Isolation of a Chinese hamster ovary cell mutant defective in intramitochondrial transport of phosphatidylserine. (57/2206)

A CHO-K1 cell mutant with a specific decrease in cellular phosphatidylethanolamine (PE) level was isolated as a variant resistant to Ro09-0198, a PE-directed antibiotic peptide. The mutant was defective in the phosphatidylserine (PS) decarboxylation pathway for PE formation, in which PS produced in the endoplasmic reticulum is transported to mitochondria and then decarboxylated by an inner mitochondrial membrane enzyme, PS decarboxylase. Neither PS formation nor PS decarboxylase activity was reduced in the mutant, implying that the mutant is defective in some step of PS transport. The transport processes of phospholipids between the outer and inner mitochondrial membrane were analyzed by use of isolated mitochondria and two fluorescence-labeled phospholipid analogs, 1-palmitoyl-2-[N-[6(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-PS (C6-NBD-PS) and C6-NBD-phosphatidylcholine (C6-NBD-PC). On incubation with the CHO-K1 mitochondria, C6-NBD-PS was readily decarboxylated to C6-NBD-PE, suggesting that the PS analog was partitioned into the outer leaflet of mitochondria and then translocated to the inner mitochondrial membrane. The rate of decarboxylation of C6-NBD-PS in the mutant mitochondria was reduced to approximately 40% of that in the CHO-K1 mitochondria. The quantity of phospholipid analogs translocated from the outer leaflet of mitochondria into inner mitochondrial membranes was further examined by selective extraction of the analogs from the outer leaflet of mitochondria. In the mutant mitochondria, the translocation of C6-NBD-PS was significantly reduced, whereas the translocation of C6-NBD-PC was not affected. These results indicate that the mutant is defective in PS transport between the outer and inner mitochondrial membrane and provide genetic evidence for the existence of a specific mechanism for intramitochondrial transport of PS.  (+info)

Qualitative and quantitative studies of autoantibodies to phospholipids in diabetes mellitus. (58/2206)

Diabetes mellitus is associated with vascular and neurological complications. We have investigated the presence of antibodies to phospholipids and to phospholipid binding plasma proteins in blood samples collected from 68 clinically and biochemically characterized type I and type II diabetic patients and from 252 healthy blood donor controls. Each sample was analysed for antibodies to three phospholipids (cardiolipin, phosphatidylserine and phosphatidylethanolamine), the antibody isotypes (IgA, IgG and IgM), and whether antibody activity was plasma protein-dependent. Patients were considered to have anti-phospholipid antibodies when one or more of these 18 tests was found above predetermined control values. The results of these experiments revealed an increased incidence of anti-phospholipid antibodies in diabetic patients compared with control subjects. The incidence of IgA isotype to phosphatidylethanolamine was higher than the incidence of other isotypes to other phospholipids, and their reactivities were independent of phospholipid-associated proteins. In addition, these antibody findings were studied for associations with prothrombin degradation products, activated factor VII and activated protein C, and with the incidence of diabetic complications. The anti-phosphatidylethanolamine antibody association with proliferative retinopathy was significant.  (+info)

Beta(2)-glycoprotein I promotes the binding of anionic phospholipid vesicles by macrophages. (59/2206)

Beta(2)-Glycoprotein I is a single-chain 50-kDa protein that circulates in plasma at a concentration of approximately 200 microg/mL. Its physiological role remains uncertain, but an important clue is the frequent presence of antibodies to this protein in patients with recurrent thrombosis. We have isolated beta(2)-glycoprotein I and examined its effect on the binding of phosphatidylserine (PS) vesicles by human monocyte-derived macrophages and by phorbol ester-stimulated THP-1 cells. beta(2)-Glycoprotein I stimulated the binding of PS vesicles by these cells in a concentration-dependent manner. Vesicles containing other anionic phospholipids, such as cardiolipin, phosphatidic acid, or cardiolipin, inhibited the binding, whereas PC vesicles had no effect. Platelet-derived microvesicles, which contain anionic phospholipid on the outer leaflet of their phospholipid bilayer, also inhibited beta(2)-glycoprotein I-dependent binding of anionic phospholipid vesicles. The binding is associated with incorporation of phospholipid in the cell membrane and internalization of beta(2)-glycoprotein I. These findings suggest a physiological function for beta(2)-glycoprotein I in the clearance of procoagulant anionic phospholipid-containing cell surfaces from the circulation.  (+info)

Ca(2+) bridges the C2 membrane-binding domain of protein kinase Calpha directly to phosphatidylserine. (60/2206)

The C2 domain acts as a membrane-targeting module in a diverse group of proteins including classical protein kinase Cs (PKCs), where it plays an essential role in activation via calcium-dependent interactions with phosphatidylserine. The three-dimensional structures of the Ca(2+)-bound forms of the PKCalpha-C2 domain both in the absence and presence of 1, 2-dicaproyl-sn-phosphatidyl-L-serine have now been determined by X-ray crystallography at 2.4 and 2.6 A resolution, respectively. In the structure of the C2 ternary complex, the glycerophosphoserine moiety of the phospholipid adopts a quasi-cyclic conformation, with the phosphoryl group directly coordinated to one of the Ca(2+) ions. Specific recognition of the phosphatidylserine is reinforced by additional hydrogen bonds and hydrophobic interactions with protein residues in the vicinity of the Ca(2+) binding region. The central feature of the PKCalpha-C2 domain structure is an eight-stranded, anti-parallel beta-barrel with a molecular topology and organization of the Ca(2+) binding region closely related to that found in PKCbeta-C2, although only two Ca(2+) ions have been located bound to the PKCalpha-C2 domain. The structural information provided by these results suggests a membrane binding mechanism of the PKCalpha-C2 domain in which calcium ions directly mediate the phosphatidylserine recognition while the calcium binding region 3 might penetrate into the phospholipid bilayer.  (+info)

Interaction of the N-terminus of sterol carrier protein 2 with membranes: role of membrane curvature. (61/2206)

Although neither the physiological function nor the mechanism of action of sterol carrier protein 2 (SCP(2)) is yet completely clear, it is thought that SCP(2) interacts with membranes to elicit its biological effects. The results presented here show that the SCP(2) N-terminus, composed of two amphipathic alpha-helices, interacted preferentially with highly curved but not lower-curvature membranes containing anionic phospholipid. CD spectra of SCP(2) showed up to 1. 2-fold increased alpha-helical content, on the interaction of SCP(2) with small unilamellar vesicles (SUV) (median radius 10-14 nm) but less with large unilamellar vesicles (LUV) (median radius 52-60 nm). Although enhanced interaction with the SUV membranes was due in part to the radius of curvature and to the greater exposure of acidic phospholipid in the outer leaflet of the bilayer, simply increasing the molar percentage of acidic phospholipid in the LUV membranes had much less effect on SCP(2) binding. A similar preferential interaction was observed with highly curved SUV as opposed to LUV for the SCP(2) N-terminal peptide (1-32)SCP(2) as well as structurally modified peptides in the order (1-32)SCP(2)=(10-32)SCP(2)>(1-24)SCP(2)>>(1-E20-32)SCP(2). The CD results were confirmed with an independent filtration binding assay, which showed that SCP(2) bound 5-fold more to SUV than LUV, whereas its N-terminal peptides bound up to 4-fold better in the order (1-32)SCP(2)=(10-32)SCP(2)>(1-24)SCP(2)>(1-E20-32)SCP(2). Finally, cholesterol potentiated the binding of SCP(2) and N-terminal peptides to anionic-phospholipid-containing SUV but not LUV. These findings were consistent with the SCP(2) N-terminus being a membrane-binding domain that was highly dependent on membrane surface curvature as well as on lipid composition.  (+info)

Lipid-dependent activation of protein kinase C-alpha by normal alcohols. (62/2206)

Significant stimulation of protein kinase C-alpha (PKCalpha) by n-alcohols was observed in characterized lipid systems composed of phosphatidylcholine/phosphatidylserine/dioleoylglycerol (PC/PS/DO). The logarithm of the alcohol concentrations to achieve half-maximal PKC stimulation (ED(50)) and of the maximal PKC stimulation by alcohols were both linear functions of alcohol chain length, consistent with the Meyer-Overton effect. Binding of phorbol esters to PKC was not significantly affected by octanol. Octanol increased, up to 4-fold, the affinity of PKC binding to the lipid bilayers in both the absence and presence of DO. However, octanol increased PKC activity much more significantly than it enhanced binding of the enzyme to the lipid bilayers, suggesting that the stimulation of PKC is not merely a reflection of the increase in PKC bilayer binding affinity. (31)P NMR experiments did not reveal formation of non-lamellar phases with octanol. Differential scanning calorimetry suggested that alcohols, like diacylglycerol, induce formation of compositionally distinct domains and the maximal enzyme activity with alcohol resided roughly in the putative domain-coexistence region. These results suggest that alcohols are mimicking diacylglycerol in activating PKC, not by binding to the high affinity phorbol ester binding site, but by altering lipid structure and by enhancing PKC-bilayer binding.  (+info)

Phosphatidylserine-mediated phagocytosis of anticancer drug-treated cells by macrophages. (63/2206)

Apoptotic cells are rapidly phagocytosed and eliminated from the organism. Although cancer cells apoptose when treated with anticancer drugs, how those cells are recognized by phagocytic cells has remained unclear. The human leukemia cell line Jurkat was cultured with doxorubicin or bufalin and induced to undergo apoptosis accompanied by phosphatidylserine externalization. When apoptotic Jurkat cells were mixed with mouse peritoneal macrophages, efficient phagocytosis was observed. Apoptosis and phagocytosis of Jurkat cells were both inhibited by Z-VAD-FMK, and phagocytosis was significantly reduced in the presence of phosphatidylserine-containing liposomes. These results suggest that anticancer drugs induce apoptosis-dependent and phosphatidylserine-mediated phagocytosis in cancer cells.  (+info)

Antioxidant action of the antiarrhythmic drug mexiletine in brain membranes. (64/2206)

Mexiletine is a class Ib antiarrhythmic drug used in the treatment of ventricular arrhythmias. The Na+ channel blocker mexiletine inhibits calcium influx in cells via decreasing reverse operation of the Na+-Ca2+ exchanger. Thus this drug is shown to protect the CNS white matter against anoxic/ischemic injury. The aim of our study was to investigate if this drug could act as an antioxidant drug as well. The antioxidant action of this drug was studied under different oxidant conditions in vitro, and thiobarbituric acid-reactive substances were measured to follow lipid peroxidation. Mexiletine inhibited iron-ascorbate-H2O2-induced lipid peroxidation in brain membranes, liver microsomes and phospholipid liposomes, being most effective in brain membranes. The inhibition was dose- and time-dependent. Mexiletine also inhibited copper-ascorbate-H2O2-induced lipid peroxidation but to a lesser extent. It is concluded that mexiletine has a dual effect toward oxidative injury in brain, both by inhibiting Na+-Ca2+ exchanger-dependent Ca2+ influx and by acting as an inhibitor of lipid peroxidation. However, as this drug is effective at millimolar concentrations, it should be considered less active than natural antioxidants that are effective at micromolar concentrations.  (+info)