Characterization of mouse phosphatidylinositol transfer protein expressed in Escherichia coli

Geijtenbeek, Teunis B. H.; Groot, Ellen de; Baal, J. van; Brunink, Freek; Westerman, Jan; Snoek, Gerry T.; Wirtz, K.W.A.

doi:10.1016/0005-2760(94)00063-8

Cited by 29 publications

(32 citation statements)

References 30 publications

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“…This protein contains PG as shown by Geijtenbeek et al (26). The average mass of PG/PI-TPR is 31 764 ( 1 Da, which is almost identical to the calculated molecular mass of 31 762 Da (26).…”

Section: Esi-tof Analysis Of the Phosphatidylcholine Transfersupporting

confidence: 76%

“…This protein contains PG as shown by Geijtenbeek et al (26). The average mass of PG/PI-TPR is 31 764 ( 1 Da, which is almost identical to the calculated molecular mass of 31 762 Da (26). The difference between the complex and the empty protein is about 735 Da, which concurs with the average molecular mass of the PG molecular species bound (see below).…”

Section: Esi-tof Analysis Of the Phosphatidylcholine Transfersupporting

confidence: 76%

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Determination of the Stability of the Noncovalent Phospholipid Transfer Protein−Lipid Complex by Electrospray Time-of-Flight Mass Spectrometry

et al. 2002

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Phosphatidylcholine transfer protein (PC-TP) containing different molecular species of PC and phosphatidylinositol transfer protein R (PI-TPR) containing either a PI, PC, or PG molecule were identified as intact complexes by nano-electrospray ionization time-of-flight mass spectrometry. The stability of these complexes in the gas phase was determined by elevating the cone voltage (cv) resulting in the appearance of the protein void of lipid. PC-TP containing a PC species carrying an sn-1 palmitoyl chain was less stable than PC-TP containing a PC species carrying an sn-1 stearoyl chain given that these complexes were dissociated for 50% at a cv of roughly 30 and 45 V, respectively. Different acyl chains on the sn-2 position did not lead to significant changes in stability of the complex. In the case of PI-TPR, the complexes containing PI and PG were dissociated for 50% at a cv of 100 V as compared to a cv of 40 V for the complex containing PC. We propose that this difference in stability is due to hydrogen bonds between the polar headgroup of PI and PG and the lipid-binding site of PI-TPR. This may explain why PI-TPR preferentially binds PI from a membrane interface.

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“…This protein contains PG as shown by Geijtenbeek et al (26). The average mass of PG/PI-TPR is 31 764 ( 1 Da, which is almost identical to the calculated molecular mass of 31 762 Da (26).…”

Section: Esi-tof Analysis Of the Phosphatidylcholine Transfersupporting

confidence: 76%

Section: Esi-tof Analysis Of the Phosphatidylcholine Transfersupporting

confidence: 76%

Determination of the Stability of the Noncovalent Phospholipid Transfer Protein−Lipid Complex by Electrospray Time-of-Flight Mass Spectrometry

et al. 2002

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“…Cloning of the cDNA encoding rat brain PI-TP␣ showed that the protein consists of 271 amino acid residues (8). The subsequent isolation of the cDNAs encoding mouse and human PI-TP␣ revealed a high homology between the different mammalian PI-TPs (about 99% amino acid sequence identity) (9,10). Furthermore, the cross-reactivity of the antibodies raised against bovine PI-TP␣ with a 35-kDa protein from other animals (e.g.…”

Section: The Addition Of Pi-tp␣ To a Total Lysate Of Myo-[mentioning

confidence: 99%

“…The cDNA encoding mouse PI-TP␣ was isolated and cloned into the pBluescript vector (9). The PI-TP␣ cDNA contains a NcoI restriction site around the translational start codon and an EcoRI and XhoI site downstream of the translational stop codon.…”

Section: Psg5-pi-tp␣ Constructmentioning

confidence: 99%

Overexpression of Phosphatidylinositol Transfer Protein α in NIH3T3 Cells Activates a Phospholipase A

Snoek¹,

Berrie²,

Geijtenbeek³

et al. 1999

Journal of Biological Chemistry

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In order to investigate the cellular function of the mammalian phosphatidylinositol transfer protein ␣ (PI-TP␣), NIH3T3 fibroblast cells were transfected with the cDNA encoding mouse PI-TP␣. Two stable cell lines, i.e. SPI6 and SPI8, were isolated, which showed a 2-and 3-fold increase, respectively, in the level of PI-TP␣. Overexpression of PI-TP␣ resulted in a decrease in the duration of the cell cycle from 21 h for the wild type Upon equilibrium labeling of the cells with myo-[ 3 H] inositol, the relative incorporation of radioactivity in the total inositol phosphate fraction was 2-3-fold increased in SPI6 and SPI8 cells when compared with wtNIH3T3 cells. A detailed analysis of the inositol metabolites showed increased levels of glycerophosphoinositol, Ins(1)P, Ins(2)P, and lysophosphatidylinositol (lyso-PtdIns) in SPI8 cells, whereas the levels of phosphatidylinositol (PtdIns) and phosphatidylinositol 4,5-bisphosphate were the same as those in control cells. The addition of PI-TP␣ to a total lysate of myo-[3 H]inositol-labeled wtNIH3T3 cells stimulated the formation of lyso-PtdIns. The addition of Ca 2؉ further increased this formation. Based on these observations, we propose that PI-TP␣ is involved in the production of lyso-PtdIns by activating a phospholipase A acting on PtdIns. The increased level of lyso-PtdIns that is produced in this reaction could be responsible for the increased growth rate and the partial loss of contact inhibition in SPI8 and SPI6 cells. The addition of growth factors (plateletderived growth factor, bombesin) to these overexpressers did not activate the phospholipase C-dependent degradation of phosphatidylinositol 4,5-bisphosphate.Phospholipid transfer proteins are proteins that are able to transfer phospholipids between membranes in vitro. A major phospholipid transfer protein in mammalian tissues is the phosphatidylinositol transfer protein (PI-TP) 1 (1). Recently, two isoforms of PI-TP have been identified (i.e. PI-TP␣ and PI-TP␤) that demonstrate differences in cellular localization and in specific lipid transfer activity (2-5).PI-TP␣ has been purified from both rat and bovine brain (6, 7). Cloning of the cDNA encoding rat brain PI-TP␣ showed that the protein consists of 271 amino acid residues (8). The subsequent isolation of the cDNAs encoding mouse and human PI-TP␣ revealed a high homology between the different mammalian PI-TPs (about 99% amino acid sequence identity) (9, 10). Furthermore, the cross-reactivity of the antibodies raised against bovine PI-TP␣ with a 35-kDa protein from other animals (e.g. rat, mouse, chicken, frog, and lizard) indicates an extensive conservation of the amino acid sequence between species (11). An exception is PI-TP from yeast (i.e. SEC14p) that has the same molecular weight as mammalian PI-TP and comparable phospholipid transfer activities yet shows no homology in the amino acid sequence (12)(13)(14).So far, very little is known about the precise cellular role of mammalian PI-TP␣. Since PI-TP␣ is able to transfer in vitro PtdIns between membrane...

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“…All other chemicals were purchased from Sigma or Fisher Scientific (Pittsburgh, PA, U.S.A.). Recombinant mouse PITP-α was prepared from inclusion bodies after expression of the cDNA in Escherichia coli [22] and was stored at k80 mC before use.…”

Section: Methodsmentioning

confidence: 99%

Activity of phosphatidylinositol transfer protein is sensitive to ethanol and membrane curvature

Komatsu

Bouma

Wirtz

et al. 2000

Biochemical Journal

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Phosphatidylinositol transfer protein (PITP) is critical for many cellular signalling and trafficking events that are influenced by ethanol. The influence of ethanol and membrane curvature on the activity of recombinant mouse PITP-α in vitro is evaluated by monitoring the transfer of phosphatidylinositol (PtdIns) from rat hepatic microsomes to unilamellar vesicles. Acute exposure to pharmacological levels of ethanol enhanced the function of PITP. Chloroform shared a similar ability to enhance function when both drug concentrations were normalized to their respective octanol/water partition coefficients, indicating that the effect is not unique to ethanol and might be common to hydrophobic solutes. Neither the PITP activity nor its ethanol enhancement was altered by using thermally pretreated (denatured) or protease-treated microsomes, indicating that the native microsomal protein structure was unlikely to be a determinant of transfer. Kinetic analyses indicated that ethanol acted by increasing the PITP-mediated flux of PtdIns from both microsomal and liposomal surfaces. The activity of PITP was strongly dependent on the lipid structure, with a steep dependence on the expressed curvature of the membrane. Activity was greatest for small, highly curved sonicated vesicles and decreased markedly for large, locally planar unilamellar vesicles. Ethanol enhanced PITP-mediated PtdIns transfer to all vesicles, but its effect was much smaller than the enhancement due to curvature, which is consistent with ethanol's comparatively modest ability to perturb membrane lipids. The ethanol efficacy observed is as pronounced as any previously described lipid-mediated ethanol action. In addition, these observations raise the possibility that PITP specifically delivers PtdIns to metabolically active membrane domains of convex curvature and/or low surface densities of lipid.

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Characterization of mouse phosphatidylinositol transfer protein expressed in Escherichia coli

Cited by 29 publications

References 30 publications

Determination of the Stability of the Noncovalent Phospholipid Transfer Protein−Lipid Complex by Electrospray Time-of-Flight Mass Spectrometry

Determination of the Stability of the Noncovalent Phospholipid Transfer Protein−Lipid Complex by Electrospray Time-of-Flight Mass Spectrometry

Overexpression of Phosphatidylinositol Transfer Protein α in NIH3T3 Cells Activates a Phospholipase A

Activity of phosphatidylinositol transfer protein is sensitive to ethanol and membrane curvature

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