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Biomedical subjects

J Westerman

Publications and source records attributed to J Westerman.

At least 37 records · Page 2Linked to original sources
PubMed1990

Proteolytic activation of a bovine brain protein with phosphatidylinositol transfer activity.

We have purified a 38 kDa protein from bovine brain which is cross-reactive with an affinity purified antibody against the 35 kDa phosphatidylinositol transfer protein from the same source. Controlled trypsinization of the 38 kDa protein yielded an immunoreactive protein of 35 kDa which displayed a 6-fold increase in phosphatidylinositol transfer activity and a 10-fold higher affinity for this phospholipid. The possibility that the 38 kDa protein is a precursor of the phosphatidylinositol transfer protein is discussed.

Animals↗
PubMed1990

Enzymatic synthesis of pyrene-labeled polyphosphoinositides and their behavior in organic solvents and phosphatidylcholine bilayers.

A method is reported for the synthesis of pyrene-labeled analogues of phosphatidylinositol 4-phosphate (Pyr-PIP) and phosphatidylinositol 4,5-biphosphate (Pyr-PIP2) from sn-2-(pyrenyl-decanoyl)phosphatidylinositol (Pyr-PI) using partially purified PI and PIP kinase preparations. Phosphorylation of Pyr-PI and Pyr-PIP was extensive (more than 50%) provided that the ATP concentration was high and that stabilizing agents such as sucrose and polyethylene glycol were present in the incubation medium. Pyr-PIP and Pyr-PIP2 were isolated by chromatography on immobilized neomycin. The identity of the products was established by thin-layer chromatography, UV-absorption spectroscopy, and spectrofluorometry. The pyrene excimer/monomer fluorescence technique revealed that, in contrast to Pyr-PI, Pyr-PIP and Pyr-PIP2 formed clusters in organic solvents. By use of the same technique for model membranes, it was shown that in phosphatidylcholine bilayers the collision frequency of the three fluorescent phosphoinositides decreased in the order PI greater than PIP greater than PIP2. Addition of Ca2+ at concentrations above 0.1 mM increased the collision frequency of Pyr-PIP2 and, to a much lesser extent, Pyr-PIP; Ca2+ had no effect on Pyr-PI.

Animals↗
PubMed1989

Transfer of cholesterol and oxysterol derivatives by the nonspecific lipid transfer protein (sterol carrier protein 2): a study on its mode of action.

The nonspecific lipid transfer protein (nsLTP) facilitates the transfer of both phospholipids and cholesterol between membrane interfaces. In this study, we have investigated the transport of 14C-labelled cholesterol, 7-ketocholesterol, 7 alpha-hydroxycholesterol and 25-hydroxycholesterol from a mixed lipid monolayer at the air/water interface to acceptor vesicles in the subphase. In the absence of nsLTP the transport of cholesterol was virtually nil, whereas the spontaneous transport of the oxysterol derivatives increased in the order 7-ketosterol less than 7 alpha-hydroxycholesterol less than 25-hydroxycholesterol. In the presence of nsLTP, the transport of both cholesterol and the oxysterol derivatives was greatly enhanced; the highest rate of transport was observed for 25-hydroxycholesterol. In the absence of vesicles, binding of cholesterol and of 25-hydroxycholesterol from the monolayer to nsLTP was negligible. Similarly, nsLTP did not bind cholesterol from radiolabeled bovine heart mitochondria under conditions where it stimulated the transfer of cholesterol to vesicles. In agreement with this failure to bind, nsLTP was unable to carry cholesterol between two separate monolayers. From the monolayer experiments it became apparent that nsLTP is highly surface-active. Measurement of the transport of cholesterol and of oxysterol derivatives by the monolayer-vesicles assay and of a series of pyrene-labeled phosphatidylcholine species by the fluorescent transfer assay showed a high correlation between the spontaneous and the nsLTP-mediated lipid transport. This supports the notion that nsLTP lowers the energy barrier for the lipid monomer-membrane interface equilibration process. In view of the above observations, we propose that nsLTP may facilitate the transfer of lipids by being part of a transient collisional complex between donor and acceptor membrane.

Animals↗
PubMed1986

Identification of an essential lysine residue in the phosphatidylcholine-transfer protein from bovine liver by modification with phenylisothiocyanate.

Modification by phenylisothiocyanate inhibits the phosphatidylcholine-transfer protein from bovine liver. Inhibition by this apolar reagent was greatly enhanced in the presence of vesicles, indicating that an effective modification of an essential lysine residue(s) from the interface may occur. Labeling with [14C]phenylisothiocyanate demonstrated that Lys55 was the major site of modification. We propose that Lys55 is part of the peptide segment that interacts with the membrane.

Androgen-Binding Protein↗
PubMed1985

The primary structure of the nonspecific lipid transfer protein (sterol carrier protein 2) from bovine liver.

The primary structure of the nonspecific lipid transfer protein (sterol carrier protein 2) from bovine liver has been determined. The protein consists of a single polypeptide chain of 121 amino acid residues with serine as the amino-terminal and alanine as the carboxy-terminal residue. The protein contains one single cysteine and tryptophan residue and lacks tyrosine, histidine and arginine.

Amino Acid Sequence↗
PubMed1983

The complete primary structure of phospholipase A2 from human pancreas.

The complete amino acid sequence of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from human pancreas was determined. The protein consists of a single polypeptide chain of 125 amino acids and has a molecular weight of 14003. The chain is cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digestion of the reduced and thialaminated derivative of the protein with clostripain, yielding three fragments. The largest fragment (residues 7-100) was further degraded both with staphylococcal proteinase and chymotrypsin. The sequence was determined by automated Edman degradation of the intact protein and of several large peptide fragments. Phospholipase A2 from human pancreas contains the same number of amino acids (125) as the enzyme from horse, while the enzymes from pig and ox contain 124 and 123 residues, respectively. The enzymes show a high degree of homology; human phospholipase differs from the other enzymes by substitutions of 26 (porcine), 28 (bovine) and 32 (equine) residues, respectively.

Amino Acid Sequence↗
PubMed1983

Identification of the lipid-binding site of phosphatidylcholine-transfer protein with phosphatidylcholine analogs containing photoactivable carbene precursors.

The lipid binding site of the phosphatidylcholine transfer protein from bovine liver has been investigated by use of phosphatidylcholine analogs which carry a diazirinophenoxy group linked to the omega-carbon of either the sn-2-[1-14C]hexanoyl (PC I) or sn-2-[1-14C]undecanoyl chain (PC II). Photolysis of the PC I(PC II)-transfer protein complex resulted in a covalent coupling of 30-40% of the label to the protein as shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Upon mild alkaline treatment of the photolysed complex the protein containing covalently coupled 14C-label was separated from the noncoupled 14C-label by gel permeation chromatography. The 14C-labeled protein was degraded with protease from Staphylococcus aureus, trypsin and cyanogen bromide and specific 14C-labeled peptides were sequenced by automated Edman degradation. Major sites of coupling shown by release of radioactivity were identified as Tyr54 and the peptide segment Val171-Phe-Met-Tyr-Tyr-Phe-Asp177. Both PC I and PC II coupled extensively to Tyr54 (90% and 50% of total labeling, respectively). The remainder of the radioactivity was released from the peptide Val171-Asp177 with a distinct difference in in the pattern of release depending on whether PC I or PC II were used. Thus, coupling occurred preferentially to Tyr175 and Asp177 with PC I while Val171 and Met173 were labeled preferentially with PC II. This shift in coupling is compatible with an increase of 0.6 nm for the sn-2-fatty-acyl chains of PC I and II, assuming that the peptide Val171-Asp177 has adopted the strongly predicted beta-strand configuration. These data have been interpreted in terms of the localization of phosphatidylcholine in the phosphatidylcholine transfer protein.

Amino Acid Sequence↗
PubMed1982

Human erythrocyte membranes are fluid down to -5 degrees C.

This first observation of the deuterium nuclear magnetic resonance (2H-NMR) spectrum of phospholipid molecules incorporated into intact human erythrocyte ghosts shows that the liquid crystalline phase is stable down to a temperature of -5 degrees C. The quality of the 3H-NMR spectra indicate that it is now possible to carry out clinical studies of erythrocyte membranes using the techniques employed in this study.

Erythrocyte Membrane↗
PubMed1982

Prediction of secondary structural elements in the phosphatidylcholine-transfer protein from bovine liver.

Secondary structural elements of the phosphatidylcholine-transfer protein from bovine liver have been predicted from its primary structure with the aid of two computerized methods. The predicted alpha-helix and beta-strand content have been compared with the values derived from circular dichroism spectra. The hydrophobicity profile (Rose plot) of the protein indicated that the supposed lipid-binding site occurs in the most hydrophobic region. The predicted secondary structural elements have been folded in a tentative model of the protein molecule according to its hydrophobicity profile.

Amino Acid Sequence↗
PubMed1981

Modification of the phosphatidylcholine-transfer protein from bovine liver with butanedione and phenylglyoxal. Evidence for one essential arginine residue.

1. Modification of arginine residues with 2,3-butanedione and phenylglyoxal completely inhibits the transfer activity of the phosphatidylcholine transfer protein from bovine liver. Removal of borate and butanedione leads to a slow reactivation of the protein. 2. Both alpha-dicarbonyl reagents modify three of the ten arginine residues present per protein molecule. The extent of modification is linearly related to the loss of activity. 3. Inactivation with butanedione is greatly diminished when the protein is bound to strongly negatively charged vesicles. Under these conditions a rapid modification of two arginine residues is observed. This suggests that the transfer protein contains one arginine residue essential for activity, probably as a binding site for the negatively charged phosphate group of the phosphatidylcholine molecule. 4. This study provides convincing evidence that arginine residues may play an essential role in phospholipidprotein interactions.

Aldehydes↗
PubMed1980

The primary structure of the phosphatidylcholine-exchange protein from bovine liver. Isolation and characterization of the staphylococcal protease peptides and the amino-acid sequence of the N-terminal half (residues 1--122).

The phosphatidylcholine exchange protein from bovine liver consists of a single polypeptide chain and has a blocked N terminus. The protein contains an estimated 244 amino acid residues in accordance with a determined molecular weight of 28000. The protease from mouse submaxillaris gland cleaved the citraconylated and S-carboxymethylated derivative of the exchange protein at one specific site (Arg14-Glu15) close to the N terminus. Analysis of the two resulting peptides showed that N-acetyl-methionine was the N-terminal residue and gave the sequence of the first 41 residues. The modified protein was also fragmented with the protease from Staphylococcus aureus. The peptides isolated represented 88% of the protein; their sequences were determined by manual and automated Edman degradation. Alignment of a number of these peptides gave the complex sequence of the N-terminal half up to position 122.

Amino Acid Sequence↗
PubMed1979

Kinetic analysis of the interaction of the phosphatidylcholine exchange protein with unilamellar vesicels and multilamellar liposomes.

The mode of action of the phosphatidylcholine exchange protein from bovine liver has been studied by using unilamellar vesicles and multilamellar liposomes both of which membranes contain phosphatidylcholine and phosphatidic acid. The protein-mediated exchange of phosphatidylcholine between vesicles and liposomes fit the kinetic model presented in a previous study [V.D. Besselaar et al. (1975) Biochemistry, 1j, 1852]. Kinetic analysis of the rates of exchange indicate that the apparent dissociation constant of the exchange protein-vesicle complex decreases with an increasing phosphatidic acid content of the vesicles. Both vesicles and liposomes of 10 mol% phosphatidic acid show the same dissociation constant; on the other hand, both the formation and the disruption of the protein-membrane complex was 50--100-times higher for the vesicles than for the liposomes. This implies that the exchange protein can discriminate between vesicles and liposomes. Equilibrium gel chromatography of a column of Bio Gel A-5m confirmed that the exchange protein binds more strongly to vesicles of an increased phosphatidic acid content. The protein-mediated exchange of phosphatidylcholine in the vesicle-liposome system demonstrates a pH optimum at 4.0 to 5.5. The kinetic analysis at pH 5.0 as compared to pH 7.4 indicates that the enhanced exchange at pH 5.0 can solely be accounted for by altered interaction of the exchange protein with the liposomes.

Carrier Proteins↗
PubMed1978

Evidence for the existence of different pools of microsomal phosphatidylinositol by the use of phosphatidylinositol-exchange protein.

1. The phosphatidylinositol-exchange protein from bovine brain was used to determine to what extent phosphatidylinositol in rat liver microsomal membranes is available for transfer. 2. The microsomal membranes used in the transfer reaction contained either phosphatidyl[2-(3)H]inositol or (32)P-labelled phospholipid. The (32)P-labelled microsomal membranes were isolated from rat liver after an intraperitoneal injection of [(32)P]P(i). The (3)H-labelled microsomal membranes and rough- and smooth-endoplasmic-reticulum membranes were prepared in vitro by the incorporation of myo-[2-(3)H]inositol into phosphatidylinositol by either exchange in the presence of Mn(2+) or biosynthesis de novo in the presence of CTP and Mg(2+). 3. Tryptic or chymotryptic treatment of the microsomes impaired the biosynthesis de novo of phosphatidylinositol. It was therefore concluded that the biosynthesis of phosphatidylinositol and/or its immediate precursor CDP-diacylglycerol takes place on the cytoplasmic surface of the microsomal membrane. 4. Under the conditions of incubation 42% of the microsomal phosphatidyl[2-(3)H]inositol was transferred with an estimated half-life of 5min; 38% was transferred with an estimated half-life of about 1h; the remaining 20% was not transferable. Identical results were obtained irrespective of the method of myo-[2-(3)H]inositol incorporation. 5. Both measurement of phosphatidylinositol phosphorus in the microsomes after transfer and the transfer of microsomal [(32)P]phosphatidylinositol indicate that phosphatidyl[2-(3)H]-inositol formed by exchange or biosynthesis de novo was homogeneously distributed throughout the microsomal phosphatidylinositol. 6. We present evidence that the slowly transferable pool of phosphatidylinositol does not represent the luminal side of the microsomal membrane; hence we suggest that this phosphatidylinositol is bound to membrane proteins.

Animals↗