scholarly journals A trypsin-sensitive, heat-labile, N-ethylmaleimide-sensitive factor in adipocyte post-microsomal supernatant which affects the assay of adipocyte glycerol phosphate acyltransferase activities

1983 ◽  
Vol 214 (1) ◽  
pp. 247-255 ◽  
Author(s):  
M H Rider ◽  
E D Saggerson
Keyword(s):  
Trypsin Treatment ◽  
Glycerol Phosphate ◽  
Acyltransferase Activity ◽  
Isolated Mitochondria ◽  
Low Concentrations ◽  
Equivalent Quantity ◽  
Sensitive Factor ◽  
Labile N ◽  
Z Protein ◽  
Coa Hydrolase

Addition of adipocyte 100 000 g post-microsomal supernatant to assays of glycerol phosphate acyltransferase in isolated mitochondria or microsomal fractions decreased activity at lower concentrations of palmitoyl-CoA. At higher concentrations of palmitoyl-CoA, activation was observed on addition of post-microsomal supernatant. The effect of post-microsomal supernatant to decrease activity at lower [palmitoyl-CoA] was abolished by heating or by trypsin treatment, and was also abolished by addition of N-ethylmaleimide to assays or by pretreatment of post-microsomal supernatant with N-ethylmaleimide. The stimulatory effect seen at higher [palmitoyl-CoA] was not sensitive to heat or trypsin treatment. The effect of post-microsomal supernatant at lower [palmitoyl-CoA] cannot be attributed to palmitoyl-CoA hydrolase activity. It was found that brief treatment of adipocyte mitochondria with low concentrations of trypsin was an effective way to remove contaminating microsomal glycerol phosphate acyltransferase activity. Adipocyte post-microsomal supernatant was more effective than an equivalent quantity of liver post-microsomal supernatant protein in decreasing adipocyte microsomal glycerol phosphate acyltransferase activity. The effects of the supernatants from both tissues were decreased by flavaspidic acid. Semi-purified Z-protein fraction from rat liver did not mimic the effect of adipocyte post-microsomal supernatant to decrease glycerol phosphate acyltransferase at lower [palmitoyl-CoA]. Post-microsomal supernatants obtained from noradrenaline-treated adipocytes were less effective than those from control cells in decreasing glycerol phosphate acyltransferase activity in microsomal fractions at lower [palmitoyl-CoA]. It is suggested that adipocyte cytosol may contain an acyl-CoA-binding protein or proteins differing from Z-protein in some respects. The physiological significance of the findings is briefly discussed.

scholarly journals The relationship between palmitoyl-coenzyme A synthetase activity and esterification of sn-glycerol 3-phosphate by the microsomal fraction of guinea-pig intestinal mucosa

1973 ◽  
Vol 132 (4) ◽  
pp. 707-715 ◽  
Author(s):  
David N. Brindley
Keyword(s):  
Guinea Pig ◽  
Intestinal Mucosa ◽  
Specific Activity ◽  
Synthetase Activity ◽  
Glycerol Phosphate ◽  
Acyltransferase Activity ◽  
Low Concentrations ◽  
System 2 ◽  
The Mean ◽  
Glycerolipid Synthesis

1. With microsomal fractions of guinea-pig intestinal mucosa the mean specific activity of palmitoyl-CoA synthetase was approx. 1.3-fold the esterification of sn-glycerol 3-phosphate with palmitoyl-CoA generated by the endogenous synthetase. The latter activity was approx. 2.5- and 5-fold that when palmitoyl-CoA was generated from palmitoylcarnitine or when it was added directly to the assay system. 2. There were significant correlations (P<0.001) between the specific activities of palmitoyl-CoA synthetase and glycerolipid synthesis from either palmitate or palmitoylcarnitine. 3. The mean molar composition of glycerolipid synthesized from palmitate or palmitoylcarnitine was approx. 18% lysophosphatidate, 75% phosphatidate and 7% neutral lipid. 4. Glycerolipid synthesis from palmitate was inhibited by 80–90% after preincubation of microsomal fractions at 37°C for 40min and was caused by inactivation of palmitoyl-CoA synthetase. 5. Addition of 100–400mm-KCl inhibited palmitoyl-CoA synthetase activity and glycerolipid synthesis from palmitate but stimulated glycerol phosphate acyltransferase activity. 6. Diversion of palmitoyl-CoA synthesized by the endogenous synthetase to palmitoylcarnitine resulted in an almost stoicheiometric decrease in glycerolipid synthesis. 7. Addition of rac-1-monopalmitin promoted utilization of palmitoyl-CoA by the monoglyceride pathway but did not inhibit phosphatidate biosynthesis. 8. With rate-limiting concentrations of CoA and Mg2+ the relative decreases in velocity for palmitoyl-CoA synthetase and glycerolipid synthesis from palmitate were almost identical. However, low concentrations of palmitate and ATP produced greater decreases in synthetase activity than in glycerolipid synthesis. 9. There appears to be a fine balance between the activities of palmitoyl-CoA synthetase and glycerol phosphate acyltransferase, with neither activity being in excess with respect to phosphatidate synthesis.

Cell surface lipids and adhesion. IV. The effects of trypsin on lipid turnover by the plasmalemma

1979 ◽  
Vol 38 (1) ◽  
pp. 283-292
Author(s):  
A.S. Curtis ◽  
O. Hill
Keyword(s):  
Free Energy ◽  
Enzyme Treatment ◽  
Embryonic Chick ◽  
Trypsin Treatment ◽  
Acyltransferase Activity ◽  
Intact Cells ◽  
Coa Ligase ◽  
Surface Lipids ◽  
Lipid Turnover ◽  
Phospholipase A2 Activity

Trypsin treatment of intact cells or isolated plasmalemmae from embryonic chick neural retinae leads to an accumulation of lysophospholipids in the plasmalemmae. Trypsin was used at activities commonly used in cell disaggregation techniques. This accumulation appears to result from the decrease in acyltransferase activity in the plasmalemma produced by enzyme treatment. Plasmalemmal CoA ligase activity is not affected by trypsin treatment. Trypsinization has little effect on plasmalemmal phospholipase A2 activity. These results are discussed in relation to (a) the effects of trypsinization on cell adhesion, and (b) the theory that cells cannot adhere to lecithins because of their fluidity or surface-free-energy values. We propose that the effects of trypsinization on adhesion may in large part be due to the effects on other plasmalemmal proteins. Similarly the inability of cells to adhere to lecithin substrates is simply explained as being due to the lysolecithin that contacting cells release from these substrates.

Acquired Hypolipoproteinemia

1992 ◽  
Vol 38 (5) ◽  
pp. 776-781 ◽  
Author(s):  
M De Buyzere ◽  
J Delanghe ◽  
C Labeur ◽  
L Noens ◽  
Y Benoit ◽  
...  
Keyword(s):  
Density Lipoprotein ◽  
Hdl Cholesterol ◽  
Acyltransferase Activity ◽  
Apo B ◽  
Familial Predisposition ◽  
Low Concentrations ◽  
Polyclonal Hypergammaglobulinemia ◽  
Severe Infections ◽  
Hematological Abnormalities

Abstract We present a six-year follow-up of a boy with a novel type of hypolipoproteinemia, with clinical and biochemical features distinct from classical hypoalphalipoproteinemias. There were abnormally low concentrations of total and high-density lipoprotein (HDL) cholesterol, apolipoprotein (apo) B, apo A-I, and apo A-II, and the phospholipids were decreased. The most striking abnormality was an extra fraction containing mainly phospholipids and apo A-I in the HDL3 subfraction. This fraction is reminiscent of concentric 20- to 50-nm-diameter lamellar phospholipid liposomes. Plasma lecithin:cholesterol acyltransferase activity was strongly decreased. We noted a persisting polyclonal hypergammaglobulinemia, hematological abnormalities (hemolytic anemia and thrombocytopenia), and a progressive splenomegaly. After the five-year follow-up, the patient had recurrent severe infections; moderate hematuria and proteinuria developed gradually. Treatment with corticosteroids and immunoglobulins improved thrombocytopenia and hypolipoproteinemia. These clinical and biochemical findings differ from those in the known primary and secondary hypo-alpha-lipoproteinemia syndromes. Although investigation of the relatives suggests a familial predisposition for hypo-alpha-lipoproteinemia, the subject's condition can be regarded as acquired.

scholarly journals Interactions of insulin, glucagon and dexamethasone in controlling the activity of glycerol phosphate acyltransferase and the activity and subcellular distribution of phosphatidate phosphohydrolase in cultured rat hepatocytes

1985 ◽  
Vol 230 (2) ◽  
pp. 525-534 ◽  
Author(s):  
R A Pittner ◽  
R Fears ◽  
D N Brindley
Keyword(s):  
Neutral Lipids ◽  
Relative Proportion ◽  
Rat Hepatocytes ◽  
Glycerol Phosphate ◽  
Acyltransferase Activity ◽  
Phosphatidate Phosphohydrolase ◽  
Cultured Rat Hepatocytes ◽  
Total Glycerol

Rat hepatocytes were incubated in monolayer culture for 8 h. Glucagon (10nM) increased the total phosphatidate phosphohydrolase activity by 1.7-fold. This effect was abolished by adding cycloheximide, actinomycin D or 500 pM-insulin to the incubations. The glucagon-induced increase was synergistic with that produced by an optimum concentration of 100 nM-dexamethasone. Theophylline (1mM) potentiated the effect of glucagon, but it did not affect the dexamethasone-induced increase in the phosphohydrolase activity. The relative proportion of the phosphohydrolase activity associated with membranes was decreased by glucagon when 0.15 mM-oleate was added 15 min before the end of the incubations to translocate the phosphohydrolase from the cytosol. This glucagon effect was not seen at 0.5 mM-oleate. Since glucagon also increased the total phosphohydrolase activity, the membrane-associated activity was maintained at 0.15 mM-oleate and was increased at 0.5 mM-oleate. This activity at both oleate concentrations was also increased in incubations that contained dexamethasone, particularly in the presence of glucagon. Insulin increased the relative proportion of phosphatidate phosphohydrolase that was associated with membranes at 0.15 mM-oleate, but not at 0.5 mM-oleate. It also decreased the absolute phosphohydrolase activity on the membranes at both oleate concentrations in incubations that also contained glucagon and dexamethasone. None of the hormonal combinations significantly altered the total glycerol phosphate acyltransferase activity. However, glucagon significantly increased the microsomal activities, and insulin had the opposite effect. Glucagon also decreased the mitochondrial acyltransferase activity. There was a highly significant correlation between the total phosphatidate phosphohydrolase activity and the synthesis of neutral lipids from glycerol phosphate and 0.5 mM-oleate in homogenates of cells from all of the hormonal combinations. Phosphatidate phosphohydrolase activity is increased in the long term by glucocorticoids and also by glucagon through cyclic AMP. In the short term, glucagon increases the concentration of fatty acid required to translocate the cytosolic reservoir of activity to the membranes on which phosphatidate is synthesized. Insulin opposes the combined actions of glucagon and glucocorticoids. The long-term events explain the large increases in the phosphohydrolase activity that occur in vivo in a variety of stress conditions. The expression of this activity depends on increases in the net availability of fatty acids and their CoA esters in the liver.

scholarly journals The control of tricarboxylate-cycle oxidations in blowfly flight muscle. The steady-state concentrations of coenzyme A, acetyl-coenzyme A and succinyl-coenzyme A in flight muscle and isolated mitochondria

1974 ◽  
Vol 142 (3) ◽  
pp. 509-519 ◽  
Author(s):  
Richard G. Hansford
Keyword(s):  
Coenzyme A ◽  
Flight Muscle ◽  
Citrate Synthase ◽  
Phormia Regina ◽  
Primary Control ◽  
Glycerol Phosphate ◽  
Acetyl Coa ◽  
Isolated Mitochondria ◽  
Tricarboxylate Cycle

(1) A ‘cycling’ method involving citrate synthase (EC 4.1.3.7) and malate dehydrogenase (EC 1.1.1.37) was modified by the inclusion of succinyl-CoA synthetase (EC 6.2.1.5) and hexokinase (EC 2.7.1.1) to permit the determination of very small amounts of succinyl-CoA in addition to CoA and acetyl-CoA. (2) Application of this technique to blowfly (Phormia regina) flight-muscle extracts reveals no change in acetyl-CoA concentration, a slight fall in CoA concentration and a rise in succinyl-CoA concentration during flight. (3) Extraction of isolated mitochondria during controlled (state 4) pyruvate oxidation reveals essentially only acetyl-CoA. Activation of respiration by ADP (state 3) or uncoupling agents leads to a fall in acetyl-CoA and a rise in CoA and succinyl-CoA content. (4) The presence of glycerol phosphate in addition to pyruvate results in a lower acetyl-CoA content in state 4. (5) It is contended that these results are consistent with a primary control of one of the reactions of the tricarboxylate cycle, rather than of pyruvate dehydrogenase, during the state 4 oxidation of pyruvate by isolated mitochondria, and that the modulation of citrate synthase activity by the ratio of acetyl-CoA/succinyl-CoA is unimportant under these conditions.

scholarly journals Effects of chronic modification of dietary fat and carbohydrate in rats. The activities of some enzymes of hepatic glycerolipid synthesis and the effects of corticotropin injection

1981 ◽  
Vol 200 (2) ◽  
pp. 265-273 ◽  
Author(s):  
N Lawson ◽  
R J Jennings ◽  
A D Pollard ◽  
R G Sturton ◽  
S J Ralph ◽  
...  
Keyword(s):  
Beef Tallow ◽  
Corn Oil ◽  
Urate Oxidase ◽  
Dihydroxyacetone Phosphate ◽  
High Fat ◽  
Glycerol Phosphate ◽  
Acyltransferase Activity ◽  
High Fat Diets ◽  
Fat Diets ◽  
Starch Diet

1. Rats were fed on diets enriched with starch, sucrose, corn oil or beef tallow for 3 weeks and the activities of various enzymes in the liver were measured. 2. The mitochondrial glycerol phosphate acyltransferase activity was lower in rats fed on the starch diet than on the two high-fat diets. 3. The non-microsomal (presumably peroxisomal) dihydroxyacetone phosphate acyltransferase activity was higher in rats fed on the starch diet and corn-oil diets than in those fed on the sucrose and beef-tallow diets. Urate oxidase activity was higher in rats fed on the starch diet than in the three other groups. There were no significant differences in the activity of acyl-CoA oxidase among the groups. 4. The activity of soluble phosphatidate phosphohydrolase was not significantly different among the dietary groups. There were increases of 3.3--4.3-fold in this activity in the dietary groups 6h after injection of corticotropin. The equivalent increases for the mitochondrial glycerol phosphate acyltransferase were 1.4--1.6 fold. 5. The corticosterone responses to the corticotropin injection were not significantly different between dietary groups. However, the corticosterone response of the rats fed on the two high-fat diets was prolonged when the rats were given an acute load of fructose [Brindley, Cooling, Glenny, Burditt & McKechnie (1981) Biochem. J. 200. 275--283]. 6. Rats fed on the high-fat diets had higher concentrations of circulating cholesterol than those fed on the starch and sucrose diets. Serum triacylglycerol concentrations were lower in the rats fed on the starch diet than in the three other groups. 7. The results are discussed in terms of the relationship between diet, hormonal balance and hepatic glycerolipid metabolism.

scholarly journals Some properties of pyruvate and 2-oxoglutarate oxidation by blowfly flight-muscle mitochondria

1972 ◽  
Vol 127 (1) ◽  
pp. 271-283 ◽  
Author(s):  
R. G. Hansford
Keyword(s):  
Oxygen Uptake ◽  
Isocitrate Dehydrogenase ◽  
Adenine Nucleotides ◽  
Slight Reduction ◽  
Glycerol Phosphate ◽  
Pyruvate Oxidation ◽  
Low Concentrations ◽  
High Concentrations ◽  
Kinetics Of ◽  
Very High

1. High rates of state 3 pyruvate oxidation are dependent on high concentrations of inorganic phosphate and a predominance of ADP in the intramitochondrial pool of adenine nucleotides. The latter requirement is most marked at alkaline pH values, where ATP is profoundly inhibitory. 2. Addition of CaCl2 during state 4, state 3 (Chance & Williams, 1955) or uncoupled pyruvate oxidation causes a marked inhibition in the rate of oxygen uptake when low concentrations of mitochondria are employed, but may lead to an enhancement of state 4 oxygen uptake when very high concentrations of mitochondria are used. 3. These properties are consistent with the kinetics of the NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) from this tissue, which is activated by isocitrate, citrate, ADP, phosphate and H+ ions, and inhibited by ATP, NADH and Ca2+. 4. Studies of the redox state of NAD and cytochrome c show that addition of ADP during pyruvate oxidation causes a slight reduction, whereas addition during glycerol phosphate oxidation causes a `classical' oxidation. Nevertheless, it is concluded that pyruvate oxidation is probably limited by the respiratory chain in state 4 and by the NAD-linked isocitrate dehydrogenase in state 3. 5. The oxidation of 2-oxoglutarate by swollen mitochondria is also stimulated by high concentrations of ADP and phosphate, and is not uncoupled by arsenate.

scholarly journals Regulation by noradrenaline of the mitochondrial and microsomal forms of glycerol phosphate acyltransferase in rat adipocytes

1983 ◽  
Vol 214 (1) ◽  
pp. 235-246 ◽  
Author(s):  
M H Rider ◽  
E D Saggerson
Keyword(s):  
Microsomal Protein ◽  
Subcellular Fractionation ◽  
Covalent Modification ◽  
Mitochondrial Activity ◽  
Glycerol Phosphate ◽  
Acyltransferase Activity ◽  
Rat Adipocytes ◽  
Wide Range ◽  
Dependent Protein ◽  
Protein Kinase Catalytic Subunits

Incubation of rat adipocytes with 1 microM-noradrenaline caused a decrease in both the N-ethylmaleimide-sensitive (microsomal) and N-ethylmaleimide-insensitive (mitochondrial) glycerol phosphate acyltransferase activities measured in homogenates from freeze-stopped cells. The effects of noradrenaline on glycerol phosphate acyltransferase activity were apparent over a wide range of concentrations of glycerol phosphate and palmitoyl-CoA. The effect of noradrenaline was reversed within cells by the subsequent addition of insulin or propranolol. Inclusion of albumin in homogenization buffers abolished the effect of noradrenaline on the N-ethylmaleimide-sensitive activity. The effect of noradrenaline on the N-ethylmaleimide-insensitive (mitochondrial) activity was, however, not abolished by inclusion of albumin in buffers for preparation of homogenates from freeze-stopped cells. Inclusion of fluoride in homogenization buffers did not alter the observed effect of noradrenaline. The inactivating effect of noradrenaline persisted through the subcellular fractionation procedures used to isolate adipocyte microsomes (microsomal fractions). The effect of noradrenaline on mitochondrial glycerol phosphate acyltransferase did not persist through subcellular fractionation. Noradrenaline treatment of cells significantly decreased the Vmax. of glycerol phosphate acyltransferase in isolated microsomes without changing the activity of NADPH-cytochrome c reductase. Glycerol phosphate acyltransferase activity in microsomes from noradrenaline-treated cells is unstable, being rapidly lost on incubation at 30 degrees C. Bivalent metal ions (Mg2+, Ca2+) or post-microsomal supernatant protected against this inactivation. Glycerol phosphate acyltransferase activity in microsomes from noradrenaline-treated cells could not be re-activated by incubation with either alkaline phosphatase or phosphoprotein phosphatase-1. Addition of cyclic AMP-dependent protein kinase catalytic subunits to adipocyte microsomes incubated with [gamma-32P]ATP considerably increased the incorporation of 32P into microsomal protein, but did not cause inactivation of glycerol phosphate acyltransferase. These findings provide no support for the proposal that inactivation of adipocyte microsomal glycerol phosphate acyltransferase by noradrenaline is through a phosphorylation type of covalent modification.

Biosynthesis of Phosphatidic Acid in Mitochondria and Microsomes Via the Acylation of sn-Glycero-3-phosphate

1972 ◽  
Vol 50 (8) ◽  
pp. 936-948 ◽  
Author(s):  
J. B. Davidson ◽  
N. Z. Stanacev
Keyword(s):  
Phosphatidic Acid ◽  
Subcellular Fraction ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Marker Enzymes ◽  
Acyltransferase Activity ◽  
Isolated Mitochondria ◽  
Direct Acylation ◽  
Submitochondrial Fractions ◽  
Nadph Cytochrome C Reductase

Nuclei-free homogenate, prepared from guinea pig livers, was fractionated into subcellular particles which were then examined for the activities of two microsomal marker enzymes, glucose-6-phosphatase and NADPH: cytochrome c reductase. In an incubation system containing sn-glycero-3-phosphate, fatty acid, and various cofactors the intracellular distribution of acyl-CoA: sn-glycero-3-phosphate acyltransferase(s) was studied and compared with the distribution of the two microsomal marker enzymes.Results obtained showed that the highest specific activity for the acylation of sn-glycero-3-phosphate was associated with the microsomal fraction and the activity in each subcellular fraction paralleled activities of the two microsomal marker enzymes. Furthermore, the amount of acyl-CoA: sn-glycero-3-phosphate acyltransferase activity observed in the mitochondrial and submitochondrial fractions could be accounted for by the content of endoplasmic reticulum as determined by the marker enzymes. This observation was also true for brain, heart, and kidney, as well as for rat liver.These results are interpreted as evidence that isolated mitochondria are unable to synthesize phosphatidic acid by direct acylation of sn-glycero-3-phosphate.

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