Studies on the formation by rat brain preparations of CDP-diglyceride from CTP and phosphatidic acids of varying fatty acid compositions

1976 ◽  
Vol 54 (3) ◽  
pp. 249-260 ◽  
Author(s):  
H. H. Bishop ◽  
K. P. Strickland
Keyword(s):  
Fatty Acid ◽  
Phosphatidic Acid ◽  
Rat Brain ◽  
Unsaturated Fatty Acids ◽  
Fatty Acid Distribution ◽  
Assay Method ◽  
Detergent Solution ◽  
Significant Activity ◽  
Ph Optimum ◽  
Phosphatidic Acids

The enzyme, CTP:phosphatidate cytidylyltransferase (EC 2.7.7.41) which catalyses formation of CDP-diglyceride from CTP and phosphatidic acid has been studied in rat brain preparations and other tissues. Improvement, as judged by the higher tissue activities obtained, in the assay method for this enzyme was achieved through use of phosphatidic acids sonicated in buffer–detergent solution saturated with ether and containing bovine serum albumin and use of short incubation times which essentially provided a measure of initial rates. The enzyme of rat brain microsomes yielded with 1,2-dioleoylphosphatidic acid as substrate a pH optimum of 6.8 with maleate buffer and optimal concentrations of 60 mM for Mg2+, 6 mM for CTP and 250 μg per 0.8 ml for phosphatidic acid. Enzyme activity was mainly located in the 90 000 × g fraction (microsomal) with small but significant activity in the 12 000 × g fraction. Comparison of activities (nanomoles CTP incorporated per milligram protein per minute) amongst tissues showed the following order: brain, 1.87; liver, 1.32; lung, 1.19; small intestine, 1.00; kidney, 0.69; heart, 0.41; diaphragm, 0.07; skeletal muscle, 0.02. Examination of the effect of varying the fatty acid composition in the phosphatidic acids added exogenously gave the following order (activities in parentheses): 1-stearoyl-2-oleoyl- (5.58), 1-oleoyl-2-stearoyl- (5.37), 1,2-dioleoyl- (4.49) 1-palmitoyl-2-oleoyl- (3.85), 1-stearoyl-2-arachidonoyl- (3.31), 1-arachidonoyl-2-stearoyl- (3.16), 1,2-diarachidonoyl- (0.72), 1,2-dicaproyl- (0.67), 1,2-dipalmitoyl- (0.67) and 1,2-distearoyl-(0.18). The single bis- and lysophosphatidic acids tested were inactive as substrates. Apart from a possible preference for one or more unsaturated fatty acids the transferase enzyme showed no selectivity in respect to the fatty acid distribution of phosphatidic acids.

On the specificity of cytidine diphosphate diglycerides in monophosphoinositide biosynthesis by rat brain preparations

1970 ◽  
Vol 48 (3) ◽  
pp. 269-277 ◽  
Author(s):  
H. H. Bishop ◽  
K. P. Strickland
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Rat Brain ◽  
Microsomal Fraction ◽  
Unsaturated Fatty Acids ◽  
Saturated Fatty Acids ◽  
Fatty Acid Distribution ◽  
Optimal Conditions ◽  
Cytidine Diphosphate ◽  
Palmitic Acids

Some properties of the enzyme cytidine diphosphate diglyceride : inositol transferase which catalyzes the reaction[Formula: see text]have been studied with various preparations from rat brain. The enzyme was found to be primarily located in the microsomal fraction (either the 12 000 × g supernatant or the 90 000 × g pellet). Optimal conditions were established for pH and concentrations of each of the reactants and Mn2+ ion. A number of cytidine diphosphate diglycerides with different fatty acids in the 1- and 2-positions were synthesized chemically and used as substrates for the above reaction. The substrates included those with the following distribution of fatty acids: 1,2-dipalmitoyl; 1,2-distearoyl; 1-palmitoyl,2-oleoyl; 1-stearoyl,2-oleoyl; 1-oleoyl,2-stearoyl and 1,2-dioleoyl. It was observed that those substrates with oleic at the 2-position were more readily utilized than those containing palmitic or stearic acids at the 2-position or at both the 1- and 2-positions. This selectivity for cytidine diphosphate diglyceride with an unsaturated fatty acid at the 2-position was consistent with the pattern of fatty acid distribution found for isolated rat brain monophosphoinositide. In the latter, saturated fatty acids (stearic and palmitic acids) were predominantly located at the 1-position and unsaturated fatty acids (20:4, arachidonic acid) were predominantly located in the 2-position.

scholarly journals Comparison of Mangalica and Hungarian Large White pigs at identical bodyweight: 2. Fatty acid regiodistribution analysis of the triacylglycerols

2010 ◽  
Vol 53 (2) ◽  
pp. 147-161 ◽  
Author(s):  
A. Szabó ◽  
P. Horn ◽  
R. Romvári ◽  
Z. Házas ◽  
H. Fébel
Keyword(s):  
Adipose Tissue ◽  
Fatty Acid ◽  
Unsaturated Fatty Acids ◽  
Fatty Acid Distribution ◽  
Direct Analysis ◽  
Distribution Analysis ◽  
Large White ◽  
White Tissue ◽  
Extrahepatic Tissues ◽  
Docosahexaenoic Acids

Abstract. At identical bodyweight values (130 kg) Mangalica and Hungarian Large White tissue (liver, kidney, heart and skeletal muscle, adipose tissue, spleen and lung) triacylglycerol (TAG) fatty acid distribution analysis was performed, to describe tissue types and possible genotype-associated differences. Tissue TAG was partially hydrolyzed with hog pancreatic lipase, and the fatty acid profile of 2-monoacylglycerols (2MAG) and TAGs was analyzed by gas-chromatography. All extrahepatic tissues provided marked central (sn-2) saturated fatty acid (SFA) (mostly palmitate) recruitment, while liver 2MAGs were mainly aclylated by unsaturated fatty acids (UFA). Inter-genotype differences were minor: in adipose tissue in Mangalica the total palmitate moiety was found in the 2MAGs, while in the liver docosapentaenoic and docosahexaenoic acids were only found in the Mangalica 2MAGs. In conclusion, the direct analysis results of the 2MAGs in six extrahepatic organs and in the liver provided evidence that the building-up of the characteristic porcine TAG structure is located at a post-hepatic site, being true for most of the organs in the pig body.

scholarly journals Substrate-specificities of acid and alkaline ceramidases in fibroblasts from patients with Farber disease and controls

1982 ◽  
Vol 205 (2) ◽  
pp. 419-425 ◽  
Author(s):  
Toru Momoi ◽  
Yoav Ben-Yoseph ◽  
Henry L. Nadler
Keyword(s):  
Fatty Acids ◽  
Unsaturated Fatty Acids ◽  
Specific Activity ◽  
Kinetic Studies ◽  
Significant Activity ◽  
Acid Ceramidase ◽  
Ph Optimum ◽  
Farber Disease ◽  
Normal Controls

The specific activity of acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) was measured at pH4.5 in normal fibroblasts and in fibroblasts from patients with Farber disease and obligate heterozygotes. Greater activity was found when the synthetically made ceramide substrates contained shorter-chain fatty acids or higher content of double bonds. Acid ceramidase activities towards N-lauroyl- (C12:0), N-myristoyl- (C14:0) and N-palmitoyl- (C16:0) sphingosine (C18:1) were respectively about 38, 26 and 6 times higher than the activity towards the N-stearoyl (C18:0) substrate. The activity towards N-linolenoylsphingosine (C18:3/C18:1), N-linoleoylsphingosine (C18:2/C18:1) and N-oleoylsphingosine (C18:1/C18:1) were respectively about 5, 4 and 3 times higher than the activity towards N-stearoylsphingosine (C18:0/C18:1). The activity towards N-stearoyldihydrosphingosine (C18:0/C18:0) was about 40% of that towards N-stearoylsphingosine. Fibroblast alkaline ceramidase possessed significant activity only towards ceramides of unsaturated fatty acids, with a pH optimum of about 9.0. Deficiency of acid ceramidase activity in fibroblasts from patients with Farber disease and intermediate activities in obligate heterozygotes were demonstrated with all ceramides examined except for N-hexanoylsphingosine (C6:0/C18:1), whereas alkaline ceramidase activity was unaffected. Comparative kinetic studies of acid ceramidase activity with N-lauroylsphingosine and N-oleoylsphingosine demonstrated about 5 (2–12)-fold and 7 (4–17)-fold higher Km values in fibroblasts from patients with Farber disease as compared with normal controls. N-Lauroylsphingosine, towards which acid ceramidase activity in control fibroblasts was about 10 times higher than that towards N-oleoylsphingosine, may serve as a better substrate for enzymic diagnosis of Farber disease as well as for further characterization of the catalytically defective acid ceramidase.

Lipid and Fatty Acid Composition of Synchronized Synechococcus leopoliensis

1989 ◽  
Vol 44 (3-4) ◽  
pp. 202-206 ◽  
Author(s):  
Günter Döhler ◽  
Günter Datz
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Chlorophyll A ◽  
Unsaturated Fatty Acids ◽  
Fatty Acid Distribution ◽  
Light Period ◽  
Lag Phase ◽  
Synechococcus Leopoliensis ◽  
Dark Regime ◽  
Total Fatty Acids

Abstract The cyanobacterium Synechococcus leopoliensis (Anacystis nidulans, strain L 1402-1) grown at 39 °C and 2 vol. % CO : could be synchronized by a light/dark regime of 3:5 h (white light intensity 1.5 × 104 erg cm-2 sec-1). Content of pigments (chlorophyll a. phycocyanin and carotenoids), R N A and proteins increased linearly up to 100% at the end of the light period while DNA synthesis was lower. Chlorophyll a synthesis was correlated to the photosystem I activity of the isolated thylakoids and to the formation of MGD G . Galacto lipids were synthesized in the light period, only. A lag phase of 2h was observed in the biosynthesis of SQDG and PG. No significant differences were found between the cell and thylakoid fractions. Palmitic (C16:0), hexadecenoic (C16:1) and octadecenoic (C18:1) acid as major com ponents accounted for more than 90% of total fatty acids in MGD G , DGDG and SQDG . PG contains a small amount of stearic (C18:0) and heptadecenoic (C17:1) acid. No significant variations in the fatty acid distribution of all lipids could be detected in the cell fraction during the division cycle. Changes in the ratio of saturated to unsaturated fatty acids were found in isolated thylakoids. only. In experiments with [14C]bicarbonate main radioactivity was measured in galacto lipids while using [14C]acetate SQDG and PG were markedly [14C]labelled. Results were discussed with reference to the findings of eucaryotic algae and the formation of photosynthetic membranes.

scholarly journals Purification of a phosphatidic-acid-hydrolysing phospholipase A2 from rat brain

1995 ◽  
Vol 306 (1) ◽  
pp. 305-309 ◽  
Author(s):  
F J Thomson ◽  
M A Clark
Keyword(s):  
Catalytic Activity ◽  
Phosphatidic Acid ◽  
Phospholipase A2 ◽  
Rat Brain ◽  
Lysophosphatidic Acid ◽  
Catalytic Properties ◽  
Gel Filtration ◽  
Gel Filtration Chromatography ◽  
Ph Optimum ◽  
Sds Page

A phosphatidic-acid-hydrolysing phospholipase A2 was purified from rat brain and characterized. This phospholipase A2 was purified by sequential cation, hydrophobic, heparin and gel-filtration chromatography. The purified protein had a mass of approximately 58 kDa as assayed by SDS/PAGE, had a pH optimum of 6.0, and was Ca(2+)-independent. This enzyme was apparently phosphatidic-acid-selective and had little measurable catalytic activity when phosphatidylcholine, phosphatidylethanolamine or diacylglycerol was used as substrate. On the basis of its physical and catalytic properties, we conclude that this phospholipase A2 is unique from those previously purified, and we speculate that it may be important for the production of the bioactive lipid lysophosphatidic acid.

LIPID SYNTHESIS IN RAT LIVER PARTICLES: DIVERSION OF SYNTHESIS FROM TRIGLYCERIDE TO LECITHIN BY THE IN VITRO ADDITION OF CYTIDINE DIPHOSPHATE CHOLINE

1962 ◽  
Vol 40 (1) ◽  
pp. 1051-1058 ◽  
Author(s):  
K. P. Strickland ◽  
R. J. Rossiter
Keyword(s):  
Fatty Acid ◽  
Phosphatidic Acid ◽  
Experimental Test ◽  
Lipid Synthesis ◽  
Phosphatidyl Inositol ◽  
Fatty Acyl ◽  
Phosphatidic Acids ◽  
Acyl Coenzyme A ◽  
Cytidine Diphosphate

When particle preparations from rat or chicken liver were incubated in a suitable medium containing α-glycerophosphate-C14, radioactivity was recovered from lecithin, phosphatidyl inositol, phosphatidic acid, and triglyceride. Whole homogenate and various particle preparations catalyzed the dephosphorylation of a number of phosphatidic acids, with the liberation of inorganic P.It is currently believed that liver preparations are capable of catalyzing the esterification of L-α-glycerophosphate, with the formation of L-α-phosphatidic acid, which subsequently may be dephosphorylated to form D-α,β-diglyceride. The diglyceride so formed may then give rise either to lecithin, by combining with phosphorylcholine from cytidine diphosphate choline, or to triglyceride, by combining with fatty acid from fatty acyl coenzyme A. If these reactions occur in liver particle preparations, it should be possible, by the addition in vitro of unlabelled cytidine diphosphate choline, to divert the synthesis of lipid from the formation of triglyceride to the formation of lecithin. In experiments designed to put this hypothesis to the experimental test, such a diversion of lipid synthesis was achieved.

Evidence that hepatic triglycerides provide acylglycerides for synthesis of bile phosphatidylcholines

1994 ◽  
Vol 267 (6) ◽  
pp. G1028-G1034
Author(s):  
G. M. Patton ◽  
J. M. Fasulo ◽  
S. J. Robins
Keyword(s):  
Fatty Acid ◽  
Phosphatidic Acid ◽  
Rat Liver ◽  
De Novo ◽  
Specific Activity ◽  
De Novo Synthesis ◽  
Chase Period ◽  
Phosphatidic Acids

To determine the biochemical origin of bile phosphatidylcholines (PCs), rat liver perfusions with 16:1 fatty acid (FA) and [3H]glycerol were performed to generate novel radiolabeled bile and liver PCs and their hepatic glyceride precursors. Results showed total equilibration of bile and liver 16:1-16:1 PC when the specific activity of precursor glycerol-3-phosphate was kept constant. However, when the specific activity of glycerol-3-phosphate decreased during the labeling period and during a prolonged chase period with 17:1 FA and nonradiolabeled glycerol, the specific activity of bile 16:1-16:1 PC was appreciably higher than this same PC in the liver and during the chase period was even higher than its hepatic 16:1-16:1 acylglycerol precursors, phosphatidic acid and diglyceride. During the chase period with 17:1 FA, new radiolabeled 16:1-17:1 PC was formed, and again the specific activity of this PC in bile was greater than this PC and 16:1-17:1 phosphatidic acid and diglyceride in the liver. Only the specific activity of liver 16:1-16:1-(FA) triglyceride equaled or was high enough to support the formation of new bile 16:1-16:1 PC. These studies indicate that bile PCs do not directly derive from preexisting hepatic PCs or by de novo synthesis through phosphatidic acids and diglycerides, but likely originate by remodeling from a pool of hepatic triglycerides.

The deacylation of phosphatidylinositol by rat brain preparations

1979 ◽  
Vol 57 (12) ◽  
pp. 1359-1367 ◽  
Author(s):  
T. Y. P. Shum ◽  
N. C. C. Gray ◽  
K. P. Strickland
Keyword(s):  
Oleic Acid ◽  
Fatty Acid ◽  
Rat Brain ◽  
Divalent Cations ◽  
Brain Homogenate ◽  
Phospholipase A ◽  
Ph Optimum ◽  
Major Activity ◽  
Nonionic Detergent

The deacylation of phosphatidylinositol (PI) in rat brain was studied in vitro. Using 1-acyl, 2-[1-14C-oleoyl] sn-glycerol-3-phosphoinositol and [U-14C]phosphatidylinositol as substrates, a release of 14C-free fatty acid was found when incubations were conducted with the PI labelled in the 2 position, both in the 12 000 – 106 000 g pellet (microsomal) and in the 106 000 g supernate prepared from rat brain homogenate. With the 106 000 g pellet the deacylation activity was linear with time up to 15 min and was directly proportional to the amount of protein added. Two pH optima were observed, one in the region of pH 7.5 which constituted the major activity and the other in the region of pH 6.0. The apparent Km for the enzyme activity at pH 7.5 was found to be 6.2 × 10−4 M and the Vmax was 1.24 nmol of [14C]oleic acid released per minute per milligram of protein. The Ca2+ ion stimulated the activity maximally at 5 mM while other divalent cations such as Mg2+, Mn2+, Cd2+, Co2+, Cu2+, Fe2+, Hg2+, Ni2+, Pb2+, and Zn2+ either partially or completely inhibited the activity. The nonionic detergent, Triton X-100, stimulated the deacylation more than twofold at a concentration of 0.01%. The sulfydryl reagents, p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetamide showed partial inhibition of the reaction which was reversed by the addition of dithiothreitol. The deacylation activity in the 106 000 g supernate from rat brain was found to be directly proportional to the amount of protein added, and to the time (up to 15 min). A pH optimum was observed in the region of pH 6.0. Substrate concentration studies showed that the apparent Km was 5.0 × 10−4 M and the Vmax was 3.93 nmol of [14C]oleic acid released per minute per milligram of protein. "lyso-PI," diacylglycerol, and fatty acid were formed at pH 6.0 and pH 7.5. The data obtained indicate that from 54 (pH 7.5) to 70% (pH 6.0) of the altered PI is due to phospholipase A2 activity, 24 (pH 6.0) to 28% (pH 7.5) is due to phospholipase "C-like" activity, and from 2 (pH 6.0) to 22% (pH 7.5) may be due to phospholipase A1 activity. These results provide evidence for the deacylation component of a deacylation–reacylation cycle for the generation of specific molecular species of PI.

LIPID SYNTHESIS IN RAT LIVER PARTICLES: DIVERSION OF SYNTHESIS FROM TRIGLYCERIDE TO LECITHIN BY THE IN VITRO ADDITION OF CYTIDINE DIPHOSPHATE CHOLINE

1962 ◽  
Vol 40 (8) ◽  
pp. 1051-1058 ◽  
Author(s):  
K. P. Strickland ◽  
R. J. Rossiter
Keyword(s):  
Fatty Acid ◽  
Phosphatidic Acid ◽  
Experimental Test ◽  
Lipid Synthesis ◽  
Phosphatidyl Inositol ◽  
Fatty Acyl ◽  
Phosphatidic Acids ◽  
Acyl Coenzyme A ◽  
Cytidine Diphosphate

When particle preparations from rat or chicken liver were incubated in a suitable medium containing α-glycerophosphate-C14, radioactivity was recovered from lecithin, phosphatidyl inositol, phosphatidic acid, and triglyceride. Whole homogenate and various particle preparations catalyzed the dephosphorylation of a number of phosphatidic acids, with the liberation of inorganic P.It is currently believed that liver preparations are capable of catalyzing the esterification of L-α-glycerophosphate, with the formation of L-α-phosphatidic acid, which subsequently may be dephosphorylated to form D-α,β-diglyceride. The diglyceride so formed may then give rise either to lecithin, by combining with phosphorylcholine from cytidine diphosphate choline, or to triglyceride, by combining with fatty acid from fatty acyl coenzyme A. If these reactions occur in liver particle preparations, it should be possible, by the addition in vitro of unlabelled cytidine diphosphate choline, to divert the synthesis of lipid from the formation of triglyceride to the formation of lecithin. In experiments designed to put this hypothesis to the experimental test, such a diversion of lipid synthesis was achieved.

In Vitro Incorporation and Metabolism of Icosapentaenoic and Docosahexaenoic Acids in Human Platelets - Effect on Aggregation

1986 ◽  
Vol 56 (01) ◽  
pp. 057-062 ◽  
Author(s):  
Martine Croset ◽  
M Lagarde
Keyword(s):  
Arachidonic Acid ◽  
Fatty Acid ◽  
Platelet Aggregation ◽  
Fatty Acid Distribution ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Aggregation Response ◽  
Docosahexaenoic Acids ◽  
Membrane Level

SummaryWashed human platelets were pre-loaded with icosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or EPA + DHA and tested for their aggregation response in comparison with control platelets. In fatty acid-rich platelets, an inhibition of the aggregation could be observed when induced by thrombin, collagen or U-46619. The strongest inhibition was observed with DHA-rich platelets and it was reduced when DHA was incorporated in the presence of EPA.Study of fatty acid distribution in cell lipids after loading showed that around 90% of EPA or DHA taken up was acylated into phospholipids and a very small amount (less than 2%) remained in their free and hydroxylated forms. DHA was more efficiently acylated into phosphatidylethanolamine (PE) than into phosphatidylinositol (PI) in contrast to what observed with EPA, and both acids were preferentially incorporated into phosphatidylcholine (PC). EPA inhibited total incorporation of DHA and increased its relative acylation into PE at the expense of PC. In contrast, DHA did not affect the acylation of EPA. Upon stimulation with, thrombin, EPA was liberated from phospholipids and oxygenated (as judged by the formation of its monohydroxy derivative) whereas DHA was much less metabolized, although consistently transferred into PE.It is concluded that EPA and DHA might affect platelet aggregation via different mechanisms when pre-loaded in phospholipids. Whereas EPA is known to alter thromboxane A2 metabolism from endogenous arachidonic acid, by competing with it, DHA might act directly at the membrane level for inhibiting aggregation.

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