The aminoacyl phosphatidyl glycerols of the plastid membrane system in the process of chloroplast biogenesis

The composition of aminoacyl phosphatidyl glycerols in maize plastids at different stages of chloroplast differentiation has been studied. In the course of incubation of 14C amino acids or 14CO2 with maize and bean seedlings in vivo the 14C amino acids were incorporated preferably into the acid phospholipid fraction, forming O-esters of amino acids with phosphatidylglycerols. The rate of lipoamino acid compounds formation increased with the chloroplast differentiation and reached its maximum in the seedlings containing chloroplasts with a developed lamellar system. Changes in the amino acid composition of 14C aminoacyl phosphatidyl glycerols were observed at all stages of chloroplast ultrastructure development.     

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.     

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.     

BIOSYNTHESIS OF SMALL MOLECULES IN CHLOROPLASTS OF HIGHER PLANTS

1. Chloroplasts of higher plants contain enzymes which permit them to synthesize many kinds of small molecules in addition to carbohydrates. 2. Either aqueous or non-aqueous techniques may be used to isolate chloroplasts. Aqueous methods permit the isolation of chloroplasts showing high rates of photosynthesis; the organelles can be purified by means of density gradients. Non-aqueously isolated chloroplasts cannot photosynthesize, but show good retention of low-molecular-weight substances and soluble enzymes. 3. Whole cells photoassimilating ¹⁴CO₂ show considerable formation of ¹⁴C-labelled amino acids and lipids, but isolated chloroplasts exhibit very poor synthesis of amino acids and lipids from ¹⁴CO₂. 4. Chloroplasts play an important rôle in reducing nitrate to ammonia. There is controversy about the presence in chloroplasts of nitrate reductase and about the mechanism of the light-dependent reduction of nitrate to nitrite; however, it is generally agreed that non-cyclic electron transport directly supports reduction of nitrite to ammonia via a chloroplastic nitrite reductase. 5. Chloroplasts actively assimilate inorganic nitrogen into amino acids. The assimilation reaction is either the reductive amination of α-ketoglutarate to glutamate or the ATP-dependent conversion of glutamate to glutamine. The enzyme glutamate synthase has recently been found to be present in chloroplasts and may play an important function in nitrogen assimilation. 6. Numerous transaminases (aminotransferases) are present in chloroplasts. 7. The source of α-keto-acid precursors of chloroplastic amino acids is unknown. It remains to be established whether chloroplasts import the required keto acids or whether some of them might be generated via an incomplete tricarboxylic-acid cycle located in the chloroplast. 8. Chloroplasts contain characteristically high levels of mono and digalactosyl diglycerides, sulpholipid and phosphatidyl glycerol. They also have large amounts of polyunsaturated fatty acids. 9. Fatty acids are synthesized by the concerted action of fatty-acid synthetase, elongases and desaturases. Two pathways have been implicated for the formation of α-linolenic acid. 10. The galactosyldiglycerides are synthesized by successive galactosylation of diglyceride. The enzymes responsible are probably located in the chloroplastic envelope. 11. The other major chloroplastic acyl lipids (sulpholipid, phosphatidylglycerol and phosphatidylcholine) have not been, as yet, synthesized de novo by means of isolated chloroplast fractions. However, indirect evidence indicates that the first two are probably formed there. 12. Chlorophyllide synthesis involves the formation of δ-aminolaevulinic acid (δALA) followed by conversion of δALA to protoporphyrin IX, which is then transformed into protochlorophyll. 13. Recent evidence favours the view that δALA synthesis is not mediated by δALA synthetase but by another pathway in which δALA can be derived from α-ketoglutarate or glutamate. It has not been established whether this pathway is localized in plastids. 14. Conversion of δALA to protoporphyrin IX is mediated by soluble enzymes of the plastid stroma. Membrane-bound enzymes mediate the conversion of protoporphyrin to protochlorophyll. 15. Carotenoids are synthesized from acetyl C_oA via geranylgeranyl-pyrophosphate and phytoene intermediates. Evidence has been obtained for both neurosporene and lycopene as precursors of the cyclic carotenoids. 16. The overall pathway of carotenoid formation is subject to photoregulation, particularly during the development of the chloroplast. 17. Carotenes are precursors of xanthophylls, the inserted oxygen being derived from molecular oxygen. 18. Chloroplasts may synthesize or interconvert gibberellin hormones.     

On the localization of polyribosomes in the system of chloroplast lamellae

From chloroplasts of 10-day-old pea seedlings exposed to the light for 19 h, two fractions have been isolated. One of them is rich in lamellae of the stroma, and the other is rich in thylakoids and fragments of the grana. These fractions have been obtained after centrifugation of chloroplasts disrupted by osmotic shock in a discontinuous sucrose gradient. The fraction containing thylakoids of grana differs from the fraction of lamellae of the stroma in its higher content of RNA and DNA as related to protein and in the capacity to incorporate intensively ¹⁴C amino acids into proteins. After its treatment with detergents (0.5% sodium deoxycholate and 0.4% Triton X-100) and repeated centrifugation in the discontinuous sucrose gradient it dissociates further into two fractions. During electron microscopic studies one of these fractions displays partially disrupted grana and the other exhibits extensive networks of polyribosomes incompletely liberated from proteins, including the de novo synthesized protein.The similar treatment of the fraction rich in lamellae of the stroma does not result in the liberation of polyribosomes.It is concluded that in this stage of chloroplast development, polyribosomes occurring in the lamellae system are localized in the thylakoids of grana and are not bound to lamellae of the stroma.     

BIOSYNTHESIS IN ISOLATED ACETABULARIA CHLOROPLASTS : I. Protein Amino Acids

The ability of chloroplasts isolated from Acetabulana mediterranea to synthesize the protein amino acids has been investigated. When this chloroplast isolate was presented with ¹⁴CO₂ for periods of 6–8 hr, tracer was found in essentially all amino acid species of their hydrolyzed protein Phenylalanine labeling was not detected, probably due to technical problems, and hydroxyproline labeling was not tested for The incorporation of ¹⁴CO₂ into the amino acids is driven by light and, as indicated by the amount of radioactivity lost during ninhydrin decarboxylation on the chromatograms, the amino acids appear to be uniformly labeled. The amino acid labeling pattern of the isolate is similar to that found in plastids labeled with ¹⁴CO₂ in vivo. The chloroplast isolate did not utilize detectable amounts of externally supplied amino acids in light or, with added adenosine triphosphate (ATP), in darkness. It is concluded that these chloroplasts are a tight cytoplasmic compartment that is independent in supplying the amino acids used for its own protein synthesis. These results are discussed in terms of the role of contaminants in the observed synthesis, the "normalcy" of Acetabularia chloroplasts, the synthetic pathways for amino acids in plastids, and the implications of these observations for cell compartmentation and chloroplast autonomy.     

Biosynthesis of aromatic amino acids by highly purified spinach chloroplasts – Compartmentation and regulation of the reactions

The ability of chloroplasts to synthesize aromatic amino acids from CO₂ was investigated using highly purified, intact spinach ( Spinacia oleracea L. cv. Viking II) chloroplasts and ¹⁴CO₂. Incorporation of ¹⁴C into aromatic amino acids was very low, however, and this was assumed to be due to lack of phosphoenolpyruvate (PEP), one of the substrates for the shikimate/arogenate pathway leading to aromatic amino acids in chloroplasts. Therefore, the glycolytic enzymes phosphoglycerate mutase (EC 2.7.5.3) and enolase (EC 4.2.1.11) were added to the ¹⁴CO₂ fixation medium in order to convert labelled 3-phosphoglycerate exported from the intact chloroplasts to 2-phosphoglycerate and PEP. In this way a part of the glycolytic pathway was reconstituted outside the chloroplasts to substitute for the cytoplasm lost on isolation. The presence of both enzymes in the medium increased incorporation of ¹⁴C into Tyr and Phe more than ten-fold and incorporation into Trp about two-fold, while total ¹³CO₂ fixation rates were not affected. Our results suggest that chloroplasts do not contain phosphoglycerate mutase or enolase, and that, in vivo, PEP is synthesized in the cytoplasm and imported to the chloroplast stroma for the biosynthesis of aromatic amino acids. The biosynthesis of all three aromatic amino acids was under feedback control. Using expected physiological concentrations (below 100 μ M ), each of the aromatic amino acids exerted a strict feedback inhibition of its own biosynthesis only.     

Involvement of ferredoxin in desaturation of lipid-bound oleate in chloroplasts

Intact spinach (Spinacia oleracea) chloroplasts, pulse-labeled with [¹⁴C]acetate, desaturate newly formed fatty acids as ester groups of monogalactosyl diacylglycerol in a subsequent chase in the dark. Rupture of pulse-labeled chloroplasts by addition of a detergent solution 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate preserves part of this desaturation activity. Direct addition of different free fatty acids together with appropriate cofactors to detergent-ruptured chloroplasts results in fatty acid labeling of monogalactosyl diacylglycerol. During subsequent incubation these lipid-linked fatty acids are desaturated, i.e. 18:1 to 18:2 and 18:3 and to a small extent also 16:0 to 16:3. The formation of 18:2 was also observed after incorporation of 18:1 into sulfolipid and phosphatidyl choline. Density gradient centrifugation separated a membrane fraction from detergent-ruptured chloroplasts which in the presence of appropriate cofactors incorporated 18:1 and 18:2 into the above-mentioned lipids. In the light, desaturation was dependent on added ferredoxin, whereas in the dark, in addition to ferredoxin NAD(P)H was also required. Preliminary evidence for the involvement of membrane-bound ferredoxin:NADP oxidoreductase (FNR) as a third component of desaturation in the dark was obtained by inhibitor studies including antibodies against FNR. Desaturation of lipid-bound 18:1 and 18:2 resembles stearoyl-ACP desaturation with respect to its requirement of reduced ferredoxin and oxygen.     

Lipid fixation during preparation of chloroplasts for electron microscopy

Reaction of osmium tetroxide with isolated spinach chloroplasts fixed completely the glycolipids, phosphatidyl glycerol, and phosphatidyl choline. Under the same reaction conditions only 30% of the chlorophyll was fixed. Reaction of potassium permanganate with isolated spinach chloroplasts fixed more than 90% of the glycolipids, phosphatidyl glycerol, and phosphatidyl choline, provided the reaction period was long enough. Potassium permanganate also fixed the chlorophyll. Reaction of osmium tetroxide and potassium permanganate with isolated (14)C-lipids from Chlorella pyrenoidosa fixed 59% and 66% of the radioactivity, respectively. The lipids that were not fixed included sterols and pigments. Electron micrographs show that chloroplasts extracted with chloroform-methanol after fixation in osmium tetroxide or potassium permanganate differ from those dehydrated with acetone mainly in that in the former, osmiophilic globules have been removed and there seems to be some fusion of the boundary membranes and grana membranes. These effects may be due to the extraction of unfixed, neutral lipids such as sterols and quinones.     

Lipid biosynthesis by chloroplasts from olive tree leaves

In vitro incubation of isolated chloroplasts from young olive tree leaves ( Olea europaea L. cv. Marteño) in acetate-1-¹⁴C showed a high labelling of saturated fatty acids (palmitic + stearic) and, above all, of the monounsatured ones (oleic); the low biosynthetic rate of α-linolenic acid being noteworthy. These fatty acids are mainly found as free ones, or incorporated in mono-and diglyceride molecules. Phosphoand galactolipids, the most abundant acyl-lipid components of chloroplast lamellae, showed low incorporation rates. The fatty acid synthesis by isolated chloroplasts depends on exogenous CoA, ATP, NADPH and, especially, on added ACP (acyl carrier protein) preparation from Escherichia coli , whereas it was strongly inhibited by Triton X-100.
In vivo experiments with acetate-1-¹⁴C infiltration into young excised leaves showed a high labelling of chloroplast phospholipids, but a low ¹⁴C incorporation into galactolipids, a remarkable feature because these latter are main components of chloroplast lamellae. The high biosynthetic rate of α-linolenic acid is noteworthy and appears mainly linked to monogalactosyldiglycerides. Also the low incorporation of saturated fatty acids to neutral lipids is remarkable. The low in vitro synthesis of α-linolenic acid in comparison with that of the in vivo conditions, suggests the existence of a cooperation between chloroplasts and other parts of the cell to carry out the synthesis of this compound.     

MALDI-TOF mass spectrometry methods for evaluation of in vitro aminoacyl tRNA production

Unnatural amino acid mutagenesis requires the in vitro production of aminoacyl tRNAs. Bacteriophage T4 RNA ligase is used to ligate a-amino-protected dCA amino acids to 74mer tRNA. Previously, there has been no facile method for evaluating the efficiency of this reaction prior to using the tRNA in translation. We report a novel use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in monitoring the formation of aminoacyl 76mer tRNA. This method is more efficient and precise than the traditional technique of gel electrophoresis. These MALDI conditions should also prove useful for analyzing aminoacyl tRNAs produced through aminoacyl tRNA synthetases and other methods.     

A model for the coevolution of the genetic code and the process of protein synthesis: Review and assessment.

The contemporary genetic code and the process of protein biosynthesis most assuredly evolved from a simpler code and process. We believe that there was obligatory coevolution of the two and that the earlier code and process must have involved a more direct linkage between the amino acids and the information macromolecule. We propose that an early form of translating existed in which amino acids were attached directly to the 'messenger' RNA along the backbone as 2'OH aminoacyl esters. These esters then condensed with each other on the RNA backbone yielding a peptide covalently attached to the RNA, without the use of tRNA's and ribosomes. THis presentation is concerned with experimental data which indicate that such a simple translation system is possible and must have involved the following steps: (1) formation of the aminoacyl adenylate anhydride, (2) transfer of the amino acid from the adenylate to immidazole, (3) transfer of the amino acid from imidazole to 2'OH groups along the backbone of RNAs, (4) condensation of the amino acids to yield peptides. Steps (1)-(3) have been confirmed in chemical systems. Our preliminary evidence indicates step (4) is also possible. The aminoacylation of polyribonucleotides and the subsequent formation of peptides is a dynamic and experimentally accessible system for studying genetic coding specfities and our present studies are now concentrated on step (4), looking for such specifities.     

Effects of prolactin and androgens on seminal vesicular lipids of castrated mature bonnet monkeys Macaca radiata

Effects of prolactin (PRL), bromocriptine (Br), testosterone propionate (TP), dihydrotestosterone (DHT) and the combination of these androgens with PRL/Br on the total lipid, total cholesterol, total glyceride glycerols, total phospholipid and their fractions in seminal vesicles of castrated mature monkeys were studied. Glyceride glycerols formed the major portion (50%) of total lipids in normal monkeys. Cholesterol and phospholipids were of equal share (25%). Esterified cholesterol formed major share (75%) of total cholesterol. Diacyl glycerol was the major (60%) glyceride glycerol and phosphatidyl choline and ethanolamine were the major phospholipid classes. Except triacyl glycerol castration markedly decreased all the lipid classes. PRL restored normal free and esterified cholesterol and phosphatidyl inositol but Br invariably decreased all the lipid classes. TP/DHT treatment stimulated the free and esterified cholesterol more than the control; it restored the normal glyceride glycerols. Phosphatidyl inositol, choline and ethanolamine were stimulated by androgens and other phospholipid classes were brought to normal. Addition of PRL + TP/DHT markedly increased esterified cholesterol, phosphatidyl inositol, choline, ethanolamine and phosphatidic acid. In all these aspects, Br counteracted the effects of androgens and PRL.     

The occurrence of phosphatidyl choline exchange protein in leaves

The transfer of phosphatidyl choline between liposomes was stimulated by the protein fractions from spinach leaves, etiolated and greening leaves of $\text{Avena}$ seedlings. This is confirmed by the transfer of [¹⁴C]phosphatidyl choline or spin-labeled phosphatidyl choline between donor and acceptor liposomes. ESR spectrum changes also indicated that no spin-labeled phosphatidyl choline was released from donor liposomes by spinach leaf protein unless acceptor liposomes were present. [¹⁴C]phospholipids were transferred from liposomes to both spinach chloroplasts and $\text{Avena}$ etiochloroplasts by phosphatidyl choline exchange protein from germinated castor bean endosperms and further from liposomes to spinach chloroplasts by spinach leaf protein. These results support the view that phosphatidyl choline in the plastid is supplied from the synthesis site, the endoplasmic reticulum, by phospholipid exchange protein.     

A model for the coevolution of the genetic code and the process of protein synthesis: Review and assessment

The contemporary genetic code and the process of protein biosynthesis most assuredly evolved from a simpler code and process. We believe that there was obligatory coevolution of the two and that the earlier code and process must have involved a more direct linkage between the amino acids and the informational macromolecule. We propose that an early form of translating existed in which amino acids were attached directly to the ‘messenger’ RNA along the backbone as 2'OH aminoacyl esters. These esters then condensed with each other on the RNA backbone yielding a peptide covalently attached to the RNA, without the use of tRNAs and ribosomes. This presentation is concerned with experimental data which indicate that such a simple translation system is possible and must have involved the following steps: (1) formation of the aminoacyl adenylate anhydride, (2) transfer of the amino acid from the adenylate to imidazole, (3) transfer of the amino acid from imidazole to 2'OH groups along the backbone of RNAs (4) condensation of the amino acids to yield peptides. Steps (1)–(3) have been confirmed in chemical systems. Our preliminary evidence indicates step (4) is also possible. The aminoacylation of polyribonucleotides and the subsequent formation of peptides is a dynamic and experimentally accessible system for studying genetic coding specifities and our present studies are now concentrated on step (4), looking for such specifities.     

A substrate-assisted concerted mechanism for aminoacylation by a class II aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRS) join amino acids to their cognate transfer RNAs, establishing an essential coding relationship in translation. To investigate the mechanism of aminoacyl transfer in class II Escherichia coli histidyl-tRNA synthetase (HisRS), we devised a rapid quench assay. Under single turnover conditions with limiting tRNA, aminoacyl transfer proceeds at 18.8 s(-)(1), whereas in the steady state, the overall rate of aminoacylation is limited by amino acid activation to a rate of 3 s(-)(1). In vivo, this mechanism may serve to allow the size of amino acid pools and energy charge to control the rate of aminoacylation and thus protein synthesis. Aminoacyl transfer experiments using HisRS active site mutants and phosphorothioate-substituted adenylate showed that substitution of the nonbridging Sp oxygen of the adenylate decreased the transfer rate at least 10 000-fold, providing direct experimental evidence for the role of this group as a general base for the reaction. Other kinetic experiments revealed that the rate of aminoacyl transfer is independent of the interaction between the carboxyamide group of Gln127 and the alpha-carboxylate carbon, arguing against the formation of a tetrahedral intermediate during the aminoacyl transfer. These experiments support a substrate-assisted concerted mechanism for HisRS, a feature that may generalize to other aaRS, as well as the peptidyl transferase center.     

Pyruvate-Derived Amino Acids in Spinach Chloroplasts : Synthesis and Regulation during Photosynthetic Carbon Metabolism

A probable carbon flow from the Calvin cycle to branched chain amino acids and lipids via phosphoenolpyruvate (PEP) and pyruvate was examined in spinach (Spinacia oleracea) chloroplasts. The interpendence of metabolic pathways in and outside chloroplasts as well as product and feedback inhibition were studied. It was shown that alanine, aromatic, and small amounts of branched chain amino acids were formed from bicarbonate in purified intact chloroplasts. Addition of PEP only favored formation of aromatic amino acids. Mechanisms of regulation remained unclear. Concentrations of PEP and pyruvate within the chloroplast impermeable space during photosynthetic carbon fixation were 15 times higher than in the reaction medium. A direct carbon flow to pyruvate was identified (0.1 micromoles per milligram chlorophyll per hour). Pyruvate was taken up by intact chloroplasts slowly, leading to the formation of lysine, alanine, valine, and leucine plus isoleucine (approximate ratios, 100-500:60-100:40-100:2-10). The K_m for the formation of valine and leucine plus isoleucine was estimated to be 0.1 millimolar. Ten micromolar glutamate optimized the transamination reaction regardless of whether bicarbonate or pyruvate was being applied. Alanine and valine formation was enhanced by the addition of acetate to the reaction mixture. The enhancement probably resulted from an inhibition of pyruvate dehydrogenase by acetyl-S-coenzyme A formed from acetate, and resulting accumulation of hydroxyethylthiamine diphosphate and pyruvate. High concentrations of valine and isoleucine inhibited their own and each others synthesis and enhanced alanine formation. When pyruvate was applied, only amino acids were formed; when complemented with bicarbonate, fatty acids were formed as well. This is probably the result of a requirement of acetyl-S-coenzyme A-carboxylase for bicarbonate.     

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.     

Active site of an aminoacyl-tRNA synthetase dissected by energy-transfer-dependent fluorescence.

Aminoacyl-tRNA synthetases establish the rules of the genetic code by catalyzing attachment of amino acids to specific transfer RNAs (tRNAs) that bear the anticodon triplets of the code. Each of the 20 amino acids has its own distinct aminoacyl-tRNA synthetase. Here we use energy-transfer-dependent fluorescence from the nucleotide probe N-methylanthraniloyl dATP (mdATP) to investigate the active site of a specific aminoacyl-tRNA synthetase. Interaction of the enzyme with the cognate amino acid and formation of the aminoacyl adenylate intermediate were detected. In addition to providing a convenient tool to characterize enzymatic parameters, the probe allowed investigation of the role of conserved residues within the active site. Specifically, a residue that is critical for binding could be distinguished from one that is important for the transition state of adenylate formation. Amino acid binding and adenylate synthesis by two other aminoacyl-tRNA synthetases was also investigated with mdATP. Thus, a key step in the synthesis of aminoacyl-tRNA can in general be dissected with this probe.