Enzymic Studies on Phosphatidic Acid Synthesis in Human Platelets

Abstract Particulate preparations of human platelets were capable of catalyzing acylation of sn-glycerol 3-phosphate by long-chain fatty acyl-CoA thioesters. The principal lipid product formed was identified as phosphatidic acid. The highest specific activity was found in the particulate fraction that sedimented between 12,000 g and 105,000 g. The reaction exhibited a broad pH optimum around 7.4-8.5. The apparent Michaelis constant for sn-glycerol 3-phosphate was 0.48 mM when 28.5 µM palmityl-CoA was used as acyl donor. Palmityl- and oleyl-CoA were better substrates than stearyl-, linoleyl-, and arachidonyl-CoA, as far as the maximal velocity was concerned. Particulate preparations of platelets from normal subjects catalyzed the incorporation of 0.271 ± 0.048 nmole of sn-glycerol 3-phosphate/min per mg of protein. The capacity of human platelets to acylate sn-glycerol 3-phosphate was approximately 40% that of human liver, as compared on the basis of the specific activity of the microsomal fraction. These results suggest that the glycerophosphate pathway makes an essential contribution to the de novo synthesis of phospholipids in human platelets.

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Thermostable glucose isomerase from psychrotolerant Streptomyces species

International Journal of Life Sciences ◽

10.3126/ijls.v1i0.2300 ◽

1970 ◽

Vol 1 ◽

pp. 6-10 ◽

Cited By ~ 3

Author(s):

Bidur Dhungel ◽

Manoj Subedi ◽

Kiran Babu Tiwari ◽

Upendra Thapa Shrestha ◽

Subarna Pokhrel ◽

...

Keyword(s):

Specific Activity ◽

Fold Increase ◽

Glucose Isomerase ◽

Enzyme Concentration ◽

Maximal Velocity ◽

Ph Optimum ◽

Streptomyces Spp ◽

Optimum Ph ◽

Optimum Reaction ◽

Sepharose 4B

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM<) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.DOI: 10.3126/ijls.v1i0.2300 Int J Life Sci 1 : 6-10

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Use of deuterium oxide to measure de novo fatty acid synthesis in normal subjects consuming diets of different fatty acid composition

Atherosclerosis ◽

10.1016/s0021-9150(99)80508-5 ◽

1999 ◽

Vol 144 ◽

pp. 130

Author(s):

S.D. Konrad ◽

S.L. Cook ◽

Y.K. Goh ◽

M.A. French ◽

M.T. Clandinin

Keyword(s):

Fatty Acid Composition ◽

Fatty Acid ◽

Acid Composition ◽

Fatty Acid Synthesis ◽

De Novo ◽

Deuterium Oxide ◽

Normal Subjects ◽

Acid Synthesis

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Purification and characterization of human platelet glutathione-S- transferase

Blood ◽

10.1182/blood.v67.6.1595.bloodjournal6761595 ◽

1986 ◽

Vol 67 (6) ◽

pp. 1595-1599

Author(s):

J Loscalzo ◽

J Freedman

Keyword(s):

Specific Activity ◽

Human Platelet ◽

Reduced Glutathione ◽

Human Platelets ◽

Glutathione S Transferase ◽

Purification And Characterization ◽

Ph Optimum ◽

Complete Purification ◽

High Concentrations

A glutathione-S-transferase was isolated and purified to homogeneity from human platelets. With a combination of ammonium sulfate fractionation and chromatographic methods, 0.2 mg of pure enzyme was obtained from 9 X 10(11) platelets with a 12% recovery. The purified enzyme had a specific activity of 7.5 U per milligram, representing an approximately 1,100-fold purification. The enzyme was found to be anionic, with an isoelectric point of 4.6. With reduced glutathione as a co-substrate, platelet glutathione-S-transferase was most active with the synthetic substrate, 1-chloro-2,4-dinitrobenzene, less active with 1,2-dichloro-4-nitrobenzene, and essentially inactive with nitroglycerin and 1,2-epoxy-3-(p-nitrophenoxy)-propane. The pH optimum for activity with glutathione and 1-chloro-2,4-dinitrobenzene was 7.0. Indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyindole-3-acetic acid), a chlorobenzene derivative, noncompetitively inhibited human platelet glutathione-S-transferase with an apparent KI of 0.23 mmol/L. This study represents the first complete purification and characterization of a glutathione-S-transferase from platelets. The presence of this enzyme in the platelet, within which high concentrations of reduced glutathione coexist, suggests the potential importance of the platelet in detoxification reactions and in the synthesis of the glutathione adducts of leukotriene metabolism.

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Triacylglycerol in the human erythrocyte: quantification and fatty acyl turnover in normal subjects and chronic alcoholics

Clinical Science ◽

10.1042/cs0800313 ◽

1991 ◽

Vol 80 (4) ◽

pp. 313-318 ◽

Cited By ~ 2

Author(s):

Brice Lerique ◽

Marguerite Gastaldi ◽

Jean Boyer

Keyword(s):

Human Erythrocyte ◽

Specific Activity ◽

Neutral Lipids ◽

Human Subjects ◽

Minor Component ◽

Normal Subjects ◽

Fatty Acyl ◽

Active Metabolites ◽

A Value ◽

Normal Human

1. Triacylglycerol in erythrocytes from normal human subjects was estimated to average 2.7 ± 0.7 nmol/1010 cells, equivalent to 0.07% of total lipids or 0.3% of neutral lipids. 2. The specific activity of triacylglycerol labelling attained by incubating intact erythrocytes with [3H]oleic acid was 10 nmol/μmol, a value 20-fold higher than that of the highest labelled phospholipid, sphingomyelin; as isolated by ultracentrifugation over a density gradient, the youngest erythrocytes exhibited a labelling rate 10-fold higher than that of older cells. 3. The triacylglycerol content was not modified in erythrocytes from chronic alcoholics, whereas the mean rate of triacylglycerol labelling was 31% (P <0.05) higher than that of control subjects, and did not normalize 4 weeks after alcohol withdrawal. 4. These results indicate that triacylglycerol, although a quantitatively minor component, is one of the most active metabolites in the lipid matrix of the human erythrocyte membrane and appears to be implicated in the membrane response to antagonistic agents.

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Prostacyclin inhibition of phosphatidic acid synthesis in human platelets is not mediated by protein kinase C

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(84)91410-4 ◽

1984 ◽

Vol 120 (1) ◽

pp. 37-44 ◽

Cited By ~ 19

Author(s):

Eduardo G. Lapetina

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phosphatidic Acid ◽

Acid Synthesis ◽

Human Platelets ◽

Kinase C

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Biosynthesis of phosphatidic acid by glycerophosphate acyltransferases in rat liver mitochondria and microsomes

Biochemistry and Cell Biology ◽

10.1139/o90-201 ◽

1990 ◽

Vol 68 (12) ◽

pp. 1380-1392 ◽

Cited By ~ 11

Author(s):

Amy Y. P. Mok ◽

William C. McMurray

Keyword(s):

Phosphatidic Acid ◽

Rat Liver ◽

Neutral Lipids ◽

Divalent Cations ◽

Liver Mitochondria ◽

Fatty Acyl ◽

Subcellular Fractions ◽

Rat Liver Mitochondria ◽

Acyl Donor ◽

Palmitoyl Carnitine

The acyltransferases that catalyze the synthesis of phosphatidic acid from labelled sn-[14C]glycero-3-phosphate and fatty acyl carnitine or coenzyme A derivatives have been shown to be present in both isolated mitochondria and microsomes from rat liver. The major reaction product was phosphatidic acid in both subcellular fractions. A small quantity of lysophosphatidic acid and neutral lipids were produced as by-products. Divalent cations had significant effects on both mitochondrial and microsomal fractions in stimulating acylation using palmitoyl CoA, but not when palmitoyl carnitine was used as the acyl donor. Palmitoyl CoA and palmitoyl carnitine could be used for acylation by both mitochondria and microsomes. Mitochondria were more permeable to palmitoyl carnitine and readily used it as the substrate for acylation. On the other hand, microsomes yielded a better rate with palmitoyl CoA and the rate of acylation from palmitoyl carnitine in microsomes was correlated with the degree of mitochondrial contamination. The enzymes were partially purified from Triton X-100 extracts of subcellular fractions. Based on the differences of substrate utilization, products formed, divalent cation effects, molecular weights, and polarity, the mitochondrial and microsomal acyltransferases appeared to be different enzymes.Key words: glycerophosphate, acyltransferase, mitochondria, microsomes, phosphatidic acid.

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Biogenesis of mitochondria. Phospholipid synthesis in vitro by yeast mitochondrial and microsomal fractions

Biochemical Journal ◽

10.1042/bj1440265 ◽

1974 ◽

Vol 144 (2) ◽

pp. 265-275 ◽

Cited By ~ 71

Author(s):

G S Cobon ◽

P D Crowfoot ◽

A W Linnane

Keyword(s):

Phosphatidic Acid ◽

Microsomal Fraction ◽

De Novo ◽

Specific Activity ◽

Neutral Lipids ◽

Mitochondrial Fraction ◽

Biogenesis Of Mitochondria ◽

Glycerolipid Synthesis ◽

Phosphatidylcholine Synthesis

The ability in vitro of yeast mitochondrial and microsomal fractions to synthesize lipid de novo was measured. The major phospholipids synthesized from sn-[2-3H]glycerol 3-phosphate by the two microsomal fractions were phosphatidylserine, phosphatidylinositol and phosphatidic acid. The mitochondrial fraction, which had a higher specific activity for total glycerolipid synthesis, synthesized phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid, together with smaller amounts of neutral lipids and diphosphatidylglycerol. Phosphatidylcholine synthesis from both S-adenosyl[Me-14C]methionine and CDP-[Me-14C]choline appeared to be localized in the microsomal fraction.

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