Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.
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PMID:Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit. 144 27

Our previous studies have shown that the DAN gene product possesses an ability to revert phenotypes of transformed rat fibroblasts and represents a candidate tumour suppressor gene for neuroblastoma. In the present study, characterisation of DAN was carried out using rat fibroblast 3Y1 cells and their DAN-overexpressor counterparts (S-9). The N-terminal region of DAN (amino acids 1-24) was highly hydrophobic and DAN protein was found to be secreted into the culture medium. When DAN was treated with PNGase F, a enzyme that cleaves most N-linked carbohydrate residues, the mobility of both cytoplasmic and secreted DAN was increased in SDS-polyacrylamide gel electrophoresis, suggesting DAN is N-glycosylated, irrespective of its localisation. When partially purified, DAN was able, when added to the culture, to suppress DNA synthesis of Rous sarcoma virus-transformed 3Y1 cells, which lack the expression of DAN.
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PMID:A product of DAN, a novel candidate tumour suppressor gene, is secreted into culture medium and suppresses DNA synthesis. 951 39