Enhancement of microsomal phosphatidate phosphohydrolase and diacylglycerol acyltransferase activity by insulin during growth of rat adipocyte precursors in culture

1979 ◽  
Vol 57 (6) ◽  
pp. 573-577 ◽  
Author(s):  
Daniel A. K. Roncari ◽  
Esther Y. W. Mack ◽  
Dominic K. Yip
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Diacylglycerol Acyltransferase ◽  
Culture Conditions ◽  
Acyltransferase Activity ◽  
Intact Cells ◽  
Triacylglycerol Synthesis ◽  
Phosphatidate Phosphohydrolase ◽  
Morphological Maturation ◽  
Adipocyte Precursor

The availability of a propagating adipocyte precursor culture system has provided the opportunity to study biochemical processes under conditions in which any known interacting influences are controlled. We have studied the activity of various triacylglycerol-biosynthetic enzymes during maturation of rat epididymal adipocyte precursors and any possible effect of insulin on enzyme activity. At certain times in culture, the specific activity of microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) and diacylglycerol acyltransferase (EC 2.3.1.20) is significantly enhanced in cells grown in the presence of added insulin. Under the culture conditions used in this study, the adipocyte precursors acquire several small lipid inclusions and become rounder, but do not assume the signet-ring appearance of mature fat cells during the first 2 weeks of monolayer confluence. Consequently, the effects of the hormone on enzyme activity become evident prior to complete morphological maturation. Phosphatidate phosphohydrolase is believed to be a rate-controlling enzyme in triacylglycerol synthesis in adipose tissue and liver. The fact that the adipocyte precursor microsomal, rather than cytosolic, phosphohydrolase is influenced by insulin suggests that the membrane-bound enzyme is the regulatory phosphohydrolase in intact cells. The enhancement of diacylglycerol acyltransferase activity may be of significance in the reesterification of fatty acids with diacylglycerols, a reaction that by passes the phosphohydrolase step. Thus, in addition to the well-known mechanisms by which insulin promotes triacylglycerol accretion in adipocytes and their precursors, the hormone significantly enhances the specific activity of critical enzymes of triacylglycerol synthesis.

Purification of liver cytosolic proteins that stimulate triacylglycerol synthesis

1981 ◽  
Vol 59 (11-12) ◽  
pp. 944-950 ◽  
Author(s):  
Daniel A. K. Roncari ◽  
Esther Y. W. Mack
Keyword(s):  
Molecular Weight ◽  
Adipose Tissue ◽  
Diacylglycerol Acyltransferase ◽  
Microsomal Enzymes ◽  
Acyltransferase Activity ◽  
Cytosolic Proteins ◽  
Heat Stable ◽  
Triacylglycerol Synthesis ◽  
Phosphatidate Phosphohydrolase ◽  
Liver Microsomal Enzymes

Heat-stable rat liver cytosolic proteins that stimulate triacylglycerol synthesis catalyzed by adipose tissue or liver microsomal enzymes have been isolated in a highly purified state. Their molecular weight ranges were found to be 35 000 – 45 000, 20 000 – 28 000, and 8000 – 12 000. The protein with molecular weight 20 000 – 28 000 and pI 7.3–7.7 was purified 4716-fold. The cytosolic proteins were stable at 85 °C for 15 min and were inactivated by proteases. They did not reveal any intrinsic phosphatidate phosphohydrolase or diacylglycerol acyltransferase activity, but led to enhanced conversion of phosphatidate to diacylgiycerol and triacylglycerol.

scholarly journals Studies on the production of glucose isomerase by Bacillus licheniformis

2015 ◽  
Vol 17 (3) ◽  
pp. 84-88 ◽  
Author(s):  
Ogbonnaya Nwokoro
Keyword(s):  
Enzyme Activity ◽  
Bacillus Licheniformis ◽  
Organic Nitrogen ◽  
Specific Activity ◽  
Inorganic Nitrogen ◽  
Glucose Isomerase ◽  
Culture Conditions ◽  
Enzyme Productivity ◽  
Carbon Substrates ◽  
Optimum Ph

Abstract This work reports the effects of some culture conditions on the production of glucose isomerase by Bacillus licheniformis. The bacterium was selected based on the release of 3.62 mg/mL fructose from the fermentation of glucose. Enzyme was produced using a variety of carbon substrates but the highest enzyme activity was detected in a medium containing 0.5% xylose and 1% glycerol (specific activity = 6.88 U/mg protein). Media containing only xylose or glucose gave lower enzyme productivies (specific activities= 4.60 and 2.35 U/mg protein respectively). The effects of nitrogen substrates on glucose isomerase production showed that yeast extract supported maximum enzyme activity (specific activity = 5.24 U/mg protein). Lowest enzyme activity was observed with sodium trioxonitrate (specific activity = 2.44 U/mg protein). In general, organic nitrogen substrates supported higher enzyme productivity than inorganic nitrogen substrates. Best enzyme activity was observed in the presence of Mg2+ (specific activity = 6.85 U/mg protein) while Hg2+ was inhibitory (specific activity = 1.02 U/mg protein). The optimum pH for best enzyme activity was 6.0 while optimum temperature for enzyme production was 50ºC.

scholarly journals Effects of streptozotocin-diabetes and insulin administration in vivo or in vitro on the activities of five enzymes in the adipose-tissue triacylglycerol-synthesis pathway

1987 ◽  
Vol 243 (1) ◽  
pp. 289-292 ◽  
Author(s):  
E D Saggerson ◽  
C A Carpenter
Keyword(s):  
Insulin Treatment ◽  
Streptozotocin Diabetes ◽  
Diacylglycerol Acyltransferase ◽  
Fatty Acyl ◽  
Diabetic Rats ◽  
Insulin Administration ◽  
Triacylglycerol Synthesis ◽  
Phosphatidate Phosphohydrolase

At 2 days after administration of streptozotocin (100 mg/kg), activities in rat epididymal fat-pads of the following enzymes were significantly decreased: fatty acyl-CoA synthetase (FAS), mitochondrial and microsomal forms of glycerolphosphate acyltransferase (GPAT), monoacylglycerolphosphate acyltransferase (MGPAT) and Mg2+-dependent phosphatidate phosphohydrolase (PPH). There were no significant changes in diacylglycerol acyltransferase or Mg2+-independent PPH. Insulin administration to diabetic rats over 2 days restored activities of FAS, both forms of GPAT, MGPAT and Mg2+-dependent PPH. Significant restoration of all five activities was also seen 2 h after a single administration of insulin, but was not observed 45 min after insulin treatment. Insulin significantly increased all five enzyme activities when adipocytes from diabetic rats were incubated for 2 h with a mixture of glucose, lactate, pyruvate and amino acids.

Diacylglycerol acyltransferase activity and triacylglycerol synthesis in germinating castor seed cotyledons

Lipids ◽  
2006 ◽  
Vol 41 (3) ◽  
pp. 281-285 ◽  
Author(s):  
Xiaohua He ◽  
Grace Q. Chen ◽  
Jiann-Tsyh Lin ◽  
Thomas A. McKeon
Keyword(s):  
Diacylglycerol Acyltransferase ◽  
Castor Seed ◽  
Acyltransferase Activity ◽  
Triacylglycerol Synthesis

β-Lactamase activity of Escherichia intermedia

1968 ◽  
Vol 14 (4) ◽  
pp. 349-354 ◽  
Author(s):  
F. M. Huber ◽  
P. G. Caltrider ◽  
Susanne Noble
Keyword(s):  
Marked Effect ◽  
Specific Activity ◽  
Culture Conditions ◽  
Physiological Age ◽  
Cephalosporin C ◽  
Intact Cells ◽  
A Cell ◽  
Rate Of Hydrolysis ◽  
Gram Negative Organisms ◽  
Derivatives Of

Escherichia intermedia was found to produce a β-lactamase that hydrolyzed several penicillin and cephalosporin C derivatives. Culture conditions affecting production of the enzyme were studied. The enzyme was non-inducible and cell-bound. Growth temperature, medium, and physiological age had a marked effect on production of the enzyme. Properties of the enzyme were studied with intact cells and with a cell-free preparation. Although the β-lactamase of E. intermedia was different in some respects to that produced by gram-positive organisms, it was found to have properties similar to the β-lactamases of gram-negative organisms. 6-Aminopenicillanic acid was cleaved by the enzyme, but 7-aminocephalosporanic acid was resistant to cleavage. The rate of hydrolysis by intact cells and the purified preparation was influenced by the type of N-acyl sidechain of 7-aminocephalosporanic acid and 6-aminopenicillanic acid. The desacetoxy derivatives of cephalosporin C and cephalothin were hydrolyzed less rapidly than the parent compounds. The specific activity of the intact cells on cephalonium and methicillin was substantially less than that of the purified preparation.

scholarly journals The effect of dietary carbohydrate and fat on the activities of some enzymes responsible for glycerolipid synthesis in rat liver

1978 ◽  
Vol 174 (2) ◽  
pp. 535-541 ◽  
Author(s):  
H P Glenny ◽  
M Bowley ◽  
S L Burditt ◽  
J Cooling ◽  
P H Pritchard ◽  
...  
Keyword(s):  
Corn Oil ◽  
Specific Activity ◽  
Male Rats ◽  
Dihydroxyacetone Phosphate ◽  
Triacylglycerol Synthesis ◽  
Choline Phosphotransferase ◽  
Phosphatidate Phosphohydrolase ◽  
Glycerolipid Synthesis ◽  
Starch Diet

1. Male rats were fed for 14 days on diets containing (by wt.) 53% of starch, or on diets in which 20% of the starch was replaced by sucrose, corn oil or lard. 2. The hepatic activities of the microsomal glycerol phosphate acyltransferase, dihydroxyacetone phosphate acyltransferase, phosphatidate cytidylyltransferase, diacylglycerol acyltransferase and choline phosphotransferase, and of the soluble phosphatidate phosphohydrolase, were measured. 3. The soluble phosphatidate phosphohydrolase activity was higher in those rats fed on lard than in those fed on the starch diet. Choline phosphotransferase activity was higher in the rats fed on corn oil than in those fed on the starch diet. 4. The rate of hepatic glycerolipid synthesis was measured in vivo 1 min after injection of [1,3-3H]glycerol and [1-14C]palmitate into the portal veins. 5. The relative rate of phosphatidylcholine synthesis in vivo was increased after feeding with corn oil and the higher specific activity of choline phosphotransferase may contribute to this result. The equivalent rate of triacylglycerol synthesis was increased by feeding with lard rather than corn oil, and the increased activity of phosphatidate phosphohydrolase may partly explain this. The latter changes probably contribute to the increased concentration of triacylglycerol which other authors have observed in the livers and sera of animals fed on saturated and monounsaturated fats.

scholarly journals The activities of lipoprotein lipase and of enzymes involved in triacylglycerol synthesis in rat adipose tissue. Effects of starvation, dietary modification and of corticotropin injection

1981 ◽  
Vol 200 (2) ◽  
pp. 285-294 ◽  
Author(s):  
N Lawson ◽  
A D Pollard ◽  
R J Jennings ◽  
M I Gurr ◽  
D N Brindley
Keyword(s):  
Adipose Tissue ◽  
Lipoprotein Lipase ◽  
Corn Oil ◽  
Diacylglycerol Acyltransferase ◽  
Dietary Modification ◽  
Dihydroxyacetone Phosphate ◽  
Glycerol Phosphate ◽  
Triacylglycerol Synthesis ◽  
Phosphatidate Phosphohydrolase ◽  
Starch Diet

1. The effects of dietary modification, including starvation, and of corticotropin injection on the activities of acyl-CoA synthetase, glycerol phosphate acyltransferase, dihydroxyacetone phosphate acyltransferase, phosphatidate phosphohydrolase, diacylglycerol acyltransferase and lipoprotein lipase were measured in adipose tissue. 2. Lipoprotein lipase activities in heart were increased and those in adipose tissue were decreased when rats were fed on diets enriched with corn oil or beef tallow rather than with sucrose or starch. The lipoprotein lipase activity was lower in the adipose tissue of rats fed on the sucrose rather than on the starch diet. 3. Rats fed on the beef tallow diet had slightly higher activities of the total glycerol phosphate acyltransferase in adipose tissue than did rats fed on the sucrose or starch diet. The diacylglycerol acyltransferase and the mitochondrial glycerol phosphate acyltransferase activities were higher for the rats fed on the tallow diet than for those fed on the corn-oil diet. 4. Starvation significantly decreased the activities of lipoprotein lipase (after 24 and 48 h), acyl-CoA synthetase (after 24 h) and of the mitochondrial glycerol phosphate acyltransferase and the N-ethylmaleimide-insensitive dihydroxyacetone phosphate acyltransferase (after 48 h) in adipose tissue. The activities of the microsomal glycerol phosphate acyltransferase, diacylglycerol acyltransferase and the soluble phosphatidate phosphohydrolase were not significantly changed after 24 or 48 h of starvation. 5. The activities of lipoprotein lipase and phosphatidate phosphohydrolase in adipose tissue were decreased 15 min after corticotropin was injected into rats during November to December. No statistically significant differences were found when these experiments were performed during March to September. These differences may be related to the seasonal variation in acute lipolytic responses. 6. These results are discussed in relation to the control of triacylglycerol synthesis and lipoprotein metabolism.

scholarly journals Solubilization and characterization of diacylglycerol acyltransferase from microspore-derived cultures of oilseed rape

1994 ◽  
Vol 304 (3) ◽  
pp. 951-958 ◽  
Author(s):  
D Little ◽  
R Weselake ◽  
K Pomeroy ◽  
T Furukawa-Stoffer ◽  
J Bagu
Keyword(s):  
Enzyme Activity ◽  
Suspension Culture ◽  
Cell Suspension ◽  
Oilseed Rape ◽  
Cell Suspension Culture ◽  
Solution Structure ◽  
Specific Activity ◽  
Diacylglycerol Acyltransferase ◽  
Dgat Activity ◽  
Protein Ratio

Particulate fractions prepared from microspore-derived (MD) embryos of oilseed rape (Brassica napus L. cv. Reston) and an embryogenic MD cell suspension culture of oilseed rape (B. napus L. cv. Jet Neuf) were used as a source of diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) for enzyme characterization and development of a solubilization procedure. DGAT activity in the 1500-100,000 g fraction from MD embryos was stimulated 4-5-fold by 3 to 4 mg of BSA/ml of reaction mixture. DGAT activity from MD embryos was stimulated 2-3-fold by fluoride salts and 1.4-fold by NaCl, whereas iodide salts caused substantial inhibition of enzyme activity. The effect of the various 1:1 electrolytes on enzyme activity appeared to be related more to their differential effects on solution structure rather than ionic strength. DGAT was solubilized from membranes of MD embryos and the cell suspension culture by about 80 and 50% respectively, using 2 M NaCl in 1% (w/v) octanoyl-N-methyl-glucamide (MEGA-8) (pH 8.0 buffer) at a detergent to protein ratio of 2:1. The specific activity of solubilized DGAT was about 2-fold greater than that of the particulate enzyme. The mechanism of solubilization appeared to be related to the lowering of the critical micellar concentration of MEGA-8 in the presence of NaCl. DGAT, solubilized from MD embryos, eluted with an M(r) of about 2 x 10(6) during gel-filtration chromatography on a Superose 6 column equilibrated in buffer containing 0.1% (w/v) MEGA-8. The solubilized enzyme exhibited optimal activity at pH 7. At concentrations above 2 microM acyl-CoA, the specificity of solubilized DGAT for oleoyl-CoA and palmitoyl-CoA was considerably greater than for stearoyl-CoA.

Triacylglycerol synthesis and diacylglycerol acyltransferase activity during skeletal myogenesis

1990 ◽  
Vol 68 (12) ◽  
pp. 1393-1401 ◽  
Author(s):  
Victor S. Sauro ◽  
Kenneth P. Strickland
Keyword(s):  
Fatty Acid ◽  
Unsaturated Fatty Acids ◽  
Diacylglycerol Acyltransferase ◽  
Skeletal Myogenesis ◽  
Acyltransferase Activity ◽  
Triacylglycerol Synthesis ◽  
Tag Accumulation ◽  
Long Chain

The role that diacylglycerol acyltransferase (DAGAT) may play in the switch in lipid metabolism from predominantly triacylglycerol- and phospholipid-synthesizing myoblasts to predominantly phospholipid-synthesizing myotubes has been studied during L6 skeletal myogenesis. Fatty acid induced triacylglycerol (TAG) accumulation in vivo was found to be optimal with long-chain, unsaturated fatty acids. The fatty acid induced TAG accumulation was significantly greater in myoblasts than that in myotubes. DAGAT activity in vitro was found to be associated with the particulate (membrane) fraction only. The inhibition by many thiol-specific reagents (N-ethylmaleimide, p-chloromercuribenzoate, iodoacetate, 5,5′-dithiobis(2-nitrobenzoic acid)) suggest that a thiol group is at or near the active site. In general, optimal DAGAT activity in vitro was observed when long-chain unsaturated acyl-CoAs and diacylglycerols (DAGs) containing long acyl chains were used as substrates for in vitro TAG synthesis (although 1,2-didecanoin was also very effective). DAGAT activity (expressed relative to DNA) was shown to decline over twofold during skeletal myogenesis when measured in the absence of exogenous DAG. However, in the presence of exogenous (1 mM) DAG, there was no significant change in DAGAT activity, suggesting that the levels of this enzyme are not altered during skeletal myogenesis. These results indicate that endogenous DAG levels are limiting TAG synthesis in L6 myotubes. However, DAG content of myotubes was significantly greater than that of myoblasts, suggesting that there may be an increased competition for DAG (perhaps owing to enhanced phospholipid synthesis) during skeletal myogenesis. The combined effects of decreased synthesis and increased degradation (reported earlier) of TAG may account for the decrease in endogenous TAG contents observed during skeletal myogenesis.Key words: diacylglycerol acyltransferase, TAG synthesis, skeletal myogenesis.

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