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Journal ArticleDOI

Enzymatic reactions on thin-layer chromatographic plates. II. Phospholipase A2 hydrolysis of phosphatidylcholine and separation of the products on a single plate.

21 May 1979-Journal of Chromatography A (J Chromatogr)-Vol. 173, Iss: 2, pp 379-387
TL;DR: The acyl group distributions in the 1- and 2-positions of hen egg phosphatidylcholine obtained from the gas-liquid chromatographic analysis of the methyl ester corresponding to the lyso and free fatty acid band agreed with those obtained by the method of Wells and Hanahan.

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Abstract: A procedure for the phospholipase A2 hydrolysis of phosphatidylcholine on a thin-layer chromatographic plate and subsequent separation of the products on the same plate is described. A 0.2-0.8-mg amount of Russell's viper venom (phospholipase A2) in 0.2 ml of 0.005 M calcium chloride solution was applied on a 0.5-mm silica gel G plate as a band over which 2-5 mg of egg phosphatidylcholine in 0.2 ml of diethyl ether containing 5% of methanol was evenly applied. After the reaction had proceeded for 15-20 min in a diethyl ether-saturated chamber at 25 degrees, the plate was developed with chloroform-methanol-water (65:25:4). The bands were identified and their contents extracted. The extent of hydrolysis under different reaction conditions was evaluated from the amount of lysophosphatidylcholine formed. Approximately 74.6% (maximum) conversion was obtained within 15 min at 25 degrees using a substrate to enzyme ratio of 4:1. The acyl group distributions in the 1- and 2-positions of hen egg phosphatidylcholine obtained from the gas-liquid chromatographic analysis of the methyl ester corresponding to the lyso and free fatty acid band agreed with those obtained by the method of Wells and Hanahan. The method is also applicable to phosphatidylethanolamine.

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Topics: Phosphatidylcholine (56%)
Citations
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Journal ArticleDOI
TL;DR: The results indicate that sPLA(2)-III can create white matter pathologies that are remyelinated by Schwann cells 2 to 3 weeks after injury, and is the first report of a reaching task being able to discriminate between various grades of cervical white matter damage and varying extents of recovery.

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Abstract: Phospholipases A2 (PLA2) are group of enzymes that hydrolyze membrane phospholipids at the sn-2 position. PLA2 are present in the brain and spinal cord and are implicated in several neurological disorders. Previously, we showed that PLA2 activity increases following traumatic spinal cord injury and injection of group III secretory PLA2 (sPLA2-III) demyelinates spinal cord axons. Here, we demonstrate that injections of sPLA2-III into the cervical dorsolateral funiculus (DLF) resulted in dose-dependent demyelination, loss of oligodendrocytes and astrocytes, as well as axonopathy. Additionally, spared axons within the lesion were remyelinated by Schwann cells between weeks 2 and 3. To assess functional loss and recovery, we employed a modified “Staircase Test” pellet retrieval device and footprint analysis of forelimb function during locomotion. Pellet retrieval assessment sensitively detected the dose dependent lesion and its recovery after sPLA2-III injections with greater sensitivity than footprint analysis. We believe that this is the first report of a reaching task being able to discriminate between various grades of cervical white matter damage and varying extents of recovery. Thus, our results indicate that sPLA2-III can create white matter pathologies that are remyelinated by Schwann cells 2 to 3 weeks after injury. Additionally, the pellet retrieval test is a sensitive and quantifiable method for assessing the dysfunction and later recovery mediated by sPLA2-III injections.

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37 citations


Journal ArticleDOI
J. Dutta1, Arun Kr. Das1, Sandip Saha1Institutions (1)
Abstract: A novel method for the lipase deacylation of triglycerides is described in which 0.1–0.2 ml of the enzyme solution in 1 M tris buffer (pH 8.2) containing 2–4 mg of protein is applied as a band on a 0.5-mm thick silica gel thin-layer chromatographic plate. Over this band, 1–4 mg of triglyceride in n-hexane is applied evenly and the plate is incubated at 40° for 1–2 min. The reaction is stopped by exposure to hydrogen-chloride vapour and the products are removed from the reaction zone by three consecutive developments in diethyl ether up to 2 cm from the line of application. These are resolved by re-developing the plate in n-hexane—diethyl ether—acetic acid (80:20:1.5) up to 14 cm. The sn-2-monoglyceride and sn-1,2(2,3)-diglyceride bands located by iodine vapour are extracted and the fatty acid compositions evaluated by gas-liquid chromatography for the determination of fatty acid distribution in the glyceride molecule. The method, when applied to groundnut oil, goat depot fat, body fat of shol fish (Channa striatus) and yeast (Rhodotorula glutinis) fat, produced representative sn-1,2(2,3)-diglycerides which can be used for the stereospecific analysis of triglyceride.

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23 citations


Journal ArticleDOI
01 Dec 1983-Lipids
TL;DR: A rapid method for the stereospecific analysis of triglycerides based on enzymatic hydrolysis on thin layer plates was applied to a number of GlyCine max, Glycine soya, Avena sativa and Avena sterilis strains and large deviations from the common triglyceride pattern were not found.

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Abstract: A rapid method for the stereospecific analysis of triglycerides based on enzymatic hydrolysis on thin layer plates was applied to a number ofGlycine max, Glycine soya, Avena sativa andAvena sterilis strains. The percentage of each fatty acid on thesn-1,sn-2- andsn-3-positions was linearly related to the total percentage of the fatty acid in the triglyceride. Large deviations from the common triglyceride pattern were not found.

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20 citations


Journal ArticleDOI
01 Dec 1985-Lipids
TL;DR: The PE component of rabbit sperm phospholipids appears to differ from that of the other cells in having the previously unreported diplasmalogen as its major constituent.

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Abstract: The question of whether diplasmalogens [1,2-di(O-1′-alkenyl) phosphatidyl derivatives] make up part of the plasmalogen component of cell phospholipids was examined using rabbit epididymal spermatozoa. These cells are readily obtained as a highly homogeneous suspension and long have been known to have high plasmalogen content. Phospholipids were determined by thin layer chromatography (TLC) with CuSO4 staining. Plasmalogens were determined by hydrolysis of the phospholipids with TCA/HCl, followed by TLC and CuSO4 staining. Ethanolamine derivatives were determined by ninhydrin. The phosphatidylethanolamine (PE) content of these cells was 29±2 μg/108 cells, 90% of which was assayed as diplasmalogen and 10% as diacyl PE. No monoplasmalogen could be detected. The presence of diplasmalogen as the major component of PE was given further support from infrared and proton nuclear magnetic resonance (1H-NMR) spectroscopy, which showed the presence of O-1′-alkenyl substituents but near absence of O-acyl substituents. The phosphatidylcholine (PC) content of the cells was 104±5 μ/108 cells, of which 50% was monoplasmalogen with the 1′-alkenyl group on the 2 position of the glycerol moiety. No diplasmalogen was found in PC. The other phospholipids in rabbit sperm were phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SP) and lysophosphatidylcholine (LPC). Phosphatidylserine (PS) and phosphatidylinositol (PI) were present at the limits of detectability of the TLC method. None of these phospholipids contained plasmalogen. The PE component of rabbit sperm phospholipids appears to differ from that of the other cells in having the previously unreported diplasmalogen as its major constituent.

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19 citations


Journal ArticleDOI
Hiromi Yoshida1, Sachiko Takagi1Institutions (1)
Abstract: Whole soybeans were exposed to microwave roasting for 6, 12, and 20 min at a frequency of 2,450 MHz and were studied not only for phospholipid composition but also for positional distribution of the fatty acids. During microwave roasting, the greatest rate of phospholipid losses (P<0.05) was observed in phosphatidylethanolamine (PE), followed by phosphatidylcholine (PC) and phosphatidylinositol (PI), respectively. Therefore, the effects of microwave roasting on the composition and positional distribution of the fatty acids are likely clearer in PE than in PC or PI. However, the principal characteristics for the positional distribution of fatty acids are still retained during microwave roasting: unsaturated fatty acids, especially linoleic, are predominantly concentrated in the 2-position, and saturated fatty acids, especially palmitic, primarily occupy the 1-position after 12 or 20 min of roasting. The results suggest that unsaturated fatty acids located in the 2-position are significantly protected from microwave roasting.

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15 citations


Cites methods from "Enzymatic reactions on thin-layer c..."

  • ...The positional distribution of fatty acids in each of the PE, PC, and PI samples isolated by preparative TLC was determined with phospholipase A 2 hydrolysis on a silica gel plate by a modification of the method of Dutta et al. ( 20 )....

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References
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Journal Article
TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.

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Abstract: Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins, a number of modified analytical procedures utilizing this reagent have been reported for the determination of proteins in serum, in antigen-antibody precipitates, and in insulin. Although the reagent would seem to be recommended by its great sensitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard to effects of variations in pH, time of reaction, and concentration of reactants, permissible levels of reagents commonly used in handling proteins, and interfering substances. Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.

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285,427 citations


Journal ArticleDOI

212 citations


Journal ArticleDOI
TL;DR: Fatty acid analysis of several lipid fractions showed that the Δ3-trans-hexadecenoic acid, present in the leaves, is concentrated almost exclusively in phosphatidyl glycerol, and degradation experiments with phospholipase A showed that this acid is located preferentially at the 2-ester position.

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Abstract: Pure phosphatidyl glycerol was obtained from spinach leaves after repeated chromatography on silica columns. Ascertainment of the configuration of the hydrolysis products formed by the action of phospholipases C (EC 3.1.4.3) and D (EC 3.1.4.4) demonstrated that this phospholipid is identical with 1,2-diacyl-glycerol-3-phosphoryl-1-glycerol. Fatty acid analysis of several lipid fractions showed that the Δ3-trans-hexadecenoic acid, present in the leaves, is concentrated almost exclusively in phosphatidyl glycerol. Degradation experiments with phospholipase A (EC 3.1.1.4) showed that this acid is located preferentially at the 2-ester position. A subfractionation of phosphatidyl glycerol was accomplished by thin-layer chromatography on silica plates impregnated with silver nitrate. A breakdown of the two fractions obtained with phospholipase A allowed the recognition of several molecular species, and 1-linolenoyl,2-Δ3-trans-hexadecenoyl-glycerol-3-phosphoryl-1′-glycerol appeared to be the major species. The results were confirmed by hydrolysis of phosphatidyl glycerol with phospholipase C and separation on impregnated adsorbents of the diglycerides formed.

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195 citations


Journal ArticleDOI

149 citations


Journal ArticleDOI
TL;DR: The composition of lecithins from lung and brain showed many similarities irrespective of animal species, which may indicate a certain degree of tissue specificity.

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Abstract: 1. 1. The molecular species composition of lecithins from lung, brain, liver and kidney derived from rat, rabbit, pig and cow was determined. 2. 2. In all animals considerable differences were found in the molecular composition of lecithins from the various tissues. The composition of lecithins from lung and brain showed many similarities irrespective of animal species. This may indicate a certain degree of tissue specificity. On the other hand, pronounced differences were observed in the lecithin composition of liver and kidney from the different animals. 3. 3. (Dipalmitoyl)-lecithin was the main lecithin present in lung, but was only a trace compound in the liver and kidney of all animals examined. No significant amounts of (distearoyl)- or (1-palmitoyl-2-stearoyl)-lecithin could be detected in any tissues investigated. 4. 4. Lecithin species containing oleic acid were found to predominate in brain. In general, the content of (1-palmitoyl-2-oleoyl)-lecithin was higher than (1-stearoyl-2-oleoyl)-lecithin. Species with linoleic acid were not significantly present in brain. 5. 5. Lecithins containing one saturated and one polyunsaturated fatty acid constituent are abundant in the liver and kidney of all animals species examined. Molecules with two identical or two different polyunsaturated acyl constituents were not encountered in significant amounts in the tissues investigated.

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142 citations


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No. of citations received by the Paper in previous years
YearCitations
20081
20071
19971
19872
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19831