Enhanced phospholipase activity in peripheral blood monocytes from patients with rheumatoid arthritis

Peripheral blood monocytes (PBM) from patients with rheumatoid arthritis (RA) produce greater amounts of prostaglandins (PG) than do control cells. To further explore the reasons for the increased PG production, we assessed the phospholipase activities in these cells. We found that PBM from patients with severe RA expressed greater phospholipase A2 (PLA2) and phospholipase C (PLC) activities than did the control cells. Enhanced PLA2 activities were observed in RA patient cells when phosphatidylcholine (PC) or phosphatidylethanolamine (PE) were used as substrates. Enhanced PLC activities also were seen when PC, PE, and phosphatidylinositol (PI) were used as labeled substrates. Increased PLC activity was observed whether linoleic acid or arachidonic acid was esterified to the 2 position of the phospholipid substrate used. Because all patients with RA were treated with nonsteroidal antiinflammatory drugs, we examined the effects of aspirin ingestion on phospholipase activities. Aspirin had no consistent effect on PLA2 activities but markedly inhibited PLC activities against PC, PI, and PE with arachidonic acid in the R2 position. That aspirin enhanced PLC activities against PC and PI with linoleic acid in the R2 position, suggests that PLC activity may be regulated in part by the R2 fatty acid. Our results indicate that increased phospholipase activities exhibited by PBM from RA patients may help explain the increased PG production by these cells. The increased phospholipase activities in PBM from RA patients do not appear to be due solely to nonsteroidal antiinflammatory drug therapy.     

Mechanisms of amphipath-induced stomatocytosis in human erythrocytes.

We studied stomatocytosis induced in human red blood cells (RBC) by vinblastine and chlorpromazine, monitoring the movements of spin-labeled phosphatidylcholine (PC*) and sphingomyelin (SM*) by electron spin resonance (ESR) spectroscopy. This technique allows determination of the fraction of labeled lipids, respectively, on the external leaflet, on the cytosol face, or trapped in endocytic vacuoles. Both vinblastine and chlorpromazine produce a time- and concentration-dependent stomatocytic shape change, which is paralleled by a shift of approximately 10% to 33% of outer leaflet SM* and PC* inward. Of this amount, 8% to 12% was trapped in endocytic vacuoles and 8% to 19% had flipped to the inner leaflet. Vanadate, while inhibiting the stomatocytosis, did not block the flip of either SM* or PC* to the inner leaflet. To explain the inhibiting effect of vanadate, as well as the adenosine triphosphate (ATP) requirement for drug-induced stomatocytosis, we propose the following model: (1) addition of amphipath partially scrambles the bilayer; and (2) the flop of phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the outer leaflet provides substrate for the aminophospholipid translocase (APLT), which flips back PS and PE inward faster than PC or SM can diffuse outward--thereby producing inner layer expansion or stomatocytosis. This role of APLT accounts for the vanadate inhibition of amphipath stomatocytosis. However, the vanadate effect can be overcome by increasing the amphipath concentration, which at such levels probably passively expands the inner leaflet.     

Identification of residues defining phospholipid flippase substrate specificity of type IV P-type ATPases

Type IV P-type ATPases (P4-ATPases) catalyze translocation of phospholipid across a membrane to establish an asymmetric bilayer structure with phosphatidylserine (PS) and phosphatidylethanolamine (PE) restricted to the cytosolic leaflet. The mechanism for how P4-ATPases recognize and flip phospholipid is unknown, and is described as the "giant substrate problem" because the canonical substrate binding pockets of homologous cation pumps are too small to accommodate a bulky phospholipid. Here, we identify residues that confer differences in substrate specificity between Drs2 and Dnf1, Saccharomyces cerevisiae P4-ATPases that preferentially flip PS and phosphatidylcholine (PC), respectively. Transplanting transmembrane segments 3 and 4 (TM3-4) of Drs2 into Dnf1 alters the substrate preference of Dnf1 from PC to PS. Acquisition of the PS substrate maps to a Tyr618Phe substitution in TM4 of Dnf1, representing the loss of a single hydroxyl group. The reciprocal Phe511Tyr substitution in Drs2 specifically abrogates PS recognition by this flippase causing PS exposure on the outer leaflet of the plasma membrane without disrupting PE asymmetry. TM3 and the adjoining lumenal loop contribute residues important for Dnf1 PC preference, including Phe587. Modeling of residues involved in substrate selection suggests a novel P-type ATPase transport pathway at the protein/lipid interface and a potential solution to the giant substrate problem.     

Trio engagement via plasma membrane phospholipids and the myristoyl moiety governs HIV-1 matrix binding to bilayers

Localization of the HIV type-1 (HIV-1) Gag protein on the plasma membrane (PM) for virus assembly is mediated by specific interactions between the N-terminal myristoylated matrix (MA) domain and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂]. The PM bilayer is highly asymmetric, and this asymmetry is considered crucial in cell function. In a typical mammalian cell, the inner leaflet of the PM is enriched in phosphatidylserine (PS) and phosphatidylethanolamine (PE) and contains minor populations of phosphatidylcholine (PC) and PI(4,5)P₂. There is strong evidence that efficient binding of HIV-1 Gag to membranes is sensitive not only to lipid composition and net negative charge, but also to the hydrophobic character of the acyl chains. Here, we show that PS, PE, and PC interact directly with MA via a region that is distinct from the PI(4,5)P₂ binding site. Our NMR data also show that the myristoyl group is readily exposed when MA is bound to micelles or bicelles. Strikingly, our structural data reveal a unique binding mode by which the 2′-acyl chain of PS, PE, and PC lipids is buried in a hydrophobic pocket whereas the 1′-acyl chain is exposed. Sphingomyelin, a major lipid localized exclusively on the outer layer of the PM, does not bind to MA. Our findings led us to propose a trio engagement model by which HIV-1 Gag is anchored to the PM via the 1′-acyl chains of PI(4,5)P₂ and PS/PE/PC and the myristoyl group, which collectively bracket a basic patch projecting toward the polar leaflet of the membrane.     

Altered plasma membrane phospholipid organization in Plasmodium falciparum-infected human erythrocytes

The intraerythrocytic development of the malaria parasite is accompanied by distinct morphological and biochemical changes in the host cell membrane, yet little is known about development-related alterations in the transbilayer organization of membrane phospholipids in parasitized cells. This question was examined in human red cells infected with Plasmodium falciparum. Normal red cells were infected with strain FCR3 or with clonal derivatives that either produce (K+) or do not produce (K-) knobby protuberances on the infected red cells. Parasitized cells were harvested at various stages of parasite development, and the bilayer orientation of red cell membrane phospholipids was determined chemically using 2,4,6-trinitrobenzene sulphonic acid (TNBS) or enzymatically using bee venom phospholipase A2 (PLA2) and sphingomyelinase C (SMC). We found that parasite development was accompanied by distinct alterations in the red cell membrane transbilayer distribution of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Increases in the exoplasmic membrane leaflet exposure of PE and PS were larger in the late-stage parasitized cells than in the early-stage parasitized cells. Similar results were obtained for PE membrane distribution using either chemical (TNBS) or enzymatic (PLA2 plus SMC) methods, although changes in PS distribution were observed only with TNBS. Uninfected cohort cells derived from mixed populations of infected and uninfected cells exhibited normal patterns of membrane phospholipid organization. The observed alterations in P falciparum-infected red cell membrane phospholipid distribution, which is independent of the presence or absence of knobby protuberances, might be associated with the drastic changes in cell membrane permeability and susceptibility to early hemolysis observed in the late stages of parasite development.     

The effect of phospholipase digestion upon the boundary lubricating ability of synovial fluid.

OBJECTIVE: To identify the boundary lubricant in synovial fluid (SF). Is synovial lubrication mediated by surface active phospholipid as opposed to mucinous glycoprotein? METHODS: A sonicated preparation of phosphatidylcholine and bovine SF were tested in vitro in a bearing of latex oscillating against polished glass under a load of 0.35 x 10(6) N/m2. The friction apparatus isolates conditions of boundary lubrication and has been validated against a cartilaginous bearing. Coefficient of friction (mu) was measured and compared against mu from physiologic saline, which served as a control. Separate digestions were carried out upon the SF with trypsin, phospholipase C, and phospholipase A2 in the presence and absence of proteolytic inhibitors. RESULTS: Digestions of bovine SF by phospholipase C in the presence of protease inhibitors did not remove boundary lubricating ability compared to an undigested control (p = 0.89). Digestion of bovine SF with trypsin removed all lubricating ability and raised friction (p = 0.004). Commercial purified phospholipase C contained trypsin-like activity when activity was tested with N alpha-benzoyl-L-arginine ethyl ester as substrate. Similar results were observed for phospholipase A2, which possesses a lower amount of trypsin activity. CONCLUSION: The results indicate that phospholipid does not play a prominent role in synovial fluid's ability to lubricate an artificial bearing. Rather, the boundary lubricating ability of SF is attributable to lubricin, a mucinous glycoprotein.     

红花总黄素对衰老模型小鼠肝线粒体的保护作用

目的　探讨小鼠肝线粒体膜磷脂组成的随龄变化及红花总黄素 (SY)对肝线粒体膜的保护作用。方法　采用HPLC法测定线粒体膜PC、PE、CL的含量 ,陈思峰法测定PLA2 活性 ,原子吸收法测定线粒体Ca2 含量。结果　衰老模型组PC、PE、CL均明显降低 ;PLA2 活性随增龄而升高 ;线粒体Ca2 含量中年组较青年组升高 (P <0 .0 5) ,而衰老模型组Ca2 含量显著低于中年组 (P <0 .0 5)。SY可升高衰老模型组PC、PE、CL含量和线粒体Ca2 含量 (P <0 .0 1 ) ;而显著降低衰老模型组PLA2 活性。结论　SY可显著改善衰老模型组肝线粒体膜磷脂的组成 ,有效维持钙稳态     

Study on membrane fluidity and erythrocyte aggregation in equine, bovine and human species

The aim of the present paper is to analyze whether membrane fluidity can be predicted from its lipid composition and to assay the possible relationship between such variable and the aggregating properties of erythrocytes from equine, bovine and human species due to the widely acknowledged differences in their tendency to form aggregates. The main difference between phospholipids from plasma membrane in these species lies in the concentration levels of sphyngomyelin (SM) and phosphatidilcoline (PC); more precisely, in the external hemilayer of the lipid bilayer. Membrane fluidity was estimated by the fluorescence polarization method, while erythrocyte aggregation was assessed by an optical method. According to our results, bovine erythrocytes containing high SM and low PC levels, presented the highest anisotropy value as well as an imperceptible aggregation value. Equine erythrocytes, which contain a considerable PC percentage and scarce SM levels, showed the lowest anisotropy value and the highest values of the aggregation parameters. Human erythrocytes presented intermediate values for both properties. Our hypothesis claims that the phospholipid composition would constitute one of the factors determining erythrocyte membrane fluidity and also taking part in the different aggregation tendency shown by equine, bovine and human species.     

The molecular species composition of phosphatidylcholine affects cellular properties in normal and sickle erythrocytes

The phosphatidylcholine specific transfer protein (PCTP) from bovine liver was used to retailor the molecular species composition of phosphatidylcholine (PC) in the membrane of normal (AA) and sickleable (SS) human erythrocytes. Changes in molecular species composition of PC altered morphology as well as cellular deformability and stability as measured with ektacytometry. In normal cells, replacement of native PC with 1-palmitoyl,2-arachidonoyl PC (PAPC) resulted in a decrease in osmotic fragility with no change in hydration, whereas replacement with 1,2-dipalmitoyl PC (DPPC) led to an increased osmotic fragility and cellular hydration. Replacement of native PC by 1-palmitoyl,2-oleoyl PC (POPC) in normal cells had no apparent effect on these parameters. In contrast, replacement of native PC in sickle cells with either PAPC, DPPC or POPC led to cellular hydration. Facilitation of PC exchange between subpopulations of SS cells separated on buoyant density also led to cellular hydration. These observations suggest that the state of hydration of sickle cells can be modified by the fatty acyl composition of PC and illustrate a a role for the lipid core in the observed permeability changes in sickle erythrocytes. They also raise the interesting possibility that the state of cellular hydration of sickle cells may be modulated by altering the molecular species composition of the membrane phospholipids.     

Hereditary Hemolytic Anemia Associated With Abnormal Membrane Lipids: Mechanism of Accumulation of Phosphatidyl Choline

In order to study the mechanism of theaccumulation of phosphatidyl choline(PC) in erythrocytes with abnormal erythrocyte phospholipids from patientswith a hereditary hemolytic anemia, thephospholipids of the erythrocytes werelabeled radioactively. Labeling of phosphatides was achieved by both passiveequilibration with preformed phosphatides, and active "acylase"-mediated incorporation of fatty acid (FA) in thepresence of glucose, ATP and coenzymeA. The labeled cells were then reincubated in fresh compatible sera and thecatabolism of the labeled erythrocytephospholipids was followed. In addition,total acylase capacity of erythrocytestroma was determined under optimalconditions in a system with excesslysophosphatide, FA, ATP, CoA, andMg⁺⁺. No differences in passive uptakeor release of phosphatides were found between the patients’ erythrocytes andcomparable reticulocyte-rich controls. Onthe other hand, overall active incorporation of FA into PC was abnormally increased in the patients’ erythrocytes,whereas incorporation of FA intophosphatidyl-ethanolamine (PE) was decreased. However, acylase capacity forboth lysophosphatidylcholine (LPC) andlysophosphatidylethanolamine (LPE) wasnormal in the patients’ cells. This apparent paradox could be explained by thesubsequent turnover of actively incorporated PC-FA which was found to bereduced. A brief labeling experiment designed to approximate pulse-chase conditions and to label primarily PC showed aconsiderable inhibition of the subsequenttransfer of PC-FA to PE upon reincubation in fresh serum. This transfer haspreviously been shown to be responsiblefor a significant portion of PC-FA catabolism. Reincubation in hyperlipemicsera obtained from patients with liverdisease or artificially enriched with PCdid not influence the abnormal outflow ofphosphatide-FA in actively labeled cells.The findings were consistent with theconcept that PC accumulated in thesecells because of a defect in the catabolismof actively incorporated PC-FA. This defect appeared to be in the transfer ofPC-FA to PE prior to final release fromthe cell. Passive exchange pathways andthe active anabolic acylase pathway werenot abnormal in these patients’ erythrocytes.

Submitted on March 4, 1971 Revised on May 4, 1971 Accepted on May 10, 1971     

The effect of glycemic control and duration of diabetes on cholesterol and phospholipid classes in erythrocytes of type I diabetes.

Abnormalities in the lipid composition of erythrocytes can alter blood rheology and viscosity. These alterations have been implicated in the pathogenesis of microvascular disease in diabetic patients. The present study was undertaken to examine whether or not long-term glycemic control or duration of diabetes has any role in the altered membrane cholesterol and phospholipid composition of erythrocytes in type I diabetes. Long-term glycemic control was assessed by measuring glycosylated hemoglobin (GHb) from diabetic patients and age-matched normal volunteers. There was no significant correlation between GHb or duration of diabetes with total cholesterol, phospholipid, and cholesterol to phospholipid molar ratios in erythrocytes of these patients. Among phospholipid classes, GHb showed a significantly negative relationship with sphingomyelin (SM) (r = .55, P less than .01) levels, but was not related to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) levels of erythrocytes. Duration of diabetes had no effect on SM, PC, or PE levels of erythrocytes.     

Studies on heterophile antibodies in rheumatoid arthritis

Sera and synovial fluids of patients with rheumatoid arthritis were studied for the presence of heterophile antibodies to sheep and bovine erythrocytes by means of hemolysis in agar gel. It was demonstrated that 18 of 146 sera had hemolytic antibody titers of 160 or more; all 18 (12%) against sheep and 8 (6%) against bovine erythrocytes. Of 31 synovial fluids examined, 5 showed hemolysin titers of 40 or more; all 5 (16%) against sheep and 3 (10%) against bovine erythrocytes. These heterophile antibodies were shown to belong to IgM and/or IgG class. Absorption and inhibition studies revealed that antibodies of 10 positive sera and 2 synovial fluids were of Forssman specificity and antibodies of 6 sera and 3 synovial fluids were of Hanganutziu-Deicher specificity. Two remaining sera were shown to contain a mixture of Forssman antibodies and immune anti-B antibodies.     

Interleukin-1-Mediated phospholipid breakdown and arachidonic acid release in human synovial cells

Objective. Interleukin-1 (IL-1), an important mediator contributing to joint destruction in rheumatoid arthritis, is known to stimulate the release of arachidonic acid (AA) and prostaglandin E₂ (PGE₂) from adherent synoviocytes. To study the intracellular pathways involved in these functions, we stimulated cultures of human synovial cells with recombinant IL-1β. Methods. AA liberation was measured after labeling synovial cells with ³H-AA, and PGE₂ levels were determined by high performance liquid chromatography or radioimmunoassay. Identification of ³H-AA-labeled phospholipids was performed by thin layer chromatography. Cell-associated phospholipase A₂ (PLA₂) enzymatic activity was determined by an assay with cell-free systems and exogenous substrates. Results. Stimulation of synovial cells with recombinant IL-1β induced a decrease in phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylethanolamine (PE), and a marked increase in cell-associated PLA₂ activity as compared with controls. In the presence of either quinacrine, an inhibitor of PLA₂ pathway activation, or neomycin, which binds to PI mono- and biphosphate thus blocking their degradation by phospholipases, AA and PGE₂ secretion were reduced in a dose-dependent manner. Kinetic studies revealed that quinacrine had little blocking activity on the IL-1-mediated AA release after 1 hour of stimulation but completely abolished it after 5 or 8 hours. In contrast, neomycin exerted a partial but significant inhibitory effect from the first hour of stimulation onward. Addition of quinacrine was also demonstrated to abolish the IL-1-induced hydrolysis of PC and PE but not PI, indicating that PC and PE are the preferred substrates for PLA₂ enzymatic activity in human synovial cells. Conclusion. Our findings strongly suggest that AA and PGE₂ production by IL-1-triggered synoviocytes are largely dependent upon PLA₂-mediated hydrolysis of PC and PE and to a lesser extent upon the earlier degradation of PI.     

Involvement of the phospholipase C second messenger system in the regulation of steroidogenesis in small bovine luteal cells

We have previously suggested that the interaction between luteinizing hormone (LH) and its receptor, in addition to stimulating adenylate cyclase, is able to trigger a negative regulatory signal at a step beyond cAMP synthesis (Benhaim et al. (1987) FEBS Lett. 223, 321-326). The present study was conducted to determine whether the phospholipase C system is involved in this phenomenon. Small bovine luteal cells from pregnant cows were incubated with phospholipase C, A23187, an ionophore of calcium and/or phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), in the presence or absence of bovine luteinizing hormone or dibutyryl cyclic AMP (dbcAMP). A23187 associated with PMA was able to mimic the stimulatory effect of phospholipase C on basal progesterone production, whereas neither A23187 nor PMA alone had any effect. In the presence of high doses of LH, phospholipase C inhibited progesterone and cAMP production in a dose-dependent manner. A23187 and PMA were able to mimic the inhibition of progesterone synthesis but stimulated LH-induced cAMP accumulation. When cells were stimulated by high doses of dbcAMP, phospholipase C and A23187 but not PMA inhibited progesterone synthesis. These observations suggest that (1) phospholipase C can mimic the post-cAMP negative regulatory signal induced in vitro by high doses of LH, in the presence of an activation of PKC; (2) phospholipase C is also able to mimic in vitro the luteolytic properties of prostaglandin F2 alpha that we previously described (Benhaim et al. (1987) Prostaglandins 33, 227-239); and (3) under basal conditions or in the presence of low doses of LH, the phospholipase C system slightly stimulates steroidogenesis.     

Thrombin infusion in endotoxin-treated rabbits reduces the plasma levels of plasminogen activator inhibitor: evidence for a protein-C- mediated mechanism [see comments]

Plasminogen activator inhibitors (PAIs) play a pivotal role in the control of fibrinolysis. The mechanisms regulating the plasma levels of PAI(s) are still unknown. We report here that the infusion of bovine thrombin (1 U/kg/min, over 60 minutes) in rabbits treated with 0.5 microgram/kg endotoxin (to induce an increase in circulating fast- acting PAI) causes a marked reduction of PAI (50% of preinfusion value), as indicated by functional assay and reverse fibrin autography. Moreover, blood clots prepared from samples obtained after thrombin infusion lysed faster than preinfusion clots when exposed, in vitro, to tissue plasminogen activator. Donor-receiver transfusion experiments showed that the half-life of circulating PAI activity was shorter in thrombin-infused rabbits than in controls (4.1 minutes versus 7.4 minutes), suggesting an accelerated clearance. As expected, thrombin infusion resulted also in activation of protein C (PC). The following observations suggest a close relationship between PC activation and PAI reduction. (1) Infusion of thrombin in rabbits made deficient in vitamin K-dependent plasma proteins by warfarin treatment did not result in modification of PAI activity. (2) Treatment of the latter animals with a barium citrate eluate (PE) of rabbit plasma restored both the anticoagulant and profibrinolytic response to thrombin. (3) Short infusion of thrombin-activated PE (containing activated PC, PCa), but not of unactivated PE, induced both anticoagulation and reduction of PAI activity. In vitro, incubation of PAI-rich rabbit serum with thrombin-activated PE and phospholipids resulted in a progressive disappearance of PAI activity with a t1/2 of 30 minutes. However, this slow inactivation rate does not fully explain the results obtained in vivo. Our data suggest that thrombin infusion in rabbits causes a reduction of circulating PAI activity and that activation of PC is the intermediary mechanism involved in this phenomenon.     

The differential processing of proenkephalin A in mouse and human breast tumour cell lines.

We have carried out an investigation into the processing of the enkephalin-like immunoreactivity reported in breast tissue using two human breast tumour cell lines and a mouse tumour cell line. A 46 kDa form of proenkephalin (PE) has been observed in the cell lysates of two human breast tumour cell lines (MCF-7, ZR-75-1) and the mouse androgen-responsive Shionogi breast carcinoma cell line (SC115). PE processing in the cell lysates of these cells was assessed by a specific met-enkephalin RIA. The basal levels of processed PE in the MCF-7, ZR-75-1 and SC115 cell lysates were 30, 30 and 76% respectively. The processing enzymes PC1 and PC2, which have been implicated in the differential processing of PE, were detected by immunoblot analysis in these cells. PC1 was found within the cell extracts of all three cell lines. PC2 was only observed in the SC115 cell line, which may account for the higher percentage of processed PE measured. The cDNA of PC2 has been transfected into ZR-75-1 cells and this was accompanied by an increase in the level of processed PE from 30 to 76%. These breast tumour cell lines may provide a useful insight into the function of enkephalin-containing peptides in breast cancer.     

Cellular responses to Pyrularia thionin are mediated by Ca2+ influx and phospholipase A2 activation and are inhibited by thionin tyrosine iodination.

Pyrularia thionin, isolated from nuts of Pyrularia pubera, is a strongly basic peptide of 47 amino acids. The amino acid sequence and configuration of its four disulfide bonds place this plant peptide, known to be hemolytic, cytotoxic, and neurotoxic, among the thionins. We report and compare several cellular responses mediated by Pyrularia thionin: hemolysis of human erythrocytes, activation of an endogenous phospholipase A2 in Swiss 3T3 cells, cytotoxicity toward HeLa and mouse B16 melanoma cells in culture, viability of rat hepatocytes and lymphocytes measured by trypan blue exclusion, and lethality in mice. Cellular responses related to ion movement include a toxin-mediated influx of Ca2+ into mouse P388 cells measured by Fura-2 fluorescence, depolarization of mouse P388 plasma membrane measured by fluorescence of bis(1,3-diethylthiobarbituric acid)trimethine oxonol (bisoxonol), and depolarization of frog (Rana pipiens) sartorius muscle determined by direct measurement of membrane potential. Graded iodination of Pyrularia thionin leads to a related loss of activity for hemolysis, phospholipase A2 activation, cytotoxicity, and lethality in mice. The mediated Ca2+ influx into and depolarization of P388 cells require Ca2+ in the external medium and are inhibited by 100 microM Ni2+. Depolarization of sartorius muscle by Pyrularia thionin also requires a functional Ca2+ channel, as shown by verapamil inhibition. This muscle depolarization also involves phospholipase A2 activation because dexamethasone and quinacrin, but not indomethacin, protect against depolarization. The IC50 values for viability of rat hepatocytes and splenic lymphocytes measured by trypan blue exclusion were 0.17 and 40 microM, respectively. The general response of cells to Pyrularia thionin involves a membrane alteration leading to depolarization and a channel-mediated influx of Ca2+. There is a related activation of phospholipase A2 that results in loss of membrane integrity, hemolysis in the case of erythrocytes, and eventually cell death. Iodination of Pyrularia thionin leads to a corresponding inhibition of all three cellular responses, which indicates an essential role for tyrosine in either maintenance of peptide structure or interaction of the peptide with cellular membranes.     

Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function

Aminophospholipid translocases (APLTs) are defined primarily by their ability to flip fluorescent or spin-labeled derivatives of phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external leaflet of a membrane bilayer to the cytosolic leaflet and are thought to establish phospholipid asymmetry in biological membranes. The identities of APLTs remain unknown, although candidate proteins include the Drs2p/ATPase II subfamily of P-type ATPases. Drs2p from budding yeast localizes to the trans-Golgi network (TGN), and here we show that this membrane contains an ATP-dependent APLT that flips 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) PS and PE derivatives from the luminal to the cytosolic leaflet. To assess the contribution of Drs2p to this activity, TGN membranes were prepared from strains harboring WT or temperature-sensitive alleles of DRS2 and null alleles of three other potential APLT genes (DNF1, DNF2, and DNF3). Assay of these membranes indicated that Drs2p was required for the ATP-dependent translocation of NBD-PS, whereas no active translocation of NBD-PE or NBD-phosphatidylcholine was detected. The specificity of Drs2p for NBD-PS suggested that translocation of PS would be required for the function of Drs2p in protein transport from the TGN. However, cho1 yeast strains that are unable to synthesize PS do not phenocopy drs2 but instead transport proteins normally via the secretory pathway. In addition, a drs2 cho1 double mutant retains drs2 transport defects. Therefore, whereas NBD-PS is a preferred substrate for Drs2p in vitro, endogenous PS is not an obligatory substrate in vivo for the role Drs2p plays in protein transport.     

Inositol phospholipid-specific phospholipase C: complete cDNA and protein sequences and sequence homology to tyrosine kinase-related oncogene products.

Antibodies against an inositol phospholipid-specific phospholipase C purified from bovine brain were used to screen rat brain lambda gt11 expression cDNA libraries. Complete sequences of three cDNA inserts yielded a cumulative sequence of 5106 base pairs. The deduced protein had 1289 amino acids with a calculated molecular weight of 148,431. The determination of an open reading frame was aided by the amino acid sequences of 21 tryptic peptides isolated from bovine brain phospholipase C. Only 9 residues of a total of 140 amino acid residues determined for the bovine enzyme were different from those deduced from the rat cDNA. Two regions of phospholipase C (amino acid residues 555-598 and 668-705) exhibited significant amino acid similarities to the products of various tyrosine kinase-related oncogenes (yes, src, fgr, abl, fps, fes, and tck). The homologous domain was located in the region that is not essential for the protein-tyrosine kinase activity but is likely to be involved in an interaction with cellular components that modulate kinase function. Therefore, this unexpected similarity raises the possibility that the 148-kDa phospholipase C and cytoplasmic tyrosine kinases are modulated by common cellular component(s).     

Alterations in sensitivity to calcium and enzymatic hydrolysis of membranes from sickle cell disease and trait erythrocytes

Normally, human erythrocytes display several responses to elevated intracellular calcium levels. These include a shape transition from discocyte to spherocyte, shedding of microvesicles into the extracellular fluid, and enhanced susceptibility to the hydrolytic action of secretory phospholipase A(2). These responses to elevated intracellular calcium were all blunted in erythrocytes containing hemoglobin S. The reduction of both the shape transition and the shedding of microvesicles were greater than the impairment of phospholipase susceptibility, and both correlated strongly with the intracellular content of hemoglobin S. In contrast to the response to elevated intracellular calcium, erythrocytes containing hemoglobin S displayed a 2.5-fold increase in basal susceptibility to phospholipase A(2) compared to control erythrocytes in the absence of ionophore. The effect was more prominent among samples from patients heterozygous for hemoglobin S than in samples from homozygous individuals. These results reveal additional abnormalities in the membranes of sickle cell erythrocytes beyond those described previously and demonstrate that red blood cells from both heterozygous and homozygous are affected. Furthermore, they suggest a possible means by which sickle cell disease and trait patients may display enhanced vulnerability to inflammatory stimuli.