Carbamoyl-phosphate synthetase (CPSase) consists of a 120-kDa synthetase domain (CPS) that makes carbamoyl phosphate from ATP, bicarbonate, and ammonia usually produced by a separate glutaminase domain. CPS is composed of two subdomains, CPS.A and CPS.B. Although CPS.A and CPS.B have specialized functions in intact CPSase, the separately cloned subdomains can catalyze carbamoyl phosphate synthesis. This report describes the construction of a 58-kDa chimeric CPSase composed of Escherichia coli CPS.A catalytic subdomains and the mammalian regulatory subdomain. The catalytic parameters are similar to those of the E. coli enzyme, but the activity is regulated by the mammalian effectors and protein kinase A phosphorylation. The chimera has a single site that binds phosphoribosyl 5 -pyrophosphate (PRPP) with a dissociation constant of 25 M. The dissociation constant for UTP of 0.23 mM was inferred from its effect on PRPP binding. Thus, the regulatory subdomain is an exchangeable ligand binding module that can control both CPS.A and CPS.B domains, and the pathway for allosteric signal transmission is identical in E. coli and mammalian CPSase. A deletion mutant that truncates the polypeptide within a postulated regulatory sequence is as active as the parent chimera but is insensitive to effectors. PRPP and UTP bind to the mutant, suggesting that the carboxyl half of the subdomain is essential for transmitting the allosteric signal but not for ligand binding.Escherichia coli carbamoyl-phosphate synthetase (CPSase 1 ; EC 6.3.5.5) consists of a 40-kDa glutaminase subunit and a 120-kDa synthetase subunit (1, 2). The glutaminase (GLN) subunit hydrolyzes glutamine to provide ammonia for the biosynthetic reaction (1-4). The synthetase subunit is composed of two 60-kDa homologous subdomains (5), CPS.A and CPS.B (Fig. 1), that catalyze carbamoyl phosphate formation from bicarbonate, ATP, and ammonia (4, 6 -13). The overall reaction involves the concerted action of CPS.A and CPS.B, each of which catalyzes two different ATP-dependent partial reactions (3, 16). The E. coli enzyme is allosterically regulated (1, 14) by metabolites from both pyrimidine and arginine biosynthetic pathways. UMP is a feedback inhibitor, while ornithine, IMP, and NH 3 activate the enzyme. The elegant three-dimensional structure of E. coli carbamoyl-phosphate synthetase has recently been solved (15) to 2.8-Å resolution.Mammalian glutamine-dependent carbamoyl-phosphate synthetase catalyzes the first committed step in the de novo pyrimidine biosynthetic pathway. The enzyme is part of a large multifunctional protein (17-20), called CAD (Fig. 1), that also catalyzes the second and third steps of the pathway. The CAD polypeptide (21-30) is organized into multiple, autonomously folded domains (Fig. 1), each with a distinct function. The CPSase activity of CAD, the major locus of regulation of the de novo pyrimidine biosynthetic pathway, is inhibited by UTP, activated by PRPP (31-37), and controlled (36) by protein kinase A phosphorylation.While the CPS.A and CPS...
XK469 is an investigational anticancer agent that exhibits antiproliferative activity in tumor-bearing animal models. We examined the drug-action profile of this agent at the molecular level regarding alterations induced in gene expression and proteins in HCT-116 human colon adenocarcinoma cells. We used a unique cDNA microarray (GeneMap TM Cancerarray) comprising 1152 human tumor-related genes and 2-D gel electrophoresis, respectively, following a 24-hour exposure to a drug concentration that killed a two-log fraction of HCT-116 clonogenic cells. Functional gene cluster profile (FGCP) analysis of the 71 out of 1152 genes that displayed a Ͼ2-fold increase or decrease in expression (over untreated control) identified a drug-specific involvement of the MAPK signal transduction pathway. MAPK signaling together with the involvement of ubiquitin proteins from 2-D gel electrophoresis suggest a novel drug-action profile at the molecular level for the in vitro antiproliferative activity of XK469. Cytometry 47: 72-79, 2002.
XK469 is an investigational anticancer agent that exhibits antiproliferative activity in tumor-bearing animal models. We examined the drug-action profile of this agent at the molecular level regarding alterations induced in gene expression and proteins in HCT-116 human colon adenocarcinoma cells. We used a unique cDNA microarray (GeneMap(TM) Cancerarray) comprising 1152 human tumor-related genes and 2-D gel electrophoresis, respectively, following a 24-hour exposure to a drug concentration that killed a two-log fraction of HCT-116 clonogenic cells. Functional gene cluster profile (FGCP) analysis of the 71 out of 1152 genes that displayed a >2-fold increase or decrease in expression (over untreated control) identified a drug-specific involvement of the MAPK signal transduction pathway. MAPK signaling together with the involvement of ubiquitin proteins from 2-D gel electrophoresis suggest a novel drug-action profile at the molecular level for the in vitro antiproliferative activity of XK469.
DNA microarray technology is one of the most important recent breakthroughs in experimental molecular biology. With evermore laboratories acquiring this highthroughput technology, the amounts of data being generated are growing extremely rapidly, and the informatics necessary for handling and analysing these data is becoming a major bottleneck. The EBI is working on the development of applications to promote and further the development of the informatics and analysis of microarray data and that is integrated with other biological resources in order to better understand the results of gene expression experiments. We are exploring the development of new techniques as well as technology transfer from marketing and telecommunications domains, e.g. application of visualisation, data mining and statistical analysis. Currently, the EBI is assessing the suitability of different data mining algorithms to microarray gene expression data, e.g. neural networks, classification trees, market basket analysis, clustering, classification, etc. As well developing Internet tools that will allow users to browse and query microarray data stored in a database, the EBI is investigating techniques and technologies that will allow direct access to the microarray information in a database over computer networks, for example CORBA and JDBC. This technology will allow developers at other sites to develop and write their own novel software tools that can perform queries and analyses on microarray data that is pulled over the Internet from the EBI microarray database.It is now becoming generally recognized that microarray technology will be a fundamental tool used in future genomics research. As the technology becomes more widely accessible, larger numbers of biologists will be able to shift their focus from the study of individual events to the analysis of complex systems and pathways. To help drive this transition, we have used novel tools for creating and reading highdensity microarrays of spotted DNAs to investigate whether conventional chemistries (for example, Southern, northern and western blots) might be applied to microarray analysis. It had previously been assumed that these well-documented methodologies could not be used for microarray analysis because: (i) spotting instruments could not produce arrays on membranes such as nitrocellulose or nylon; and (ii) these membranes were thought to produce levels of fluorescence which would make analysis impossible. We will show data developed using a Pin-and-Ring TM spotting system (the GMS 417 Arrayer) and an epi-fluorescent confocal laser microscope that employs Flying Objective TM scanning technology (the GMS 418 Array Scanner), demonstrating that conventional chemistries can be used for microarray analysis. We believe that demonstration of the feasibility of this approach will facilitate the migration of more biologists toward the use of microarray technology, as they will now be able to use commercially available instrumentation and familiar methodologies for highly parallel genomic ana...
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