm2 s-1, and f/f0 1.21).(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:The dihydroorotase domain of the multifunctional protein CAD. Subunit structure, zinc content, and kinetics. 287 Oct 22

Improved methodologies are described which allow the measurement of the part-reactions, with glutamine or ammonia as nitrogen donor, of mammalian carbamoyl-phosphate synthase II (EC 6.3.5.5) through the incorporation of [14C]bicarbonate into either carbamoyl phosphate or carbamoylaspartate. The enzyme is part of the multifunctional polypeptide (CAD) which also comprises the pyrimidine-biosynthetic enzymes aspartate transcarbamoylase (EC 2.1.3.2) and dihydro-orotase (EC 3.5.2.3). The conformational stability of the carbamoyl-phosphate synthase was investigated through the inactivation of the part-reactions which occurred during incubation at 37 degrees C. The domain involved in the removal of the amide N from glutamine was more thermolabile than the ammonia-dependent synthase moiety. The former activity was stabilized in the presence of sodium aspartate or MgATP, whereas the latter was stabilized by MgATP and MgUTP. Binding of MgUTP and MgATP to CAD restricted the initial proteolysis by trypsin and elastase of one or both regions linking the carbamoyl-phosphate synthase domain to the other major domains. A model is described to account for both aspects of nucleotide binding to CAD; these stabilizing effects may be important in the cell, where similar concentrations of nucleotides are found.
...
PMID:Nucleotide ligands protect the inter-domain regions of the multifunctional polypeptide CAD against limited proteolysis, and also stabilize the thermolabile part-reactions of the carbamoyl-phosphate synthase II domains within the CAD polypeptide. 363 65

A strategy is described to locate the carbonyl position in oxofatty acids by utilizing charge-remote fragmentations of various molecular ions that are desorbed by fast atom bombardment (FAB). Oxofatty acids were cationized with alkali metal ions (Li+, Na+, K+, Rb+, and Cs+) to form [M + 2Met-H]+ or alkaline earth metal ions (Mg2+, Ca2+, Sr2+ or Ba2+) to from [M + Met-H]+ in the gas phase. The cationized acids undergo charge-remote fragmentations upon high-energy activation, giving a product-ion pattern that has a gap corresponding to the oxo position and bordered by two high-intensity peaks. One of the peaks corresponds to an ion that is formed by the cleavage of the C-C bond beta to the oxo position and proximal to the charge (beta ion), whereas the other is formed from the cleavage of the C-C bond gamma to the oxo position and distal to the charge (gamma' ion). The oxo position is easily determined by identifying the gap and the beta and gamma' ions. Furthermore, there are two competing patterns of fragments in a CAD spectrum of an oxofatty acid or ester [M + Li]+ ion. These arise because Li+ attaches to either the oxo or the carboxylic end, as was confirmed by ab initio molecular orbital calculations. The results demonstrate that control of the fragmentation can be guided by an understanding of metal-ion affinities. Collisional activation of the anionic carboxylates gives results that are similar to those for positive ions, showing that the process is not related to the charge status. Collisional activation of [M + H]+ ions does not give structural information because the charge migrates, leading to charge-mediated fragmentations.
...
PMID:Structural determination of oxofatty acids by charge-remote fragmentations. 987 59

Cell lines derived from tumors engineered in the CNS offer promise as models of specific neuronal cell types. CAD cells are an unusual subclone of a murine cell line derived from tyrosine hydroxylase (TH) driven tumorigenesis, which undergoes reversible morphological differentiation on serum deprivation. Using single-cell electrophysiology we have examined the properties of ion channels expressed in CAD cells. Despite relatively low resting potentials, CAD cells can be induced to fire robust action potentials when mildly artificially hyperpolarized. Correspondingly, voltage-dependent sodium and potassium currents were elicited under voltage clamp. Sodium currents are TTX sensitive and exhibit conventional activation and inactivation properties. The potassium currents reflected two pharmacologically distinguishable populations of delayed rectifier type channels while no transient A-type channels were observed. Using barium as a charge carrier, we observed an inactivating current that was completely blocked by nimodipine and thus associated with L-type calcium channels. On differentiation, three changes in functional channel expression occurred; a 4-fold decrease in sodium current density, a 1.5-fold increase in potassium current density, and the induction of a small noninactivating barium current component. The neuronal morphology, excitability properties, and changes in channel function with differentiation make CAD cells an attractive model for study of catecholaminergic neurons.
...
PMID:Voltage-dependent ion channels in CAD cells: A catecholaminergic neuronal line that exhibits inducible differentiation. 1111 Aug 18

The purpose of this study was to investigate whether there are differences in the expression of the endothelin (ET) system in the peripheral vasculature of diabetic African-American (AA) and Caucasian (CA) patients. Tibial artery specimens were obtained from diabetic (MD = 8 and CAD = 5) and non-diabetic (AAND = 6 and CAND = 5) patients undergoing lower limb amputation. The gene expression of ET-1 precursor (PPET-1), ET(A)R and ET(B)R was determined by a reverse transcriptase polymerase chain reaction technique. PPET-1 and ET(A)R expression was up-regulated 4- and 3-fold, respectively, in both AA and CA diabetics (P<.05 vs non-diabetics). ET(B)R mRNA was significantly lower in AA diabetic patients. Function of ET-1 and ET receptors was assessed by vascular contractility assays. Vascular relaxation in response to sodium nitroprusside in arteries precontracted with ET-1 was significantly lower in AA (58% +/- 9) as compared to CA diabetics (74% +/- 5) (P<.05). In conclusion, this study demonstrated that the ET system is altered in favor of the contractile phenotype in AA diabetics and may contribute to the increased incidence of vascular complications in this population.
...
PMID:Selective downregulation of endothelin-B receptors in diabetic African-American patients. 1191 20

Low-grade inflammatory activity is associated with an increased risk for ischaemic coronary events. sPLA(2) (secretory non-pancreatic type II phospholipase A(2)) serum activity is increased in chronic inflammatory diseases and may also contribute to atherogenesis. Since the endothelium is a major target for inflammatory cytokines, we hypothesized that elevated serum activity of sPLA(2) is associated with an impaired vasodilator function in patients with documented CAD (coronary artery disease). Endothelium-dependent (acetylcholine, 10-50 microg/min) and endothelium-independent (sodium nitroprusside, 2-8 microg/min) FBF (forearm blood flow) responses were measured by venous occlusion plethysmography in 50 male patients with angiographically documented CAD. sPLA(2) serum activity was inversely correlated with acetylcholine-induced FBF responses ( r =-0.36; P <0.05). In addition, there was a significant correlation between sPLA(2) and CRP (C-reactive protein; r =0.33, P <0.02). In contrast, FBF responses to sodium nitroprusside did not correlate with sPLA(2) serum activity. In order to identify independent predictors of an impaired endothelium-dependent vasodilator function in patients with CAD, a multivariate analysis was performed including the inflammatory serum markers as well as classical risk factors of CAD. This analysis demonstrated that both sPLA(2) ( P <0.05) and CRP serum levels ( P <0.05) were the only significant independent predictors of an impaired acetylcholine-induced FBF response. In conclusion, elevated sPLA(2) serum activity is associated with a significant impairment in systemic endothelial vasodilator function in patients with CAD. The identification of sPLA(2) as a novel independent predictor for endothelial dysfunction provides another important clue to link a systemic marker of inflammation with coronary atherosclerotic disease.
...
PMID:Elevated secretory non-pancreatic type II phospholipase A2 serum activity is associated with impaired endothelial vasodilator function in patients with coronary artery disease. 1474 Oct 42

Oligonucleosomal fragmentation of nuclear DNA is the late-stage apoptosis hallmark. In apoptotic mammalian cells the fragmentation is catalyzed by DFF40/CAD DNase primarily activated by caspase 3 through the site-specific proteolytic cleavage of DFF45/ICAD. A deletion in the casp3 gene of human breast adenocarcinoma MCF-7 results in lack of procaspase 3 in these cells. The absence of caspase 3 in MCF-7 leads to disability to activate oligonucleosomal DNA fragmentation in TNF-alpha induced cell death. In this study, sodium palmitate was used as an apoptotic stimulus for MCF-7. It has been shown that palmitate but not TNF-alpha induces both apoptotic changes in nuclei and oligonucleosomal DNA fragmentation in casp3-mutated MCF-7. Activation and accumulation of 40-50 kD DFF40-like DNases in nuclei of palmitate-treated apoptotic MCF-7 were detected by SDS-DNA-PAGE assay. Microsomal fraction of apoptotic MCF-7 does not contain any detectable DNases, but activates 40-50 kD nucleases when incubated with human placental chromatin. Furthermore, microsomes of apoptotic MCF-7 induce oligonucleosomal fragmentation of chromatin in a cell-free system. Both the activation of DNases and chromatin fragmentation are suppressed in the presence of the caspase 3/7 inhibitor Ac-DEVD-CHO. Microsome-associated caspase 7 is suggested to play an essential role in the induction of oligonucleosomal DNA fragmentation in casp3-deficient MCF-7 cells.
...
PMID:Oligonucleosomal DNA fragmentation in MCF-7 cells undergoing palmitate-induced apoptosis. 1475 30

Evidence suggests that flavonoid-containing diets reduce cardiovascular risk, but the mechanisms responsible are unclear. In the present study, we sought to determine the effect of flavanol-rich cocoa on vascular function in individuals with CAD (coronary artery disease). Forty subjects (61+/-8 years; 30 male) with CAD were recruited to a 6-week randomized double-blind placebo-controlled study. Subjects consumed either a flavanol-rich chocolate bar and cocoa beverage daily (total flavanols, 444 mg/day) or matching isocaloric placebos daily (total flavanols, 19.6 mg/day) for 6 weeks. Brachial artery FMD (flow-mediated dilation) and SAC (systemic arterial compliance) were assessed at baseline, 90 min following the first beverage and after 3 and 6 weeks of daily consumption. Soluble cellular adhesion molecules and FBF (forearm blood flow) responses to ACh (acetylcholine chloride; 3-30 microg/min) and SNP (sodium nitroprusside; 0.3-3 microg/min) infusions, forearm ischaemia and isotonic forearm exercise were assessed at baseline and after 6 weeks. FMD, SAC and FBF responses did not differ between groups at baseline. No acute or chronic changes in FMD or SAC were seen in either group. No difference in soluble cellular adhesion molecules, FBF responses to ischaemia, exercise, SNP or ACh was seen in the group receiving flavanol-rich cocoa between baseline and 6 weeks. These data suggest that over a 6-week period, flavanol-rich cocoa does not modify vascular function in patients with established CAD.
...
PMID:Acute and chronic effects of flavanol-rich cocoa on vascular function in subjects with coronary artery disease: a randomized double-blind placebo-controlled study. 1655 Dec 72

The anti-epileptic drug (R)-lacosamide ((2R)-2-(acetylamino)-N-benzyl-3-methoxypropanamide (LCM)) modulates voltage-gated sodium channels (VGSCs) by preferentially interacting with slow inactivated sodium channels, but the observation that LCM binds to collapsin response mediator protein 2 (CRMP-2) suggests additional mechanisms of action for LCM. We postulated that CRMP-2 levels affects the actions of LCM on VGSCs. CRMP-2 labeling by LCM analogs was competitively displaced by excess LCM in rat brain lysates. Manipulation of CRMP-2 levels in the neuronal model system CAD cells affected slow inactivation of VGSCs without any effects on other voltage-dependent properties. In silico docking was performed to identify putative binding sites in CRMP-2 that may modulate the effects of LCM on VGSCs. These studies identified five cavities in CRMP-2 that can accommodate LCM. CRMP-2 alanine mutants of key residues within these cavities were functionally similar to wild-type CRMP-2 as assessed by similar levels of enhancement in dendritic complexity of cortical neurons. Next, we examined the effects of expression of wild-type and mutant CRMP-2 constructs on voltage-sensitive properties of VGSCs in CAD cells: 1) steady-state voltage-dependent activation and fast-inactivation properties were not affected by LCM, 2) CRMP-2 single alanine mutants reduced the LCM-mediated effects on the ability of endogenous Na(+) channels to transition to a slow inactivated state, and 3) a quintuplicate CRMP-2 alanine mutant further decreased this slow inactivated fraction. Collectively, these results identify key CRMP-2 residues that can coordinate LCM binding thus making it more effective on its primary clinical target.
...
PMID:In silico docking and electrophysiological characterization of lacosamide binding sites on collapsin response mediator protein-2 identifies a pocket important in modulating sodium channel slow inactivation. 2053 11

The development of strategies based on mass spectrometry to help for deep structural analysis of acidic oligosaccharides remains topical. We thus examined the dissociation behavior of deprotonated ions of heparin-derived di- to tetra-saccharides under UV irradiation at 220 nm. Depending on the ionization state of the carboxylic groups, an oxidized species issued from electron photodetachment was observed in complement to photoinduced fragmentation of precursor ions. The influence of the charge location in the oligosaccharide dianions on the balance between photodissociation and electron photodetachment is examined and a way to direct the relaxation pathways, (i.e., dissociation versus electron detachment), is proposed using sodium adducts. The oxidized species was subjected to activated-electron photodetachment (activated-EPD) leading to complementary informative fragment ions to those issued from photodissociation. Directed photoinduced dissociation at 220 nm and activated-EPD should complement the more conventional CAD and IRMPD activation modes for deeper structural analysis of acidic oligosaccharides-derived anions.
...
PMID:Photoinduced dissociation of heparin-derived oligosaccharides controlled by charge location. 2093 74


1 2 3 Next >>

---