Evidence for a lack of phospholipid transfer protein in the stroma of spinach chloroplasts

The possible activity of phospholipid transfer protein in stroma extracts from spinach leaf has been investigated. Stroma, prepared from purified intact chloroplasts, was dialyzed and passed through various chromatography columns. None of the protein fractions eluted was able to stimulate the transfer of phosphatidylglycerol (PG) or phosphatidylcholine (PC) from liposomes to mitochondria, suggesting the lack of phospholipid transfer protein in the stroma from mature spinach chloroplasts.     

Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive

Plasma phospholipid transfer protein (PLTP) plays an important role in the maintenance of plasma high-density lipoprotein (HDL) content and remodeling of HDL in the circulation. In the present study we have used different fractionation methods to investigate the distribution of PLTP in human plasma. A novel enzyme-linked immunosorbent assay developed during the study allowed for simultaneous assessment of both PLTP mass and activity in the fractions obtained. Size-exclusion chromatography and plasma fractionation by nondenaturing polyacrylamide gel electrophoresis (PAGE) yielded similar results demonstrating that PLTP associates in native plasma with two distinct particle populations, while ultracentrifugation with high salt leads to detachment of PLTP from lipoprotein particles and loss of a majority of its phospholipid transfer activity. Interestingly, analysis of the size-exclusion chromatography fractions demonstrated that PLTP exists in the circulation as an active population that elutes in the position of HDL corresponding to an average molecular mass of 160+/-40 kDa and an inactive form with an average mass of 520+/-120 kDa. The inactive fraction containing approximately 70% of the total PLTP protein eluted between HDL and low density lipoprotein (LDL). Thus, the two PLTP pools are associated with different types of lipoprotein particles, suggesting that the PLTP activity in circulation is modulated by the plasma lipoprotein profile and lipid composition.     

Effects of phospholipid composition on the metabolism of triacylglycerol, cholesteryl ester and phosphatidylcholine from lipid emulsions injected intravenously in rats

Lipid emulsions were prepared with a similar size and lipid composition to natural lymph chylomicrons, but in which the surface phospholipid was either egg phosphatidylcholine, dioleoyl-, dimyristoyl-, dipalmitoyl- or 1-palmitoyl-2-oleoylphosphatidylcholine (EYPC, DOPC, DMPC, DPPC or POPC). When injected into the bloodstream of conscious rats, the emulsions containing EYPC or POPC were metabolized similarly to natural chylomicrons, consistent with rapid lipoprotein lipase-mediated hydrolysis of triacylglycerols, followed by hepatic uptake of the remnants derived from the emulsions. Phospholipids from the injected emulsions were removed more slowly and became associated with the high-density lipoprotein fractions of the plasma. Emulsions containing DPPC were metabolized differently. Triacylglycerols disappeared very slowly from plasma, indicating lack of hydrolysis by lipoprotein lipase, and phospholipid radioactivity did not transfer to high-density lipoprotein. With emulsions containing DMPC, the plasma removal rates for emulsion triacylglycerols and cholesteryl esters were fast, but phospholipid radioactivity failed to transfer to the high-density lipoprotein fractions of plasma. With DOPC emulsions, clearances were slower than EYPC or POPC emulsions, but transfer to high-density lipoproteins was efficient. Therefore, an unsaturated chain at the glycerol 2-position was necessary for rapid hydrolysis by lipoprotein lipase and for efficient transfer of phospholipids to high-density lipoproteins. With an unsaturated chain at the glycerol 2-position, a saturated chain at the glycerol 1-position optimized the rate of remnant removal from the plasma.     

A new fluorimetric method to measure protein-catalyzed phospholipid transfer using 1-acyl-2-parinaroylphosphatidylcholine

A new, simple and versatile method to measure phospholipid transfer has been developed, based on the use of a fluorescent phospholipid derivative, 1-acyl-2-parinaroylphosphatidylcholine. Vesicles prepared of this phospholipid show a low level of fluorescence due to interactions between the fluorescent groups. When phospholipid transfer protein and vesicles consisting of non-labeled phosphatidylcholine are added the protein catalyzes an exchange of phosphatidylcholine between the labeled donor and non-labeled acceptor vesicles. The insertion of labeled phosphatidylcholine into the non-labeled vesicles is accompanied by an increase in fluorescence due to abolishment of self-quenching. The initial rate of fluorescence enhancement was found to be proportional to the amount of transfer protein added. This assay was applied to determine the effect of membrane phospholipid composition on the activity of the phosphatidylcholine-, phosphatidylinositol- and non-specific phospholipid transfer proteins. Using acceptor vesicles of egg phosphatidylcholine and various amounts of phosphatidic acid it was observed that the rate of phosphatidylcholine transfer was either stimulated, inhibited or unaffected by increased negative charge depending on the donor to acceptor ratio and the protein used. In another set of experiments acceptor vesicles were prepared of phosphatidylcholine analogues in which the ester bonds were replaced with ether bonds or carbon-carbon bonds. Assuming that only a strictly coupled exchange between phosphatidylcholine and analogues gives rise to the observed fluorescence increase, orders of substrate preference could be established for the phosphatidylcholine- and phosphatidylinositol transfer proteins.     

Phospholipid composition and phospholipid asymmetry of ram spermatozoa plasma membranes

The phospholipid composition of ram spermatozoa plasma membranes has been investigated. An exclusively high participation of the choline- and ethanolamine-plasmalogens in the phosphatidylcholine and phosphatidylethanolamine fractions has been established. Phosphatidylcholine of ram spermatozoa plasma membranes contains a great amount of polyunsaturated fatty acids. The phospholipid distribution in spermatozoa plasma membrane was investigated. It was established that the choline containing phospholipids are situated mainly in the outer membrane lipid monolayer, whereas diphosphatidylglycerol and phosphatidylserine are localized predominantly in the inner monolayer. The rest of the phospholipids are evenly distributed among the two monolayers. Ram spermal plasma membranes exhibit high phospholipase A2 activity.     

The effect of carbon and nitrogen sources on bovicin HC5 production by Streptococcus bovis HC5

Aims:  To investigate the effect of media composition and agroindustrial residues on bovicin HC5 production by Streptococcus bovis HC5.
Methods and Results:  Batch cultures of S. bovis HC5 were grown in basal medium containing different carbon and nitrogen sources. The activity of cell-free and cell-associated bovicin HC5 was determined in culture supernatants and acidic extracts obtained from cell pellets, respectively. Streptococcus bovis HC5 produced bovicin using a variety of carbon and nitrogen sources. The highest specific activity was obtained in media containing 16 g l⁻¹ of glucose, after 16 h of incubation. The peak in cell-free and cell-associated bovicin HC5 activity was detected when S. bovis HC5 cultures reached stationary phase. The bovicin HC5 specific activity and bacterial cell mass increased approximately 3-fold when yeast extract and trypticase (0·5 and 1·0 g l⁻¹, respectively) were added together to the basal medium. Streptococcus bovis HC5 cultures produced bovicin HC5 in cheese whey and sugar cane juice and maximal volumetric productivity was obtained after 12 h of incubation.
Conclusions:  Streptococcus bovis HC5 is a versatile lactic acid bacterium that can utilize several carbon and nitrogen sources for bovicin HC5 production. This bacterium could be a useful model to study bacteriocin production in the rumen ecosystem.
Significance and Impact of the Study:  The use of agroindustrial residues as carbon sources could have an economical impact on bovicin HC5 production. To our knowledge, this is the first report to show the use of sugar cane juice for bacteriocin production by lactic acid bacteria.     

Concentration of neutral lipids in the phospholipid surface of substrate particles determines lipid transfer protein activity

To better understand the mechanism of lipid transfer protein (LTP) action and the effects of altered lipoprotein composition on its activity, we evaluated the dependence of LTP activity on the concentrations of cholesteryl ester (CE) and/or triglyceride (TG) in the phospholipid bilayer of substrate particles. Phosphatidylcholine (PC)-cholesterol liposomes containing up to 2 mole% TG and/or CE were prepared by cholate dialysis and used as either the donor of lipids to, or the acceptor of lipids from, low density lipoproteins (LDL). CE or TG transfer from liposomes of varying neutral lipid content to LDL showed saturation kinetics with an apparent Km of less than or equal to 0.2 mole%. Throughout this concentration-dependent response. PC transfer, which depended on the same LTP-donor particle binding interactions as those required for neutral lipid transfer, was essentially unchanged. Lipid transfer in the reverse direction (from LDL to liposomes of varying neutral lipid content) followed the same kinetics showing that transfer between the two particles is tightly coupled and bidirectional. When liposomes contained both TG and CE, these lipids competed for transfer in a manner analogous to that previously noted with lipoprotein substrates. In conclusion, CE and TG transfer activities are determined by the concentration of these lipids in the phospholipid surface of donor and acceptor particles. At low TG and CE concentrations, LTP bound to the liposome surface as indicated by PC transfer, but only a portion of these interactions actually facilitated a neutral lipid transfer event. Thus, the overall rate of neutral lipid transfer, and the competition between TG and CE for transfer, depend on the concentrations of these lipids in the phospholipid layer.     

Characterization of Aspergillus oryzae fermentation extract effects on the rumen fungus Neocallimastix frontalis,EB 188. Part 2. Carbon source utilization and effects on zoospore production

The effect of a commercial Aspergillus oryzae fermentation extract on the utilization of carbon source and zoospore production by the rumen fungus Neocallimastix frontalis EB 188 was determined. In addition, the composition of a soluble extract prepared from the commercial product was analyzed. This extract was added to N. frontalis EB 188 cultures grown on a variety of substrates and periodically assayed for protein, enzymes, zoospore production, and carbon source utilization. The powdered product contained 93% dry matter, more than 3,000 A. oryzaespores per gram, and did not contain strong buffers or high concentrations of salt. Measurable concentrations of DNA, protein, carbohydrate and several enzymes including cellulase and amylase were also found. Soluble extract increased fungal physiology and treated cultures produced significantly higher levels of supernatant protein and enzymes including amylase, cellulase and beta-glucosidase. The fungal response depended on culture carbon source. However, culture zoospore production was increased regardless of substrate provided. Culture utilization of glucose was more rapid in treated cultures, yet high levels of the extract greatly inhibited glucose utilization.     

The phospholipid composition of embryonic chick liver microsomes

1. The phospholipid composition of hepatic microsomal fractions from different developmental stages of embryonic chick was established. The major components were phosphatidylcholine (approx. 66%), phosphatidylethanolamine plus phosphatidylserine (approx. 21%) and sphingomyelin (approx. 9%). 2. There were no significant changes in the phospholipid composition during embryonic development from 9 to 20 days. 3. When microsomal subfractions were prepared it was found that the smooth-microsomal fractions (Ia and Ib) had a significantly greater sphingomyelin content than the rough-microsomal fraction (II). This was compensated by a lower phosphatidylcholine content in fractions Ia and Ib and an increase of phosphatidylcholine in fraction II. 4. The significance of the differences in the phospholipid composition of smooth and rough microsomes is discussed with particular reference to the origin and interrelation of smooth and rough endoplasmic reticulum.     

Intracellular localization of phospholipid transfer activity in Rhodopseudomonas sphaeroides and a possible role in membrane biogenesis.

The cellular content of phospholipid transfer activity in Rhodopseudomonas sphaeroides was examined as a function of both oxygen partial pressure and light intensity used for growth. Cells grown under high light conditions (100 W/m2) had over two times the cellular level of phospholipid transfer activity when compared with cells grown under other conditions. Although cells grown under low light conditions (3 W/m2) had the lowest amount of total phospholipid transfer activity, they had the highest level (49%) of membrane-associated transfer activity. The soluble phospholipid transfer activity was further localized into periplasmic and cytoplasmic fractions. The distribution of phospholipid transfer activity in cells grown under medium light intensity (10 W/m2) was calculated as 15.1% membrane-associated, 32.4% in the periplasm, and 52.5% in the cytoplasm. The phospholipid transfer activities in the periplasmic and cytoplasmic fractions had distinctly different properties with respect to their molecular weights (56,000 versus 27,000) and specificities of transfer (phosphatidylethanolamine greater than phosphatidylglycerol versus phosphatidylglycerol greater than phosphatidylethanolamine).     

Evidence of peroxisomes and peroxisomal enzyme activities in the oleaginous yeast Apiotrichum curvatum

The presence of peroxisomes and peroxisomal enzyme activities were investigated in the oleaginous yeast Apiotrichum curvatum ATCC 20509 (formerly Candida curvata D.) Catalase, a marker enzyme for peroxisomes, was measured in cell-free extracts prepared by sonication. The nature of the carbon and nitrogen sources in the growth medium greatly affected catalase activity. Cells grown on corn oil had high specific activity of catalase, but those grown on glucose, sucrose, or maltose had low specific activity. High specific activity of catalase was measured in cultures grown on media that supported poor growth (with soluble starch as carbon source or with methylamine, urea, or asparagine as nitrogen source). Peroxisomes from cells grown on corn oil were separated from other subcellular fractions in a discontinuous sucrose gradient. Major peaks of activity of fatty acid beta-oxidation and of two key enzymes in the glyoxylate cycle were found in fractions containing peroxisomes, but not in fractions corresponding to the mitochondria. Peroxisomal beta-oxidation showed equivalent activity with palmitoyl CoA or n-octanoyl CoA as substrate. Mitochondria did not seem to contain NAD-linked glutamate dehydrogenase. Peroxisomes with a homogeneous matrix and core surrounded by a single-layer membrane were observed with an electron microscope in cells grown on corn oil, but not in those grown on glucose. Staining with 3,3'-diaminobenzidine revealed that catalase activity was located in peroxisomes. Peroxisomes in this oleaginous yeast play important roles in lipid metabolism.     

Characterization of<Emphasis Type="Italic"> Aspergillus oryzae</Emphasis> fermentation extract effects on the rumen fungus<Emphasis Type="Italic"> Neocallimastix frontalis</Emphasis>, EB 188. Part 1. Zoospore development and physiology

Experiments were performed to determine the effect of Aspergillus oryzae (AO) fermentation extract on zoospore development in the rumen fungus Neocallimastix frontalis EB 188. Powdered product, or liquid extract prepared from such powder, was added at the recommended value for supplementation in dairy cattle. Stationary and stirred cultures were periodically sampled and assayed for extracellular and intracellular protein and enzymes, gas production, zoospore production and maturation, and carbon source utilization. Soluble extract increased fungal physiology when grown in stirred vessels or stationary cultures. Treated cultures produced higher levels of enzymes (nearly double). Mobile zoospores matured into germination entities more rapidly in treated cultures, and when powdered product was used, nearly 3 times more motile zoospores were produced at 56 h of fungal growth. Levels of the intracellular enzyme malate dehydrogenase increased by 6-fold in the presence of powdered product. Product wheat bran carrier used as soluble extract or powder had very little effect on fungal cultures. Medium cellulose was completely hydrolyzed in all cultures but this occurred earlier in those containing AO treatment.     

Role of substrate in determining the phospholipid specificity of protein kinase C activation

The phospholipid selectivity of protein kinase C (PKC) activation was examined by using two substrates, histone and a random copolymer of lysine and serine [poly(lysine, serine)] (PLS), plus phospholipids provided as vesicles or as Triton-mixed micelle preparations. The results indicated that substrate-phospholipid interaction was an essential component of PKC activation and that many in vitro properties of PKC activation are attributable to this interaction. The substrate histone interacted with phospholipid-Triton mixed micelles containing phosphatidylserine (PS), but not with those containing phosphatidylinositol (PI) or phosphatidylglycerol (PG). In direct correlation, only PS-Triton mixed micelles were effective in supporting PKC activity. Also, the minimum PS composition (4 mol % in Triton) required to induce significant histone-PS interaction coincided with the minimum composition required for phosphorylation of histones. Moreover, the PS composition required for maximum activity varied with the histone concentration of the reaction. In contrast to histone, PLS interacted with phospholipid-Triton mixed micelles containing either PS, PI, or PG, and all these mixed micelles supported the phosphorylation of PLS. In fact, by selection of appropriate experimental conditions (e.g., concentration of substrate and phospholipid), any of the three mixed micelles could appear the most effective in supporting PKC activity. Phospholipid vesicles containing PS, PG, or PI were found to interact with both histone and PLS and to support the activity of PKC. Physical properties of the solution and conditions used for preparation of phospholipid vesicles had considerable influence on PKC activation. At high phospholipid concentrations, vesicles containing PS, PI, or PG supported the activity of PKC to essentially the same level, provided that the physical differences among the phospholipid vesicles were minimized.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipids of dystrophic and normal mouse muscle: whole tissue and particulate fractions

Myofibrillar, mitochondrial, and microsomal fractions were prepared from normal and dystrophic mouse limb muscle by differential centrifugation and analyzed for phospholipids and cholesterol. Fatty acids and aldehydes of neutral lipids and of phospholipids from whole muscle and particulate fractions were also determined. Normal microsomes contained more lecithin and less total ethanolamine phospholipids and cardiolipin than mitochondria. The myofibrils had an intermediate phospholipid composition, but their cholesterol-phospholipid ratio was smaller than that of the other two fractions. Except for an increased percentage of phosphatidalethanolamine in the dystrophic mitochondria, only the composition of the dystrophic microsomes differed from normal by containing less lecithin but more total ethanolamine phospholipid, phosphatidalethanolamine, sphingomyelin, and cholesterol. No significant differences were found in the fatty acid composition of neutral lipid extracts from normal and dystrophic preparations, but there was a significant decrease in the percentage of 22:6 in phospholipids from both dystrophic whole muscle and microsomes (-25% and -37%, respectively), whereas the 20:4 content was unaltered. By contrast, the percentages of 18:0 and total fatty aldehyde increased significantly. Phospholipid extracts from all dystrophic samples showed a significant decrease in 16:0 and an increase in 18:1 as compared with the normal.     

Interaction of bovine brain phospholipid exchange protein with liposomes of different lipid composition

The major phospholipid exchange protein from bovine brain catalyzes the transfer of phosphatidylinositol and phosphatidylcholine between rat liver microsomes and sonicated liposomes. The effect of liposomal lipid composition on the transfer of these phospholipids has been investigated. Standard liposomes contained phosphatidylcholine-phosphatidic acid (98:2, mol%); in general, phosphatidylcholine was substituted by various positively charged, negatively charged, or zwitterionic lipids. The transfer of phosphatidylinositol was essentially unaffected by the incorporation into liposomes of phosphatidic acid, phosphatidylserine, or phosphatidylglycerol (5–20 mol%) but strongly depressed by the incorporation of stearylamine (10–40 mol%). Marked stimulation (2–4-fold) of transfer activity was observed into liposomes containing phosphatidylethanolamine (2–40 mol%). The inclusion of sphingomyelin in the acceptor liposomes gave mixed results: stimulation at low levels (2–10 mol%) and inhibition at higher levels (up to 40 mol%). Cholesterol slightly diminished transfer activity at a liposome cholesterol/phospholipid molar ratio of 0.81. Similar effects were noted for the transfer to phosphatidylcholine from microsomes to these various liposomes. Compared to standard liposomes, the magnitude of $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ tended to increase for liposomes which depressed phospholipid transfer and to decrease for those which stimulated; little change was observed in the values of $\text{V}$. Single phospholipid liposomes of phosphatidylinositol were inhibitory when added to standard liposomes.     

Sunflower seed oil and oleic acid utilization for the production of rhamnolipids by Thermus thermophilus HB8

The potential production of rhamnolipids was demonstrated using the thermophilic eubacterium Thermus thermophilus HB8 and sunflower seed oil or oleic acid as carbon sources. Sunflower seed oil was directly hydrolyzed by secretion of lipase and became a favorable carbon source for rhamnolipids production. Rhamnolipids levels were attainted high values, comparable to those produced by Pseudomonas strains from similar sources. Rhamnolipids synthesis in oleic acid exhibited a long period of induction, while in sunflower seed oil, the synthesis is more rapid. Glucose resulted in a more protracted period of rhamnolipids production after exhaustion of each or both carbon sources. Both mono- and di-rhamnolipids were identified by thin-layer chromatography (TLC) in the total rhamnolipids extract. The molecular composition of the produced biosurfactant was evaluated by Fourier transform infrared (FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and LC-MS analysis. Furthermore, secretion of rhamnolipids was confirmed on agar plates. The antimicrobial activity of rhamnolipids was detected against the bacterium Micrococcus lysodeikticus using a lysoplate assay. These results demonstrate that rhamnolipids produced in these substrates can be useful in both environmental and food industry applications by using cheap oil wastes. The alternative use of this thermophilic microorganism opens a new perspective concerning the valorization of wastes containing plant oils or frying oils to reduce the cost of rhamnolipids production.     

Increased endoplasmic reticulum content of Phanerochaete chrysosporium INA-12 by inositol phospholipid precursor in relation to peroxidase excretion

Summary Haem protein excretion (i.e., lignin and manganese-dependent peroxidases) by Phanerochaete chrysosporium INA-12 was improved in response to an exogenous supply of phospholipid components (inositol and linoleic acid) as well as phosphatidylinositol (PI). Maximal enzyme productions were 46.3 and 21.1 nkat · ml^–1, respectively, in inositol cultures after 3 days incubation. Cellular compartment determination by marker enzymes revealed that the enhancement of protein excretion with inositol was correlated with a proliferation of endoplasmic reticulum; cytosol and mitochondrial activity did not change. In contrast, in the presence of linoleic acid and PI total cellular activity was increased. In culture containing inositol, the intracellular phospholipid composition of strain INA-12 mycelium exhibited a two fold enrichment in PI at the expense of phosphatidylserine and its derives, phosphatidylethanolamine and phosphatidylcholine. Offprint requests to: M. Asther     

Influence of carbon source on α-amylase production by Aspergillus oryzae

The influence of the carbon source on alpha-amylase production by Aspergillus oryzae was quantified in carbon-limited chemostat cultures. The following carbon sources were investigated: maltose, maltodextrin (different chain lengths), glucose, fructose, galactose, sucrose, glycerol, mannitol and acetate. A. oryzae did not grow on galactose as the sole carbon source, but galactose was co-metabolized together with glucose. Relative to that on low glucose concentration (below 10 mg/l), productivity was found to be higher during growth on maltose and maltodextrins, whereas it was lower during growth on sucrose, fructose, glycerol, mannitol and acetate. During growth on acetate there was no production of alpha-amylase, whereas addition of small amounts of glucose resulted in alpha-amylase production. A possible induction by alpha-methyl-D-glucoside during growth on glucose was also investigated, but this compound was not found to be a better inducer of a-amylase production than glucose. The results strongly indicate that besides acting as a repressor via the CreA protein, glucose acts as an inducer.     

Localization of phospholipid biosynthetic enzyme activities in cell-free fractions derived from Rhodopseudomonas sphaeroides

The phospholipid biosynthetic enzyme activities: CDP-diglyceride synthetase, phosphatidylglycerophosphate synthetase, PGP phosphatase, phosphatidylserine (PS) synthase, PS decarboxylase, and S-adenosyl-L-methionine:phosphatidylethanolamine (AdoMet:PE) N-methyltransferase were detected in crude cell-free extracts of Rhodopseudomonas sphaeroides. CDP-diglyceride synthetase and phosphatidylglycerophosphate synthetase co-enriched with penicillin-binding protein activity, a known cytoplasmic membrane marker, throughout fractionation of cell-free extracts of both chemoheterotrophically and photoheterotrophically grown cells. PS decarboxylase also co-enriched with the cytoplasmic membranes in fractions derived from chemoheterotrophically and photoheterotrophically grown cells, but substantially greater quantities of PS decarboxylase activity was found in the chromatophores derived from photoheterotrophically grown cells than could be accounted for by cytoplasmic membrane contamination of this sample. PS synthase (60% of the recovered activity) and S-adenosyl-L-methionine:phosphatidylethanolamine N-methyltransferase (90% of the recovered activity) were found in the supernatant fraction after high speed centrifugation of crude cell lysates, suggesting that these enzyme activities were not tightly membrane associated. The localization of phospholipid biosynthetic enzyme activity in R. sphaeroides is discussed in terms of the biosynthesis of the photosynthetic membranes.