scholarly journals A new assay procedure for monoglyceride acyltransferase

1974 ◽  
Vol 141 (2) ◽  
pp. 407-411 ◽  
Author(s):  
Verena J. Short ◽  
David N. Brindley ◽  
Raymond Dils
Keyword(s):  
Enzyme Activity ◽  
Guinea Pig ◽  
Intestinal Mucosa ◽  
Microsomal Fraction ◽  
Assay System ◽  
Acyltransferase Activity ◽  
Palmitoyl Carnitine ◽  
Assay Procedure ◽  
Acyl Acceptors

1. A new assay system is described for monoglyceride acyltransferase (acylglycerol palmitoyltransferase, EC 2.3.1.22) in which palmitoyl-CoA is generated from palmitoyl-(-)-carnitine. 2. With the microsomal fraction from homogenates of guinea-pig intestinal mucosa, the Vmax. of this enzyme decreased with different acyl acceptors in the order 2-monopalmitoylglycerol>2-hexadecylglycerol>rac-1-monopalmitoylglycerol. 3. There were highly significant correlations between the monoglyceride acyltransferase activity as measured with these three substrates. This demonstrates that each of these substrates can be used to measure the same enzyme activity. 4. The advantages of using generated palmitoyl-CoA with 2-hexadecylglycerol and rac-1-monopalmitoylglycerol as model substrates for this enzyme are 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.

scholarly journals Acylation of 1-alkenyl-glycerophosphocholine and 1-acyl-glycerophosphocholine in guinea pig heart

1986 ◽  
Vol 236 (2) ◽  
pp. 481-487 ◽  
Author(s):  
G Arthur ◽  
P C Choy
Keyword(s):  
Heat Treatment ◽  
Guinea Pig ◽  
Strong Evidence ◽  
Microsomal Fraction ◽  
Kinetic Studies ◽  
Guinea Pig Heart ◽  
Acyltransferase Activity ◽  
Unsaturated Acyl ◽  
Important Pathway ◽  
High Degree

The deacylation-reacylation process has been shown to be an important pathway for phospholipids to attain the desired acyl groups at the C-2 position. The acylation of 1-acyl-glycerophosphocholine (-GPC) in mammalian hearts has been well documented, but the acylation of 1-alkenyl-GPC has not been described. In this paper, we demonstrate the presence of acyl-CoA: 1-alkenyl-GPC acyltransferase for the acylation of 1-alkenyl-GPC in mammalian hearts; the highest activity is found in guinea pig heart. The guinea pig heart 1-alkenyl-GPC acyltransferase has only 10-40% of the 1-acyl-GPC acyltransferase activity, and both activities are located in the microsomal fraction. However, these two enzymes respond differently to cations, detergents and heat treatment, and the two enzymes also display different acyl specificity. Kinetic studies indicate that both reactions could not be accommodated by the same catalytic site. The results provide strong evidence that the two activities are from separate and distinct proteins. The specificity of 1-alkenyl-GPC acyltransferase for unsaturated species of acyl-CoA may play an important role in the maintenance of the high degree of unsaturated acyl groups found in guinea pig heart plasmalogens.

scholarly journals The acylation of lysophosphoradylglycerocholines in guinea-pig heart mitochondria

1987 ◽  
Vol 242 (1) ◽  
pp. 171-175 ◽  
Author(s):  
G Arthur ◽  
L L Page ◽  
C L Zaborniak ◽  
P C Choy
Keyword(s):  
Guinea Pig ◽  
Fatty Acyl ◽  
Heart Mitochondria ◽  
Acyl Donor ◽  
Mitochondrial Enzyme ◽  
Microsomal Enzymes ◽  
Guinea Pig Heart ◽  
Acyltransferase Activity ◽  
Equal Importance ◽  
Acyl Acceptors

The importance of the deacylation-reacylation pathway for attaining the desired fatty acid composition in microsomal phospholipids has been well established. It is not clear, however, whether this mechanism is of equal importance in mitochondria. The absence of acyltransferase activity in mammalian heart mitochondria has been reported in a number of studies. In the present study we report the presence of acyltransferase activities for lysophosphoradylglycerocholines in guinea-pig heart mitochondria. This enzyme showed properties that were considerably different from those of the microsomal enzymes. Of all the acyl-CoAs tested (C18:0, C18:1, C18:2 and C20:4) the mitochondrial enzyme utilized only linoleoyl-CoA as fatty acyl donor and utilized both 1-acyl-sn-glycero-3-phosphocholine and 1-alkenyl-sn-glycero-3-phosphocholine as fatty acyl acceptors. The presence of significant quantities of fatty acids other than linoleate at the C-2 position of mitochondrial acylglycerophosphocholines, coupled with the specificity of the enzyme for linoleoyl-CoA, suggest that, in addition to reacylation, other mechanisms play a significant role in producing the molecular composition of these phospholipids found in the mitochondria.

INCORPORATION OF IODIDE INTO ORGANIC COMPOUNDS IN THE GUINEA PIG THYROID

1963 ◽  
Vol 43 (1) ◽  
pp. 110-118 ◽  
Author(s):  
R. Ekholm ◽  
T. Zelander ◽  
P.-S. Agrell
Keyword(s):  
Guinea Pig ◽  
Protein Binding ◽  
Organic Compounds ◽  
Guinea Pigs ◽  
Microsomal Fraction ◽  
Thyroid Tissue ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Hydrolysis Of

ABSTRACT Guinea pigs, kept on a iodine-sufficient diet, were injected with Na131I and the thyroids excised from 45 seconds to 5 days later. The thyroid tissue was homogenized and separated into a combined nuclear-mitochondrial-microsomal fraction and a supernatant fraction by centrifugation at 140 000 g for one hour. Protein bound 131iodine (PB131I) and free 131iodide were determined in the fractions and the PB131I was analysed for monoiodotyrosine (MIT), diiodotyrosine (DIT) and thyroxine after hydrolysis of PB131I. As early as only 20 minutes after the Na131I-injection almost 100% of the particulate fraction 131I was protein bound. In the supernatant fraction the protein binding was somewhat less rapid and PB131I values above 90% of total supernatant 131I were not found until 3 hours after the injection. In all experiments the total amount of PB131I was higher in the supernatant than in the corresponding particulate fraction. The ratio between supernatant PB131I and pellet PB131I was lower in experiments up to 3 minutes and from 2 to 5 days than in experiments of 6 minutes to 20 hours. Hydrolysis of PB131I yielded, even in the shortest experiments, both MIT and DIT. The DIT/MIT ratio was lower in the experiments up to 2 hours than in those of 3 hours and over.

scholarly journals Adrenal microsomal C19-steroid 5α-reductase activity in the Snell transplantable rat adrenocortical tumour 494 and the effect of oestradiol, testosterone propionate and adrenocorticotrophin in intact and gonadectomized rats

1973 ◽  
Vol 132 (2) ◽  
pp. 293-300 ◽  
Author(s):  
Paul V. Maynard ◽  
Euan H. D. Cameron
Keyword(s):  
Enzyme Activity ◽  
Steroid Hormones ◽  
Microsomal Fraction ◽  
Testosterone Propionate ◽  
Reductase Activity ◽  
Intact Control ◽  
Adrenal Tissue ◽  
Males And Females ◽  
Adrenocortical Tumour ◽  
Hormonal Treatments

The C19-steroid 5α-reductase activity in the microsomal fraction of rat adrenal tissue under various hormonal treatments was examined. In intact control rats the activity is similar in both males and females, and after gonadectomy it is markedly increased. Treatment with oestradiol (150μg/day per animal for 7 days) or testosterone propionate (2mg/day per animal for 7 days) lowered the activity of 5α-reductase in castrated animals to approximately the values for intact animals in both sexes, and in intact animals the activity was also decreased by these treatments. The enzyme activity was also decreased by adrenocorticotrophin treatment but to a lesser extent than by the steroid hormones. The activity of the 5α-reductase enzyme in the Snell adrenocortical tumour 494 is very low when incubated as a whole homogenate, but the activity in microsomal material of the tumour was measured and unexpectedly found to be similar to that in intact controls.

scholarly journals Effect of dexamethasone on mannolipid synthesis by hepatocytes prepared from control and inflamed rats

1984 ◽  
Vol 219 (2) ◽  
pp. 429-436 ◽  
Author(s):  
M Sarkar ◽  
S Mookerjea
Keyword(s):  
Enzyme Activity ◽  
Microsomal Fraction ◽  
Fold Increase ◽  
Cell Homogenate

Hepatocytes were prepared from control and inflamed rats. Mannose incorporation into dolichol monophosphate mannose in homogenate and microsomal fraction of the hepatocytes was increased 2-fold over the controls 24 h after induction of inflammation by turpentine injection. Incubation of hepatocytes from both control and inflamed rats with 0.1-10 microM-dexamethasone produced a 1.5-fold increase of dolichol phosphate mannose formation, whereas, 100 microM-dexamethasone decreased its formation. The increase in the ratio of dolichol phosphate mannose formation in inflamed over controls was virtually eliminated when the cell homogenate assay mixtures included 30 nmol of exogenous dolichol phosphate. This supports the earlier suggestion that the increase in the enzyme activity in inflammation could be due to higher concentrations of endogenous dolichol phosphate [Coolbear & Mookerjea (1981) J. Biol. Chem. 256, 4529-4535]. In contrast, the increase in the ratio of dolichol phosphate mannose formation between dexamethasone-treated and untreated hepatocytes remained unchanged when increasing concentrations of exogenous dolichol phosphate were added to the assays. This suggests that the increase in glycosylation of dolichol phosphate in dexamethasone-treated hepatocytes is probably due to the increased mannosyltransferase activity, rather than due to higher concentrations of endogenous dolichol phosphate in these cells.

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