PHOSPHOLIPID METABOLISM IN LIVER SLICES: LABELLING OF PHOSPHOLIPIDS WITH ACETATE-1-C14

1956 ◽  
Vol 34 (1) ◽  
pp. 429-440 ◽  
Author(s):  
Dorothy L. Kline ◽  
H. A. DeLuca
Keyword(s):  
Fatty Acids ◽  
Gas Phase ◽  
Time Course ◽  
Specific Activity ◽  
Phospholipid Metabolism ◽  
Great Decrease ◽  
Lipid Fractions ◽  
Optimum Ph ◽  
Specific Activities ◽  
Guinea Pig Liver

A study has been made of the labelling of the phospholipids, the fatty acids from the acetone-soluble lipid, and the non-esterified cholesterol in slices of rat and guinea pig liver respiring in a suitably buffered Krebs–Ringer medium containing acetate-1-C14. The time course of the reactions and the effects of the concentration of potassium ion and the pH of the incubating medium have been defined. For phospholipid and fatty acids of the acetone-soluble lipid, the optimum pH was in the range 6.8–7.4, whereas for cholesterol there was a much sharper optimum at pH 6.6–6.8. When the oxygen of the gas phase was replaced with nitrogen, the labelling of all three lipid fractions was abolished. The addition of glucose to the incubating medium slightly increased the labelling of the phospholipids and the fatty acids of the acetone-soluble lipid, but had no consistent effect on the labelling of the non-esterified cholesterol. Purification of the cholesterol by the method of bromination and debromination caused only a slight change in specific activity, indicating that the cholesterol was not contaminated with large amounts of companion substances with specific activities greatly different from that of the cholesterol itself. The addition of cyanide, fluoride, iodoacetate, or 2,4-dinitrophenol to the incubating medium caused a great decrease in the labelling of all fractions studied. With the exception of 2,4-dinitrophenol, the inhibitors were used in concentrations that inhibit the oxygen consumption. Malonate inhibited the incorporation of acetate-1-C14 into cholesterol, but did not affect the labelling of the phospholipids. When the acetate-1-C14 was replaced with other C14-labelled precursors, good labelling of phospholipids was observed with glycine-2-C14, glycerol-1-C14, and fructose-C11, but not with formate-C14, lactate-1-C14, or glucose-C14. The cholesterol was not significantly labelled from any of the precursors other than acetate-1-C14.

PHOSPHOLIPID METABOLISM IN LIVER SLICES: LABELLING OF PHOSPHOLIPIDS WITH ACETATE-1-C14

1956 ◽  
Vol 34 (3) ◽  
pp. 429-440 ◽  
Author(s):  
Dorothy L. Kline ◽  
H. A. DeLuca
Keyword(s):  
Fatty Acids ◽  
Gas Phase ◽  
Time Course ◽  
Specific Activity ◽  
Phospholipid Metabolism ◽  
Great Decrease ◽  
Lipid Fractions ◽  
Optimum Ph ◽  
Specific Activities ◽  
Guinea Pig Liver

A study has been made of the labelling of the phospholipids, the fatty acids from the acetone-soluble lipid, and the non-esterified cholesterol in slices of rat and guinea pig liver respiring in a suitably buffered Krebs–Ringer medium containing acetate-1-C14. The time course of the reactions and the effects of the concentration of potassium ion and the pH of the incubating medium have been defined. For phospholipid and fatty acids of the acetone-soluble lipid, the optimum pH was in the range 6.8–7.4, whereas for cholesterol there was a much sharper optimum at pH 6.6–6.8. When the oxygen of the gas phase was replaced with nitrogen, the labelling of all three lipid fractions was abolished. The addition of glucose to the incubating medium slightly increased the labelling of the phospholipids and the fatty acids of the acetone-soluble lipid, but had no consistent effect on the labelling of the non-esterified cholesterol. Purification of the cholesterol by the method of bromination and debromination caused only a slight change in specific activity, indicating that the cholesterol was not contaminated with large amounts of companion substances with specific activities greatly different from that of the cholesterol itself. The addition of cyanide, fluoride, iodoacetate, or 2,4-dinitrophenol to the incubating medium caused a great decrease in the labelling of all fractions studied. With the exception of 2,4-dinitrophenol, the inhibitors were used in concentrations that inhibit the oxygen consumption. Malonate inhibited the incorporation of acetate-1-C14 into cholesterol, but did not affect the labelling of the phospholipids. When the acetate-1-C14 was replaced with other C14-labelled precursors, good labelling of phospholipids was observed with glycine-2-C14, glycerol-1-C14, and fructose-C11, but not with formate-C14, lactate-1-C14, or glucose-C14. The cholesterol was not significantly labelled from any of the precursors other than acetate-1-C14.

THE EFFECT OF HOMOGENIZATION ON FREE AND ESTERIFIED FATTY ACID POOLS IN ADIPOSE TISSUE

1965 ◽  
Vol 43 (2) ◽  
pp. 271-280 ◽  
Author(s):  
David Rubinstein ◽  
Anna M. Daniel ◽  
Sylvester Chiu ◽  
John C. Beck
Keyword(s):  
Fatty Acids ◽  
Adipose Tissue ◽  
Fatty Acid ◽  
Incubation Medium ◽  
Specific Activity ◽  
Tissue Cell ◽  
Intact Tissue ◽  
Adipose Tissue Cell ◽  
Specific Activities ◽  
Presence And Absence

The effect of homogenization of adipose tissue on fatty acid pools was studied with palmitate-1-C14 in the presence and absence of epinephrine. Addition of epinephrine to intact tissue in an incubation medium high in FFA increases the specific activity of the tissue FFA. When the tissue is incubated in a medium low in FFA, epinephrine induces an increase in the concentration and radioactivity of the tissue FFA. Epinephrine decreases the esterification of palmitate-1-C14 by intact tissue, regardless of the FFA concentration in the medium. This decrease is unrelated to the specific activities of either the medium or the tissue FFA. In homogenates, the decrease in incorporation of palmitate-1-C14 is proportional to the decrease in the specific activity of the FFA induced by epinephrine. Under the influence of epinephrine, FFA released from adipose tissue that was previously charged with palmitate-1-C14 have a specific activity about six times as great as the glyceride fatty acids. This difference is abolished by homogenization of the tissue. These results suggest that the newly synthesized triglycerides exist as a separate pool and are more readily hydrolyzed, thereby contributing FFA to an intracellular FFA pool. The existence of multiple pools of glycerides and FFA in the adipose tissue cell is dependent on the architecture of the cell.

INVESTIGATIONS INTO THE PRODUCTION OF LIPIDS BY SUBMERGED CULTURES OF THE MUSHROOM TRICHOLOMA NUDUM: II. STUDIES WITH UNIFORMLY LABELLED GLUCOSE

1962 ◽  
Vol 40 (1) ◽  
pp. 857-867 ◽  
Author(s):  
D. C. Leegwater ◽  
B. M. Craig
Keyword(s):  
Fatty Acids ◽  
Linoleic Acid ◽  
Specific Activity ◽  
Neutral Lipids ◽  
Basal Medium ◽  
Submerged Cultures ◽  
Intact Cells ◽  
Lipid Fractions ◽  
Time Specific ◽  
Low Nitrogen

Tricholoma nudum mycelium, grown in a medium with a low nitrogen content, was adapted to a basal medium containing glucose and incubated with glucose-U-C14for 25 hours. The time – specific activity relations of the major fatty acids produced were determined for both the neutral lipids and the phospholipids. The results confirm a previous finding that in the phospholipids of T. nudum linoleic acid becomes labelled under the conditions used at almost the same rate as palmitic, stearic, or oleic acid, but that in the neutral lipids the specific activity of this acid initially lags considerably behind those of the latter three. It is postulated that this phenomenon is due to a difference in the relative sizes of the linoleic acid pools involved in the synthesis of the two lipid fractions. Only part of the material used for the formation of the fatty acids is derived directly from the substrate, the remainder apparently being provided by material already present in the mycelium. The various results, together with those of earlier workers, are critically evaluated, and it is concluded that the order in which fatty acids are labelled in tracer studies with intact cells is determined to a large extent by the conditions of the experiment and does not necessarily provide information on possible product–precursor relations.

Incorporation of Na-1-14C-acetate into the fatty acids of two insect parasites (Hymenoptera) reared on different hosts

1971 ◽  
Vol 49 (10) ◽  
pp. 1297-1300 ◽  
Author(s):  
J. S. Barlow ◽  
G. K. Bracken
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Dietary Fat ◽  
Palmitoleic Acid ◽  
Galleria Mellonella ◽  
Specific Activity ◽  
Host Tissue ◽  
Metabolic Rates ◽  
Specific Activities ◽  
Insect Parasites

Sodium-1-14C-acetate was injected into larvae of the parasite, Exeristes comstockii, reared on Galleria mellonella or on Lucilia sericata. The concentration and specific activities of the fatty acids in the larvae were measured 24 h later. The concentration of palmitoleic acid was 10 times greater in C. comstockii when it grew on L. sericata but the specific activity of this fatty acid was the same on either host. It is concluded that the level of palmitoleic acid and probably other fatty acids in the parasite is controlled predominantly by changes in metabolic rates which are regulated by the concentration of the fatty acid in the parasite's diet, that is, host tissue. Direct deposition of dietary fat would not accomplish this result.Another parasite, Itoplectus conquisitor, reared on G. mellonella was also examined.

scholarly journals PURIFICATION AND SOME PROPERTIES OF ALPHA-AMYLASE OF PEAR FRUIT

HortScience ◽  
1991 ◽  
Vol 26 (6) ◽  
pp. 723E-723
Author(s):  
Shiao J. Li ◽  
Tim Facteau ◽  
Paul M. Chen
Keyword(s):  
Ammonium Sulfate ◽  
Time Course ◽  
Specific Activity ◽  
Alpha Amylase ◽  
Acetate Buffer ◽  
Pear Fruit ◽  
Ammonium Sulfate Precipitation ◽  
Freeze Dried ◽  
Optimum Ph ◽  
The Difference

Alpha-amylase was purified from freeze-dried pear fruit by extraction at pH 7.40 with Tris, Acetate and Imidazole buffer followed by differential ammonium sulfate precipitation and desalting column. The specific activity of the enzyme was increased 5.68 fold during purification. The optimum pH was 5.64 in Acetate buffer. The difference in the time course of alpha-amylase was observed between freeze-dried and fresh samples.

Phospholipid metabolism in Plasmodium-infected erythrocytes: guidelines for further studies using radioactive precursor incorporation

Parasitology ◽  
1989 ◽  
Vol 98 (3) ◽  
pp. 351-357 ◽  
Author(s):  
H. J. Vial ◽  
M. L. Ancelin ◽  
M. J. Thuet ◽  
J. R. Philippot
Keyword(s):  
Fatty Acids ◽  
Time Course ◽  
De Novo ◽  
Plasmodium Knowlesi ◽  
Lipid Synthesis ◽  
Experimental System ◽  
Phospholipid Metabolism ◽  
Intracellular Release ◽  
Phosphatidylcholine Biosynthesis ◽  
Lysophospholipase Activity

SUMMARYThe biosynthesis of phospholipids is extensive in Plasmodium knowlesi-infected simian erythrocytes due to the synthesis of membranes by this single-cell eukaryote in a host erythrocyte devoid of any pathway for lipid biosynthesis. In the present paper, we show that the incorporation of [3H]glycerol, which reflects de novo biosynthesis, is better studied at 300 μM−1 mM than at the trace doses, since this non-physiological precursor does not modify the amount of phosphatidylcholine biosynthesis from [3H]choline. Time-course incorporation of radioactive glycerol, oleate, lysophosphatidylcholine, choline, and inositol in RPMI 1640 medium containing nutrients for lipid synthesis showed that the optimum incubation time for phospholipid studies is 60–90 min, after which radioactive incorporation slows considerably. On the other hand, studies with [14C]serine revealed that incubation for 2–3 h is necessary for isotopic labelling of phosphatidylcholine via phosphatidylserine decarboxylation and phosphatidylethanolamine N-methylation. Incorporation of the various fatty acids into individual lipids was related to the molecular species composing each of them. Studies with [14C palmitoyl] lysophosphatidylcholine showed a very fast intracellular release of radioactive fatty acids, which indicates a potent lysophospholipase activity. Taken together, these data define the indispensable conditions for an experimental system suitable for further studies.

A dual cofactor-specific isocitrate dehydrogenase fromPythium ultimum

1996 ◽  
Vol 42 (12) ◽  
pp. 1241-1247 ◽  
Author(s):  
Hakryul Kim ◽  
Zahid Mozaffar ◽  
John D. Weete
Keyword(s):  
Fatty Acids ◽  
Ion Exchange ◽  
Molecular Mass ◽  
Isocitrate Dehydrogenase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Pythium Ultimum ◽  
Isocitric Dehydrogenase ◽  
Regulatory Enzymes ◽  
Optimum Ph

Isocitrate dehydrogenase is considered to be one of the key regulatory enzymes in the conversion of glucose into fatty acids by oleaginous microorganisms. A dual coenzyme-specific isocitrate dehydrogenase (EC 1.1.1.41) (IDH) was isolated from the primitive fungus Pythium ultimum and purified by 211-fold by sequential ion-exchange, affinity, and gel filtration chromatographies. Specific activity of the partially purified enzyme was 76.2 μmol/(min∙mg protein) with NAD+and 40% less active with NADP+. Optimum pH for activity was 8.5–9.5. Kmvalues for threo-D-isocitrate and NAD+were 0.031 and 0.55 mM, respectively. The estimated molecular mass of the IDH was 96 kDa under nondenaturing conditions and 48 kDa under denaturing conditions, suggesting that the enzyme is composed of two subunits of the same size. The enzyme was relatively stable up to 55 °C, but no activity was detected after exposure to 65 °C for 15 min. Mg2+or Mn2+were required for activity.Key words: isocitric dehydrogenase, Pythium ultimum, dual cofactor specific, oleaginicity.

Aspects of circadian periodic changes in phosphorus metabolism in mice

1964 ◽  
Vol 206 (3) ◽  
pp. 589-598 ◽  
Author(s):  
Walter Nelson
Keyword(s):  
Inorganic Phosphate ◽  
Time Course ◽  
Specific Activity ◽  
Kinetic Studies ◽  
Phospholipid Metabolism ◽  
Liver Phospholipid ◽  
Circadian Periodicity ◽  
Relative Specific Activity ◽  
Similar Explanation ◽  
Periodic Changes

Kinetic studies on the P32 content of mouse brain phosphorus fractions following the intraperitoneal injection of P32-labeled orthophosphate were performed during two selected segments of the 24-hr time scale. The results of these studies suggest that the circadian periodicity in relative specific activity of brain phospholipids is probably a consequence of a variation in the extent of P32 incorporation into brain inorganic phosphate and is not indicative of a periodicity in the rate of intermediary phospholipid metabolism. Data on the postinjection time course of the specific activities of plasma inorganic phosphate, liver inorganic phosphate, and liver phospholipid suggest a similar explanation for the circadian periodic changes in relative specific activity of liver phospholipids. A circadian rhythm of inorganic phosphate concentration in mouse plasma is demonstrated. Consideration of a mathematical model suggests that the observed within-day variation in P32 distribution and the circadian periodicity of phospholipid relative specific activity in liver and brain are a consequence of this periodicity in plasma phosphate content. A significant within-day variation in total phosphorus content of liver was observed. The disturbance of mice incident to P32 injection has a marked effect on the plasma content of inorganic phosphate and corticosterone.

INVESTIGATIONS INTO THE PRODUCTION OF LIPIDS BY SUBMERGED CULTURES OF THE MUSHROOM TRICHOLOMA NUDUM: II. STUDIES WITH UNIFORMLY LABELLED GLUCOSE

1962 ◽  
Vol 40 (7) ◽  
pp. 857-867 ◽  
Author(s):  
D. C. Leegwater ◽  
B. M. Craig
Keyword(s):  
Fatty Acids ◽  
Linoleic Acid ◽  
Specific Activity ◽  
Neutral Lipids ◽  
Basal Medium ◽  
Submerged Cultures ◽  
Intact Cells ◽  
Lipid Fractions ◽  
Time Specific ◽  
Low Nitrogen

Tricholoma nudum mycelium, grown in a medium with a low nitrogen content, was adapted to a basal medium containing glucose and incubated with glucose-U-C14for 25 hours. The time – specific activity relations of the major fatty acids produced were determined for both the neutral lipids and the phospholipids. The results confirm a previous finding that in the phospholipids of T. nudum linoleic acid becomes labelled under the conditions used at almost the same rate as palmitic, stearic, or oleic acid, but that in the neutral lipids the specific activity of this acid initially lags considerably behind those of the latter three. It is postulated that this phenomenon is due to a difference in the relative sizes of the linoleic acid pools involved in the synthesis of the two lipid fractions. Only part of the material used for the formation of the fatty acids is derived directly from the substrate, the remainder apparently being provided by material already present in the mycelium. The various results, together with those of earlier workers, are critically evaluated, and it is concluded that the order in which fatty acids are labelled in tracer studies with intact cells is determined to a large extent by the conditions of the experiment and does not necessarily provide information on possible product–precursor relations.

An International Collaborative Study to Investigate Standardisation of Hirudin Potency

1993 ◽  
Vol 69 (05) ◽  
pp. 430-435 ◽  
Author(s):  
Colin Longstaff ◽  
Man-Yu Wong ◽  
Patrick J Gaffney
Keyword(s):  
Specific Activity ◽  
Collaborative Study ◽  
Residual Activity ◽  
Quality Standard ◽  
Upper Limit ◽  
Assay Procedure ◽  
Specific Activities ◽  
International Collaborative ◽  
Range Of Values ◽  
International Collaborative Study

SummaryAn international collaborative study has been carried out to investigate the reproducibility of hirudin assays in 13 laboratories using four recombinant hirudins and one natural, sulphated product. A simple assay procedure was proposed involving the titration of α-thrombin with inhibitor and measurement of residual activity using a chromogenic substrate. A standard α-thrombin preparation was supplied to ensure that this reagent was of uniform quality throughout the study. The method appeared to present no difficulties and laboratories reported similar potencies for the 5 hirudin samples, in line with expected values. This gave 200–222 Thrombin Inhibitory Units/ampoule (TIU/ampoule) of lyophilised hirudin, with geometric coefficient of variation (gcv) values ranging from 10.15–15.97%. This corresponds to specific activities of approximately 14,300–15,900 TIU/mg protein. This is close to the upper limit of previously reported values of specific activity. We conclude that the precision of this determination compared with the wider range of values in the literature (8,000–16,000 thrombin inhibitory units [TIU]/mg) results from the use of good quality standard α-thrombin by all laboratories. This study has important implications for hirudin standardisation.

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