In Vitro Fatty Acid Synthesis and Complex Lipid Metabolism in the Cyanobacterium, Anabaena Variabilis: II. Acyl Transfer and Complex Lipid Formation

Ozone exposure has been shown to increase the loss of K from Chlorella cells due to an increase in passive permeability and a depolarization of membrane potential. One factor which likely influences or can be influenced by these changes is the energy state of the cell. To study this relationship, various indicators of cell energy status were examined in the presence and absence of O₃.

The active uptake of chloride and deoxyglucose is nearly completely inhibited by O₃ at a dose at which cellular death, measured by plating efficiency, is minimal. Glucose-stimulated respiration, dependent upon ATP/ADP balance, is depressed to a greater degree than endogenous respiration in ozonated cells. Total ATP and glucose-6-phosphate levels also decrease but not as rapidly, and labeled intermediates of glucose metabolism are lost.

Thus, exposure to O₃ results in a depletion of the cell's energy reserves as substantiated by changes observed in processes which both utilize and generate ATP. This loss in energy reserves occurs at the same exposure level of O₃ as do the changes in passive transport properties. Thus, we cannot tell which occurs first; and the processes seem to be linked with respect to O₃ injury.

     

Lipid Biosynthesis in the Blue-Green Alga (Cyanobacterium), Anabaena variabilis III. UDPglucose:Diacylglycerol Glucosyltransferase Activity in vitro

The synthesis of glyceroglycolipids was studied in membraneand soluble fractions of Anabaena variabilis. The membrane fractionexhibited a high activity of UDPglucose: diacylglycerol glucosyltransferase,but practically no activity of UDPgalactose: diacylglycerolgalactosyltransferase. The glucosyltransferase activity wasmaximal at about pH 7.0 and dependent on Mg²⁺ The Michaelisconstant (K_m) for UDPglucose was 45?10^–6 M. The solublefraction catalyzed the incorporation of galactose from UDP galactoseinto digalactosyl diacylglycerol. These in vitro results werecompatible with the biosynthetic pathway of glyceroglycolipidsin this alga that we previously elucidated on the basis of tracerexperiments in vivo. ¹ Present address: Department of Biology, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan. (Received June 1, 1982; Accepted July 1, 1982)     

Lipid biosynthesis in the blue-green alga,Anabaena variabilis: II. Fatty acids and lipid molecular species

The biosynthesis of lipid molecular species was studied in Anabaena variabilis by pulse-labeling with NaH¹⁴CO³ and chasing. The experimental results indicate that the primary products of lipid biosynthesis are 1-stearoyl-2-palmitoyl species of monoglucosyl diacylglycerol, phosphatidylglycerol and sulfoquinovosyl diacylglycerol. In monoglucosyl diacylglycerol, stearic acid is desaturated rapidly to oleic acid and further to linoleic acid, whereas palmitic acid is hardly desaturated to palmitoleic acid. The stearoyl-palmitoyl, oleoyl-palmitoyl and linoleoyl-palmitoyl species of monoglucosyl diacylglycerol are converted to the corresponding species of monogalactosyl diacylglycerol. Desaturation of the fatty acids also takes place in monogalactosyl diacylglycerol. At 38° C the stearoyl-palmitoyl species is converted to oleoyl-palmitoyl, then to either linoleoyl-palmitoyl or oleoyl-palmitoleoyl, and finally to linoleoyl-palmitoleoyl species, and at 22°C the stearoyl-palmitoyl molecular species is sequentially converted to oleoyl-palmitoyl, linoleoyl-palmitoyl, linolenoyl-palmitoyl and linolenoyl-palmitoleoyl species. The molecular species of digalactosyl diacylglycerol are synthesized from the corresponding species of monogalactosyl diacylglycerol. Desaturation does not seem to occur in digalactosyl diacylglycerol. In phosphatidylglycerol and sulfoquinovosyl diacylglycerol, stearic acid is desaturated to oleic and to linolenic acids at 38° C, and further to linoleic acid at 22° C, whereas palmitic acid is hardly desaturated.     

Ischemia-Induced Alterations in Lipid Metabolism of the Gerbil Cerebral Cortex: I. Changes in Free Fatty Acid Liberation

Abstract: Does the impaired lipid metabolism during nonlethal transient ischemia truly recover within a few hours after recirculation? In an attempt to answer this question, we first investigated the time course of the changes in the amount and composition of free fatty acids (FFAs) accumulated during 5-min ischemia and after various postischemic recirculation durations (3 min, 1 h, 24 h, 3 days, and 6 days) in the gerbil cerebral cortex. Then those of FFAs liberated in response to the second 5-min ischemia at various recirculation intervals (3 min, 1 h, 3 days, and 6 days) following the initial one were also measured to evaluate the changes in the cellular response. The former study disclosed that the FFA levels transiently returned to the control levels at 1-h recirculation, increased again a few days after the onset of recirculation, followed by the final return to the control levels after 6-day recirculation. The latter study disclosed that the cellular response to the second ischemia was quite different from that to the initial one even after 6-day recirculation, suggesting that membrane lipid metabolism had not yet been recovered even at such a late period. We discuss the significance of the alterations in lipid metabolism.     

Studies on the Temperature Shift-induced Desaturation of Fatty Acids in Monogalactosyl Diacylglycerol in the Blue-green Alga (Cyanobacterium), Anabaena variabilis

The desaturation of fatty acids in the monogalactosyl diacylglycerolupon a downward shift in temperature was studied under variousconditions in Anabaena variabilis. The following conclusionsare drawn from the experimental results. (1) The desaturationof palmitic to palmitoleic acids after the temperature shiftfrom 38 to 22°C occurs in the dark as well as in the light.The desaturations of oleic to linoleic and of linoleic to linolenicacids after the temperature shift are stimulated by illumination.(2) The C₁₆ and C₁₈ acids are desaturated to different degreesdepending on the magnitude of the temperature shift. (3) Thedesaturations require molecular oxygen. (4) Syntheses of RNAand proteins are involved in the mechanism for the temperatureshift-induced desaturation of fatty acids. (Received May 27, 1981; Accepted July 7, 1981)     

Fatty Acid Acylation of Rat Brain Myelin Proteolipid Protein In Vitro: Identification of the Lipid Donor

The immediate acyl chain donor for fatty acid esterification of proteolipid protein (PLP) was identified in an in vitro system. Rat brain total membranes, after removal of crude nuclear and mitochondrial fractions, were incubated with radioactive acyl donors, extracted with chloroform/methanol, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of [3H]palmitic acid, CoA, ATP, and Mg2+, acylation of endogenous PLP occurred at a linear rate for at least 2 h. The radioactivity was associated with the protein via an ester linkage, mainly as palmitic acid. Omission of ATP, CoA, Mg2+, or all three reduced fatty acid incorporation into PLP to 44, 27, 8, and 4%, respectively, of the values in the complete system. Incubation of the membrane fraction with [3H]palmitoyl-CoA in the absence of CoA and ATP led to highly labeled PLP. These data demonstrate that activation of free fatty acid is required for acylation. Phospholipids and glycolipids were not able to acylate the PLP directly. Finally, when isolated myelin was incubated with [3H]palmitoyl-CoA in the absence of cofactors, only PLP was labeled, thus confirming the identity of palmitoyl-CoA as the direct acyl chain donor and suggesting that the acylating activity and the PLP pool available for acylation are both in the myelin.     

Lipid-Linked Desaturation of Palmitic Acid in Monogalactosyl Diacylglycerol in the Blue-Green Alga (Cyanobacterium) Anabaena variabilis Studied in Vivo

The mechanism of desaturation of palmitic acid in monogalactosyldiacylglycerol in Anabaena variabilis was studied by labelingin vivo with ¹³C and mass spectrometry. When the cells werefed with [¹³C]Na₂CO₃ for 2.5 h, 19% of the palmitic, but virtuallynone of the palmitoleic, acid at the C-2 position of the lipidwas enriched with ¹³C. During subsequent incubation for 7.5h, the [¹³C]palmitic acid was desaturated to [¹³C]palmitoleicacid. Mass spectrometric analysis of the 2-acylglycerol moietyof the lipid indicated that [¹³C]palmitoyl-[¹³C]glycerol and[¹²C]palmitoyl-[¹²C]glycerol were converted to [¹³C]palmitoleoyl-[¹³C]glyceroland [¹²C]palmitoleoyl-[¹²C]glycerol, respectively. These resultssuggest that the palmitic acid was converted to palmitoleicacid in vivo by lipid-linked desaturation but not via a pathwayconsisting of deacylation, desaturation and reacylation. ⁴Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan ⁵Present address: Department of Physiological Chemistry andNutrition, Faculty of Medicine, University of Tokyo, Hongo,Tokyo 113, Japan (Received December 7, 1985; Accepted April 16, 1986)     

Studies on the Induction of Reversible Hydrogenase from Cyanobacterium Anabaena variabilis and its Properties

The reversible hydrogenase in vegetative cells of A. variabilis cultured on NH4+ or N-free medium was induced by sparging with N2 for 24 hours under light. Both anaerobic condition and illumination appear to be necessary for the induction of hydrogenase in this algae. The properties of the hydrogenase in cell-free extract obtained from the cells grown on two nitrogen sources are similar: (1) Both the enzymes are able to evolve H2 in the presence of reduced methyl viotogen as electron donor, and to uptake H2 in the presence of benzyl viologen as electron acceptor. (2) The enzymes posses the thermal stability and are stable to O2. (3) The optimum pH required for H2 evolution activity of the enzymes is 7.0–7 5. (4) The Km of the enzymes obtained from NH4+ grown cells and N-free grown cells is 300 mmol/l and 295 mmol/l, respectively. So the high Km measured here suggests that the enzymes in both cases function physiologically as H2 evolution. (5) The activities of both enzymes are inhibited by CO but are not affected by C2H2. The induced H2 evolution activity of the reversible hydrogenase in cells grown on NH4+ reached 1530 nmol H2/mg dry wt, h, which was 3 to 5 times higher than from cells grown on N-free medium. Our experiment results indicate that the appearance of heterocysts of A. variabilis cultured on N-free medium affects the synthesis of reversible hydrogenase and the regulation of its activity.     

Control of Fatty Acid Metabolism I. Induction of the Enzymes of Fatty Acid Oxidation in Escherichia coli

Escherichia coli grows on long-chain fatty acids after a distinct lag phase. Cells, preadapted to palmitate, grow immediately on fatty acids, indicating that fatty acid oxidation in this bacterium is an inducible system. This hypothesis is supported by the fact that cells grown on palmitate oxidize fatty acids at rates 7 times faster than cells grown on amino acids and 60 times faster than cells grown on a combined medium of glucose and amino acids. The inhibitory effect of glucose may be explained in terms of catabolite repression. The activities of the five key enzymes of beta-oxidation [palmityl-coenzyme A (CoA) synthetase, acyl-CoA dehydrogenase, enoyl-CoA hydrase, beta-hydroxyacyl-CoA dehydrogenase, and thiolase] all vary coordinately over a wide range of activity, indicating that they are all under unit control. The ability of a fatty acid to induce the enzymes of beta-oxidation and support-growth is a function of its chain length. Fatty acids of carbon chain lengths of C(14) and longer induce the enzymes of fatty acid oxidation and readily support growth, whereas decanoate and laurate do not induce the enzymes of fatty acid oxidation and only support limited growth of palmitate-induced cells. Two mutants, D-1 and D-3, which grow on decanoate and laurate were isolated and were found to contain constitutive levels of the beta-oxidation enzymes. Short-chain fatty acids (相似文献   

Metabolism of sulfur compounds by whole filaments and heterocysts of Anabaena variabilis.

Filaments of the heterocyst-forming cyanobacterium Anabaena variabilis reproduced 35SO4(2)-, incorporating 35S into cysteine, methionine, glutathione, sulfolipid, and several unidentified metabolites. The majority of the incorporated label accumulated in reduced glutathione. Heterocysts isolated from labeled filaments contained the same major labeled products. Isolated, metabolically active heterocysts were unable to reduce 35SO4(2)-, but were able to incorporate 35S2- into cysteine and glutathione. The results suggest that the initial activation of SO4(2)- occurs in vegetative cells and that some reduced forms, possibly including S20, are translocated into heterocysts.     

Fat Metabolism in Higher Plants: XLV. Some Factors Regulating Fatty Acid Synthesis by Isolated Spinach Chloroplasts

In the biosynthesis of fatty acids from 1-¹⁴C-acetate by intact spinach chloroplasts, ATP and Triton X-100 exert opposing effects on the conversion of palmitic acid to stearic acid; thus, ATP decreases the conversion and Triton X-100 increases the conversion. Changes in the availability of photosynthetically generated reduced nicotinamide adenine dinucleotide phosphate apparently does not markedly affect the C₁₆-C₁₈ ratio. Various H₂O₂-generating systems, such as viologen dyes, inhibit oleate synthesis from acetate and cause stearate to accumulate. Catalase partially reverses the effect of these days.     

Changes in Lipid and Fatty Acid Metabolism of Vicia faba Mesophyll Protoplasts

The metabolism of the major polar and neutral lipids of Viciafaba protoplasts isolated from ¹⁴CO₂-fed leaves has been examined.The results show large losses in the radioactivity found inphosphatidylcholine and monogalactosyldiacylglycerol while thatof phosphatidylglycerol was stable. This loss was accountedfor by a rapid increase in the ¹⁴C content of the neutral lipids,particularly the triacylglycerols. Analysis of the fatty acidradioactivity in the lipids suggests that protoplast isolationinhibited fatty acid desaturation on phosphatidylcholine andpossibly on other lipids. These results also suggest a roleof phosphatidylcholine in the donation of fatty acids for triacylglycerolsynthesis in mesophyll protoplasts. The results are discussedin terms of the regulation of lipid metabolism and protoplastbiology. (Received April 20, 1984; Accepted August 27, 1984)     

In Vitro Metabolism of Mevalonic Acid in the Bovine Retina

Bovine retinas were incubated with 3RS-[5-3H]-mevalonic acid under conditions similar to those previously shown to support opsin biosynthesis in vitro. TLC of the total lipids indicated the formation of numerous radiolabeled components, including sterols, hydrocarbons, and "fatty acid-like material." The nonsaponifiable lipids were analyzed by TLC, GLC, and chromatography on columns of silicic acid-Super Cel, silica gel G-Super Cel-silver nitrate, and alumina-Super Cel-silver nitrate. The major nonsaponifiable components had the chromatographic properties of squalene and "methylated sterols" (i.e., C30, C29, and C28 monohydroxy sterols). Cholesterol represented no more than 1% of the total radioactivity in the nonsaponifiable lipid fraction. The "fatty acid-like material" was derivatized with diazomethane, and the resulting methyl esters were analyzed by GLC before and after catalytic hydrogenation. The radioactivity did not correspond to the normal fatty acids endogenous to the retina, but rather had the chromatographic properties of C15 and C20 isoprenoid acids. These results obtained with intact retinas are consistent with our previous observations concerning mevalonic acid metabolism in cell-free homogenates of bovine retinas.     

Light-Induced Proton Release by the Cyanobacterium Anabaena variabilis: Dependence on CO(2) and Na

Light-induced acidification by the cyanobacterium Anabaena variabilis is biphasic (a fast phase I and slow phase II) and shown to be sodium-dependent with an optimum concentration of 40 to 60 millimolar Na⁺. Cells grown under low CO₂ concentrations at pH 9 (i.e. mainly HCO₃⁻ present in the medium) exhibited the slow phase II of proton efflux only, while cells grown under low CO₂ concentrations at pH 6.3 (i.e. CO₂ and HCO₃⁻ present) exhibited both phases. Light-induced proton release of phase I was dependent on inorganic carbon available in the bathing medium with an apparent K_m for CO₂ of 20 to 70 micromolar. As was concluded from the CO₂ dependence of acidification measured at different pH of the bathing medium, bicarbonate inhibited phase-I acidification noncompetetively. Acidification was inhibited by acetazolamide, an inhibitor of carbonic anhydrase. Apparently, acidification of phase I is due to a light-dependent uptake of CO₂ being converted to HCO₃⁻ by a carbonic anhydrase-like function of the HCO₃⁻-transport system (M Volokita, D Zenvirth, A Kaplan, L Reinhold 1984 Plant Physiol 76: 599-602) before or during entering the cell, thus releasing one proton per CO₂ converted to HCO₃⁻.     

Glyoxylate Induced Changes in the Carbon and Nitrogen Metabolism of the Cyanobacterium Anabaena cylindrica

Addition of millimolar concentrations of glyoxylate to nitrogen-fixing cultures of Anabaena cylindrica, grown aerobically in the light, caused the following effects: an increase in the number of glycogen granules and in the excretion of carbohydrates; a decreased phycocyanin concentration, but an increase in the chlorophyll a to phycocyanin ratio. Also, an enhancement in the carbon to nitrogen ratio was noted, but this was restored if NH₄⁺ was added simultaneously. The most pronounced effect of glyoxylate addition was a 20-fold increase in the glycine pool. The effect of glyoxylate on N₂ fixation (acetylene reduction) was enhanced at high light intensities, but it did not affect the in vitro ribulose-1,5-bisphosphate carboxylase activity. However, addition of millimolar concentrations of glycolate did not cause changes in nitrogenase activity, CO₂ fixation, and NH₃ release comparable to those caused by glyoxylate. The primary mechanism of action of glyoxylate appears to be within the glycolate pathway of the vegetative cells and metabolically downstream from glycolate.     

Amino Acid Derivatives as Palmitoylethanolamide Prodrugs: Synthesis,In Vitro Metabolism and In Vivo Plasma Profile in Rats

Palmitoylethanolamide (PEA) has antinflammatory and antinociceptive properties widely exploited in veterinary and human medicine, despite its poor pharmacokinetics. Looking for prodrugs that could progressively release PEA to maintain effective plasma concentrations, we prepared carbonates, esters and carbamates at the hydroxyl group of PEA. Chemical stability (pH 7.4) and stability in rat plasma and liver homogenate were evaluated by in vitro assays. Carbonates and carbamates resulted too labile and too resistant in plasma, respectively. Ester derivatives, prepared by conjugating PEA with various amino acids, allowed to modulate the kinetics of PEA release in plasma and stability in liver homogenate. L-Val-PEA, with suitable PEA release in plasma, and D-Val-PEA, with high resistance to hepatic degradation, were orally administered to rats and plasma levels of prodrugs and PEA were measured at different time points. Both prodrugs showed significant release of PEA, but provided lower plasma concentrations than those obtained with equimolar doses of PEA. Amino-acid esters of PEA are a promising class to develop prodrugs, even if they need further chemical optimization.     

Adaptation of the Cyanobacterium Anabaena variabilis to Low CO(2) Concentration in Their Environment

The rate of adaptation of high CO₂ (5% v/v CO₂ in air)-grown Anabaena to a low level of CO₂ (0.05% v/v in air) was determined as a function of O₂ concentration. Exposure of cells to low (2.6%) O₂ concentration resulted in an extended lag in the adaptation to low CO₂ concentration. The rate of adaptation following the lag was not affected by the concentration of O₂. The length of the lag period is markedly affected by the O₂/CO₂ concentration ratio, indicating that the signal for adaptation to low CO₂ may be related to the relative rate of ribulose-1,5-bisphosphate carboxylase/oxygenase activities, rather than to CO₂ concentration proper. This suggestion is supported by the observed accumulation of phosphoglycolate following transfer of cells from high to low CO₂ concentration.     

Aluminum Effects on Uptake and Metabolism of Phosphorus by the Cyanobacterium Anabaena cylindrica

Aluminum severely affects the growth of the cyanobacterium Anabaena cylindrica and induces symptoms indicating phosphorus starvation. Preor post-treating the cells with high (90 micromolar) phosphorus reduces the toxicity of aluminum compared to cells receiving a lower orthophosphate concentration. In this study aluminum (ranging from 9 to 36 micromolar) and phosphorus concentrations were chosen so that the precipitation of insoluble AIPO₄ never exceeded 10% of the total phosphate concentration. The uptake of ³²P-phosphorus is not disturbed by aluminum either at high (100 micromolar) or low (10 micromolar) concentrations of phosphate. Also, the rapid accumulation of polyphosphate granules in cells exposed to aluminum indicates that the incorporation of phosphate is not disturbed. However, a significant decrease in the mobilization of the polyphosphates is observed, as is a lowered activity of the enzyme acid phosphatase, in aluminum treated cells. We conclude that aluminum acts on the intracellular metabolism of phosphate, which eventually leads to phosphorus starvation rather than on its uptake in the cyanobacterium A. cylindrica.     

L-Pyroglutamic Acid Inhibits Energy Production and Lipid Synthesis in Cerebral Cortex of Young Rats In Vitro

In the present study we investigated the effects of L-pyroglutamic acid (PGA), which predominantly accumulates in the inherited metabolic diseases glutathione synthetase deficiency (GSD) and -glutamylcysteine synthetase deficiency (GCSD), on some in vitro parameters of energy metabolism and lipid biosynthesis. We evaluated the rates of CO₂ production and lipid synthesis from [U-¹⁴C]acetate, as well as ATP levels and the activities of creatine kinase and of the respiratory chain complexes I-IV in cerebral cortex of young rats in the presence of PGA at final concentrations ranging from 0.5 to 3 mM. PGA significantly reduced brain CO₂ production by 50% at the concentrations of 0.5 to 3 mM, lipid biosynthesis by 20% at concentrations of 0.5 to 3 mM and ATP levels by 52% at the concentration of 3 mM. Regarding the enzyme activities, PGA significantly decreased NADH:cytochrome c oxireductase (complex I plus CoQ plus complex III) by 40% at concentrations of 0.5–3.0 mM and cytochrome c oxidase activity by 22–30% at the concentration of 3.0 mM, without affecting the activities of succinate dehydrogenase, succinate:DCPIP oxireductase (complex II), succinate:cytochrome c oxireductase (complex II plus CoQ plus complex III) or creatine kinase. The results strongly indicate that PGA impairs brain energy production. If these effects also occur in humans, it is possible that they may contribute to the neuropathology of patients affected by these diseases.