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J Westerman

Publications and source records attributed to J Westerman.

At least 19 recordsLinked to original sources
PubMed2002

Determination of the stability of the noncovalent phospholipid transfer protein-lipid complex by electrospray time-of-flight mass spectrometry.

Phosphatidylcholine transfer protein (PC-TP) containing different molecular species of PC and phosphatidylinositol transfer protein alpha (PI-TPalpha) 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-TPalpha, 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-TPalpha. This may explain why PI-TPalpha preferentially binds PI from a membrane interface.

Androgen-Binding Protein↗
PubMed2002

Clofibrate-induced relocation of phosphatidylcholine transfer protein to mitochondria in endothelial cells.

The phosphatidylcholine transfer protein (PC-TP) is a specific transporter of phosphatidylcholine (PC) between membranes. To get more insight into its physiological function, we have studied the localization of PC-TP by microinjection of fluorescently labeled PC-TP in foetal bovine heart endothelial (FBHE) cells and by expression of an enhanced yellow fluorescent protein-PC-TP fusion protein in FBHE cells, human umbilical vein endothelial cells, and HepG2 cells. Analysis by confocal laser scanning microscopy showed that PC-TP was evenly distributed throughout the cytosol with an apparently elevated level in nuclei. By measuring the fluorescence recovery after bleaching it was established that PC-TP is highly mobile throughout the cell, with its transport into the nucleus being hindered by the nuclear envelope. Given the proposed function of PC-TP in lipid metabolism, we have tested a number of compounds (phorbol ester, bombesin, A23187, thrombin, dibutyryl cyclic AMP, oleate, clofibrate, platelet-derived growth factor, epidermal growth factor, and hydrogen peroxide) for their ability to affect intracellular PC-TP distribution. Only clofibrate (100 microM) was found to have an effect, with PC-TP moving to mitochondria within 5 min of stimulation. This relocation did not occur with PC-TP(S110A), lacking the putative protein kinase C (PKC)-dependent phosphorylation site, and was restricted to the primary endothelial cells. Relocation did not occur in HepG2 cells, possibly due to the fact that clofibrate does not induce PKC activation in these cells.

Androgen-Binding Protein↗
PubMed2001

Activation of phosphatidylinositol transfer protein alpha and beta isoforms from inclusion bodies.

Fully active phosphatidylinositol transfer protein (PI-TP) isoforms alpha and beta have been obtained from Escherichia coli inclusion bodies. Folding and activation of PI-TPalpha was achieved in the presence of DiC7:0-phosphatidylcholine-Triton X-114 (PtdCho-TX114) mixed micelles. Replacement of DiC7:0-PtdCho with the natural ligands of PI-TPalpha, i.e. long-chain PtdCho and phosphatidylinositol, did not stimulate activation. Efficient activation of PI-TPalpha required a low temperature (4 degrees C), the presence of dithiothreitol, and was achieved at a relatively high protein concentration (i.e. up to 500 microg ml(-1)). The inclusion bodies yielded 10 mg homogeneous PI-TPalpha per liter of E. coli culture. Conditions for full activation of PI-TPbeta were similar to those for PI-TPalpha except that long-chain PtdCho-TX114 mixed micelles and a very low protein concentration (i.e. 10 microg ml(-1)) were required. In contrast to PI-TPalpha, PI-TPbeta lost its lipid transfer activity within a few days. This inactivation could be prevented by addition of beta-alanine. In summary, despite 94% sequence similarity, PI-TPalpha and PI-TPbeta display a striking difference both in their preference for the PtdCho acyl chain length required for activation, and in their conformational stability after folding.

Amino Acid Sequence↗
PubMed2001

The binding of phosphatidylcholine to the phosphatidylcholine transfer protein: affinity and role in folding.

Bovine liver phosphatidylcholine transfer protein (PC-TP) has been expressed in Escherichia coli and purified to homogeneity from the cytosol fraction at a yield of 0.45 mg PC-TP per 10 mg total cytosolic protein. In addition, active PC-TP was obtained from inclusion bodies. An essential factor in the activation of PC-TP was phosphatidylcholine (PC) present in the folding buffer. PC-TP from the cytosol contains phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) with a preference for the di-monounsaturated species over the saturated species as determined by fast atom bombardment mass spectrometry (FAB-MS). By incubation with microsomal membranes the endogenous PE and PG were replaced by PC. Relative to the microsomal PC species composition, PC-TP bound preferentially C16:0/C20:4-PC and C16:0/C18:2-PC (twofold enriched) whereas the major microsomal species C18:0/C18:1-PC and C18:0/C18:2-PC were distinctly less bound. PC-TP is structurally homologous to the lipid-binding domain of the steroidogenic acute regulatory protein (Nat. Struct. Biol. 7 (2000) 408). Replacement of Lys(55) present in one of the beta-strands forming the lipid-binding site, with an isoleucine residue yielded an inactive protein. This suggests that Lys(55) be involved in the binding of the PC molecule.

Androgen-Binding Protein↗
PubMed2000

The protein kinase C-dependent phosphorylation of serine 166 is controlled by the phospholipid species bound to the phosphatidylinositol transfer protein alpha.

The charge isomers of bovine brain PI-TPalpha (i.e. PI-TPalphaI containing a phosphatidylinositol (PI) molecule and PI-TPalphaII containing a phosphatidylcholine (PC) molecule) were phosphorylated in vitro by rat brain protein kinase C (PKC) at different rates. From the double-reciprocal plot, it was estimated that the V(max) values for PI-TPalphaI and II were 2.0 and 6.0 nmol/min, respectively; the K(m) values for both charge isomers were about equal, i.e. 0.7 micrometer. Phosphorylation of charge isomers of recombinant mouse PI-TPalpha confirmed that the PC-containing isomer was the better substrate. Phosphoamino acid analysis of in vitro and in vivo (32)P-labeled PI-TPalphas showed that serine was the major site of phosphorylation. Degradation of (32)P-labeled PI-TPalpha by cyanogen bromide followed by high pressure liquid chromatography and sequence analysis yielded one (32)P-labeled peptide (amino acids 104-190). This peptide contained Ser-148, Ser-152, and the consensus PKC phosphorylation site Ser-166. Replacement of Ser-166 with an alanine residue confirmed that indeed this residue was the site of phosphorylation. This mutation completely abolished PI and PC transfer activity. This was also observed when Ser-166 was replaced with Asp, implying that this is a key amino acid residue in regulating the function of PI-TPalpha. Stimulation of NIH3T3 fibroblasts by phorbol ester or platelet-derived growth factor induced the rapid relocalization of PI-TPalpha to perinuclear Golgi structures concomitant with a 2-3-fold increase in lysophosphatidylinositol levels. This relocalization was also observed for Myc-tagged wtPI-TPalpha expressed in NIH3T3 cells. In contrast, the distribution of Myc-tagged PI-TPalpha(S166A) and Myc-tagged PI-TPalpha(S166D) were not affected by phorbol ester, suggesting that phosphorylation of Ser-166 was a prerequisite for the relocalization to the Golgi. A model is proposed in which the PKC-dependent phosphorylation of PI-TPalpha is linked to the degradation of PI.

Animals↗
PubMed1999

High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2).

Binding of fluorescent fatty acids to bovine liver non-specific lipid-transfer protein (nsL-TP) was assessed by measuring fluorescence resonance energy transfer (FRET) between the single tryptophan residue of nsL-TP and the fluorophore. Upon addition of pyrene dodecanoic acid (Pyr-C12) and cis-parinaric acid to nsL-TP, FRET was observed indicating that these fatty acids were accommodated in the lipid binding site closely positioned to the tryptophan residue. Substantial binding was observed only when these fatty acids were presented in the monomeric form complexed to beta-cyclodextrin. As shown by time-resolved fluorescence measurements, translocation of Pyr-C12 from the Pyr-C12-beta-cyclodextrin complex to nsL-TP changed dramatically the direct molecular environment of the pyrene moiety: i.e. the fluorescence lifetime of the directly excited pyrene increased at least by 25% and a distinct rotational correlation time of 7 ns was observed. In order to evaluate the affinity of nsL-TP for intermediates of the beta-oxidation pathway, a binding assay was developed based on the ability of fatty acyl derivatives to displace Pyr-C12 from the lipid binding site as reflected by the reduction of FRET. Hexadecanoyl-CoA and 2-hexadecenoyl-CoA were found to bind readily to nsL-TP, whereas 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA bound poorly. The highest affinities were observed for the very-long-chain fatty acyl-CoA esters (24:0-CoA, 26:0-CoA) and their enoyl derivatives (24:1-CoA, 26:1-CoA). Binding of non-esterified hexadecanoic acid and tetracosanoic acid (24:0) was negligible.

Carrier Proteins↗
PubMed1997

High-frequency oscillatory ventilation for adult respiratory distress syndrome--a pilot study.

OBJECTIVE: To evaluate the safety and effectiveness of high-frequency oscillatory ventilation using a protocol designed to recruit and maintain optimal lung volume in patients with severe adult respiratory distress syndrome (ARDS). SETTING: Surgical and medical intensive care units in a tertiary care, military teaching hospital. DESIGN: A prospective, clinical study. PATIENTS: Seventeen patients, 17 yrs to 83 yrs of age, with severe ARDS (Lung Injury Score of 3.81 +/- 0.23) failing inverse ratio mechanical conventional ventilation (PaO2/FiO2 ratio of 68.6 +/- 21.6, peak inspiratory pressure of 54.3 +/- 12.7 cm H2O, positive end-expiratory pressure of 18.2 +/- 6.9 cm H2O). INTERVENTIONS: High-frequency oscillatory ventilation was instituted after varying periods of conventional ventilation (5.12 +/- 4.3 days). We employed lung volume recruitment strategy that consisted of incremental increases in mean airway pressure to achieve a PaO2 of > or = 60 torr (> or = 8.0 kPa), with an FiO2 of < or = 0.6. MEASUREMENTS AND MAIN RESULTS: High-frequency oscillator ventilator settings (FiO2, mean airway pressure, pressure amplitude of oscillation [delta P] frequency) and hemodynamic parameters (cardiac output, oxygen delivery [DO2]), mean systemic and pulmonary arterial pressures, and the oxygenation index (oxygenation index = [FiO2 x mean airway pressure x 100]/PaO2) were monitored during the transition to high-frequency oscillatory ventilation and throughout the course of the high-frequency protocol. Thirteen patients demonstrated improved gas exchange and an overall improvement in PaO2/FiO2 ratio (p < .02). Reductions in the oxygenation index (p < .01) and FiO2 (p < .02) at 12, 24, and 48 hrs after starting high-frequency oscillatory ventilation were observed. No significant compromise in cardiac output or DO2 was observed, despite a significant increase in mean airway pressure (31.2 +/- 10.3 to 34.0 +/- 6.7 cm H2O, p < .05) on high-frequency oscillatory ventilation. The overall survival rate at 30 days was 47%. A greater number of pretreatment days on conventional ventilation (p < .009) and an entry oxygenation index of > 47 (sensitivity 100%, specificity 100%) were associated with mortality. CONCLUSIONS: High-frequency oscillatory ventilation is both safe and effective in adult patients with severe ARDS failing conventional ventilation. A lung volume recruitment strategy during high-frequency oscillatory ventilation produced improved gas exchange without a compromise in DO2. These results are encouraging and support the need for a prospective, randomized trial of algorithm-controlled conventional ventilation vs. high-frequency oscillatory ventilation for adults with severe ARDS.

Adolescent↗
PubMed1996

Fluorescently labeled phosphatidylinositol transfer protein isoforms (alpha and beta), microinjected into fetal bovine heart endothelial cells, are targeted to distinct intracellular sites.

Upon permeabilization of Swiss mouse 3T3 fibroblasts, an isoform of phosphatidylinositol transfer protein (PI-TP) was preferentially retained, a major part of which was associated with the perinuclear Golgi system (K. J. de Vries, A. Momchilova-Pankova, G. T. Snoek, and K. W. A. Wirtz, Exp. Cell Res. 215, 109-113, 1994). In the present study, the intracellular localization of this isoform (PI-TP beta) and the regular form (PI-TP alpha) was investigated in fetal bovine heart endothelial cells by microinjection of fluorescently labeled analogs followed by confocal laser scanning microscopy. The PI-TP alpha and PI-TP beta used were purified from bovine brain cytosol and covalently labeled with sulfoindocyanine dyes. By this novel method it was found that PI-TP beta was preferentially associated with perinuclear membrane structures whereas PI-TP alpha was predominantly present in the nucleus and in the cytoplasm. This intracellular localization was confirmed by indirect immunofluorescence indicating that the fluorescently labeled PI-TP alpha and PI-TP beta were targeted to the same sites as their endogeneous counterparts.

Animals↗
PubMed1996

Phosphatidylcholine transfer protein from bovine liver contains highly unsaturated phosphatidylcholine species.

The phosphatidylcholine transfer protein (PC-TP) from bovine liver contains one molecule of non-covalently bound PC. In order to gain more insight into the physiological function of PC-TP, PC was extracted from bovine liver PC-TP and its molecular species composition identified by fast atom bombardment mass spectrometry. The prevailing molecular species were C18:0/C18:1-, C18:0/C18:2-, C18:0/C20:4-, C18:0/20:5- and C18:0/C22:5-PC accounting for 85% of the PC species present. This molecular species composition is not representative for what is present in bovine liver where these species account for 43% of the total PC content [Montfoort et al. (1971) Biochim. Biophys. Acta 231, 335-342]. Another striking observation is that PC species carrying a palmitoyl chain at the sn-1 position are nearly absent, despite these species being abundantly present in bovine liver. This study suggests that PC-TP could play a role in the metabolism of highly unsaturated, stearoyl-containing PC species.

Androgen-Binding Protein↗
PubMed1995

An isoform of the phosphatidylinositol-transfer protein transfers sphingomyelin and is associated with the Golgi system.

An isoform of the phosphatidylinositol-transfer protein (PI-TP) was identified in the cytosol fraction of bovine brain. This protein, designated PI-TP beta, has an apparent molecular mass of 36 kDa and an isoelectric point of 5.4. The N-terminal amino acid sequence (21 residues) is 90% similar to that of bovine brain PI-TP, henceforth designated PI-TP alpha (molecular mass 35 kDa and pI 5.5). As observed for PI-TP alpha, PI-TP beta has a distinct preference for phosphatidylinositol over phosphatidylcholine. In addition, it expresses a high transfer activity towards sphingomyelin. PI-TP alpha lacks this activity completely. By indirect immunofluorescence we demonstrated that, in Swiss mouse 3T3 fibroblasts, PI-TP beta is preferentially associated with the Golgi system whereas PI-TP alpha is predominantly present in the cytoplasm and the nucleus. In cytosol-depleted HL60 cells, both PI-TP alpha and PI-TP beta were equally effective at reconstituting guanosine 5'-[gamma-thio]triphosphate-mediated phospholipase C beta activity.

3T3 Cells↗
PubMed1995

A sphingomyelin-transferring protein from chicken liver. Use of pyrene-labeled phospholipid.

A phospholipid transfer protein was purified from chicken liver which, in addition to phosphatidylinositol (PI) and phosphatidylcholine (PC), carries sphingomyelin (SM) between membranes. For comparison, the PI-transfer protein from chicken liver only carries PI and PC. Specificity was established by use of phospholipids that carry a pyrene-labeled acyl chain. Based on the N-terminal sequence and Western blot analysis we conclude that this protein is an isoform of the PI-transfer protein. At increasing length of the pyrene-labeled acyl chain, the isoform expresses a high activity toward SM, a low activity toward PI, and virtually no activity toward PC.

Amino Acid Sequence↗
PubMed1994

In vivo turnover of phospholipids in rabbit erythrocytes.

The rate of phospholipid turnover in erythrocyte membranes in vivo has been studied using a recently developed procedure (Kuypers, F.A., Easton, E.W., van den Hoven, R., Wensing, T., Roelofsen, B., Op den Kamp, J.A.F. and van Deenen, L.L.M. (1985) Biochim. Biophys. Acta 819, 170-178). The technique is based on the application of phospholipid transfer proteins in order to introduce trace amounts of radiolabelled phospholipids in the membrane of isolated erythrocytes, followed by re-injection of the erythrocytes into the bloodstream of the animal. The most abundant species of the phosphatidylcholine (PC) class, 1-palmitoyl,2-linoleoyl PC, has, on the basis of loss of the radioactivity in its fatty acyl part, a relatively high turnover with a half-time value of 1.5 days. Other PC species studied exhibit more moderate turnover rates of about 5 days for 1-palmitoyl,2-oleoyl PC and 1-stearoyl,2-arachidonoyl PC. Dipalmitoyl PC, labelled in the polar headgroup, turns over at a slow rate with a half-time value of 9 days. From these data and the relative abundance of the various species, it can be calculated that, on a daily basis in vivo, about one third of the total PC pool in rabbit erythrocyte membranes is replaced and/or modified by de-/reacylation. The only phosphatidylethanolamine (PE) species studied so far, 1-palmitoyl,2-arachidonoyl PE, appeared to be renewed at a relatively low rate with a half-time value of 12 days. The data demonstrate that the in vivo turnover values of phospholipids in the erythrocyte membrane may depend on their polar head group structure, their localization in the membrane and, to a large extent, on their fatty acid composition.

Animals↗
PubMed1994

Characterization of mouse phosphatidylinositol transfer protein expressed in Escherichia coli.

The cDNA encoding mouse phosphatidylinositol transfer protein (PI-TP) was isolated by means of reverse transcriptase polymerase chain reaction. The nucleotide sequence of this cDNA has a high similarity (98%) with that of rat PI-TP; the predicted amino acid sequence is 99.6% identical to that of rat PI-TP. The cDNA encoding mouse PI-TP was cloned into the expression vector pET3d and the Escherichia coli strain BL21(DE3) was transformed with the resulting plasmid. After induction of the bacteria with isopropyl-beta-D-thiogalactopyranoside, PI-TP was efficiently expressed in the E. coli strain. It was estimated that 5% of the total soluble cell protein consisted of PI-TP. The recombinant mouse PI-TP was purified from the bacterial lysate in four steps: ammonium sulphate precipitation, anion-exchange chromatography, heparin-Sepharose affinity and gel filtration chromatography. Fractionation on the heparin-Sepharose affinity column yielded two forms: PI-TP Hepa1 and Hepa2. These two proteins have the same molecular mass of 35 kDa, both contain a phosphatidylglycerol molecule and both are recognized by anti-PI-TP antibody. Both recombinant proteins have an isoelectric point of 5.4 as compared to 5.5 for bovine brain PI-TP. Sequence analysis of the first 25 N-terminal amino acid residues showed that both forms are identical, except that PI-TP Hepa1 contains the initiator methionine which is lacking from PI-TP Hepa2. The two PI-TP forms have similar phospholipid-binding and transfer activity, comparable to that of bovine brain PI-TP. Both forms and bovine brain PI-TP are phosphorylated equally well in a Ca2+/phospholipid-dependent way by protein kinase C.

Amino Acid Sequence↗
PubMed1993

Characterization of phosphatidylinositol-4-phosphate 5-kinase activities from bovine brain membranes.

Phosphatidylinositol-4-phosphate (PtdIns(4)P) kinase activity associated with bovine brain membranes, was released by NaCl treatment and partially purified by chromatography on phosphocellulose, phenylsepharose, Ultrogel AcA44, DEAE-cellulose and ATP-agarose. The final preparation contained a 6333-fold purified protein fraction with a specific activity of 171 nmol.min-1 x mg-1. Under conditions where this PtdIns(4)P kinase activity (PtdIns(4)P kinase activity b) did not bind to DEAE-cellulose, a PtdIns(4)P kinase activity purified earlier (Moritz, A., De Graan, P.N.E., Ekhart, P.F., Gispen, W.H. and Wirtz, K.W.A. (1990) J. Neurochem. 54, 351-354) does bind (PtdIns(4)P kinase activity a). Both enzyme activities specifically used PtdIns(4)P as substrate and phosphorylated the inositol moiety at the 5'-position. PtdIns(4) kinase activity a has an apparent Km of 18 microM for PtdIns(4)P whereas PtdIns(4)P kinase activity b has a Km of 4 microM. All other measured kinetic parameters (i.e., Km for ATP, Mg(2+)-dependence, pH optimum, activation by phosphatidylserine and inhibition by phosphatidylinositol 4,5-bisphosphate) were similar for both enzyme activities.

Animals↗
PubMed1993

Phosphorylation and redistribution of the phosphatidylinositol-transfer protein in phorbol 12-myristate 13-acetate- and bombesin-stimulated Swiss mouse 3T3 fibroblasts.

By immunofluorescence microscopy it was shown that the phosphatidylinositol-transfer protein (PI-TP) becomes associated with the Golgi membranes when confluent (quiescent) Swiss mouse 3T3 fibroblast cells are stimulated with phorbol 12-myristate 13-acetate (PMA) and bombesin. Dibutyryl cyclic AMP or dexamethasone had no effect on the intracellular redistribution of PI-TP. In exponentially growing cells and in serum-starved (semi-quiescent) cells, PI-TP is already associated with Golgi structures. Stimulation of semi-quiescent cells by PMA resulted in a rapid redistribution of PI-TP. A similar yet slower response was observed after stimulation with bombesin. Stimulation of semi-quiescent 3T3 cells by PMA significantly increased the phosphorylation of PI-TP, as shown by immunoprecipitation of PI-TP from pre-labelled cells. No significant increase in phosphorylation of PI-TP was observed after stimulation of these cells by bombesin. Purified PI-TP was shown to be a substrate for protein kinase C in vitro. The possibility that the phosphorylation of PI-TP after activation of protein kinase C is involved in the observed redistribution of PI-TP is discussed.

3T3 Cells↗