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PubMed1986

Influence of buffer composition, membrane lipids and proteases on the kinetics of reconstituted cytochrome-c oxidase from bovine liver and heart.

Isolated cytochrome-c oxidases from bovine heart and liver were reconstituted in liposomes with asolectin and the kinetics of cytochrome c oxidation were measured under various uncoupled conditions. With 40 mM KCl, 10 mM Hepes, pH 7.4, the liver enzyme showed a higher Vmax in the polarographic but a lower Vmax in the photometric assay. With 125 mM phosphate buffer at pH 6.0 both enzymes revealed identical kinetics. Reconstitution with pure phosphatidylcholine leads to a low activity, which is specifically stimulated for the heart enzyme by inclusion of 10% cardiolipin. Proteoliposomes of both enzymes prepared with asolectin have a high activity, which is unaffected by cardiolipin. Exchanging the intraliposomal buffer, Hepes, for phosphate causes an opposite change of the Vmax and a similar change of the Km for both enzymes suggesting a conformational change of the extraliposomal binding domain for cytochrome c through the membrane. Proteases change the kinetics of both enzymes, but to a different degree. The data indicate a complex and tissue-specific influence of nucleus-coded subunits on the catalytic activity of cytochrome-c-oxidase.

Animals↗
PubMed1994

Nitric oxide reductase from Pseudomonas stutzeri, a novel cytochrome bc complex. Phospholipid requirement, electron paramagnetic resonance and redox properties.

The nitric oxide reductase (NOR) from Pseudomonas stutzeri is a cytochrome bc complex which shows on SDS/PAGE two subunits with apparent molecular masses of 17 kDa and 38 kDa. Two other species of approximately 45 kDa and 74-78 kDa represent the undissociated enzyme complex and an aggregate of the cytochrome b subunit, respectively. The cytochrome b subunit is highly hydrophobic and results in aberrant electrophoretic mobility. The stability of the enzyme in various detergents and at different pH was investigated. The highest specific activity of 60 mumol NO min-1 mg-1 protein was obtained after electrophoresis in the presence of laurylpropanediol-3-phosphorylcholine ether. Purified NOR contained cardiolipin, phosphatidylglycerol, and phosphatidylethanolamine, the latter as the major component. A phospholipid was required for high catalytic activity with either cardiolipin or phosphatidylglycerol increasing the activity of the enzyme as isolated by a factor of up to 5. Free fatty acids inhibited NOR, with cis-9-octadecenoic acid (oleic acid) showing the most pronounced effect. Certain detergents substituted for the phospholipid requirement of NOR. The enzyme, as isolated, in 0.1% Triton X-100, 20 mM Tris/HCl pH 8.5, exhibited a complex set of EPR resonances at low magnetic field, with a prominent peak at g 6.34 resulting from Fe(III) high-spin cytochrome b. The second prominent feature arose from a low-spin Fe(III) heme center with strong lines at apparent g values of 3.02 and 2.29, and a broad resonance at g approximately 1.5 which we assigned to the cytochrome c component of the enzyme. From spin quantitation and computer simulations of the various EPR signals a ratio close to 1:1 for the low-spin/high-spin heme centers in NOR was estimated. Shifting the pH from 8.5 to 5.0, replacing Triton X-100 by other detergents, or adding soybean phospholipids to the protein, led to pronounced changes of the EPR signals in the g = 6 region. In contrast, the strong inhibitor oleic acid did not cause significant spectral changes. NOR which had been reduced by L-ascorbate/phenazine methosulfate prior to incubation with its substrate NO gave the characteristic Fe(II) nitrosyl triplet centered at g approximately 2.01, with a hyperfine splitting of 1.70 mT. In the absence of dioxygen, NOR was quantitatively reduced by either sodium dithionite, or photochemically with deazaflavin and oxalate; the enzyme was reoxidizable by ferricyanide in a fully reversible reaction. Spectroelectrochemical oxidoreductive titrations gave E'o (versus standard hydrogen electrode) = +322 mV for the cytochrome b and +280 mV for the cytochrome c component.

Chromatography, Gel↗
PubMed1996

Clinical associations of anti-lamin autoantibodies.

BACKGROUND: The clinical associations of anti-lamin autoantibodies were first described in 1973. Since then a number of individual case reports and two small series have been published. These have suggested an association with connective tissue disorders and autoimmune liver disease. AIMS: To identify the clinical and laboratory associations of anti-lamin autoantibodies in an Australian population. METHODS: Retrospective review of routine antinuclear antibody testing between 1990-1994 for characteristics linear staining of nuclear envelope on indirect immunofluorescence on HEp-2 cells with clinical status defined by retrospective review of case records. RESULTS: Twenty-eight patients were identified and the clinical status of 27 patients defined. Eleven patients had associated IgG anti-cardiolipin antibodies; anti-phospholipid syndrome was present in nine. Seven further patients had liver disease; five had autoimmune liver disease, with associated autoantibodies. The remaining nine patients had a diverse group of diseases. There was no correlation between the titre of the autoantibody and clinical status. An association with anti-cardiolipin antibodies was found although the cause remains obscure. CONCLUSION: Anti-lamin autoantibodies, as identified by indirect immunofluorescence, are associated with a diverse group of diseases but particularly with anti-phospholipid syndrome and liver disease. Testing for anti-phospholipid antibodies and more specific markers of systemic lupus erythematosus and autoimmune disease, for example anti-dsDNA antibodies, anti-smooth muscle antibodies and anti-mitochondrial antibodies should be pursued when anti-lamin autoantibodies are detected.

Adult↗
PubMed1992

Lipid composition in synaptic and nonsynaptic mitochondria from rat brains and effect of aging.

The cholesterol, phospholipid, and fatty acid compositions in synaptic and nonsynaptic mitochondria from rat brains and the effect of aging were studied. Both cholesterol and phospholipid contents were found to be significantly different in synaptic compared to nonsynaptic mitochondria. In both types of brain mitochondria, aging decreases the cholesterol content by 27% and the phospholipid content by approximately 12%. The difference between these decreases observed in the organelles causes decreases in the cholesterol/phospholipid molar ratios for synaptic and nonsynaptic mitochondria of 17 and 19%, respectively. Also, the phospholipid composition is significantly different in synaptic compared to nonsynaptic mitochondria. Among phospholipids, only the cardiolipin fraction showed a significant decrease (26%) in nonsynaptic mitochondria from the brains of aged rats. Instead, the fatty acid composition was not significantly different in synaptic compared to nonsynaptic mitochondria. The 21% aging decrease in linoleic acid (18:2), observed only in nonsynaptic mitochondria, may be related to a decrease in cardiolipin, which contains a large amount of this fatty acid.

Aging↗
PubMed2005

Hepatitis C-associated autoimmunity in patients coinfected with HIV.

BACKGROUND: Hepatitis C virus (HCV) infection is associated with multiple extrahepatic manifestations. It is unclear to what extent extrahepatic manifestations occur in HIV/HCV coinfection. METHODS: We prospectively assessed cross-sectional frequencies of autoimmune manifestations in HIV/HCV-coinfected patients (n=98), HIV-mono-infected (n=45) and HCV-mono-infected patients (n=78). Diagnostic vasculitis scores, HCV and HIV loads, CD4 cell counts, thyroid-, cardiolipin-, non-organ-specific tissue antibodies (nuclear, smooth muscle, anti-liver-kidney-microsome, neutrophil-cytoplasmic) and cryoglobulins were determined. RESULTS: Synergistic effects of HCV and HIV infection were observed with respect to the prevalence of antibodies against thyroglobulin (HCV infection 15.4%, HIV infection 8.8%, HIV/HCV coinfection 30.6%; P<0.001) and cardiolipin antibodies (HCV infection 9.0%, HIV infection 31%, HIV/HCV coinfection 46%; P<0.001). Cryoglobulinemia type III, was significantly associated with HCV infection (HCV, 25.6%; HIV/HCV, 20.4%) but not with HIV infection (4.4%, P<0.05). Rheumatoid factor was commonly detected in patients with HCV infection (48%), but occurred considerably less frequently in patients with HIV infection (4.4%) or HIV/HCV coinfection (9.5%, P<0.01). CONCLUSION: HIV coinfection appears to differentially modulate the frequency of HCV-related autoimmunity. However, autoimmunity is rarely accompanied by clinical manifestations.

Adult↗
PubMed1988

Antinuclear autoantibodies in sera of healthy pregnant women and their offspring.

The presence of autoantibodies in the fetus has previously been investigated in the offspring of mothers with autoimmune diseases, but not in the offspring of healthy pregnant women. Employing the ELISA method, we examine four SLE-associated auto-antibodies (anti-dsDNA, anti-ssDNA, anti-poly(I), and anti-cardiolipin) in sera obtained from 196 healthy pregnant women and their offspring. All detected autoantibodies in maternal and blood cord sera were of the IgM isotype. Thirty-four maternal sera (17.3%) were positive for one or more tested autoantibodies: 16 (8.1%) for one autoantibody alone, 17 (8.6%) for two autoantibodies, and one (0.5%) for three autoantibodies; 1.5%, 5.6%, 9.6%, and 10.2% of the maternal sera were positive for autoantibodies against dsDNA, ssDNA, poly(I), and cardiolipin, respectively. In only one blood cord serum sample was an autoantibody (IgM anti-ssDNA) detected. This infant was born to a healthy seronegative mother. The finding that all offspring of IgM seropositive mothers are IgM seronegative is not surprising since maternal IgM autoantibodies do not cross the placenta. The finding of an IgM seropositive infant born to an IgM seronegative mother may indicate that the fetus is capable of self-production of autoantibodies.

Adult↗
PubMed1995

Anticardiolipin antibodies in patients with pregnancy loss induce factor Xa production in the presence of beta 2-glycoprotein I.

PROBLEM: Anticardiolipin antibodies (aCL) are commonly associated with recurrent pregnancy loss, though the mechanism is uncertain. Some investigators have indicated that aCL may be directed at a complex made up of cardiolipin and a blood anticoagulant, beta 2-glycoprotein I (beta 2GPI). We therefore investigated the effects of beta 2GPI-dependent aCL IgG enriched fractions, isolated from sera of patients with pregnancy losses, on blood coagulation. METHOD: beta 2GPI-dependent aCL were prepared from sera of three women with second trimester pregnancy losses, by cardiolipin affinity column chromography, following by anti-beta 2GPI affinity column chromatography. The effects of beta 2GPI and beta 2GPI-dependent aCL on the activation of factor X in vitro were examined. RESULTS: beta 2GPI inhibited the activation of factor X and beta 2GPI-dependent aCL blocked this inhibitory effect in a dose dependent manner. CONCLUSION: These results imply the possibility of beta 2GPI-dependent aCL induce hypercoagulation or thrombus by blocking the inhibitory effect of beta 2GPI on activation of factor X, which may result in pregnancy loss.

Abortion, Habitual↗
PubMed1998

Mitochondrial decay in aging. Reversal through supplementation of acetyl-L-carnitine and N-tert-butyl-alpha-phenyl-nitrone.

We show that mitochondrial function in the majority of hepatocytes isolated from old rats (24 mo) is significantly impaired. Mitochondrial membrane potential, cardiolipin levels, respiratory control ratio, and overall cellular O2 consumption decline, and the level of oxidants increases. To examine whether dietary supplementation of micronutrients that may have become essential with age could reverse the decline in mitochondrial function, we supplemented the diet of old rats with 1% (w/v) acetyl-L-carnitine (ALCAR) in drinking water. ALCAR supplementation (1 month) resulted in significant increases in cellular respiration, mitochondrial membrane potential, and cardiolipin values. However, supplementation also increased the rate of oxidant production, indicating that the efficiency of mitochondrial electron transport had not improved. To counteract the potential increase in oxidative stress, animals were administered N-tert-butyl-alpha-phenyl-nitrone (30 mg/kg) (PBN) with or without ALCAR. Results showed that PBN significantly lowered oxidant production as measured by 2,7'-dichlorofluorescin diacetate (DCFH), even when ALCAR was coadministered to the animals. Thus, dietary supplementation with ALCAR, particularly in combination with PBN, improves mitochondrial function without a significant increase in oxidative stress.

Acetylcarnitine↗
PubMed2005

Role of mitochondria in toxic oxidative stress.

Oxidative stress and mitochondrial oxidative damage have been implicated in the etiology of numerous common diseases. The critical mitochondrial events responsible for oxidative stress-mediated cell death (toxic oxidative stress), however, have yet to be defined. Several oxidative events implicated in toxic oxidative stress include alterations in mitochondrial lipids (e.g., cardiolipin), mitochondrial DNA, and mitochondrial proteins (eg. aconitase and uncoupling protein 2). Furthermore, recent findings indicate the enrichment of mitochondrial membranes with vitamin E protects cells against the toxic effects of oxidative stress. This review briefly summarizes the role of these mitochondrial events in toxic oxidative stress, including: 1) the protective role of mitochondrial vitamin E in toxic oxidative stress, 2) the role of mitochondrial DNA in toxic oxidative stress, 3) the interaction between cardiolipin and cytochrome c in mitochondrial regulation of apoptosis, 4) the role of mitochondrial aconitase in oxidative neurodegeneration, and 5) the role of mitochondrial uncoupling protein 2 in the pathogenesis of type 2 diabetes.

Aconitate Hydratase↗
PubMed1975

Extracellular lipids of Cladosporium (Amorphotheca) resinae grown on glucose or on n-alkanes.

Cladosporium (Amorphotheca) resinae was grown in shake culture on glucose, n-dodecane, or n-hexadecane. Growth was most rapid on glucose, and more acid accumulated in the medium than in n-alkane-grown cultures. Neutral lipid was the major lipid fraction and triglycerides were the only extracellular neutral lipids detected. Dodecanoic (lauir) acid was the predominant fatty acid (greater than 60%) in neutral lipids from all three media, with lesser amounts of tetradecanoic, hexadecanoic, and octadecanoic acids. Extracellular phospholipids identified were phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and cardiolipin or a cardiolipin-like compound. Phospholipids from all three media contained dodecanoic acid as their principle fatty acid. Dodecanoic acid was the only extracellular free fatty acid detected. Glucose medium contained acetic, glyoxylic, and glycolic acids and an unidentified organic acid which may contribute to the lower pH in cultures after growth on glucose. In all classes of extracellular lipids the fatty acids do not correspond to the fatty acids previously determined to be associated with cellular lipids. Moreover, the fatty acids of extracellular lipids do not reflect the chain length of the n-alkane growth substrate.

Acetates↗
PubMed1995

Studies of the specificity and cross-reactions of antibodies to lipid A found in juvenile arthritis.

This work was started to determine whether the immunoglobulin G (IgG) reactions with monophosphoryl lipid A (MPL) found in children with arthritis were due to contaminants, a specific site on lipid A, or polyspecific binding. Different lots of MPL were examined by electrophoresis and immunoblot. Competitive inhibition of enzyme-linked immunosorbent assays (ELISAs) by analogs of MPL and biologic materials of clinical interest was used to determine the specificity of the binding site and potential cross-reactions. IgG in all patient sera tested reacted with a single band just < 6.5 kDa on immunoblots of all lots of MPL tested. The ELISAs were inhibited best by analogs of lipid A with an exposed diglucosamine core and intact polar domains. The anti-MPL was also inhibited by fetal bovine collagen types I and II and in some instances by cardiolipin, but not by keratan sulfate, proteoglycan, or DnaK heat shock protein. Lot variation was a persistent technical problem, but no protein contaminant could be demonstrated in any lot. The ELISA and immunoblot results confirmed each other. Immunoblots detected a single band of MPL reactive with IgG. This antibody remains of interest because of its disease association and correlations and because it cross-reacts with collagen and cardiolipin.

Adult↗
PubMed1972

Effect of glycerol deprivation on the phospholipid metabolism of a glycerol auxotroph of Staphylococcus aureus.

A study of the effects of glycerol deprivation on the content and metabolism of the phospholipids of a glycerol auxotroph of Staphylococcus aureus showed that (i) there was an increase in the proportions of lysylphosphatidylglycerol (LPB) and a concomitant decrease in the proportion of phosphatidylglycerol. The total phospholipid content per sample and the proportion of cardiolipin did not change, but the phosphatidic acid increased transiently and then fell to pretreatment levels. (ii) The loss of (32)P from the lipids during the chase in a pulse-chase experiment was essentially the same in phosphatidylglycerol, cardiolipin, and phosphatidic acid during glycerol deprivation or growth in the presence of glycerol. LPG lost half the radioactivity in slightly more than two doubling times when grown with glycerol. In the absence of glycerol, (32)P accumulated in LPG for about 20 min and then stopped, after which time there was no apparent turnover. (iii) During glycerol deprivation, the initial (32)P incorporation decreased sixfold compared to that of the control with glycerol. The initial incorporation into LPG decreased only 2.5-fold, whereas that of PG decreased 45-fold. (iv) During glycerol deprivation, the free fatty acid content increased from 1.2 to 12.5% of the total extractable fatty acids and then slowly decreased. The increase was largely iso- and anti-iso-branched 21-carbon-atom fatty acids. In glycerol-supplemented cultures, the major fatty acids were branched 14- to 18-carbon fatty acids. The decrease in longer chain free fatty acids after 60 min represented their esterification into lipids. (v) During glycerol deprivation ribonucleic acid synthesis and cell growth continued for 40 min and protein synthesis continued for 90 min. Then synthesis and growth stopped. (vi) After the addition of glycerol to glycerol-deprived cells, (32)P and (14)C-glycerol were incorporated into the phospholipids without lag; ribonucleic acid, protein synthesis, and cell growth began after a 5- to 10-min lag at the pretreatment rate. The initial rate of lipid synthesis after the addition of glycerol was three times greater than the growth rate. This rapid rate continued for about 25 min until the lipid content and proportions of LPG and phosphatidylglycerol were restored.

Bacterial Proteins↗
PubMed1976

Thermal adaptation in yeast: growth temperatures, membrane lipid, and cytochrome composition of psychrophilic, mesophilic, and thermophilic yeasts.

The temperature limits of growth of a number of yeast species were examined, and on this basis the organisms were classified into different thermal categories. The following species were examined: Leucosporidium frigidum and Leucosporidium nivalis, psychrophilic, temperature limits of growth, -2 to 20 degrees C; Canadian lipolytica mesophilic, temperature limits of growth, 5 to 35 degrees Candida parapsilosis and Saccharomyces telluris, thermotolerant, temperature limits of growth, 8 to 42 degrees C; Torulopsis bovina and Candida slooffi, thermophilic, temperature limits of growth, 25 to 45 degrees C and 28 to 45 degrees C, respectively. The membrane lipid and cytochrome composition of mitochrondrial fractions isolated from these yeasts were compared. There was a direct correlation between the growth temperature and the degree of membrane of lipid unsaturation; the lower the temperature, the greater the degree of lipid unsaturation. The membrane lipid composition of the thermophilic yeasts were distinguished by the high percentage (30 to 40%) of saturated fatty acid, as compared with the mesophilic and psychrophilic yeasts. The latter contained approximately 90% unsaturated fatty acid, 55% of which was linolenic acid, C alpha-18:3. Changes in phospholipid composition in relation to temperature were also noted. The respiratory-deficient thermophile, C. slooffi, was characterized by the absence of cardiolipin (sensitivity 0.1 mug of phosphorus) and cytochrome aa3. The absence of conventional mitochondrial structures in this thermophilic microorganism is tentatively suggested although low concentrations of cytochromes b, c, and c1 were detected by low-temperature spectroscopy. On the other hand, the respiratory-competent thermophile, T. bovina, was characterized by a high cardiolipin (25% of the total phospholipid) and cytochrome aa3 content (1 nmol/mg of mitochrondrial protein). Low-temperature spectra showed the presence of one b-type cytochrome in the thermophilic yeasts, two b-type cytochromes in the mesophilic yeasts, and three b-type cytochromes in the psychrophilic yeasts. It was concluded that a knowledge of the properties of the biological membrane is fundamental to an understanding of the ability of a microorganism to grow and reproduce in different temperature environments.

Adaptation, Physiological↗
PubMed1979

Protein-associated lipid of Bacillus stearothermophilus.

The composition and patterns of metabolism of phospholipids isolated as part of a lipid-depleted membrane fragment (LDM fragment) and associated with the membrane adenosine triphosphatase complex have been compared with those of the bulk membrane phospholipid. The bulk lipid was extracted from washed membranes with sodium cholate. The LDM fragments, which contained a portion of the electron transport system and the membrane adenosine triphosphatase complex, were purified by chromatography with Sepharose 6B. The LDM fragment preparations contained 0.10 +/- 0.02 mumol of lipid phosphorus per mg of protein, compared with 0.54 +/- 0.05 mumol of lipid phosphorus per mg of protein for washed membranes. The phospholipid associated with the LDM fragments consisted of 78 +/- 4% cardiolipin, 7 +/- 1% phosphatidylglycerol, and 15 +/- 3% phosphatidylethanolamine. Changes in the total membrane lipid composition (produced by culture conditions) did not alter the phospholipid composition of the LDM fragments. The adenosine triphosphate complex was separated from the other components of the LDM fragments by suspension of the fragments in 1% Triton X-100 and precipitation with antibody specific for the F(1) component of the adenosine triphosphatase complex. The phospholipid isolated with the adenosine triphosphatase complex consisted of 86% cardiolipin, 8% phosphatidylglycerol, and 6% phosphatidylethanolamine. In pulse-chase experiments with (32)P and [2-(3)H]glycerol, the labeling patterns of the phosphatididylglycerol and phosphatidylethanolamine associated with the LDM fragments were different from those of the bulk membrane phosphatidylglycerol and phosphatidylethanolamine. It was concluded that at least a portion of the phospholipid isolated with the LDM fragments was part of a native lipid-protein complex.

Adenosine Triphosphatases↗
PubMed1983

Lipid composition of Zymomonas mobilis: effects of ethanol and glucose.

Zymomonas mobilis is an alcohol-tolerant microorganism which is potentially useful for the commercial production of ethanol. This organism was found to contain cardiolipin, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine as major phospholipids. Vaccenic acid was the most abundant fatty acid, with lesser amounts of myristic, palmitic, and palmitoleic acids. No branched-chain or cyclopropane fatty acids were found. Previous studies in our laboratory have shown that ethanol induces the synthesis of phospholipids enriched in vaccenic acid in Escherichia coli (L. O. Ingram, J. Bacteriol. 125:670-678, 1976). The fatty acid composition of Z. mobilis, an obligately ethanol-producing microorganism, represents an extreme of the trend observed in E. coli. In Z. mobilis, vaccenic acid represents over 75% of the acyl chains in the polar membrane lipids. Glucose and ethanol had no major effect on the fatty acid composition of Z. mobilis. However, both glucose and ethanol caused a decrease in phosphatidylethanolamine and phosphatidylglycerol and an increase in cardiolipin and phosphatidylcholine. Ethanol also caused a dose-dependent reduction in the lipid-to-protein ratios of crude membranes. The lipid composition of Z. mobilis may represent an evolutionary adaptation for survival in the presence of ethanol.

Bacteria↗
PubMed1990

Interaction of RecA protein with acidic phospholipids inhibits DNA-binding activity of RecA.

The RecA protein of Escherichia coli binds specifically to acidic phospholipids such as cardiolipin and phosphatidylglycerol. This binding appears to be affected by the presence of divalent cations such as Ca2+ and Mg2+. The interaction leads to the inhibition of RecA binding to at least two different conformations of DNA, single-stranded DNA and left-handed Z-DNA, thus suggesting that the phospholipids interact at the DNA-binding site of the RecA protein. Inclusion of a nucleotide cofactor [adenosine 5'-O-(gamma-thiotriphosphate)] in the reactions did not prevent the inhibition of DNA-binding activities of RecA protein by the phospholipids. The interaction of RecA protein with cardiolipin and phosphatidylglycerol, which represent two of the three major phospholipids of the E. coli membrane, may be physiologically important, as it provides a possible mechanism for the RecA-membrane association during the SOS response. These observations raise the possibility that the Z-DNA-binding activity of RecA protein is merely a manifestation of its phospholipid-binding property.

DNA↗
PubMed1993

Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixed micelles and in biological membranes.

Listeria monocytogenes secretes a phospholipase C (PLC) which has 39% amino acid sequence identity with the broad-specificity PLC from Bacillus cereus. Recent work indicates that the L. monocytogenes enzyme plays a role during infections of mammalian cells (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). The homogeneous enzyme has a specific activity of 230 mumol/min/mg when phosphatidylcholine (PC) is dispersed in sodium deoxycholate. With phospholipid-Triton X-100 mixed micelles, the enzyme had a broad pH optimum between 5.5 and 8.0, and the rates of lipid hydrolysis were in the following order: PC > phosphatidylethanolamine (PE) > phosphatidylserine > sphingomyelin >> phosphatidylinositol (PI). Activity on PC was stimulated 35% by 0.5 M NaCl and 60% by 0.05 mM ZnSO4. When Escherichia coli phospholipids were dispersed in Triton X-100, PE and phosphatidylglycerol, but not cardiolipin, were hydrolyzed. The enzyme was active on all phospholipids of vesiculated human erythrocytes including PI, which was rapidly hydrolyzed at pH 7.0. PI was also hydrolyzed in PI-PC-cholesterol liposomes by the nonspecific PLC from L. monocytogenes and by the homologous enzyme from B. cereus. The water-soluble hydrolysis product was identified as inositol-1-phosphate. For the hydrolysis of human erythrocyte ghost phospholipids, a broad pH optimum was also observed. 32P-labelled Clostridium butyricum protoplasts, which are rich in ether lipids, were treated with PLC. The enzyme hydrolyzed the plasmalogen form of PE, its glycerol acetal, and cardiolipin, in addition to PE. I-, Cl- and F- stimulated activity on either PC- Triton X-100 mixed micelles or human erythrocyte ghosts, unlike the enzyme from B. cereus which is strongly inhibited by halides. Tris-HCl, phosphate, and calcium nitrate had similar inhibitory effects on the enzyme on the enzymes from L. monocytogenes and B. cereus.

Cell Membrane↗
PubMed1997

Structural characterization of molecular phospholipid species in cytoplasmic membranes of the cell wall-less Streptomyces hygroscopicus L form by use of electrospray ionization coupled with collision-induced dissociation mass spectrometry.

A comparative analysis of the lipid compositions and fatty acids in the cytoplasmic membranes of Streptomyces hygroscopicus and its stable cell wall-less L form has been carried out to detect the differences which may be involved in the altered properties of the L-form membranes. Because only quantitative differences could be found (8), we analyzed the lipid components at the molecular level. Electrospray ionization (ESI), collision-induced dissociation (CID), and tandem mass spectrometry (MS-MS) were used for qualitative detection and quantitative determination of the molecular lipid species in phosphatidylethanolamine (PE1), lyso-cardiolipin (LCL), and cardiolipin (CL). Each phospholipid, isolated by preparative high-performance liquid chromatography showed several homologous molecular ion groups (PE1, four groups; LCL, six groups; CL, six groups) in the negative ESI-MS spectra. The sizes of the peaks represent their relative amounts in the corresponding phospholipid classes. Structural details about individual components of the molecular ion groups were obtained by mass selection and CID with MS-MS. Product ions derived from CID (daughter ions) give information about the molecular weights of the acyl constituents. The qualitative and quantitative compositions of the molecular species were determined by combining the data from the fatty acid pattern obtained by gas chromatography (GC), the relative quantities of the molecular ion groups, and the acyl constituents detected in these molecular ions. Because the ESI-MS-CID-MS data do not allow us to distinguish between n, iso, and anteiso fatty acids of the same molecular weight, it has been assumed that the ratio of these equal-numbered fatty acids determined by GC analysis of the isolated fatty acids is also present in the CID-MS peaks. In this way, 18 species were found in PE1, 43 species were estimated in LCL, and 59 species were ascertained for CL.

Cell Membrane↗