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P A Siegenthaler

Publications and source records attributed to P A Siegenthaler.

At least 19 recordsLinked to original sources
PubMed2001

Three distinct lipid kinase activities are present in spinach chloroplast envelope membranes: phosphatidylinositol phosphorylation is sensitive to wortmannin and not dependent on chloroplast ATP.

Chloroplast envelope membranes display properties that are important in lipid synthesis, regulation of metabolites, and protein transport, as well as in signal transduction. The recent discovery showing that phosphorylation of lipids occurs in envelope membranes provides a new approach for understanding the role of chloroplast lipids in these processes. The present investigation shows that three major lipid kinase activities are at least present in envelope membranes. These activities greatly depend on external conditions, such as pH, ATP concentrations, temperature, and chloroplast ATP and wortmannin sensitivity. Two types of phosphorylated lipid couples displayed similar intrinsic responses toward these biochemical parameters, namely phosphatidic acid (PA) and its lysoderivative (LPA) and monogalactosyl-phosphate-diacylglycerol (MGpDG) and its lysoderivative (LMGpDG), but not phosphatidylinositol-monophosphate (PIP) and its lysoderivative (LPIP). Phosphorylation of phosphatidylinositol was not dependent on chloroplast ATP, but was sensitive toward wortmannin in intact chloroplasts and outer envelope membrane vesicles.

Adenosine Triphosphate↗
PubMed2000

Lipid phosphorylation in chloroplast envelopes. Evidence for galactolipid CTP-dependent kinase activities.

Lipid phosphorylation takes place within the chloroplast envelope. In addition to phosphatidic acid, phosphatidylinositol phosphate, and their corresponding lyso-derivatives, we found that two novel lipids underwent phosphorylation in envelopes, particularly in the presence of carrier-free [gamma-(32)P]ATP. These two lipids incorporated radioactive phosphate in chloroplasts in the presence of [gamma-(32)P]ATP or [(32)P]P(i) and light. Interestingly, these two lipids were preferentially phosphorylated in envelope membranes in the presence [gamma-(32)P]CTP, as the phosphoryl donor, or [gamma-(32)P]ATP, when supplemented with CDP and nucleoside diphosphate kinase II. The lipid kinase activity involved in this reaction was specifically inhibited in the presence of cytosine 5'-O-(thiotriphosphate) (CTPgammaS) and sensitive to CTP chase, thereby showing that both lipids are phosphorylated by an envelope CTP-dependent lipid kinase. The lipids were identified as phosphorylated galactolipids by using an acid hydrolysis procedure that generated galactose 6-phosphate. CTPgammaS did not affect the import of the small ribulose-bisphosphate carboxylase/oxygenase subunit into chloroplasts, the possible physiological role of this novel CTP-dependent galactolipid kinase activity in the chloroplast envelope is discussed.

Chloroplasts↗
PubMed2000

The topology of phosphatidylglycerol populations is essential for sustaining photosynthetic electron flow activities in thylakoid membranes.

The transmembrane distribution of phosphatidylglycerol (PG) was determined in rightside-out (RO) and inside-out vesicles (IO) obtained by fragmentation of spinach thylakoids in a Yeda press, followed by partition in an aqueous dextran-polyethyleneglycol two-phase system. Using the phospholipase A(2) from porcine pancreas to digest selectively PG molecules in the outer monolayer (exposed to the incubation medium) of the membrane, we found the molar outside/inside distribution to be 70/30+/-5 in RO and 40/60+/-3 in IO. The transmembrane distribution of PG in IO was the opposite of that in intact thylakoids (molar ratio 58/42+/-3). The phospholipid population which sustained most of the uncoupled photosystem II electron flow activity was localized in the inner monolayer (exposed to the thylakoid lumen) of both thylakoid and RO membranes. In contrast, the activity in IO membranes was highly dependent on the PG population located in the outer monolayer. This finding brings the first direct demonstration of the dependence of the photosynthetic electron flow activity on the integrity of the inner topological pool of PG in the thylakoid membrane.

Animals↗
PubMed2000

Do glycerolipids display lateral heterogeneity in the thylakoid membrane?

The lateral heterogeneity of lipids in the thylakoid membrane has been questioned for over 20 yrs. It is generally believed that glycerolipids are asymmetrically distributed within the plane of the membrane. In the present investigation, we isolated several thylakoid membrane domains by using sonication followed by separation in an aqueous dextran-polyethylene glycol two-phase system. This technique, which avoids detergent treatments, allowed us to obtain stroma and grana lamellae vesicles as well as grana central core and grana margin vesicles from thylakoids. The relative distribution of the four lipid classes, i.e., monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol, and phosphatidylglycerol, was found to be statistically identical in all four thylakoid fractions and in whole thylakoids. Similarly, the relative amount of fatty acids in each individual lipid and the eight main phosphatidylglycerol molecular species was identical in all thylakoid membrane fractions tested as well as in the intact thylakoid membrane. Based on presently available procedures for obtaining thylakoid subfractions that are unable to discriminate microdomains within the membrane, it is concluded that glycerolipids are evenly distributed within the plane of the thylakoid membrane. These data are discussed in terms of "bulk" and "specific" lipids.

Glycerides↗
PubMed1997

Evidence for lipid kinase activities in spinach chloroplast envelope membranes.

Three spinach chloroplast envelope membrane preparations (i.e. whole, outer and inner membranes) were incubated in the presence of [gamma-32P]ATP. After lipid extraction and separation by TLC, four main phosphorylated lipids were detected by autoradiography in whole envelope preparations. These phospholipids were identified by comparing their Rf with that of lipid markers and by a deacylation procedure. They were found to be phosphatidic acid (PA) and lyso-PA, L-alpha-phosphatidyl-inositol 4-monophosphate (PIP) and lyso-PIP. These lipids were not equally distributed in the outer and inner envelope membranes. Chloroplast envelope membranes were verified not to be contaminated by plasma membranes. It is concluded that lipid kinase activities are associated with spinach chloroplast envelope membranes.

Adenosine Triphosphate↗
PubMed1996

Cyclodextrins: a new tool for the controlled lipid depletion of thylakoid membranes.

Cyclodextrins (CDs) have been used in controlled lipid depletion of thylakoid membranes avoiding the use of either detergents or lipolytic enzymes. Spinach thylakoid membranes were first treated with different CDs under various conditions. After removal of the CDs by washing, the amounts of mono-- and digalactosyldiacylglycerol (MGDG and DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), protein, pigment and plastoquinone remaining in the membranes were determined. The main results, obtained with alpha-CD and heptakis-(2,6-di-O-methyl)-beta-CD (DM-beta-CD), were as follows. (1)Acyl lipids were removed from thylakoid membranes by both CDs (DM-beta-CD being more efficient than alpha-CD; the extent of removal depended on both CD and chlorophyll concentrations. (2) alpha-CD presented a higher selectivity towards lip classes than did DM-beta-CD, but in both cases the removal order was SQDG > PG > MGDG > DGDG. (3) alpha-CD showed a preference for those lipids containing saturated 16-carbon acyl chains whereas DM-beta-CD was essentially insensitive to the fatty acid composition of the lipids. (4) The protein, chlorophyll and carotenoid contents of thylakoids were not affected by CD treatments. (5) Plastoquinones were removable but in small amounts only and with a low efficiency (DM-beta-CD > alpha-CD). (6) For all lipid classes, the extent of lipid removal was higher at 0 degrees than at 20 degrees C. (7) The presence of MgCl(2) reduced the removal of PG and SQDG but not affect galactolipid depletion levels. (8) Staple lipid depletion levels in thylakoid membranes were reached after 5-10 min of CD treatment at 0 degrees C. (9) Of the four CDs tested, only three (alpha-CD, beta-CD, and DM-beta-CD) promoted lipid depletion whereas one (hydroxypropyl-beta-CD) failed completely to do so. It is concluded that CD-mediated lipid removal provides a valuable and versatile tool to achieve controlled and specific lipid depletions in biological membranes. A few examples of the consequences of a CD-induced lipid depletion on fluorescence and electron transport properties of thylakoids are given to show the usefulness of CDs in the investigation of structure-function relationship in photosynthetic membranes.

Carotenoids↗
PubMed1996

Phosphatidylglycerol molecular species of photosynthetic membranes analyzed by high-performance liquid chromatography: theoretical considerations.

A reversed-phase high-performance liquid chromatography technique was developed to separate, identify, and quantify individual phosphatidylglycerol (PG) molecular species in thylakoid membranes isolated from higher plant leaves. PG was first separated by thin-layer chromatography; then the dinitrobenzoyl derivatives of diacylglycerols produced after phospholipase C hydrolysis of PG were separated by a C18 reversed-phase column and detected at 254 nm. A linear response of the detector was observed in the range of 0.025 to 12 nmol of PG molecular species. It was established that there was an excellent correlation (r = 0.996) between the carbon and double-bond number in the aliphatic residues and the relative retention time of dinitrobenzoyl derivatives. A new equivalent carbon number value (ECN*) which takes into consideration the number of cis-(nc) and trans-(nt) double bonds per molecular species was defined as ECN* = CN - 2nc - nt, where CN is the number of carbon atoms in the aliphatic residues. The logarithm of the retention time increased linearily as a function of ECN* value. However, in this type of correlation, it may happen that two molecular species of PG having distinct relative retention times had the same ECN* value. In this case, the two molecular species can be identified by the linear correlation (r = 1) existing between the reciprocal of the relative retention time and the number of double bonds (0 < or = n < or = 3) in the separate 18:n/delta 3-trans-hexadecenoic acid -16:1(3t)- and 18:n/16:0 molecular species series. The advantages of this method are good separation, cohort elution time, quantitative precision, and predictable retention times of PG molecular species from chloroplast membranes. The method has been used routinely to identify the ten PG molecular species of thylakoid membranes in squash, potato, lettuce, and spinach leaf: 18:3/16:1(3t), 18:3/16:0, 18:2/16:1(3t), 18:2/16:0, 18:1/16:1(3t), 18:1/16:0, 18:0/16:1(3t), 18:0/16:0, 16:0/16:1(3t), and 16:0/16:0.

Chloroplasts↗
PubMed1995

(Galacto) lipid export from envelope to thylakoid membranes in intact chloroplasts. II. A general process with a key role for the envelope in the establishment of lipid asymmetry in thylakoid membranes.

The transfer of organelle of newly synthesized lipid molecules from inner envelope to thylakoid membranes, as well as their subsequent transbilayer distribution in these membranes, have been studied in intact chloroplasts isolated from young and mature spinach, young pea and mature lettuce leaves, using a recently developed methodology (Rawyler, A., Meylan, M. and Siegenthaler, P.A. (1992) Biochim. Biophys. Acta 1104, 331-341). Three radiolabelled precursors were used. UDP-[14C]galactose allowed to follow the fate of mono- and digalactosyldiacylglycerol (MGDG and DGDG) made from polyunsaturated, preexisting diacylglycerol (DAG), whereas [14C]acetate and [14C]glycerol 3-phosphate were used to follow the fate of MGDG and phosphatidylglycerol (PG), respectively, after de novo synthesis. MGDG, DGDG and PG molecules assembled at the envelope level were found to be exportable to thylakoids in amounts strictly proportional to the amounts synthesized, provided that the necessary substrates were not limiting. Lipid export was class-selective; under our conditions, as much as 50-80% of the MGDG, 87% of the PG and 20-30% of the DGDG synthesized were exported to thylakoids. However, within the MGDG class labelled from [14C]acetate, there was hardly any selectivity in the export of its various molecular species. For MGDG, the proportionality coefficient, which reflects the efficiency of the export process, was higher in chloroplasts from young than from mature leaves, and higher in spinach than in pea and lettuce. Temperature affected the efficiency of galactolipid export in a class-dependent way. MGDG synthesis and export had similar Q10 values of about 4 in young and 3 in mature spinach leaves, while the Q10 of DGDG export was higher than that of its synthesis. In most cases, the transmembrane distribution of labelled lipids in thylakoids was found to match closely the corresponding distribution of mass, regardless of plant age and species and of incubation time and temperature. In some cases however, small but significant differences occurred between the label and the mass transbilayer distributions of MGDG (labelled molecules more inwardly oriented), DGDG and PG (more outwardly oriented). We propose a general model in which the thylakoid lipid asymmetry is primarily preestablished in the chloroplast envelope by the topography of its lipid-synthesizing enzymes, together with the occurrence of relatively fast lateral diffusion and translocation rates of the newly synthesized lipids. Transient fusions between inner envelope and thylakoid membranes would allow lipid export by lateral diffusion and build the observed lipid asymmetry in the latter.

Age Factors↗
PubMed1992

Galactolipid export from envelope to thylakoid membranes in intact chloroplasts. I. Characterization and involvement in thylakoid lipid asymmetry.

The galactolipid transfer from inner envelope to thylakoid membranes has been studied in intact spinach chloroplasts. Plastids, isolated from mature leaves, were dark-incubated in the presence of UDP-[14C]galactose. After various synthesis periods at 5 or 25 degrees C, intact plastids were reisolated and osmotically lysed. Thylakoid membranes were then prepared by a special procedure which removed greater than or equal to 99% of the envelope amount initially present. Under these conditions, purified thylakoids were found to contain radiolabelled MGDG and DGDG, indicating that galactolipids were exported from the inner envelope. The amounts exported were proportional to the amounts synthesized. About 55% of the MGDG and 25% of the DGDG synthesized in plastids were transferred to thylakoids, irrespectively of incubation time or temperature. The MGDG/DGDG radioactivity ratio was 7 in intact plastids and 18 in thylakoids, suggesting a preferential export of MGDG. Purified thylakoid membranes were then submitted to a lipolytic treatment designed to discriminate between the MGDG and DGDG pools belonging to the outer (stroma-facing) or to the inner monolayer. The radioactivity present in the lyso-products (corresponding to the outer pools) and in the residual parent lipids (corresponding to the inner pools) was measured. The labelled MGDG showed a transmembrane outside:inside distribution (mol%) of 50:50, which differed from the native (mass) MGDG asymmetry of 64:36. In contrast, the label and mass asymmetries of DGDG gave the same value of 15:85. These label distributions were affected neither by incubation time (from 5 to 90 min) nor by temperature (from 5 to 25 degrees C). We discuss the possibilities that transient fusions between the stroma-facing monolayers of the inner envelope and of the thylakoid membrane, and/or galactolipid transfer protein(s), together with lipid translocating activities in thylakoids, may account for the galactolipid export observed in mature spinach chloroplasts.

Biological Transport↗
PubMed1989

A single and continuous spectrophotometric assay for various lipolytic enzymes, using natural, non-labelled lipid substrates.

A rapid, continuous spectrophotometric assay for measuring the amount and activity of several lipolytic enzymes is described. It is based on the metachromatic properties of the cationic dye safranine, and makes use of the fact that an adequate combination of a lipolytic enzyme with one of its substrates leads to a change in the net negative charge at the lipid/water interface, which is monitored by the absorbance change of safranine. Utilizing this method, most lipolytic enzymes can be detected in very low amounts (milliunit or less) in about 1 min without employing radiolabelled lipids or synthetic lipid analogues. Over a wide range of enzyme concentrations, there is a good linearity between the initial hydrolysis rate (determination by the safranine method) and the amount of enzyme. The versatility of the assay is illustrated by examples showing how phospholipase A2, triacylglycerol hydrolase, phospholipase D or phospholipase C (either general or phosphatidylinositol-specific) activities can be detected, either separately or sequentially. Due to its high sensitivity, simplicity, and rapidity, this assay should find its main application in monitoring column effluents during the purification steps of lipolytic enzymes.

Animals↗
PubMed1988

Development of Oat Prothylakoids into Thylakoids during Greening Does Not Change Transmembrane Galactolipid Asymmetry but Preserves the Thylakoid Bilayer.

The lipase from Rhizopus arrhizus and the lipolytic acyl hydrolase from potato tubers have been used to determine the transmembrane distribution of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) in prothylakoids and thylakoids from oat (Avena sativa). Both galactolipids were found to be asymmetrically distributed. The molar outside/inside distribution was 70 +/- 8/30 +/- 8 for MGDG and 10 +/- 4/90 +/- 4 for DGDG in the prothylakoid membrane. Mature thylakoids presented a similar distribution, i.e. 63 +/- 4/37 +/- 4 for MGDG and 12 +/- 3/88 +/- 3 for DGDG. This distribution has been assessed under a variety of different conditions, namely (a) in media favoring thylakoid stacking or unstacking and inducing various membrane surface potentials, (b) in the presence of defatted bovine serum albumin which removed free fatty acids and partially lyso-galactolipids, (c) under various temperature conditions which resulted in different hydrolysis rates and degrees of fluidity of the membrane, and (d) in the presence of different enzyme concentrations which influenced the hydrolysis rate. The above distribution was found to be independent of the type of conditions used. Nonbilayer forming/bilayer forming lipid ratios suggest that both monolayers of the prothylakoid and the inner monolayer of oat thylakoid membranes should display lamellar structures (e.g. ratios <2.5). In contrast the outer monolayer of the thylakoid membrane should display non-lamellar configurations (e.g. ratio >2.5). Thus, it is concluded that the incorporation of chlorophyll-protein complexes into the nascent thylakoid membrane modifies neither the galactolipid nor the phospholipid transmembrane distribution. However, these complexes appear to be crucial to preserve a bilayer configuration to the greening membrane which, otherwise, would adopt nonlamellar structures. The possible origin of galactolipid transversal asymmetry which appears very early during the biogenesis of oat thylakoid membranes is discussed.

Journal Article↗
PubMed1984

Purification of peroxisomes and mitochondria from spinach leaf by percoll gradient centrifugation.

A procedure was developed to purify simultaneously peroxisomes and mitochondria from spinach (Spinacia oleracea L.) leaf under isoosmotic and low viscosity conditions. This method involved differential centrifugation and density gradient centrifugation on four layers of Percoll. Chlorophyll-free preparations of highly intact and active organelles were obtained and cross-contamination was negligible. Both organelles were stable for several hours, even if they remained in Percoll. Purified mitochondria were able to carry out the oxidation of different substrates with excellent respiratory control and ADP:O ratios. The method described in the present work was also suitable to purify mitochondria and peroxisomes from potato (Solanum tuberosum L.) tubers.

Journal Article↗
PubMed1982

Alteration in the acyl lipid composition of thylakoids induced by aging and its effect on thylakoid structure.

The alteration in both the lipid composition and chlorophyll proteins obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of detergent solubilized thylakoids was investigated on differentially aged spinach (Spinacia oleracea L.) chloroplasts. Freshly isolated material demonstrated seven major bands upon electrophoretic fractionation. Membranes aged in vitro showed a diminution and/or a disappearance of some bands concomitant with changes in the acyl lipid composition of these membranes. The extent of these changes was influenced by the purity of the preparation. Low temperature fluorescence measurements (77K) showed that upon aging, the photochemical capacity of photosystem II decreased prior to alterations in the molecular organization of the photochemical apparatus as indicated by the energy distribution between the two photosystems.The results are discussed in terms of whether the series of changes in the acyl lipid composition upon aging is related to the variations in the electrophoretic pattern of the chlorophyll proteins.

Journal Article↗
PubMed1981

Transmembrane distribution of phospholipids and their involvement in electron transport, as revealed by phospholipids A2 treatment of spinach thylakoids.

Thylakoid membranes were treated with either pancreatic or snake venom phospholipase A2, and the residual phospholipid content of these membranes was determined and compared to the rates of Photosystem II and/or Photosystem I electron transports. The hydrolysis curves of both phosphatidylglycerol and phosphatidylcholine displayed a first, rapid phase which was almost temperature-insensitive, followed by a second, slower phase which depended strongly on the temperature. When pancreatic phospholipase A2 had access either to the outer face or to both faces of the thylakoid membrane, either only part of or all the phospholipids, respectively, could be hydrolysed. These results were interpreted as indicating an asymmetric distribution of phospholipids across the thylakoid membrane, phosphatidylglycerol and phosphatidylcholine being preferentially located in the outer and the inner layer, respectively. When acting on uncoupled thylakoid membranes, phospholipase A2 exerted an inhibitory effect on Photosystem II activity and a stimulatory effect on Photosystem I activity. The involvement of phosphatidylcholine and of phosphatidylglycerol in electron transport activities of Photosystem II and of Photosystem I are discussed with special reference to the role of the external and internal pools of these phospholipids.

Animals↗
PubMed1980

Rapid analysis of membrane lipids using a combination of thin-layer chromatography and scanning of photographic negatives.

A simple method is proposed for the analysis of the distribution and changes in membrane lipids subjected to different treatments (lipolytic, aging, etc.). This technique involves only one thin-layer chromatographic step followed by a scanning of the photographic negative of the charred thin layer. This method is time saving, inexpensive and does not require the technical skill usually demanded in lipidology. The precision of this method is compared with that obtained with the classical TLC-GC method: its variability is roughly twice that of the TLC-GC method.

Chloroplasts↗
PubMed1980

Role of lipids in functions of photosynthetic membranes revealed by treatment with lipolytic acyl hydrolase.

1. Thylakoid membranes and subchloroplast particles I and II enriched in photosystem I and photosystem II, respectively, were treated with a potato lipolytic acyl hydrolase. 2. In the thylakoid membrane fraction, this treatment inhibited electron flows involving both photosystems and the associated photophosphorylations. However, electron flows involving either photosystem I or photosystem II were still preserved. The treatment of thylakoid membranes by lipolytic acyl hydrolase brought about a temporal convergence of different events such as maximal activity of reduced dichloroindophenol-supported electron flows, complete inhibition of photophosphorylations and electron transport activities through photosystem II + I, onset of the decay N,N,N',N-tetramethyl-rho-phenylenediamine-supported activity of photosystem activity and of the stimulation of photosystem activity (from reduced dichloroindophenol to NADP+ by exogenous plastocyanin. 3. Lipolytic acyl hydrolase catalyzed a limited hydrolysis of each lipid but in a stepwise manner, the galactolipids being attacked before the ionic lipids. The extent of the hydrolysis was not more than 50% for each lipid. Most of the hydrolytic process occurred before any significant change in photochemical activities could be observed. 4. In subchloroplast particles I, a treatment by lipolytic acyl hydrolase did not greatly affect the electron transport whilst lipid hydrolysis was almost complete. 5. In subchloroplast particles II, neither the electron flow activities nor lipid content were significantly altered by lipolytic acyl hydrolase. 6. The sites of lipolytic acyl hydrolase action appeared to be localized between plastoquinones and P700. It is suggested that it is not possible to establish a quantitative and/or temporal correlation between the extent of lipid hydrolysis and the inhibition of photochemical activities. 7. The profile of the hydrolysis of lipids in thylakoid membranes suggests that ionic lipids are less accessible to lipolytic acyl hydrolase than galactolipids.

Chloroplasts↗
PubMed1978

Chemical Cross-linking of Neighboring Thylakoid Membrane Polypeptides.

Cross-linking between protein components of whole spinach (Spinacia oleracea var. Nobel) thylakoids and of photosystem I- and II-enriched thylakoid fractions has been produced by reaction with the bifunctional imidoester dimethyl-3,3'-dithiobispropionimidate dihydrochloride as well as by the oxidation of intrinsic sulfydryl groups with an orthophenanthrolinecupric ion complex. The mixture of membrane proteins and their cross-linked products has been analyzed by two-dimensional sodium dodecyl sulfate electrophoresis, with a reductive cleavage step of the cross-linkages before the second dimension. Cross-linked aggregates up to a molecular weight of about 130 kilodaltons (kD) were analyzed, and it was inferred that the polypeptides appearing together in the same aggregates were neighbors within the membrane.In thylakoids as well as in isolated photosystem fractions, oligomers were formed by cross-linking polypeptides of the 60 to 90 kD range, among them the polypeptides of the chlorophyll-protein complex I. Polypeptides of 46, 19, and 12 kD were cross-linked to these complexes. Polypeptides of 25 and 22 kD, which are related to the chlorophyll-protein complex II, were cross-linked in thylakoids as well as in photosystem II fractions, suggesting that in the membrane these molecules are close together. In photosystem II fractions an oligomer having a molecular weight of about 60 kD was formed by cross-linking several polypeptides of different molecular weights: 40, 25, and 22 kD.Our cross-linking experiments show that protein interactions in the thylakoid membrane occurred mainly among the polypeptides of the two chlorophyll-protein complexes, thus suggesting an oligomeric nature of these apoproteins.

Journal Article↗