Fatty-acid-induced release of manganes from chloroplasts

The effect of both endogenous and exogenous unsaturated free fatty acids on manganese release from chloroplasts of chill-resistant (spinach) and chill-sensitive (tomato, bean) plants was studied. The level of endogenous free fatty acids increased 2–3-fold during cold and dark storage of leaves of chill-sensitive plants and was accompanied by depletion of about 60% of total chloroplast manganese content. Similar effects were observed when accumulation of free fatty acids in chloroplasts was achieved by storage of growing tomato plants for a few days in the dark at room temperature. In contrast, the cold and dark treatment of leaves of chill-resistant plant (spinach) affected neither free fatty acid, manganese levels nor Hill-reaction activity in chloroplasts. Incubation of chloroplasts of both chill-sensitive and chill-resistant plants with bean leaf galactolipase resulted in an accumulation of free fatty acids and a release of approx. 60% of total manganese content. The same amount of total manganese content was released following 3 h incubation of chloroplasts with linolenic acid at fatty acid/chlorophyll ratio (w/w, 2:1–10:1). The efficiency of C₁₈ unsaturated fatty acids/linolenic, linoleic, oleic on manganese release from chloroplasts was established in decreasing order C_18:3 > C_18:2 > C_18:1. The results indicate that the inhibitory effect of both endogenous and exogenous fatty acids on Hill reaction depends on the release from chloroplasts of functionally active, loosely bound manganese. Thus, similarly to both Tris and hydroxylamine treatments of chloroplasts, the incubation of chloroplast preparations with unsaturated fatty acids may be a useful tool for manganese depletion of chloroplasts.     

Photosynthetic apparatus in chilling-sensitive plants

The composition of free fatty acids (FFA) in relation to Hill reaction activity and photoperoxidation of lipids was studied in chloroplasts isolated from fresh, cold and dark-stored as well as illuminated leaves of Lycopersicon esculentum Mill., Phaseolus vulgaris L. and Cucumis sativus L. Following the cold and dark-storage of leaves the loss of Hill reaction activity is accompanied by approximately a 5-fold increase in the amount of FFA and by an increase in the percentage of unsaturated FFA, particularly that of linolenic acid. Illumination of the cold- and dark-stored leaves restores both Hill reaction activity and the content and composition of chloroplast FFA. Following the second and third cycles of cold storage and illumination of leaves the percentage of unsaturated fatty acids in chloroplasts increases while that of saturated ones decreases despite of the significant restoration of Hill reaction activity. Since the illumination of cold-stored leaves results in peroxidation of inhibitory fatty acids it seems likely that this phenomenon could, at least partially, be responsible for the restoration of Hill reaction activity. Inhibition of Hill reaction activity by exogenous linolenic acid in chloroplasts of fresh, cold-stored as well as cold-stored and illuminated leaves could be reversed following the incubation of chloroplast suspension with BSA, however only to a value measured in the absence of unsaturated fatty acid. All these results indicate that the inhibition of Hill reaction activity due to the cold and dark storage of leaves is caused by both inhibitory FFA released from chloroplast lipids as well as by damage to the thylakoid structure affecting the electron transport within photosystem II.Abbreviations BSA bovine serum albumin - DCIP 2,6-dichlorophenolindophenol - DGDG digalactosyl diglyceride - HEPES 2-(4(2-hydroxyethyl)-piperazinyl) ethanesulfonic acid - FFA free fatty acids - MDA malondialdehyde - MGDG monogalactosyldiglyceride - TBA thiobarbituric acid - Tris tris-(Hydroxymethyl)aminomethane     

Stages of uptake and incorporation of micellar palmitic acid by hamster proximal intestinal mucosa

The stages of uptake and incorporation of micellar palmitic acid by hamster proximal intestinal mucosa were investigated by incubation of everted sacs at 4 degrees C and 37 degrees C for 2, 5, 10, and 15 min in a micellar solution (10 micro moles of [1-(14)C]palmitic acid, 10 micro moles of monoolein, and 100 micro moles of sodium taurodeoxycholate) and subsequent serial rinsing of the sacs in ice-cold solutions as follows: one 20-sec rinse in unlabeled micellar solution, five 1-min rinses in Krebs-Ringer buffer (0.15 m, pH 6.3), and ten 2-min rinses in 2.5% albumin solution. The fatty acid-solubilizing capacity of all the rinsing solutions was always in excess of the amounts of radioactive palmitic acid released during each rinse. Radioactivity was determined in the tissue homogenates, rinsing solutions, and serosal fluids. The results indicate that a significant proportion of radioactive palmitic acid taken up by the sacs during the short incubation was released into the rinsing solutions. Rinsing in Krebs-Ringer buffer resulted in release of 15.5 +/- 2.4% of the labeled fatty acid, and this fraction was independent of the temperature of incubation. In contrast, the amounts of palmitic acid released in albumin were significantly greater and were markedly dependent on the temperature of incubation; a total of 48.6 +/- 7.0% and 26.3 +/- 5.1% was released from sacs incubated at 4 degrees C and 37 degrees C, respectively. While the proportion of radioactive palmitic acid in the free fatty acid fraction of the tissue after the rinsing sequence remained reasonably constant regardless of the temperature and duration of incubation, the radioactivity of the esterified palmitic acid in the tissue was much greater in the sacs incubated at 37 degrees C and tended to increase linearly up to 10 min of incubation. A highly significant inverse relationship was found between the fraction of radioactive palmitic acid released by rinsing in albumin and the fraction of the label in the tissue esterified fatty acids. The results suggest that the initial uptake of micellar fatty acid by intestinal mucosa may involve reversible binding to superficial sites with at least two strengths of binding: a weak, temperature-independent binding which could be easily dissociated by rinsing in Krebs-Ringer buffer, and a stronger, temperature-dependent binding which could be dissociated by rinsing in albumin, but not in Krebs-Ringer buffer. Analogous binding of micellar palmitic acid occurred in a brush border preparation of proximal intestine which was devoid of any fatty acid esterifying activity. This suggested that the reversible binding of fatty acid by the intestinal mucosa may be a property of its superficial components, namely the glycocalyx or microvillous membranes, and that it may be independent of the esterifying capacity of the tissue.     

Chemical composition of lipophilic extractives released during the hot water treatment of wheat straw

Treatment of wheat straw with hot water at 80-95 degrees C for 0.5 h at pH 6.0-8.0 released 41.0-53.0% of the original lipophilic extractives. The chemical compositions of six lipophilic extractives were determined by GC on a medium-length high-temperature capillary column without derivatization, thus giving a method for direct determination of individual components of free fatty and resin acids, sterols, waxes, sterol esters, and triglycerides. The extracts contained 68.7-75.8% lipophilic substances, comprising mainly free fatty acids (25.8-48.4%), waxes (9.4-27.0%), sterols (4.1-8.0%), triglycerides (3.3-11.0%), and sterol esters (2.6-5.1%). Minor amounts of diglycerides (0.3-0.5%), resin acid (0.5-3.1%), and phenolic compounds (0.9-3.6%) were also quantitatively determined in the extractives.     

Mechanism of release of integral proteins from rat liver microsomal membranes

The release of three integral enzymatic activities (NADH- and NADPH-cytochrome c reductase and 5'-nucleotidase) and total protein from washed rat liver microsomal membranes, upon simple incubation at 37 degrees C in aqueous media, was investigated. Release does not depend on contaminating proteases and is enhanced by alkaline pH. Total protein and enzyme release is consistent with a loss of phospholipids which are not recovered in the soluble phase. Following incubation at pH 9.0 large amounts of free fatty acids were recovered in the soluble phase, accounting for a ratio of 1/1 (w/w) with released protein. This evidence, together with the data available about densities (1.07-1.08 g/ml) and molecular weights (1 700 000-700 000) of the released enzymes, suggests that they are solubilized from microsomal membranes in the form of mixed micelles mostly formed by free fatty acids and integral proteins, probably owing to the activity of endogenous phospholipases on membrane lipids. Release of total protein and enzymatic activities is decreased by Ca2+, whose possible role in the phenomenon is discussed.     

Changes in lipid composition of tritosomes during lysis

Membrane phospholipids and free fatty acids were analyzed after lysis of lysosomes which had engulfed Triton WR 1339 (tritosomes). Significant elevations of lysophosphatidyl choline and lysophosphatidyl ethanolamine were seen. Phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin decreased significantly. Triglyceride also fell. The released free fatty acids paralleled closely the liberation of β-glucuronidase from the tritosomes. Thus, lipases hydrolyzed endogenous phospholipids and neutral lipids of tritosomes during incubation at 37°C at pH 5.0 to produce significant elevations of lysophospholipids and free fatty acids. The known surface-active properties of these latter compounds raise the possibility of their participation in the process of lysis of the tritosomes.     

Studies on oxidized low density lipoproteins. Controlled oxidation and a prostaglandin artifact

Low density lipoproteins (LDL), isolated by ultracentrifugal flotation, were oxidized (LDLOXID) slowly during dialysis against 0.15 M NaCl and subsequent incubation in 96% air-4% CO2 at 37 degrees C. Butylated hydroxytoluene prevented LDL oxidation. LDL preparations from different sera were oxidized at different rates and the degree of lipid peroxidation was controlled by varying the incubation time. Mild oxidation did not alter the electrophoretic mobility of the LDLOXID preparations. LDLOXID contained lipid peroxides in neutral lipids, had increased amounts of lysophosphatidylcholine, and contained a number of complex oxidation products that were generated from the oxidation of free fatty acids. These oxidation products included large amounts of soluble material that cross-reacted with antibodies to PGE2 but not 6-keto-PGF1 alpha. The amount of cross-reacting material was proportional to the degree of lipid peroxidation. Cross-reacting material in LDLOXID preparations was evidently formed from the oxidation of free fatty acids released from LDL, since cross-reacting material was also formed when a synthetic fat emulsion was oxidized in the presence of free arachidonic acid.     

Effects of cloprostenol administration on neutral lipid and prostaglandin F metabolism by porcine luteal tissue

The effects of Cloprostenol administration on porcine luteal lipid metabolism, progesterone production, and prostaglandin F production were examined in 32 pigs at day 12 of the estrous cycle. Pigs were killed between 0 and 18 hours after treatment. Recovered luteal tissue was incubated at 0 C and at 37 C in the absence and presence of dibutyryl cyclic AMP and indomethacin. Net release of progesterone from luteal tissue was depressed within 1 hour after Cloprostenol treatment whereas net release of prostaglandin F was accelerated 4 hours after Cloprostenol treatment. Inclusion of dibutyryl cyclic AMP in the incubation media did not alter progesterone production by did enhance prostaglandin F production at 0 and 1 hour after Cloprostenol treatment. Inclusion of indomethacin in the incubation media completely inhibited the Cloprostenol-induced acceleration of luteal PGF production. Cloprostenol treatment increased luteal triglycerides and decreased luteal free cholesterol and cholesterol esters within 1 hr after treatment. Arachidonic acid percentages in free fatty acids and triglycerides were also increased within 1 hr after treatment. When 37 C and 0 C incubations were compared, luteal accumulation of free fatty acids was maximum at 1 hr after Cloprostenol treatment. accumulation of triglycerides in luteal tissie was comparatively uniform at all times examined during the first 18 hr after Cloprostenol treatment. Comparison of 37 C and 0 C incubations further revealed that luteal triglycerides were active in accumulation of arachidonic acid. Inclusion of dibutyryl cyclic AMP and/or indomethacin in the incubation media did not alter luteal lipid contents or fatty acid compositions. Blood plasma progesterone was depressed at 4 hours after Cloprostenol whereas 13,14-dihydro-15-keto-prostaglandin F2a was elevated at 18 hours after treatment. Blood plasma free fatty acids increased 330 percetn at 4 hours and free fatty acid compositions also changed at this time. In both luteal tissue and blood plasma, changes in steroid and fatty acid metabolism occurred prior to changes in prostaglandin metabolism, suggesting that Cloprostenol induced functional luteal regression prior to altering prostaglandin metabolism.     

Effect of temperature on fatty acid composition in each lipid fraction of Spirometra erinaceieuropaei plerocercoids

The effects of temperature and host fatty acids on the fatty acid contents of Spirometra erinaceieuropaei plerocercoids were investigated to clarify their role in sparganosis. After 24 hr incubation at 18 C in host snake serum, omega6 series fatty acids, especially arachidonic acid in the phospholipid fraction of the plerocercoids, increased compared with those of plerocercoids incubated at 37 C. The changes in the ratio of polyunsaturated to saturated fatty acids in the phospholipid fraction of plerocercoids incubated in physiological saline for 6 hr at 10 C were almost the same as the changes at 37 C. The ratio of polyunsaturated to saturated fatty acids of the triglyceride fraction showed almost opposite change versus the phospholipid fraction. The percentage of arachidonic acid in the phospholipid fraction of plerocercoids increased during the first 3 hr of incubation and then decreased, regardless of temperature. At 37 C, the percentage of arachidonic acid in the free fatty acid fraction fell for the first 3 hr of incubation and was significantly elevated at the end of the 6-hr incubation. At 10 C, however, arachidonic acid in the free fatty acid fraction decreased for the first hour of incubation, increased at 3 hr of incubation, then decreased again. These results suggest that fatty acids of the plerocercoids are frequently exchanged between fractions. Plerocercoids can mobilize arachidonic acid to the free fatty acid fraction more quickly at lower temperature than at higher temperature. They may utilize mobilized arachidonic acid early in the infection stage to produce prostaglandins. Alternatively, they can incorporate arachidonic acid into the phospholipid fraction again when arachidonic acid is readily available in the environment.     

Fatty acid release in incubations of serum with synaptosome and myelin subfractions of brain

To study lipid breakdown in brain membranes following hemorrhage, synaptosome and myelin fractions isolated from rat brain were incubated with rat serum. After 3 h in vitro at 37 degrees C, 0.43 and 0.26 mumol of fatty acid were released in incubations containing synaptosomes (1.37 mumols phospholipid) or myelin (1.23 mumols phospholipid), respectively, in the presence of 0.25 mL serum. Less than 0.05 mumol of fatty acid was liberated in incubations containing only serum, synaptosomes, or myelin. For synaptosomes and serum, docosahexaenoate was the principal fatty acid released (28 mol% of total) after 3 h of incubation. This fatty acid and arachidonate made up 43 mol% of the liberated fatty acid. The presence of free docosahexaenoate was of interest, as this fatty acid is particularly enriched in phosphatidylserine and phosphatidylethanolamine, phospholipids found in the cytoplasmic half of the synaptosomal plasma membrane and in interior synaptosomal membranes. In incubations of serum and myelin, oleate was the major free fatty acid produced in 30 min to 3 h of incubation (29-35 mol% of total). After 3 h, docosahexaenoate contributed 20 mol% to the total. The release of fatty acids from the membranes may be mediated by serum phospholipase(s) or possibly by activated endogenous lipolytic activities.     

Non-enzymatic heme formation in the presence of fatty acids and thiol reductants

Non-enzymatic heme formation from equimolar amounts of porphyrin and iron was investigated. When mesoporphyrin IX and iron citrate were incubated with oleic acid and dithiothreitol at 37 degrees C in vacuo, mesoheme was formed in a high yield. When protoporphyrin IX and deuteroporphyrin IX were used, protoheme and deuteroheme were formed, respectively. Cysteine or 2-mercaptoethanol instead of dithiothreitol also resulted in the formation of heme. Linoleic acid was as effective as oleic acid, but at 37 degrees C, saturated fatty acids and phospholipids gave low yields. When incubation was at 70 degrees C saturated fatty acids as well as unsaturated fatty acids produced a large amount of heme. The optimum pH was 8.8. By increasing the concentration of Triton X-100 to 0.1%, heme formation decreased, and at concentrations above this level, completely disappeared. The conditions of non-enzymatic heme reaction presented here seem to be useful in elucidation of the mechanism of metalloporphyrin formation.     

Characterization of a protein fatty acylesterase present in microsomal membranes of diverse origin

A microsomal activity of baby hamster kidney cells which cleaves ester-type bound fatty acids from acyl proteins in vitro has been characterized. This activity is also present in microsomal membranes from pig liver, calf kidney, and human mucous cells. Cell free deacylation is described for the Semliki Forest virus acyl proteins E1 and E2 and the precursor of E2 designated p62. Acyl chain cleavage operates with both exogenous and endogenous viral acyl protein substrates. The in vitro cleavage requires microsomes solubilized by detergents of which various kinds are equally effective (Nonidet P-40, Tween 20, sodium deoxycholate, Triton X-100, or octyl-beta-D-glucoside). If microsomes are boiled for 15 min prior to the incubation, deacylation is abolished completely and no radioactivity is released from the palmitoylated acyl proteins during incubation with either detergents or microsomes alone. No changes in the molecular structure of the deacylated Semliki Forest virus proteins were detected, and the cleavage product was identified as free fatty acid. Deacylation is time- and temperature-dependent and can be enhanced by increasing the concentration of microsomal protein in the incubation mixture. It is completely inhibited under acidic conditions (pH 5) and at low temperature (4 degrees C). Deacylation also occurs in the presence of EDTA and bivalent cations such as Mg2+, Mn2+, and Ca2+ which influence the reaction marginally. On the other hand, fatty acid release is drastically reduced with a mixture of Co2+, Zn2+, and Hg2+ ions. The activity is not identical with protein fatty acyltransferase operating in the reverse direction, since a partially purified preparation of this acyltransferase failed to cleave fatty acids from fatty acylated substrate proteins. Taken together, these data lead us to postulate an enzymatic activity which cleaves fatty acids from ester-type fatty acylated proteins, and we propose to designate this enzyme a protein fatty acylesterase.     

Influences of incubation temperature and various saccharides on the production of organic acids and gases by gut microbes of rainbow trout Oncorhynchus mykiss in a micro-scale batch culture

We studied the influence of incubation temperature and additional saccharides on the metabolism of hindgut microbes of the rainbow trout Oncorhynchus mykiss in a 50 microl-scale batch culture system. Intestinal contents of rainbow trout reared at 15 degrees C were incubated with glucose, lactosucrose, sodium alginate or colloidal chitin (each 10 g/l) at 15 degrees C or 25 degrees C for 12 h. Levels of organic acids at 0 h and 12 h of incubation were quantified with HPLC. We also monitored gas release from these cultures during incubation. The main product was iso-butyric acid, except for the cultures with colloidal chitin where no net production of organic acids was observed. We detected higher levels of iso-butyric acid in cultures with lactosucrose than in the other cultures. Net production of this acid was less in cultures with colloidal chitin than in blank cultures. The volume of released gas was larger when incubated at 25 degrees C than at 15 degrees C. Cultures with colloidal chitin released more gas than blank cultures when they were incubated at 15 degrees C. Cultures with sodium alginate released less gas than blank cultures irrespective of incubation temperature. These results indicate that the hindgut microbes of this carnivorous fish mainly produce branched-chain fatty acids, very likely by microbial digestion of nitrogenous materials rather than saccharides. However, additional saccharides affected production of branched-chain fatty acids. The influence of incubation temperature in the present study also suggested that the environmental temperature of host fish should affect microbial digestion in the fish gut.     

Myelin Proteolipid Protein Contains Thioester-Linked Fatty Acids

Myelin proteolipid protein (PLP) is known to contain long-chain, covalently bound fatty acids. Previous studies, including our own, have suggested the occurrence of an oxyester type of linkage between fatty acids and PLP. However, we found that protein-SH groups are required in the acylation reaction, suggesting the possible presence of thioesters. In the present study, we have examined the nature of the acyl-PLP linkages by determining whether free thiol groups are generated on removal of fatty acids. Incubation of reduced and carboxyamidomethylated proteolipid apoprotein (RCM-APL) with 0.2 M hydroxylamine and [14C]iodoacetamide at pH 7.5 and 37 degrees C resulted in the release of fatty acids and the concomitant labeling of newly formed thiol groups. Incubation with Tris or methylamine at pH 7.5 failed to remove fatty acids and generate free -SH groups. The possibility that on treatment buried thiol groups became exposed was essentially excluded because (1) similar results were obtained in 2-chloroethanol, a solvent in which acylated and deacylated PLP have the same conformation, and (2) small PLP peptides were labeled only in the presence of hydroxylamine. On incubation with [14C]methylamine at pH 9.0, RCM-APL was not labeled, thus excluding the occurrence of intramolecular thiol esters. On the other hand, fatty acids were released as radioactive N-methyl fatty acylamide, indicating the presence of intermolecular thioesters between fatty acids and protein. These results demonstrate that a large proportion of fatty acids covalently bound to PLP are liked to -SH groups.     

Effects of growth regulators and light on chloroplasts NAD(P)H dehydrogenase activities of senescent barley leaves

The activities NADH and NADPH dehydrogenases were measured with ferricyanide as electron-acceptor (NADH-FeCN-ox and NADPH-FeCN-ox, respectively) in mitochondria-free chloroplasts of barley leaf segments after receiving various treatments affecting senescence. NADPH-FeCN-ox declined during senescence in the dark, in a way similar to chlorophyll and Hill reaction, and increased when leaf segments were incubated at light. These results suggest that NADPH-FeCN-ox is related to some photosynthetic electron transporter activity (probably ferredoxin-NADP⁺ oxidoreductase). In contrast, NADH-FeCN-ox is notably stable during senescence in the dark and at light. This activity increased during incubation with kinetin or methyl-jasmonate (Me-JA) but decreased when leaf segments were treated with abscisic acid (ABA). The effects of the inhibitors of protein synthesis cycloheximide and chloramphenicol suggest that the changes of NAD(P)H dehydrogenase activities may depend on protein synthesis in chloroplasts. In senescent leaf, chloroplast NADH dehydrogenase might be a way to dissipate NADH produced in the degradation of excess carbon which is released from the degradation of amino acids.Abbreviations ABA abscisic acid - DCPIP 2,6-dichlorophenol-indo-phenol - DOC deoxycholate - Me-JA methyl jasmonate - NADH-FeCN-ox NADH ferricyanide oxidoreductase - NADPH-FeCN-ox NADPH ferricyanide oxidoreductase     

Lipid biosynthesis by chloroplasts isolated from developing Zea mays

Fatty acid biosynthesis by isolated plastids has been examined in relation to chloroplast development and differentiation in leaves of maize plants grown in light for 7 days. Biosynthesis of fatty acids from acetate by proplastids prepared from the basal regions of the leaf was low and mainly palmitate was synthesized. The greatly increased utilization of acetate for fatty acid biosynthesis as the plastids increased in size was due to an increased synthesis of oleate. The maximum synthesis of total fatty acids and monoenoic fatty acids was obtained in chloroplasts prepared from leaf tissue 6–8 cm from the base of the plant where granal formation was most active. Fully-developed chloroplasts prepared from distal regions of the leaf were less active in fatty acid biosynthesis. Maize chloroplasts failed to synthesize fatty acids when isolated by methods commonly used to prepare active spinach chloroplasts. The method of isolation which included a density gradient gave a high proportion of Class I chloroplasts from maize leaves and incorporated up to about 10% of the acetate used. Biosynthesis of unsaturated fatty acids, especially with chloroplasts prepared from the most mature tissue, was increased by the addition of both mitochondrial and microsomal fractions. Increases in polyunsaturated fatty acids were also obtained but the proportions in the newly-synthesized fatty acids were well below the endogenous levels. Monoenoic synthesis was greatly stimulated by increasing the pH in the range 7·0–8·0 and also the highest proportions of unsaturated fatty acids were obtained at short incubation times.     

Kinetic properties of arachidonoyl-coenzyme A synthetase in rat brain microsomes

Free arachidonic acid is released rapidly in the brain at the onset of ischemia and during convulsions. The transient nature of this phenomenon indicates the existence of an active reacylation system for this fatty acid, likely an arachidonoyl-CoA synthetase-arachidonoyl transferase. The first of these enzymatic activities in brain microsomes was studied and it was found that [1-14C]arachidonic acid is rapidly activated and shows an absolute requirement for ATP and CoA. MgCl2 enhances this activity 10-fold. The optimum pH is 8.5, and the apparent Km values for the radiolabeled substrate, ATP, CoA, and MgCl2 are 36, 154, 8, and 182 microM, respectively. The apparent Vmax is 32.4 nmol/min/mg protein for arachidonic acid. The presence of Triton X-100 (0.1%) in the assay medium caused a significant reduction in apparent Km (9.4 microM) and Vmax (25.7 nmol/min/mg protein) values. The enzymatic activity is thermolabile with a T1/2 of less than 1 min at 45 degrees C and a maximal activity at 40 degrees C. The breaking point or transition temperature is 25 degrees C in an Arrhenius plot. The activation energies were 95 kJ/mol from 0 to 25 degrees C and 30 kJ/mol from 25 to 40 degrees C. Fatty acid competition studies showed inhibition by unlabeled docosahexaenoic and arachidonic acids with a Ki of 31 and 37 microM, respectively, in the absence and 18 and 7.7 microM in the presence of Triton X-100. Palmitic acid and oleic acid slightly inhibited the reaction whereas linoleic acid inhibited it to a moderate extent. It is concluded that this very active enzyme can activate arachidonic acid as well as docosahexaenoic acid in brain microsomes. In addition, this reaction may be involved in regulating the pool size of these free fatty acids in brain by rapid removal through activation, thus limiting eicosanoid formation. Moreover, the rapid formation of polyenoic acyl-coenzyme A may participate in the retention of essential fatty acids in the central nervous system.     

Release of free fatty acids from Ehrlich ascites tumor cells

Ehrlich ascites tumor cells release free fatty acids (FFA) during in vitro incubation in media that contain albumin. The released FFA are derived by lipolysis from endogenous lipid esters. Addition of glucose to the incubation medium greatly decreases the quantity of fatty acid released by the cells. Cyanide, which inhibits endogenous lipid oxidation but not lipolysis, increases the quantity of fatty acid released to media containing albumin and causes free fatty acid to accumulate in the cells in the absence of exogenous albumin. The release of fatty acid, either preformed or derived by lipolysis during prolonged incubations, occurs under conditions of net fatty acid uptake from the incubation medium. Net release of fatty acid from the cell occurs only when fatty acid-extracted albumin is present in the extracellular medium; extrapolation of the data suggests that net release will not occur under physiological conditions. It is postulated that free fatty acid uptake and release are independent processes, the direction of net fatty acid movement being determined by the relationship between cellular free fatty acid concentration (regulating efflux) and the molar ratio of free fatty acid to albumin in the extracellular medium (regulating uptake).     

Phospholipid-deacylating enzymes of rat stomach mucosa

1. Rat stomach mucosa exhibited three distinguishable phospholipid-deacylating enzyme activities: lysophospholipase, phospholipase A1 and phospholipase A2. 2. The lysophospholipase hydrolyzed 1-palmitoyl lysophosphatidylcholine to free fatty acid and glycerophosphorylcholine. This enzyme had an optimum pH of 8.0, was heat labile, did not require Ca2+ for maximum activity and was not inhibited by bile salts or buffers of high ionic strength. 3. Phospholipase A2 and phospholipase A1 deacylated dipalmitoyl phophatidylcholine to the corresponding lyso compound and free fatty acid. The specific activity of phospholipase A2 was 2--4-fold higher than that of phospholipase A1 under all the conditions tested. Both activities were enhanced 4--7.5-fold in the presence of bile salts at alkaline pH and 11-18-fold at acidic pH. 4. In the absence of bile salts, phospholipase A1 exhibited pH optima at 6.5 and 9.5 and phospholipase A2 at pH 6.5, 8.0 and 9.5. The pH optima for phospholipase A1 were shifted to pH 3.0, 6.0 and 9.0 in presence of sodium taurocholate; the activity was detected only at a single pH of 9.5 in the presence of sodium deoxycholate and at pH 10.0 in the presence of sodium glycocholate. Phospholipase A2 optimum activity was displayed at pH 3.0, 6.0 and 8.0 in presence of taurocholage, pH 7.5 and 9.0, in presence of glycocholate and only at pH 9.0 in presence of deoxycholate. 5. Ca2+ was essential for optimum activity of phospholipases A1 and A2. But phospholipase A1 lost complete activity in presence of 0.5 mM ethyleneglycolbis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) at pH 6.0, whereas phospholipase A2 lost only 50%. 6. Phospholipases A1 and A2 retained about 50% of their activities by heating at 75 degrees for 10 min. At 100 degrees, phospholipase A1 retained 22% of its activity, whereas phospholipase A2 retained only 7%.