In vivo incorporation of acetate into the acyl moieties of polar lipids from etiolated leek seedlings

Seven-day-old leek seedlings actively synthesize lipids in vivo from [1-¹⁴C]acetate, both in the light and in the dark. In the dark, phospholipid synthesis is more effective than galactolipid synthesis. Whatever the time of acetate incorporation by the etiolated seedlings, very long chain fatty acids having from 20 to 26 carbon atoms are found in all the polar lipids, including the acyl-CoAs. All of the labelled very long chain fatty acids incorporated into the polar lipids are saturated. On the other hand, the labelled C₁₈-fatty acids are unsaturated in phospholipids and galactolipids and almost no label is found in the saturated or unsaturated C₁₈-fatty acids of the acyl-CoAs.     

Acyl-CoA dependent acylation of phospholipids in the chloroplast envelope

Acyl-CoAs are substrates for acyl lipid synthesis in the endoplasmic reticulum. In addition, they may also be substrates for lipid acylation in other membranes. In order to assess whether lipid acylation may have a role in plastid lipid metabolism, we have studied the incorporation of radiolabelled fatty acids from acyl-CoAs into lipids in isolated, intact pea chloroplasts. The labelled lipids were phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol and free fatty acids. With oleoyl-CoA, the fatty acid was incorporated preferably into the sn-2 position of PC and the acylation activity mainly occurred in fractions enriched in inner chloroplast envelope. Added lysoPC stimulated the activity. With palmitoyl-CoA, the fatty acid was incorporated primarily into the sn-1 position of PG and the reaction occurred at the surface of the chloroplasts. As chloroplast-synthesized PG generally contains 16C fatty acids in the sn-2 position, we propose that the acylation of PG studied represents activities present in a domain of the endoplasmic reticulum or an endoplasmic reticulum-derived fraction that is associated with chloroplasts and maintains this association during isolation. This domain or fraction contains a discreet population of lipid metabolizing activities, different from that of bulk endoplasmic reticulum, as shown by that with isolated endoplasmic reticulum, acyl-CoAs strongly labelled phosphatidic acid and phosphatidylethanolamine, lipids that were never labelled in the isolated chloroplasts.     

Lipid synthesis in germinating Tradescantia pollen

Synthesis of neutral and polar lipids in pollen of Tradescantia paludosa during germination and tube growth was studied by the incorporation of acetate-[1-¹⁴C] into lipids in the presence and absence of inhibitors of RNA and protein synthesis. The proteins required for the synthesis of both neutral lipids and phospholipids are not made de novo during germination but are already present in the mature ungerminated pollen grain and they are functionally stable during the first 2 hr of pollen growth.     

In vivo utilization ofd(−)-3-hydroxybutyrate by chick brain and spinal cord

The in vivo utilization ofd-3-hydroxy[3-¹⁴C]butyrate for oxidation in the whole animal and for lipid and amino acid synthesis in brain and spinal cord of overnight-fasted 15-day-old chicks has been measured. Appreciable amounts of injected 3-hydroxy[3-¹⁴C]butyrate were expired as¹⁴CO₂ one hour after injection, the total amount of which increased with increasing dosages. Lipid synthesis was high in both brain and spinal cord. Free, cholesterol and phospholipids were the main lipids labeled in both, tissues, increasing with time after injection up to 120 min. The incorporation of radioactivity into triglycerides, esterified cholesterol and free fatty acids was not time-dependent. Increased concentrations of 3-hydroxybutyrate gave rise to higher synthetic rates both in brain and spinal cord The rate of amino acid synthesis was slightly higher in brain than in spinal cord. Glutamate was always the major amino acid formed.     

Quantitative evaluation of two pathways for ethanolamine phosphoglyceride biosynthesis in rat brainin vivo

[³H]Ethanolamine and [³²P]orthophosphate were injected intraventricularly into adult female rats. At varying time intervals after the injection (1–10 min), the animals were killed by means of a microwave apparatus, and phosphorylethanolamine and ethanolamine phosphoglycerides were extracted from the brains and counted after separation. The kinetic constants for phosphorylethanolamine incorporation into ethanolamine lipids were calculated both from³H data and from³²P data. From our results, it seems that base exchange reactions for ethanolamine incorporation into ethanolamine lipids are a pathway active in brainin vivo.     

Starch synthesis and carbohydrate oxidation in amyloplasts from developing wheat endosperm

The rates of incorporation of various metabolites into starch by isolated amyloplasts from developing endosperm of spring wheat (Triticum aestivum L. cv. Axona) were examined. Of the metabolites tested that were likely to be present in the cytosol at concentrations sufficient to sustain starch synthesis, only glucose 1-phosphate (Glc1P) supported physiologically relevant rates of starch synthesis. Incorporation of Glc1P into starch was both dependent on the presence of ATP and intact organelles. The rate of incorporation of hexose into starch became saturated at a Glc1P concentration of less than 1 mol·m^-3 in the presence of 1 mol·m^-3 ATP. Starch synthesis from 5 mol · m^-3 ADP-glucose supplied to the organelles occurred at rates 15-fold higher than from similar concentrations of Glc1P, but it is argued that this is probably of little physiological relevance. The net incorporation of hexose units into starch from GlclP was inhibited 50% by 100 mmol.m^-3 carboxyatractyloside. Carbohydrate oxidation in the amyloplast was stimulated by the addition of 2-oxoglutarate and glutamine, and in such circumstances incorporation of¹⁴C-labelled metabolites into starch was reduced. Glucose 6-phosphate proved to be a better substrate for oxidative pathways than Glc1P. Our results suggest that Glc1P is the primary substrate for starch synthesis in developing wheat endosperm, and that ATP required for starch synthesis is imported via an adenylate translocator.     

Factors influencing the abundance and metabolic capacities of microorganisms in Eastern Coastal Plain sediments

The abundance and metabolic capacities of microorganisms residing in 49 sediment samples from 4 boreholes in Atlantic Coastal Plain sediments were examined. Radiolabeled time-course experiments assessing in situ mirobial capacities were initiated within 30 min of core recovery. Acetate (1-¹⁴C- and³H-) incorporation into lipids, microbial colony forming units, and nutrient limitations were examined in aliquots of subsurface sediments. Water-saturated sands exhibited activity and numbers of viable microorganisms that were orders of magnitude greater than those of the low permeability dense clays. Increased radioisotope utilization rates were observed after 6–24-h incubation times when sediments were amended with additional water and/or nutrients. Supplements of water, phosphate, nitrate, sulfate, glucose, or minerals resulted in the stimulation of microbial activities, as evidenced by the rate of acetate incorporation into microbial lipids. Additions of water or phosphate resulted in the greatest stimulation of microbial activities. Regardless of depth, sediments that contained >20% clay particles exhibited lower activities and biomass densities, and greater stimulation with abundant water supplementation than did sediments containing >66% sands and hydraulic conductivities > 200 μm sec.^?1.     

Microbial activities in deep subsurface environments

Activities of microorganisms residing in terrestrial deep subsurface sediments were examined in 46 sediment samples from three boreholes. Radiolabeled time course experiments assessing in situ microbial activities were initiated within 30 min of core recovery. [1‐C⁴] Acetate incorporation into lipids, [ methyl‐³H] thymidine incorporation into DNA, [2‐¹⁴C]acetate, and [U‐¹⁴C]glucose mineralization in addition to microbial enrichment and enumeration studies were examined in surface and subsurface sediments. Surface soils contained the greatest biomass and activities, followed by the shallow aquifer zones. Water‐saturated subsurface sands exhibited three to four orders of magnitude greater activity and culturable microorganisms than the dense clay zones, which had low permeability. Regardless of depth, sediments that contained more than 20% clays exhibited the lowest activities and culturable microorganisms.     

Synthesis of medium-chain fatty acids and their incorporation into triacylglycerols by cell-free fractions fromCuphea embryos

During their rapid maturation period, seeds of Cuphea wrightii A. Gray mainly accumulate medium-chain fatty acids (C₈ to C₁₄) in their storage lipids. The rate of lipid deposition (40–50 mg·d^–1·(g fresh weight)^–1) is fourfold higher than in seeds of Cuphea racemosa (L. f.) Spreng, which accumulate long-chain fatty acids (C₁₆ to C₁₈). Measurements of the key enzymes of fatty-acid synthesis in cell-free extracts of seeds of different maturities from Cuphea wrightii show that malonyl-CoA synthesis may be a triggering factor for the observed high capacity for fatty-acid synthesis. Experiments on the incorporation of [1-¹⁴C]acetate into fatty acids by purified plastid preparations from embryos of Cuphea wrightii have demonstrated that the biosynthesis of medium-chain fatty acids (C₈ to C₁₄) is localized in the plastid. Thus, in the presence of cofactors for lipid synthesis (ATP, NADPH, NADH, acyl carrier protein, and sn-glycerol-3-phosphate), purified plastid fractions predominantly synthesized free fatty acids, 30% of which were of medium chain length. Transesterification of the freshly synthesized fatty acids to coenzyme A and recombination with the microsomal fraction of the embryo homogenate induced triacylglycerol synthesis. It also stimulated fatty-acid synthesis by a factor 2–3 and increased the relative amount of medium-chain fatty acids bound to triacylglycerols, which corresponded to about 60–80% in this lipid fraction.Abbreviations ACP acyl carrier protein - FW fresh weight This work was supported by the Bundesminister für Forschung und Technologie. The authors thank S. Borchert for her suggestions for plastid preparation.     

Quantitative evaluation of two pathways for phosphatidylcholine biosynthesis in rat brain in vivo

[Me-³H] choline and [³²P] orthophosphate were injected intraventricularly into adult female rats. After variable intervals from injection (1–10 min) the animals were sacrificed by means of a microwave apparatus, and phosphorylcholine and choline phosphoglycerides extracted from brain and counted for radioactivity content after separation. The kinetic constants (K) for phosphorylcholine incorporation into lipids were determined both for [³²P] and [³H] labeling. From the data obtained by these procedures it is concluded that base-exchange reactions for choline incorporation into lipids are operating in rat brain in vivo and that they represent a rapidly equilibrating system.     

Incorporation of14C-radioactivity into various lipid classes at different growth-stages of culturedLaminaria japonica

A study is reported on the incorporation of¹⁴C-acetate into lipid classes from three different growth stages ofLaminaria japonica, a species long used for food in Japan. This was done because of the possible utilization of its lipids.Radioactivity incorporated into whole lipids in the three growth stages under the same experimental conditions (10 °C, 500 lux) increased with maturity of the thalli. The radioactivity was found mainly in PC, TG and 1,2-DG and subsequently distributed into other lipid classes (PG,PI,PE,MGDG,SQDG and DGDG) to a lesser extent. The incorporation patterns of the former group were similar at all stages, but those of the latter group differed slightly according to growth stage.In juvenile thalli,¹⁴C was incorporated to a much higher extent into PG, MGDG, PI and fucosterol than PE, SQDG, DGDG and MG, while the¹⁴C-incorporation into MG, SQDG, DGDG and PS in the mature growth stage was higher than into the other lipid classes. The absolute level of incorporation was higher for all these compounds in mature thalli than the thalli of other growth stages.     

Solubilization of the enzyme catalyzing CDP-diglyceride-independent incorporation of myo-inositol into phosphatidyl inositol and its comparison to CDP-diglyceride:inositol transferase.

A solubilized preparation with activity for catalyzing the incorporation of free myo-inositol into phosphatidyl inositol was obtained from a rat liver microsomal fraction. The incorporation took place both in the presence and in the absence of cytidine diphosphodiglyceride (CDP-DG). The pH optimum of the incorporation in the absence of CDP-DG was 7.4–7.5, while that of the incorporation in its presence was 8.5–8.6. The incorporation in the absence of CDP-DG was activated by Mn²⁺ but not by Mg²⁺, while that in the presence of CDP-DG was activated by either Mn²⁺ or Mg²⁺. These results indicated that the incorporation in the absence of CDP-DG and the incorporation in its presence were catalyzed by different enzymes. Before Solubilization, the CDP-DG-independent enzyme was bound to endoplasmic reticulum. The CDP-DG-dependent enzyme also was bound mainly to endoplasmic reticulum and, to a minor extent, to plasma membrane. The CDP-DG-independent enzyme was more easily solubilized by sodium cholate than the CDP-DG-dependent enzyme. There were also differences between these two enzyme activities of the solubilized preparation with respect to their sensitivity to various detergents and their dependence on exogenous lipids. The CDP-DG-independent incorporation was inhibited by CDP-DG, by some nucleotides, and by phosphatidyl serine, while the CDP-DG-dependent incorporation was not inhibited by these substances. Both activities were both inhibited by thiol-reactive compounds.     

Acetate formation from CO and CO2 by cell extracts of Peptostreptococcus productus (strain Marburg)

Cell extracts of Peptostreptococcus productus (strain Marburg) obtained from CO grown cells mediated the synthesis of acetate from CO plus CO₂ at rates of 50 nmol/min × mg of cell protein. ¹⁴CO was specifically incorporated into C₁ of acetate. No label exchange occurred between ¹⁴C₁ of acetyl-CoA and CO, indicating that ¹⁴CO incorporation into acetate was by net synthesis rather than by an exchange reaction. In acetate synthesis from CO plus CO₂ the latter substrate could be replaced to some extent by formate or methyl tetrahydrofolate as the methyl donor. The methyl group of methyl cobalamin was incorporated into acetate ony at very low activities. The cell extracts contained high levels of enzyme activities involved in acetate or cell carbon synthesis from CO₂. The following enzymic activities were detected: CO: methyl viologen oxidoreductase, formate dehydrogenase, formyl tetrahydrofolate synthetase, methenyl tetrahydrofolate cyclohydrolase, methylene tetrahydrofolate dehydrogenase, methylene tetrahydrofolate reductase, phosphate acetyltransferase, acetate kinase, hydrogenase, NADPH: benzyl viologen oxidoreductase, and pyruvate synthase. Some kinetic and other properties were studied.     

Control of fatty acid incorporation in membrane phospholipids. X-ray-induced changes in fatty acid uptake by tumor cells

Lymphosarcoma cells isolated from the spleens of tumor-bearing mice were used to study the effect of a low dose of X-rays (5 Gy) on the incorporation of [³H]palmitate and [¹⁴C]arachidonate into the lipids of the tumor cells. Palmitate and arachidonate were rapidly incorporated especially into the phospholipids of the cells. Between one and three hours after the start of the incubation with radiactive palmitate 80–90% of the label of the total lipids was found in the phospholipid fraction. Already after a few minutes of incubation with radioactive arachidonate, about 95% of the label was incorporated in the phospholipids. Irradiation caused a small but significant increase in the rate of fatty acid incorporation for both fatty acids. Concomitantly, a significantly increased amount of fatty acid was removed from the medium by the cells as a result of the irradiation, and the specific radioactivity of the free fatty acids in the cells was found to be enhanced. The radiation effect on the tumor cells could be mimicked by a hypotonic treatment. The magnitude of the radiation-induced stimulation of the fatty acid incorporation was similar to that of the hypotonically induced effect. Cells which had received a hypotonic treatment before the irradiation, did not show an additional radiation-induced enhancement of fatty acid incorporation into the cellular lipids. When the cells were incubated with serum albumin loaded with a relatively large (non-physiological) amount of complexed fatty acids (fatty acid: albumin molar ratio, $\text{ν}\text{= 3.7}$), no radiation effect on the fatty acid incorporation could be detected. It is concluded that hypotonic treatment, irradiation, and increased supply of exogenous fatty acids all lead to an enhanced flux of fatty acids into the cells. These results confirm our previous suggestion that the uptake of fatty acids through the plasma membrane is the rate-limiting step in the fatty acid incorporation into the phospholipids and that ionizing radiation is one of the means to enhance fatty acid uptake through the plasma membrane leading to an increased incorporation into the phospholipids.     

Chloroplast activities of dark-imbibed and photoinduced spores of the fernOnoclea sensibilis

Summary Chloroplast activities of dark-imbibed (non-germinating) and photoinduced (germinating) spores of the sensitive fern,Onoclea sensibilis were compared to gain insight into the germination process. There were no changes in the number of chloroplasts or in the chlorophyll contents of the spore during dark-imbibition and during the early phase of germination. Levels of increase in the Chloroplast DNA content of dark-imbibed and photoinduced spores were nearly the same and were associated with autoradiographic incorporation of [³H]thymidine into the cytoplasm. However, incorporation of the label into the nucleus occurred only during photoinduction of spores. Analysis of Chloroplast and nuclear DNA contents by dot-blot hybridization with labeled gene-specific probes has confirmed that chloroplast DNA content of the spore increases during dark-imbibition and photoinduction, while increase in nuclear DNA occurs only in photoinduced spores. Chloroplasts isolated from dark-imbibed and photoinduced spores incorporated [³H]TTP into an acid-insoluble fraction identified as DNA. The results show that physiological activities of chloroplasts of dark-imbibed and photoinduced spores ofO. sensibilis are similar and support an exclusive role for nuclear DNA synthesis in spore germination.     

EFFECTS OF OXYGEN INDUCED CONVULSIONS ON THE UPTAKE OF ACETATE INTO RAT BRAIN LIPIDS1,2

Abstract— Rats were exposed to 5 atmospheres absolute of oxygen, and [1-¹⁴C]acetate was injected into the jugular vein either before or at the onset of electroencephalogram-defined convulsions. Levels of ¹⁴C observed 2.2 min after the injection were reduced in the total lipids of brain and elevated in the blood of convulsed rats when compared to the nonconvulsed controls. These differences between convulsed and nonconvulsed animals were less pronounced when measured 15 and 60 min after injection. Convulsions did not change the amount of ¹⁴C incorporated into the total lipids of plasma during the 60 min period studied. Six fractions obtained from total lipid extracts of brain by TEAE-cellulose showed similar ¹⁴C distributions in convulsed and control animals. The results suggest that oxygen-induced convulsions cause an impaired utilization of systemically administered acetate for fatty acid incorporation into the lipids of brain.     

Effects of ethanol on the remodeling of neutral lipids and phospholipids in brain mitochondria and microsomes

We have analyzed the effects of ethanol in vitro on the remodeling of neutral lipids and phospholipids in mitochondria and microsomes isolated from chick brain. We used three different fatty acyl-CoAs of similar chain lengths but different degrees of unsaturation. Our results demonstrate the existence of active mechanisms for acyl-CoA transfer into neutral lipids and phospholipids in both mitochondria and microsomes. The profile of fatty acid incorporation was clearly different according to the membrane and lipid fraction in question. Thus, in mitochondrial lipids, the remodeling processes showed a clear preference for the saturated fatty acid whilst the polyunsaturated one was the preferred substrate for microsomal lipid acylation. With regard to the effects of ethanol in vitro, we were able to demonstrate that exposure of the membrane to ethanol led to an increase in the incorporation of polyunsaturated fatty acid into triacylglycerol (TG) in both mitochondria and microsomes, indicating that it directly stimulates the acylation of diacylglycerol (DG) to give TG. This effect may then contribute to the widely reported stimulation of TG biosynthesis in cases of both acute and chronic ethanol ingestion. It is noteworthy that the exposure of microsomes to ethanol in vitro also stimulated the incorporation of oleoyl-CoA into the aminophospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS). We also demonstrate that both mitochondria and microsomes synthesize fatty acid ethyl esters (FAEEs) from fatty acyl-CoA, although there is a clear difference in preference for the fatty acid used as substrate in the esterification of the alcohol. Thus, mitochondria were capable of forming FAEEs from the polyunsaturated fatty acid whilst in microsomes the saturated fatty acid was the preferred substrate. In both types of membrane, FAEE production was lowest with the monounsaturated fatty acyl-CoA.     

Incorporation of Labeled Precursors into, and Accumulation of, Triglycerides and Phosphoglycerides at Selected Stages of Fungal Growth

There was greater incorporation of [2^?14C] acetate and of [6^?14C] glucose into phosphoglycerides than into triglycerides, of 1 1/2, 2, 3, 4 and 6 day old mycelial sample of the fungus Glomerella cingulata. Maximum incorporation into both classes of lipids occurred in young mycelial samples (2 to 3 days of age) which had a high content of total nitrogen. The five sets of mycelial samples all contained somewhat larger quantities of phosphoglycerides than triglycerides, and changes in content of both classes of lipids were similar in pattern to changes in content of total nitrogen. Incorporation, accumulation and total nitrogen of the mycelial samples, decreased at 4 days but increased again by 6 days. The apparent turnover of the triglycerides and phosphoglycerides was qualitatively similar although there was greater apparent turnover of phosphoglycerides than triglycerides; the similarity in patterns of apparent turnover was inferred to be a consequence of acyl exchange between labeled and unlabeled triglycerides and phosphoglycerides. There was greater incorporation of [2^?14C] acetate into the acyl than into the glyceryl moieties of both classes of lipids but greater incorporation of [6^?14C] glucose into the glyceryl than acyl moieties. With both precursors, the glyceryl and acyl moieties of the phosphoglycerides were more heavily labeled than corresponding moieties of the triglycerides.     

Caractéristiques de l'incorporation d'acides aminés par les polysomes isolés du latex d'Hevea brasiliensis

Amino acid incorporation by latex polysomes needs the presence of cofactors (K⁺, Mg²⁺), cytoplasmic serum and GTP. It is abolished by RNase but not by chloramphenicol. Poly U increases the incorporation rate.     

Cerebral lipid metabolism in experimental hyperphenylalaninaemia: incorporation of 14C-labelled glucose into total lipids

—1. Effects of the administration of phenylalanine to rats on incorporation in vivo or in vitro of [U-¹⁴C]glucose into cerebral lipids were studied during the first 5–10 days of postnatal development. In addition, the effects of added phenylalanine and its deaminated metabolites on incorporation of [U-¹⁴C]glucose by homogenates into lipids of developing rat brain were investigated. Hyperphenylalaninaemia reduced incorporation both in vivo and in vitro of [U-¹⁴C]glucose into cerebral lipids. 2. Phenylalanine or tyrosine added in vitro at concentrations equivalent to those in the brain of the hyperphenylalaninaemic rat (0-1 μmole/ml incubation medium) did not inhibit incorporation of [U-¹⁴C)glucose into lipids, although at much higher concentrations of phenylalanine (36 μumoles/ml incubation medium) slight inhibition (10 per cent) of incorporation of [U-¹⁴C]glucose into lipids was observed. 3. In contrast, the deaminated metabolites in general exerted greater inhibitory effects at lower concentrations. Phenyllactic acid, in comparison to phenylpyruvic and phenyl-acetic acid, was the most potent inhibitor of the incorporation in vitro of [U-¹⁴C]glucose into cerebral lipids. These results indicated that these metabolites of phenylalanine were the more potent inhibitors of cerebral lipid metabolism in immature animals.