Functional role of aminoacyl phosphatidylglycerols in the lamellar system of chloroplasts]

The role of 14C-aminoacyl-tRNAs in the formation of aminoacyl phosphatidyl glycerols in isolated chloroplasts of haricot bean leaves was studied. The formation of 14C-aminoacyl-tRNAs was more intensive in the case when 14C-aminoacyl phosphatidyl glycerols were the source of amino acids. On incubation of lamellae with 14C-aminoacyl phosphatidyl glycerols, 14C-amino acids proved to be incorporated intensively in protein of the lamellae. Membrane-bound chloroplast ribosome-like particles were observed on the outermost thylakoid membranes of the grana stacks as well as on the stroma thylakoids. It is concluded that aminoacyl phosphatidyl glycerols play an important role in lateral transport of amino acids within the chloroplasts lamellar system.     

Study of amino acid composition and biosynthesis of protein components of the membrane system of corn plastids during biogenesis of chloroplasts]

The biosynthesis of membrane proteins in maize plastids at different stages of differentiation of the chloroplast lamellar system was studied. Prolamellar and lamellar system preparations were isolated from maize plastids, disintegrated by osmotic shock under hypotonic conditions. Changes in the amino acid composition of 14C membrane proteins were observed at all stages of chloroplast ultrastructure formation. The maximal level of the apolar amino acids was observed in the membrane fraction of chloroplasts. Washed membranes from maize proplastids and chloroplasts can be resolved into at least 14 protein bands on formic acid--urea polyacrylamide gel. It is pointed out that biogenesis process leads to the increase of lipophylic protein content in the chloroplast lamellae fraction.     

Lipid and Fatty Acid composition of chloroplast envelope membranes from species with differing net photosynthesis

Lipid and fatty acid compositions were determined for chloroplast envelope membranes isolated from spinach (Spinacia oleracea L.), sunflower (Helianthus annuus L.), and maize (Zea mays L.) leaves. The lipid composition was similar in sunflower, spinach, and undifferentiated maize chloroplast envelope membranes and different in maize mesophyll chloroplast envelope membranes. The predominant lipid constituents in all envelope membranes were monogalactosyldiglyceride (27 to 46%), digalactosyldiglyceride (18 to 33%), and phosphatidylcholine (7 to 30%). The fatty acid composition was also similar in sunflower and spinach chloroplast envelope membranes in comparison to those from maize. The major acyl fatty acids of the chloroplast envelope membrane were palmitic (C_16:0, 41 and 36%) and linolenic (C_18:3, 29 and 40%) acids for spinach and sunflower; palmitic (77%) and stearic (C_18:0, 12%) acids for young maize; and palmitic (61%), stearic (14%), and linolenic (13%) acids for mature maize. The differences in lipid and acyl fatty acid compositions among these plants which vary in their rates of net photosynthesis were largely quantitative rather than qualitative.     

Lipid composition in leaves of cucumber genotypes as affected by different temperature regimes and grafting

The lipid composition of leaves has been investigated in different genotypes of cucumber ( Cucumis sativus L.), which differ in temperature requirement for cultivation. In addition the effects of hardening by low but non-chilling temperature, soil heating and grafting (on the chilling-resistant C. ficifolia L.) on lipid composition have been studied. Content and composition of phospholipids and sterols were determined as well as phospholipid/sterol ratio, and fatty acid composition of total lipids and the different phospholipids.
The effects of genetic differentiation and of the various culture treatments on lipid composition of the leaves were very different. Genetic differentiation was evident as higher levels of Iinolenic acid in several phospholipids in the more cold-tolerant cultivars. Hardening the plants by low temperature resulted in a higher phospholipid level (especially phosphatidyl choline), more unsaturated phospholipid, and lowering of the sterol/phiospholipid ratio, all properties which may contribute to a higher membrane fluidity and lower growth temperature limit. Soil healing reduced the phospholipid level of the leaves slightly, and a higher content of 3- trans -hexadece-noic acid in phosphatidyl glycerol was observed. Grafting cucumber on the cold-resistant rootstock of C. ficifolia also raised the level of trans -hexadecenoic acid in phosphatidyl glycerol. The role of this fatty acid in the functioning of the chloroplast is discussed.     

Changes in Pool Sizes of Free Amino Acids and Amides in Leaves and Plastids of Zea mays during Leaf Development

The concentrations of free amino acids and amides within isolated maize (Zea mays L.) plastids were determined and compared with concentrations in the leaf tissue. The concentrations were different for each individual amino acid and varied between 1 and 10 millimolar. At five different developmental stages concentrations in the plastids were greater than those in the intact leaf tissue. During development, from the proplastid stage to the mature chloroplast, the amount of each amino acid per plastid remained relatively constant, but there were decreases in concentrations of plastid amino acids resulting from the developmental increase in plastid volume. In proplastids, the free amino acids were present in greater concentrations than those previously found to inhibit partially amino acid-synthesizing enzymes located in chloroplasts. In the chloroplasts, the molarities of the free amino acids were within the range known to inhibit amino acid-synthesizing enzymes.     

Phospholipid Alterations During Growth of Escherichia coli

As cultures of Escherichia coli progressed from the exponential growth phase to the stationary growth phase, the phospholipid composition of the cell was altered. Unsaturated fatty acids were converted to cyclopropane fatty acids, and phosphatidyl glycerol appears to have been converted to cardiolipin. With dual isotope label experiments, the kinetics of synthesis of cyclopropane fatty acid for each of the phospholipids was examined in vivo. The amount of cyclopropane fatty acid per phospholipid molecule began to increase in phosphatidyl ethanolamine at a cell density below the density at which this increase was observed in phosphatidyl glycerol or cardiolipin. The rate of this increase in phosphatidyl glycerol or in cardiolipin was faster than the rate of increase in phosphatidyl ethanolamine. After a few hours of stationary-phase growth, all the phospholipids were equally rich in cyclopropane fatty acids. It is suggested that the phospholipid alterations observed are a mechanism to protect against phospholipid degradation during stationary phase growth. Cyclopropane fatty acid synthetase activity was assayed in cultures at various stages of growth. Cultures from all growth stages examined had the same specific activity in crude extracts.     

Phospholipid biosynthesis in the lungs in a chronic inflammatory bronchopulmonary process

Glycerokinase, glycerophosphate dehydrogenase, glycerophosphate acyltransferase activity, glycerophosphate, dioxiacetonphosphate level and in vivo incorporation of (U-14C)-glucose into the lung phospholipid structure were studied in normal rats and in conditions of chronic bronchopulmonary inflammation. The inhibition of glycerokinase and glycolytic ways of glycerophosphate formation was demonstrated. The data obtained have shown that glucose incorporation into phosphatidyl cholines, phosphatidyl glycerols and phosphatidyl ethanolamines was decreased, while the incorporation of radioactivity into the sphingomyelins and lysophosphatidyl cholines was significantly activated.     

Lipids of the primary intracellular symbiote of the pea aphid, Acyrthosiphon pisum

The lipid composition of isolated primary symbiotes was investigated. The symbiotes were found to possess phosphatidyl ethanolamine, phosphatidyl choline, cholesterol, and glycerides. The fatty acids esterified to the various glycerides and glyceride derivatives were found to be predominantly C10, C12, C13, and C14 saturated components.The lipids of the pea aphid were also investigated as a measure of the purity of the symbiote preparation. The same classes of compounds were found, but the fatty acid complement was found to be different. The fatty acids were found to be predominantly C14, C18, C18:1, C18:2, and C18:3. A carotenoid-like pigment, whose absorption maximum in benzene was 460 nm, was also present.The possible significance of the various lipids to the systematic classification of the symbiotes is discussed, along with some insight into what may be an apparent dependency factor of the symbiote for the host.     

Stearoyl-CoA desaturase 1 genotype and stage of lactation influences milk fatty acid composition of Canadian Holstein cows

Single nucleotide polymorphisms in the coding region of the bovine stearoyl-CoA desaturase 1 gene have been predicted to result in p.293A (alanine at amino acid 293) and p.293V (valine at amino acid 293) alleles at the stearoyl-CoA desaturase1 locus. The objectives of this study were to evaluate the extent to which genotypes at the stearoyl-CoA desaturase 1 locus and stage of lactation influence milk fatty acid composition in Canadian Holstein cows. Cows with the p.293AA genotype had higher C10 index, C12 index and C14 index and higher concentrations of C10:1 (10 carbon fatty acid with one double bond), C12:1 (12 carbon fatty acid with one double bond) and myristoleic acid (C14:1) compared with the p.293AV or p.293VV cows. Cows had higher C18 index and total index, and lower C10 index, C12 index, C14 index and CLA index during early lactation compared with the subsequent lactation stages. Early lactation was also characterized by higher concentrations of oleic acid (C18:1 cis -9), vaccenic acid (C18:1 trans -11), linoleic acid (C18:2), monounsaturated fatty acids and total polyunsaturated fatty acids, and lower concentrations of capric acid (C10:0), C10:1, lauric acid (C12:0), C12:1, myristic acid (C14:0), myristoleic acid (C14:1), palmitic acid (C16:0) and total saturated fatty acids compared with the subsequent lactation stages. Neither the stearoyl-CoA desaturase 1 genotype nor the stage of lactation had an influence on conjugated linoleic acid concentrations in milk.     

Fatty Acid Distribution in Glycolipids and Phospholipids in Cotyledons of Germinating Soybeans

This investigation was conducted to observe changes in the fatty acid distributions of glycolipids (GL) and phospholipids (PL) in cotyledons of soybean seeds which were germinated either in the dark or the light at 28°C for 8 days. The GL isolated from the total lipids of cotyledons at different germinating stages were : acyl sterylglycoside (ASG), monogalactosyl diglyceride (MGD), digalactosyl diglyceride (DGD) and sulfolipid (SL). The PL isolated from the same total lipids as described above were : diphosphatidyl glycerol (DPG), phosphatidic acid (PA), phosphatidyl ethanolamine (PE), phosphatidyl glycerol (PG), phosphatidyl choline (PC) and phosphatidyl inositol (PI).

During germination of soybean seeds, the content of linoleic and linolenic acids in MGD or DGD was markedly higher than that of the other GL. The positional distribution of fatty acids in PE, PC and PI was shown in all PL, in which saturated fatty acids, especially palmitic acid, were highly concentrated in position 1 and unsaturated fatty acids, especially linoleic acid, mainly occupied position 2. A remarkable difference in the changing patterns of fatty acid composition, which depended on the germinating conditions tested, was observed between GL and PL. The changes in fatty acid composition of GL were more marked in the light-grown seedlings than in the dark-grown, whereas those of PL were more remarkable in the latter than in the former. Therefore, the positional distribution of fatty acids in PL was more evident in the light-grown seedlings than in the dark-grown ones.

These results suggest the metabolic fate of GL and PL in cotyledons of soybean seeds, probably owing to the differences in the two germinating conditions tested.     

CmaE: a transferase shuttling aminoacyl groups between carrier protein domains in the coronamic acid biosynthetic pathway

During the biosynthesis of the cyclopropyl amino acid coronamic acid from l-allo-Ile by the phytotoxic Pseudomonas syringae, the aminoacyl group covalently attached to the pantetheinyl arm of CmaA is shuttled to the HS-pantetheinyl arm of the protein CmaD by the aminoacyltransferase CmaE. CmaE will only recognize deacylated CmaA for initial complexation. The aminoacyl group becomes covalently attached to the active site Cys of CmaE and can then be transferred out to the holo pantetheinylated form of CmaD. Both l-Val/l-[14C]Val exchange studies and MALDI-TOF support a reversible shuttling process. Aminoacylated-S-CmaE will transfer the l-Val moiety to the HS-pantetheinyl arm of other T domains, including CytC2, BarA, and ArfA C2-A2-T2 but not to free HS-pantetheine. CmaD could be loaded with other amino acids, for example, l-Leu and l-Thr, by the action of heterologous donor T domains containing alternative aminoacyl groups. Additionally, CmaE is able to accept l-Phe as a substrate when presented on CmaD and is able to load this aminoacyl moiety onto heterologous T domains, expanding the potential for CmaE to be used as a tool for generating chemical diversity within an NRPS assembly line.     

Alterations in the phospholipid composition of Escherichia coli B during growth at different temperatures

The major phospholipid classes of Escherichia coli B, phosphatidyl ethanolamine, cardiolipin, and phosphatidyl glycerol, were quantitated at different stages of the growth cycle. The organisms were incubated at both 27 and 37 C. Significant differences were observed both in the amounts of total lipid phosphorus per gram (dry weight) of cells and in the relative percentages of the individual phospholipids. At 37 C the total amount of lipid phosphorus decreased significantly throughout the growth cycle. However, at 27 C total lipid phosphorus accumulated. The patterns of the three major phospholipid classes of Escherichia coli exhibited complex quantitative changes. In addition, some evidence based on glycerol to phosphate molar ratios indicated that phosphatidyl glycerolphosphate replaced phosphatidyl glycerol during the late growth stages of E. coli B when grown at 27 C. A comparative analysis of phospholipid and fatty acid patterns led to a hypothesis attempting to explain some reported variations in the lipid composition of E. coli under different conditions of growth.     

Synthesis of lipids from acetate by human preputial and abdominal skin in vitro

Lipogenesis in vitro from acetate-1-(14)C was studied in human preputial skin and abdominal skin. Radioactive lipids were separated by column chromatography on Florisil and by thin-layer chromatography on silica gel. Radioactivity was incorporated chiefly into the triglyceride, sterol, and polar lipid fractions, while lesser amounts of (14)C were found in the hydrocarbon, wax, diglyceride, monoglyceride, and fatty acid fractions; labeling of steryl esters was minimal. On thin-layer chromatography, the radioactive polar lipids had mobilities similar to lysolecithin, phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidic acid. The radioactive fatty acids of the different lipid fractions were separated by gas-liquid chromatography. The major (14)C-labeled acids were 16:0 and 18:0. Radioactivity was also detected in acids 14:0, 15:0, 16:1, 18:1, 18:2, 20:0, 20:1, 22:0, 24:0, 24:1, and 26:0. No radioactivity could be detected in arachidonic acid, although this fatty acid comprises 9% of the chromatographed fatty acids. The pattern of incorporated (14)C was different from the percentage mass composition of the fatty acids. Skin is therefore active in the biosynthesis of a wider variety of lipids than previously demonstrated.     

The incorporation of [14C]palmitic acid into the lipids of mouse brain in vivo

Abstract— The half-life of free [¹⁴C]palmitic acid injected intracerebrally into C57BL/10J mice was less than 5 min. The rapid disappearance of radioactivity as palmitic acid was accompanied by increases in the radioactivity of the phosphatidic acids and the diacyl-glycerols. The peak specific radioactivity of the diacylglycerols occurred at about 6-8 min after injection. The triacylglycerols, phosphatidyl ethanolamines and phosphatidyl cholines exhibited increasing amounts of radioactivity during the first 40 min. At 160 min after injection, the distribution of radioactivity was similar to the pattern observed at 12 h. The biosynthetic pathway through the phosphatidic acids and the diacylglycerols to triacylglycerols, phosphatidyl ethanolamines and phosphatidyl cholines is apparently the major pathway in vivo for the esterification of free fatty acids in the brain.     

Pattern of CO2 fixation during vegetative development and gametic differentiation in Chlamydomonas reinhardtii

The rate of C¹⁴O₂ incorporation into amino acids and organic acids in C. reinhardtii is a function of particular stages of development in the life cycle of the alga. Gametic differentiation in nitrogen free medium is accompanied by a reduced rate of amino acid synthesis and a higher synthesis of organic acids than that found for the cells undergoing vegetative development. The addition of ammonium to differentiating gametes results in an increased synthesis of amino acids, particularly the basic ones, and a concomitant reduction in organic acid synthesis.     

The manipulation of polar head group composition of phospholipids in the wheat Miranovskaja 808 affects frost tolerance

Caryopses of the frost-resistant cultivar of the wheat Triticum aestivum L., Miranovskaja 808, were germinated and grown in the presence of various concentrations of choline chloride. Changes in the composition of leaf total phospholipids and leaf total fatty acids at two extreme temperatures (25°C and 2°C) as well as changes in frost resistance were followed. A choline chloride concentration-dependent accumulation of phosphatidyl choline was observed in the leaves. Seedlings grown at 2°C accumulated more phosphatidyl choline at each choline chloride concentration than those grown at 25°C. There was an inverse relationship between the contents of phosphatidyl choline and phosphatidic acid in the leaves. Neither the temperature nor choline chloride seemed to affect fatty-acid composition. Modification of polar-head group composition of phospholipids affected frost tolerance: Seedlings grown in the presence of 15 mM choline chloride at 25°C exhibited a freezing resistance equal to that of hardened controls. The data indicate that the polar-head group composition of membrane phospholipids in plants can be easily manipulated and point to the importance of phosphatidyl choline in cold adaptation processes.     

Lipid composition of chloroplast membranes from weed biotypes differentially sensitive to triazine herbicides

Chloroplasts were isolated from triazine-sensitive and triazine-resistant biotypes of common groundsel (Senecio vulgaris L.), common lambsquarter (Chenopodium album L.), and redroot pigweed (Amaranthus retroflexus L.). Chloroplast lipids were extracted and analyzed for differences among sensitive and resistant biotypes. The distribution of lipid between major lipid classes differed in chloroplasts from resistant and susceptible biotypes. Chloroplasts from resistant biotypes contained higher proportions of monogalactosyl diglyceride and phosphatidyl ethanolamine and lower proportions of digalactosyl diglyceride and phosphatidyl choline than did chloroplasts from susceptible biotypes. Monogalactosyl diglyceride and phosphatidyl ethanolamine were also quantitatively higher in membranes of resistant versus susceptible biotypes. The major lipid classes of resistant chloroplast membranes contained lipids comparatively richer in unsaturated fatty acids with the exceptions of digalactosyl diglyceride from all three biotypes and phosphatidyl ethanolamine from common groundsel. Results correlated changes in triazine sensitivity with qualitative and quantitative differences in the lipid composition of chloroplast membranes.     

Non-ribosomal biosynthesis of the cyclic octadecapeptide alamethicin.

The biosynthesis of the cyclic octadecapeptide, alamethicin, in a cell-free system of Trichoderma viride has been investigated. It was shown that nucleic acid- and ribo-some-free extracts of Trichoderma viride could catalyze alamethicin biosynthesis. Puromycin, erythromycin and RNAse did not inhibit this synthesis. The Sephadex G 200 filtrate contains a fraction (Kav=0.1) that catalyzes the biosynthesis of alamethicin and shows an ATP-32PPi exchange with 6 of the 8 constituent amino acids of alamethicin. The activated amino acids are bound to the enzyme as aminoacyl adenylates and as thiolesters in a proportion of 1 : 1. About 50% of each bound amino acid could be split off with 7% TCA. The TCA-stable bound amino acid could be split by mercury acetate, hydroxylamine and performic acid. N-ethylmaleimide blocked the binding of 50% of the amino acids to the enzyme, proving that some of the amino acids first bound as aminoacyl adenylates are then transferred into a thiolester bond.     

Primary structure of the maize NADP-dependent malic enzyme

Chloroplast-localized NADP-dependent malic enzyme (EC 1.1.1.40) (NADP-ME) provides a key activity for the carbon 4 fixation pathway. In maize, nuclear encoded NADP-ME is synthesized in the cytoplasm as a precursor with a transit peptide that is removed upon transport into the chloroplast stroma. We present here the complete nucleotide sequence for a 2184-base pair full-length maize NADP-ME cDNA. The predicted precursor protein is 636 amino acids long with a Mr of 69,800. There is a strong codon bias found in the amino-terminal portion of NADP-ME that is present in genes for the other enzymes of the C-4 photosynthetic pathway. The NADP-ME transit peptide has general features common to other known chloroplast stroma transit peptides. Comparison of mature maize NADP-ME to the amino acid sequences of known malic enzymes shows two conserved dinucleotide-binding sites. There is a third highly conserved region of unknown function. On the basis of amino acid sequence similarity, the maize chloroplastic enzyme is more closely related to eukaryotic cytosolic isoforms of malic enzyme than to prokaryotic isoforms. We discuss the functional and evolutionary relationship between the chloroplastic and cytosolic forms of NADP-ME.