Potent, Orally Active, Competitive N-Methyl-D-aspartate (NMDA) Receptor Antagonists Are Substrates for a Neutral Amino Acid Uptake System in Chinese Hamster Ovary Cells

1995 ◽  
Vol 38 (11) ◽  
pp. 1955-1965 ◽  
Author(s):  
Jia-He Li ◽  
Christopher F. Bigge ◽  
Rufus M. Williamson ◽  
Susan A. Borosky ◽  
Mark G. Vartanian ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nmda Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Nmda Receptor Antagonists ◽  
Uptake System ◽  
Ovary Cells ◽  
Orally Active

scholarly journals Substrate protection against inactivation of the mammalian polyamine-transport system by 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide

1996 ◽  
Vol 319 (1) ◽  
pp. 21-26 ◽  
Author(s):  
Krikor TOROSSIAN ◽  
Marie AUDETTE ◽  
Richard POULIN
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Dependent Manner ◽  
Temperature Dependent ◽  
Concentration Dependent Manner ◽  
Ovary Cells ◽  
Polyamine Transport ◽  
Substrate Protection

Mammalian polyamine transporters have not thus far been biochemically characterized. Since essential carboxy groups in the polyamine carrier might participate in the transport process, the ability of two different carbodi-imides to affect [3H]spermidine uptake was assessed in Chinese hamster ovary cells. Both the hydrophobic 1,3-dicyclohexylcarbodi-imide (DCC) and the more polar 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (EDC) irreversibly inhibited spermidine transport with EC50 values of 11±4 and 96±16 µM after 30 min at 22 °C respectively. Prior treatment with EDC in the absence of substrate decreased both the Vmax and Km for spermidine uptake in a time- and concentration-dependent manner. Spermidine-transport inactivation by EDC (1 mM) was temperature-dependent, with 60 and 90% inhibition observed after 10 min at 22 and 37 °C respectively. Spermine (10 µM) almost fully protected against spermidine-transport inactivation by EDC at 22 °C, and decreased the rate of inactivation at 37 °C by about 80%. Putrescine, spermidine and spermine were all effective in protecting against EDC-mediated inactivation of [3H]spermidine and [3H]putrescine uptake at 22 °C with EC50 values estimated at 10, 1 and less than 1 µM respectively. The nucleophile glycine ethyl ester (up to 50 mM) prevented the inhibition brought about by 1 mM EDC. Inhibition by 1 mM EDC was greater at pH 7.2 than at pH 5.8 (89±3 compared with 44±5%), whereas the converse was true for 100 µM DCC (81±3 compared with 92±5%). On the other hand, spermine did not protect against inactivation of spermidine uptake by DCC. Moreover, DCC, but not EDC, inhibited Na+-dependent amino acid uptake. The present data indicate that (i) EDC and DCC inhibit polyamine transport through distinct mechanisms, (ii) substrate binding occludes one or several carboxy groups lying in a polar environment of the carrier and (iii) these carboxyl residues might be activated by EDC to cross-link a neighbouring nucleophile side group, resulting in a conformation of the polyamine carrier which is inactive for transport.

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells

1986 ◽  
Vol 6 (6) ◽  
pp. 1926-1935
Author(s):  
P J Mitchell ◽  
G Urlaub ◽  
L Chasin
Keyword(s):  
Amino Acid ◽  
Dihydrofolate Reductase ◽  
Splice Site ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Splice Sites ◽  
Ovary Cells ◽  
Splicing Mutations ◽  
Intron 4

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.

scholarly journals Molecular Cloning of a Functional Allatostatin Gut/Brain Receptor and an Allatostatin Preprohormone from the SilkwormBombyx mori

2001 ◽  
Vol 276 (50) ◽  
pp. 47052-47060 ◽  
Author(s):  
Thomas Secher ◽  
Camilla Lenz ◽  
Giuseppe Cazzamali ◽  
Gunnar Sørensen ◽  
Michael Williamson ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Cloning ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Peptide Hormone ◽  
Functional Expression ◽  
Bar Gene ◽  
Ovary Cells ◽  
Intron Phasing ◽  
Insect Gut

The cockroach-type or A-type allatostatins are inhibitory insect neuropeptides with the C-terminal sequence Tyr/Phe-X-Phe-Gly-Leu-NH2. Here, we have cloned an A-type allatostatin receptor from the silkwormBombyx mori(BAR). BAR is 361 amino acid residues long, has seven transmembrane domains, shows 60% amino acid residue identity with the firstDrosophilaallatostatin receptor (DAR-1), and 48% identity with the secondDrosophilaallatostatin receptor (DAR-2). The BAR gene has two introns and three exons. These two introns coincide with and have the same intron phasing as two introns in the DAR-1 and DAR-2 genes, showing that the three receptors are not only structurally but also evolutionarily related. Furthermore, we have cloned aBombyxallatostatin preprohormone that contains eight different A-type allatostatins. Chinese hamster ovary cells permanently transfected with BAR DNA react on the addition of 4 × 10−9mBombyxA-type allatostatins with a second messenger cascade (measured as bioluminescence), showing that BAR is a functional A-type allatostatin receptor. Southern blots suggest thatBombyxhas at least one other BAR-related gene in addition to the BAR gene described in this paper. Northern blots and quantitative reverse transcriptase-polymerase chain reaction of different larval tissues show that BAR mRNA is mainly expressed in the gut and to a much lesser extent in the brain. To our knowledge, this is the first report on the molecular cloning and functional expression of an insect gut/brain peptide hormone receptor.

scholarly journals Human PIG-U and Yeast Cdc91p Are the Fifth Subunit of GPI Transamidase That Attaches GPI-Anchors to Proteins

2003 ◽  
Vol 14 (5) ◽  
pp. 1780-1789 ◽  
Author(s):  
Yeongjin Hong ◽  
Kazuhito Ohishi ◽  
Ji Young Kang ◽  
Satoshi Tanaka ◽  
Norimitsu Inoue ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Identity ◽  
Complementation Group ◽  
Long Chain Fatty Acid ◽  
Ovary Cells ◽  
Eukaryotic Proteins ◽  
Gpi Transamidase

Many eukaryotic proteins are anchored to the cell surface via glycosylphosphatidylinositol (GPI), which is posttranslationally attached to the carboxyl-terminus by GPI transamidase. The mammalian GPI transamidase is a complex of at least four subunits, GPI8, GAA1, PIG-S, and PIG-T. Here, we report Chinese hamster ovary cells representing a new complementation group of GPI-anchored protein-deficient mutants, class U. The class U cells accumulated mature and immature GPI and did not have in vitro GPI transamidase activity. We cloned the gene responsible, termed PIG-U, that encoded a 435-amino-acid hydrophobic protein. The GPI transamidase complex affinity-purified from cells expressing epitope-tagged-GPI8 contained PIG-U and four other known components. Cells lacking PIG-U formed complexes of the four other components normally but had no ability to cleave the GPI attachment signal peptide. Saccharomyces cerevisiae Cdc91p, with 28% amino acid identity to PIG-U, partially restored GPI-anchored proteins on the surface of class U cells. PIG-U and Cdc91p have a functionally important short region with similarity to a region conserved in long-chain fatty acid elongases. Taken together, PIG-U and the yeast orthologue Cdc91p are the fifth component of GPI transamidase that may be involved in the recognition of either the GPI attachment signal or the lipid portion of GPI.

scholarly journals cDNA cloning and characterization of guinea-pig leukotriene B4 receptor

1999 ◽  
Vol 342 (1) ◽  
pp. 79-85 ◽  
Author(s):  
Kazuyuki MASUDA ◽  
Takehiko YOKOMIZO ◽  
Takashi IZUMI ◽  
Takao SHIMIZU
Keyword(s):  
Amino Acid ◽  
Guinea Pig ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Leukotriene B4 ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Residues ◽  
Ovary Cells ◽  
Hek 293 ◽  
Leukotriene B4 Receptor

The cDNA for leukotriene B4 (LTB4) receptor (BLT) was cloned from a guinea-pig leucocyte cDNA library. The cloned receptor cDNA encodes 348 amino acid residues and shares 73% identity with the amino acid sequence of human BLT. Northern blot analysis showed the highest expression of the receptor mRNA in leucocytes, followed by lung and spleen. The membrane fractions of HEK-293 and Cos-7 cells transfected with the cDNA showed specific LTB4-binding activities, with Kd values of 0.27 and 0.17 nM respectively. Xenopus laevis oocytes injected with the cRNA of guinea-pig BLT showed LTB4-induced Cl- currents, indicating that the cloned receptor is functional. LTB4 is metabolized to 20-hydroxy-LTB4 and then to 20-carboxy-LTB4, a transformation considered as a major inactivation pathway of the compound. Using the cloned receptor, we analysed the agonistic effects of LTB4 and these two metabolites. 20-Carboxy-LTB4 is a much weaker agonist, with a Kd value higher than that of LTB4 by three orders of magnitude, corresponding to a much weaker chemotactic activity. Although 20-hydroxy-LTB4 is as potent as LTB4 in inhibiting [3H]LTB4 binding and cAMP formation, it is less potent than LTB4 in the mobilization of intracellular Ca2+ and the chemotaxis of Chinese hamster ovary cells expressing the guinea-pig BLT. The present study demonstrated that although LTB4 and 20-hydroxy-LTB4 bind to the receptor with similar affinities, they do differ in activating intracellular signalling.

AMINO ACID CHANGES AT LYS-158 THAT ALTER THE SENSITIVITY OF SCUPA TO CLEAVAGE BY PLASMIN

1987 ◽  
Author(s):  
G F VOVIS ◽  
J MAO ◽  
R BROEZE ◽  
D ABERCROMBIE ◽  
P PUMA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fibrinolytic Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Position ◽  
Peptide Bond ◽  
Site Directed Mutagenesis ◽  
Chromogenic Substrate ◽  
Amidolytic Activity ◽  
Ovary Cells

Plasmin converts Scu-PA to two-chain urokinase by hydrolyzing the lys-158/ile-159 peptide bond. Using site directed mutagenesis, the codon at amino acid position 158 was changed from one that codes for lysine to one that codes for either alanine, glutamic acid, or methionine. These DNA constructions were expressed and amplified in Chinese hamster ovary cells. The resulting protein products were isolated and characterized ip vitro. Under conditions where Scu-PA is completely converted by plasmin to two chain urokinase, none of these derivatives were cleaved by plasmin. However, all of these molecules, including Scu-PA, were cleaved by thrombin. These derivatives exhibited low amidolytic activity as determined by using the chromogenic substrate S2444 and low fibrinolytic activity as determined by using fibrin plates. All three derivatives activated either glu-plasminogen or lys-plasminogen to plasmin ip vitro but at rates significantly slower than that seen with Scu-PA.

Cytoplasmic domains of GpIbα and GpIbβ regulate 14-3-3ζ binding to GpIb/IX/V

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 551-557 ◽  
Author(s):  
Shuju Feng ◽  
Nicolaos Christodoulides ◽  
Julio C. Reséndiz ◽  
Michael C. Berndt ◽  
Michael H. Kroll
Keyword(s):  
Shear Stress ◽  
Amino Acid ◽  
Fusion Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Structural Determinants ◽  
Ovary Cells ◽  
Induced Aggregation

Shear stress causes the platelet glycoprotein (Gp) Ib/IX/V to bind to von Willebrand factor, resulting in platelet adhesion. GpIb/IX/V also functions to stimulate transmembranous signaling, leading to platelet activation and the expression of a ligand-receptive GpIIb-IIIa complex. The highly conserved cytoplasmic domain of GpIb binds directly to a dimeric 14-3-3 adapter protein ζ isoform. To explore structural determinants of GpIb/IX/V binding to 14-3-3ζ, the authors examined 14-3-3ζ interactions with GpIb and GpIbβ in heterologous cells and platelets. Truncations of GpIb at amino acid 542 or 594, or deletions of residues 542 through 590, inhibited binding of 14-3-3ζ. Deletion of GpIb from Trp570 to Ser590 eliminated 14-3-3ζ binding, and deletion of the sequence from Arg542-Trp570 enhanced binding of 14-3-3ζ to GpIb. All GpIb mutations that eliminated GpIb binding to the GST-14-3-3ζ fusion protein also eliminated GpIbβ binding to the fusion protein. Forskolin treatment of Chinese hamster ovary cells expressing wild-type GpIb/β/IX resulted in the phosphorylation of GpIbβ associated with enhanced binding of GpIbβ to GST-14-3-3ζ fusion protein and increased 14-3-3ζ coimmunoprecipitated with GpIb. When intact human platelets aggregated in response to 90 dynes/cm2 shear stress, 14-3-3ζ disassociated from GpIb. Prostacyclin treatment of platelets inhibited shear stress-induced aggregation and the release of 14-3-3ζ from GpIb. These data demonstrate that amino acid residues in the cytoskeletal interaction domains of GpIb regulate 14-3-3ζ binding to GpIb/β/IX, and suggest that protein kinase A-dependent phosphorylation of GpIbβ enhances 14-3-3ζ binding to the GpIb/IX/V complex in human platelets.

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