Transfection of Na,K-ATPase α-subunit: regulation of enzyme abundance

1995 ◽  
Vol 73 (5-6) ◽  
pp. 261-268 ◽  
Author(s):  
Nabila M. Shanbaky ◽  
Thomas A. Pressley
Keyword(s):  
Gene Transfer ◽  
Mammalian Cells ◽  
Specific Activity ◽  
Partial Replacement ◽  
Protein Abundance ◽  
Kidney Cells ◽  
Dna Encoding ◽  
Α Subunit ◽  
Rapid Degradation ◽  
Transfected Cells

DNA-mediated gene transfer into mammalian cells was used as a model for investigating the regulation of Na,K-ATPase abundance. Complementary DNA encoding the catalytic α1-subunit from rat was introduced into ouabain-sensitive monkey kidney cells, and transfectants were selected by their ability to survive in normally cytotoxic concentrations of ouabain. The overall specific activity of Na,K-ATPase in the membranes of transfectants was not significantly different from that in control cells, suggesting that there was a partial replacement, rather than an addition, of introduced α1 for the endogenous subunit in the functional enzyme. Immunoblotting with specific antibodies confirmed the similarities in overall α abundance between control and transfected cells. Hybridization analysis of total RNA, however, revealed a higher abundance of the mRNA encoding total α1 in transfected cells. The results suggest that endogenous and introduced α-subunit compete for a limited amount of β, with rapid degradation of unassembled subunits.Key words: DNA-mediated gene transfer, immunoblots, protein abundance, subunit assembly.

scholarly journals Control of the CDPethanolamine pathway in mammalian cells: effect of CTP:phosphoethanolamine cytidylyltransferase overexpression and the amount of intracellular diacylglycerol

2004 ◽  
Vol 379 (3) ◽  
pp. 711-719 ◽  
Author(s):  
Onno B. BLEIJERVELD ◽  
Wil KLEIN ◽  
Arie B. VAANDRAGER ◽  
J. Bernd HELMS ◽  
Martin HOUWELING
Keyword(s):  
Phospholipase C ◽  
Rat Liver ◽  
Mammalian Cells ◽  
Specific Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Ovary Cells ◽  
Transfected Cells ◽  
And Control

For an insight regarding the control of PtdEtn (phosphatidylethanolamine) synthesis via the CDPethanolamine pathway, rat liver cDNA encoding ECT (CTP:phosphoethanolamine cytidylyltransferase) was transiently or stably transfected in Chinese-hamster ovary cells and a rat liver-derived cell line (McA-RH7777), resulting in a maximum of 26- and 4-fold increase in specific activity of ECT respectively. However, no effect of ECT overexpression on the rate of [3H]ethanolamine incorporation into PtdEtn was detected in both cell lines. This was explored further in cells overexpressing four times ECT activity (McA-ECT1). The rate of PtdEtn breakdown and PtdEtn mass were not changed in McA-ECT1 cells in comparison with control-transfected cells. Instead, an accumulation of CDPethanolamine (label and mass) was observed, suggesting that in McA-ECT1 cells the ethanolaminephosphotransferase-catalysed reaction became rate-limiting. However, overexpression of the human choline/ethanolaminephosphotransferase in McA-ECT1 and control-transfected cells had no effect on PtdEtn synthesis. To investigate whether the availability of DAG (diacylglycerol) limited PtdEtn synthesis in these cells, intracellular DAG levels were increased using PMA or phospholipase C. Exposure of cells to PMA or phospholipase C stimulated PtdEtn synthesis and this effect was much more pronounced in McA-ECT1 than in control-transfected cells. In line with this, the DAG produced after PMA exposure was consumed more rapidly in McA-ECT1 cells and the CDPethanolamine level decreased accordingly. In conclusion, our results suggest that the supply of CDPethanolamine, via the expression level of ECT, is an important factor governing the rate of PtdEtn biosynthesis in mammalian cells, under the condition that the amount of DAG is not limiting.

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

scholarly journals A protocol for rapid degradation of endogenous transcription factors in mammalian cells and identification of direct regulatory targets

2021 ◽  
Vol 2 (2) ◽  
pp. 100530
Author(s):  
Hillary M. Layden ◽  
Nicholas A. Eleuteri ◽  
Scott W. Hiebert ◽  
Kristy R. Stengel
Keyword(s):  
Transcription Factors ◽  
Mammalian Cells ◽  
Rapid Degradation

Analysis by mRNA levels of the expression of six G protein α-subunit genes in mammalian cells and tissues

1991 ◽  
Vol 1094 (2) ◽  
pp. 193-199 ◽  
Author(s):  
Jermelina Linor R. Garibay ◽  
Tohru Kozasa ◽  
Hiroshi Itoh ◽  
Toshihiko Tsukamoto ◽  
Masaaki Matsuoka ◽  
...  
Keyword(s):  
G Protein ◽  
Mammalian Cells ◽  
Mrna Levels ◽  
Α Subunit ◽  
G Protein Α Subunit

scholarly journals Replication Past the -Radiation-Induced Guanine-Thymine Cross-Link G[8,5-Me]T by Human and Yeast DNA Polymerase

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Paromita Raychaudhury ◽  
Ashis K. Basu
Keyword(s):  
Dna Polymerase ◽  
Mammalian Cells ◽  
Translesion Synthesis ◽  
Kidney Cells ◽  
Equal Frequency ◽  
Cross Link ◽  
Dominant Mutation ◽  
Human Embryonic Kidney Cells ◽  
Radiation Induced

-Radiation-induced intrastrand guanine-thymine cross-link, G[8,5-Me]T, hinders replicationin vitroand is mutagenic in mammalian cells. Herein we reportin vitrotranslesion synthesis of G[8,5-Me]T by human and yeast DNA polymerase (hPol and yPol ). dAMP misincorporation opposite the cross-linked G by yPol was preferred over correct incorporation of dCMP, but further extension was 100-fold less efficient for :A compared to :C. For hPol , both incorporation and extension were more efficient with the correct nucleotides. To evaluate translesion synthesis in the presence of all four dNTPs, we have developed a plasmid-based DNA sequencing assay, which showed that yPol was more error-prone. Mutational frequencies of yPol and hPol were 36% and 14%, respectively. Targeted was the dominant mutation by both DNA polymerases. But yPol induced targeted in 23% frequency relative to 4% by hPol . For yPol , targeted and constituted 83% of the mutations. By contrast, with hPol , semi-targeted mutations (7.2%), that is, mutations at bases near the lesion, occurred at equal frequency as the targeted mutations (6.9%). The kind of mutations detected with hPol showed significant similarities with the mutational spectrum of G[8,5-Me]T in human embryonic kidney cells.

scholarly journals Placental Lactogen-I (PL-I) Target Tissues Identified with an Alkaline Phosphatase-PL-I Fusion Protein

1998 ◽  
Vol 46 (6) ◽  
pp. 737-743 ◽  
Author(s):  
Heiner Müller ◽  
Guoli Dai ◽  
Michael J. Soares
Keyword(s):  
Alkaline Phosphatase ◽  
Fusion Protein ◽  
Colorimetric Assay ◽  
Biological Activities ◽  
Placental Lactogen ◽  
Kidney Cells ◽  
Fetal Kidney ◽  
Transfected Cells ◽  
Tissue Sections ◽  
Labeling System

The rat placenta expresses a family of genes related to prolactin (PRL). Target tissues and physiological roles for many members of the PRL family have yet to be determined. In this investigation we evaluated the use of an alkaline phosphatase (AP) tag for monitoring the behavior of a prototypical member of the PRL family, placental lactogen-I (PL-I). A probe was generated consisting of a fusion protein of human placental AP and rat PL-I (AP-PL-I). The AP-PL-I construct was stably expressed in 293 human fetal kidney cells, as was the unmodified AP vector that served as a control. AP activity was monitored with a colorimetric assay in conditioned medium from transfected cells. Immunoreactivity and PRL-like biological activities of the AP-PL-I fusion protein were demonstrated by immunoblotting and the Nb2 lymphoma cell proliferation assay, respectively. AP-PL-I specifically bound to tissue sections known to express the PRL receptor, including the ovary, liver, and choroid plexus. Binding of AP-PL-I to tissues was specific and could be competed with ovine PRL. The results indicate that AP is an effective tag for monitoring the behavior of PL-I and suggest that this labeling system may also be useful for monitoring the actions of other members of the PRL family.

scholarly journals Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259837
Author(s):  
Zora Novakova ◽  
Daria Khuntsaria ◽  
Marketa Gresova ◽  
Jana Mikesova ◽  
Barbora Havlinova ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Mammalian Cells ◽  
Specific Activity ◽  
Eukaryotic Cells ◽  
Post Translational Modifications ◽  
Protoporphyrinogen Oxidase ◽  
Intracellular Targeting ◽  
Alternative Systems ◽  
Protein Functions ◽  
The Impact

Human protoporphyrinogen oxidase IX (hPPO) is an oxygen-dependent enzyme catalyzing the penultimate step in the heme biosynthesis pathway. Mutations in the enzyme are linked to variegate porphyria, an autosomal dominant metabolic disease. Here we investigated eukaryotic cells as alternative systems for heterologous expression of hPPO, as the use of a traditional bacterial-based system failed to produce several clinically relevant hPPO variants. Using bacterially-produced hPPO, we first analyzed the impact of N-terminal tags and various detergent on hPPO yield, and specific activity. Next, the established protocol was used to compare hPPO constructs heterologously expressed in mammalian HEK293T17 and insect Hi5 cells with prokaryotic overexpression. By attaching various fusion partners at the N- and C-termini of hPPO we also evaluated the influence of the size and positioning of fusion partners on expression levels, specific activity, and intracellular targeting of hPPO fusions in mammalian cells. Overall, our results suggest that while enzymatically active hPPO can be heterologously produced in eukaryotic systems, the limited availability of the intracellular FAD co-factor likely negatively influences yields of a correctly folded protein making thus the E.coli a system of choice for recombinant hPPO overproduction. At the same time, PPO overexpression in eukaryotic cells might be preferrable in cases when the effects of post-translational modifications (absent in bacteria) on target protein functions are studied.

scholarly journals GCN2 phosphorylation of eIF2α activates NF-κB in response to UV irradiation

2005 ◽  
Vol 385 (2) ◽  
pp. 371-380 ◽  
Author(s):  
Hao-Yuan JIANG ◽  
Ronald C. WEK
Keyword(s):  
Uv Irradiation ◽  
Mammalian Cells ◽  
Transcriptional Control ◽  
Control Cell ◽  
Nuclear Factor Κb ◽  
Initiation Factor ◽  
Α Subunit ◽  
Eif2α Phosphorylation ◽  
Eif2α Kinase ◽  
Secondary Function

In response to UV irradiation, mammalian cells elicit a gene expression programme designed to repair damage and control cell proliferation and apoptosis. Important members of this stress response include the NF-κB (nuclear factor-κB) family. However, the mechanisms by which UV irradiation activates NF-κB are not well understood. In eukaryotes, a variety of environmental stresses are recognized and remediated by a family of protein kinases that phosphorylate the α subunit of eIF2 (eukaryotic initiation factor-2). In the present study we show that NF-κB in MEF (murine embryo fibroblast) cells is activated by UV-C and UV-B irradiation through a mechanism requiring eIF2α phosphorylation. The primary eIF2α kinase in response to UV is GCN2 (general control non-derepressible-2), with PEK/PERK (pancreatic eIF2α kinase/RNA-dependent-protein-kinase-like endoplasmic-reticulum kinase) carrying out a secondary function. Our studies indicate that lowered protein synthesis accompanying eIF2α phosphorylation, combined with eIF2α kinase-independent turnover of IκBα (inhibitor of κBα), reduces the levels of IκBα in response to UV irradiation. Release of NF-κB from the inhibitory IκBα would facilitate NF-κB entry into the nucleus and targeted transcriptional control. We also find that loss of GCN2 in MEF cells significantly enhances apoptosis in response to UV exposure similar to that measured in cells deleted for the RelA/p65 subunit of NF-κB. These results demonstrate that GCN2 is central to recognition of UV stress, and that eIF2α phosphorylation provides resistance to apoptosis in response to this environmental insult.

scholarly journals Components of gating charge movement and S4 voltage-sensor exposure during activation of hERG channels

2013 ◽  
Vol 141 (4) ◽  
pp. 431-443 ◽  
Author(s):  
Zhuren Wang ◽  
Ying Dou ◽  
Samuel J. Goodchild ◽  
Zeineb Es-Salah-Lamoureux ◽  
David Fedida
Keyword(s):  
Mammalian Cells ◽  
Voltage Sensor ◽  
Delayed Rectifier ◽  
Charge Movement ◽  
Α Subunit ◽  
Time Constants ◽  
Gating Charge ◽  
Activation Gating ◽  
Pore Opening ◽  
Herg Channels

The human ether-á-go-go–related gene (hERG) K+ channel encodes the pore-forming α subunit of the rapid delayed rectifier current, IKr, and has unique activation gating kinetics, in that the α subunit of the channel activates and deactivates very slowly, which focuses the role of IKr current to a critical period during action potential repolarization in the heart. Despite its physiological importance, fundamental mechanistic properties of hERG channel activation gating remain unclear, including how voltage-sensor movement rate limits pore opening. Here, we study this directly by recording voltage-sensor domain currents in mammalian cells for the first time and measuring the rates of voltage-sensor modification by [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET). Gating currents recorded from hERG channels expressed in mammalian tsA201 cells using low resistance pipettes show two charge systems, defined as Q1 and Q2, with V1/2’s of −55.7 (equivalent charge, z = 1.60) and −54.2 mV (z = 1.30), respectively, with the Q2 charge system carrying approximately two thirds of the overall gating charge. The time constants for charge movement at 0 mV were 2.5 and 36.2 ms for Q1 and Q2, decreasing to 4.3 ms for Q2 at +60 mV, an order of magnitude faster than the time constants of ionic current appearance at these potentials. The voltage and time dependence of Q2 movement closely correlated with the rate of MTSET modification of I521C in the outermost region of the S4 segment, which had a V1/2 of −64 mV and time constants of 36 ± 8.5 ms and 11.6 ± 6.3 ms at 0 and +60 mV, respectively. Modeling of Q1 and Q2 charge systems showed that a minimal scheme of three transitions is sufficient to account for the experimental findings. These data point to activation steps further downstream of voltage-sensor movement that provide the major delays to pore opening in hERG channels.

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