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

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Query: EC:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Wheat-germ extract for cell-free protein synthesis was condensed with ultrafiltration membranes of which the molecular cut-off values were 10 kDa, 100 kDa, and 300 kDa. Reaction conditions of the cell-free system were optimized for the condensed extracts, which needed a higher concentration of creatine phosphate than the uncondensed one, probably due to the increased activity of degradation of ATP and GTP. By using the condensed extract and optimized reaction conditions, the rate of protein synthesis was increased 2- to 3-fold compared with using an uncondensed extract, and about 10-fold compared with conventional conditions. Condensation of the extract with the 300-kDa membrane showed the highest productivity, which was about 30 micrograms dihydrofolate reductase protein ml-1 h-1. The final amount of synthesized protein was one third of that of a continuous-flow cell-free (CFCF) system reported by Endo et al. [J. Biotechnol., 25, 221-230 (1992)] but the productivity was 5-fold higher than that obtained by the CFCF system.
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PMID:An increased rate of cell-free protein synthesis by condensing wheat-germ extract with ultrafiltration membranes. 776 55

Reaction conditions of cell-free protein synthesis using wheat germ extract were examined to prolong the period of protein synthesis in a batch reaction. By optimizing conditions for ATP regeneration system involved in the cell-free system, protein synthesis continued about 4 hours, so that about 17 micrograms dihydrofolate reductase protein was obtained in 1 ml of a reaction mixture. It suggests that maintaining ATP concentration is the primary requirement for long-life cell-free protein synthesis.
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PMID:Prolonged cell-free protein synthesis in a batch system using wheat germ extract. 776 17

Protein import into yeast mitochondria is mediated by the four outer membrane receptors Mas70p, Mas37p, Mas20p, and Mas22p. These receptors may function as two subcomplexes: a Mas37p/Mas70p heterodimer and an acidic complex consisting of Mas20p and Mas22p. To assess the relative contribution of these subcomplexes to precursor binding, we allowed different precursors to bind to the surface of deenergized mitochondria, then reenergized the mitochondria and measured the chase of the bound precursors into the organelles. Productive binding of several precursors with a positively charged amino-terminal matrix targeting sequence, such as SU9-DHFR, hsp60, and mitochondrial cpn10, was strongly inhibited by salt, by low concentrations of a mitochondrial presequence peptide, and by a deletion of Mas20p, but was independent of Mas37p/Mas70p. In contrast, productive binding of the ADP/ATP carrier was not inhibited by salt, the presequence peptide, or a deletion of Mas20p, but was strongly dependent on Mas37p/Mas70p. The precursors of alcohol dehydrogenase III and the Rieske iron-sulfur protein had binding properties between these two extremes. The productively bound precursor of cpn10 could be cross-linked to Mas20p. We conclude that Mas20p binds mitochondrial precursor proteins through electrostatic interactions with the positively charged presequence, whereas Mas37p/Mas70p may recognize some feature(s) of the mature part of precursor proteins.
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PMID:The yeast mitochondrial protein import receptor Mas20p binds precursor proteins through electrostatic interaction with the positively charged presequence. 789 Jun 75

Symbionin, a GroEL homologous molecular chaperone produced by an intracellular symbiont of the pea aphid, is autocatalytically phosphorylated in vitro at elevated temperatures. The phosphorylated symbionin showed a potent suppressive activity in spontaneous refolding of chemically denatured dihydrofolate reductase. When the 32P-labeled autophosphorylated symbionin was incubated with ADP, a portion of the radioactivity was transferred to ADP, suggesting that the autocatalytically phosphorylated symbionin contains high-energy phosphate bonds. It was also shown that when symbionin was incubated with [gamma-32P]ATP and GDP, a large amount of the radioactivity was found in GTP, indicating that phosphate transfer between ATP and GDP is catalyzed by symbionin. These results suggested that in the endosymbiotic system symbionin functions as not only a molecular chaperone but also an energy-coupling protein.
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PMID:Chaperonin produced by an intracellular symbiont is an energy-coupling protein with phosphotransferase activity. 790 96

The mouse mdr gene family is composed of three members designated mdr1, mdr2, and mdr3. A full-length mdr2 complementary DNA clone has been introduced in an amplifiable eukaryotic expression vector (pEMC2b1) which directs amplification and overexpression of a bicistronic mdr2-dihydrofolate reductase mRNA after stepwise methotrexate selection of transfected mutant dihydrofolate reductase Chinese hamster ovary DUK cells. Independent cell clones expressing low to high amounts of mdr2 cellular mRNA and Mdr2 protein in their membrane fraction could be obtained by this selection procedure. Comparison of drug survival characteristics of cell clones expressing similar amounts of either Mdr1 or Mdr2 proteins revealed that Mdr1 but not Mdr2 could confer readily detectable levels of colchicine or vinblastine resistance. Labeling experiments using membrane-enriched fractions and a photoactivatable analogue of ATP showed that the Mdr2 protein was properly inserted in the membrane of transfected cells and could bind this ligand with an apparent affinity similar to that of Mdr1. However, labeling studies with the photoactivatable drug analogue iodoarylazidoprazosin showed considerably reduced binding of this ligand to Mdr2 as compared to Mdr1. Our findings demonstrate that Mdr2 cannot confer drug resistance and suggest that this inability is linked to reduced drug binding to the Mdr2 protein.
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PMID:The inability of the mouse mdr2 gene to confer multidrug resistance is linked to reduced drug binding to the protein. 791 94

Chaperonins are oligomeric protein complexes that play an essential role in the cell, mediating ATP-dependent polypeptide chain folding in a variety of cellular compartments. They appear to bind early folding intermediates, preventing their aggregation; in the presence of MgATP and a cochaperonin, bound polypeptides are released in a stepwise manner, associated with folding to the native state. Chaperonin complexes appear in the electron microscope as cylindrical structures, usually composed of two stacked rings, each containing, by negative staining, an electron dense central "hole" approximately 6.0 nm in diameter. We sought to identify the site on the Escherichia coli chaperonin groEL, where the "molten globule"-like intermediate of dihydrofolate reductase (DHFR) becomes bound, by examining in the scanning transmission electron microscope complexes formed between groEL and DHFR molecules bearing covalently crosslinked 1.4-nm gold clusters. In top views of the groEL complexes, gold densities were observed in the central region; in side views, the densities were seen at the end portions of the cylinders, corresponding to positions within the individual rings. In some cases, two gold densities were observed in the same groEL complex. We conclude that folding intermediates are bound inside central cavities within individual chaperonin rings. In this potentially sequestered location, folding intermediates with a compact conformation can be bound at multiple sites by surrounding monomeric members of the ring; localization of folding within the cavity could also facilitate rebinding of structures that initially fail to incorporate properly into the folding protein.
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PMID:A polypeptide bound by the chaperonin groEL is localized within a central cavity. 809 82

The role of ATP in the matrix for the import of precursor proteins into the various mitochondrial subcompartments was investigated by studying protein translocation at experimentally defined ATP levels. Proteins targeted to the matrix were neither imported or processed when matrix ATP was depleted. Import and processing of precytochrome b2 (pb2), a precursor carrying a bipartite presequence, into the intermembrane space was also strongly dependent on matrix ATP. Preproteins, consisting of 220 or more residues of pb2 fused to dihydrofolate reductase, showed the same requirement for matrix ATP, whereas the import of shorter fusion proteins (up to 167 residues of pb2) was largely independent of matrix ATP. For those intermembrane-space-targeted proteins that did need matrix ATP, the dependence could be relieved either by unfolding these proteins prior to import or by introducing a deletion into the mature portion of the protein thereby impairing the tight folding of the cytochrome b2 domain. These results suggest the following: (a) The import of matrix-targeted preproteins, in addition to a membrane potential delta psi, requires matrix ATP [most likely to facilitate reversible binding of mitochondrial heat-shock protein 70 (mt-Hsp70) to incoming precursors], for two steps, securing the presequence on the matrix side of the inner membrane and for the completion of translocation; (b) in the case of intermembrane-space-targeted precursors with bipartite signals, the function of ATP/mt-Hsp70 is not obligatory, as components of the intermembrane-space-sorting pathway may substitute for ATP/mt-Hsp70 function (however, if a tightly folded domain is present in the precursor, ATP/mt-Hsp70 is indispensable); (c) unfolding on the mitochondrial surface of tightly folded segments of preproteins is facilitated by matrix-ATP/mt-Hsp70.
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PMID:The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space. 811 2

The presecretory protein ppcecDHFR, a hybrid between preprocecropin A and dihydrofolate reductase, is transported into mammalian microsomes post-translationally, i.e. independently of ribosome and signal recognition particle. Upon staging the transport process, stably folded ppcecDHFR bound to mammalian microsomes and subsequently translocated across the membrane. Membrane association depended on the signal peptide but involved neither ATP nor an N-ethylmaleimide-sensitive microsomal protein. Membrane insertion of bound ppcecDHFR did not necessitate unfolding of the DHFR domain but depended on ATP and an N-ethylmaleimide-sensitive microsomal protein. Completion of translocation relied on unfolding of the DHFR domain. Thus mammalian microsomes have the capability of transporting a bound and folded precursor protein, i.e. to trigger unfolding of a precursor protein on the membrane surface.
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PMID:A stably folded presecretory protein associates with and upon unfolding translocates across the membrane of mammalian microsomes. 811 98

We report the sequence of an 18,002 bp DNA fragment from the right arm of Saccharomyces cerevisiae chromosome XI. This segment contains nine complete open reading frames (ORFs), YKR401 to YKR409, and part of another ORF, YKR400, covering altogether 87.2% of the entire sequence. One of them, YKR400, encodes an NAD-dependent 5,10-methylene-tetrahydrofolate dehydrogenase. YKR404, YKR405 and YKR406 correspond to the previously characterized HBS1, MRP-L20 and PRP16 genes, coding for a translation elongation factor, a mitochondrial ribosomal protein and an ATP-binding protein, respectively. The putative product of YKR407 contains the zinc-binding region signature of neutral zinc metallopeptidases. The five other ORFs do not show significant homology to any known protein.
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PMID:The complete sequence of an 18,002 bp segment of Saccharomyces cerevisiae chromosome XI contains the HBS1, MRP-L20 and PRP16 genes, and six new open reading frames. 820 64

We have performed experiments which demonstrate that puromycin inhibits the import of proteins into mitochondria in in vitro reactions containing mitochondria isolated from the yeast Saccharomyces cerevisiae and precursor proteins synthesized in a nuclease-treated rabbit reticulocyte lysate. Puromycin inhibited the import of several precursor proteins including; a fusion protein consisting of the first 22 N-terminal residues of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase, both a destabilized and truncated form of this same fusion protein, the beta-subunit of the yeast mitochondrial F1-ATPase and yeast alcohol dehydrogenase III. The insertion of the yeast outer mitochondrial protein porin was not inhibited by puromycin. Puromycin-induced import inhibition could be overcome by adding additional ATP to the import reactions. However, if access of ATP to the mitochondrial matrix was prevented by blocking the adenine nucleotide translocase with carboxyatractyloside, ATP addition was unable to overcome the inhibitory effect of puromycin on protein import. Collectively, these results demonstrate that puromycin inhibits protein import into mitochondria by interfering with an ATP-dependent step in the import process and that the ATP-dependent component in the reaction is located inside the inner mitochondrial membrane. In addition to supporting the view that ATP is required in the matrix for efficient protein import, these results may provide a useful tool for identifying the ATP-binding components of the import apparatus.
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PMID:Puromycin inhibits protein import into mitochondria by interfering with an intramitochondrial ATP-dependent reaction. 833 41


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