Monoglyceride Acyltransferase Research Articles

Impaired enterocyte triglyceride synthesis after trauma-hemorrhage and resuscitation.

Although gut lipid absorptive capacity is depressed after trauma and hemorrhagic shock, it remains unknown whether this is associated with an impairment in the enterocyte triglyceride (TG) synthesis, and if so, whether villus or crypt cells participate in the lipid absorption under such conditions. To study this, midline laparotomy (i.e., trauma induction) was performed in the rats, and then the animals were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of shed blood volume was returned in the form of Ringer's lactate. The rats were then resuscitated with four times the volume of maximal bleedout with Ringer's lactate over 60 min. For lipid loading test, 1 mL of olive oil was given intraduodenally as a bolus injection upon the completion of resuscitation. The enterocytes along the crypt-villus axis from the proximal small intestine were isolated at either 0, 1.5, or 5 h after fat feeding. The intracellular and pre-chylomicron TG contents as well as the microsomal enzyme activities associated with TG synthesis were determined thereafter. The results indicated that intracellular and pre-chylomicron TG contents and lipid esterifying enzyme (i.e., acyl-coenzyme A:monoglyceride acyltransferase) activity decreased significantly in villus tip cells after trauma-hemorrhage and resuscitation. The intracellular TG content and lipid esterifying enzyme activity in crypt cells after trauma and hemorrhagic shock were also reduced. However, there was no significant decrease in pre-chylomicron TG content in the crypt cells isolated from trauma-hemorrhaged rats. It is unlikely that crypt cells participate in lipid transport after trauma and hemorrhagic shock. In contrast, the TG synthesis in villus tip cells decreased significantly following trauma-hemorrhage and resuscitation, which may contribute, at least in part, to the impaired gut lipid absorptive capacity observed under such conditions.

In vivo studies on stereospecificity of the monoglyceride kinase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase and CDP-diglyceride synthase of Streptococcus mutans BHT using the stereoisomers of the ether lipid, dodecylglycerol

Streptococcus mutans BHT metabolizes radioactive 3-dodecyl- sn-glycerol (sn-3-DDG) almost exclusively to lysophosphatidic acid, phosphatidic acid and 1,3-diradyl- sn-glycerol, whereas the cells of this organism metabolize 1-dodecyl- sn-glycerol ( sn-1- DDG) to all of the glycerol lipids of S. mutans BHT, with the largest amounts incorporated into phosphatidylglycerol and diradylglycerol (mostly the 1,2- but also the 1,3-isomer). (The common names of lipids, such as phosphatidic acid, are used in the broader sense to mean that the lipid may contain alkyl as well as acyl groups.) The addition of an equivalent amount of nonradioactive sn-3- DDG to radioactive sn-1- DDG causes more of the radioactivity to accumulate at phosphatidic acid. These results indicate that the monoglyceride kinase (EC 2.7.1.94), lysophosphatidic acid acyltransferase (EC 2.3.1.40) and the monoglyceride acyltransferase (EC 2.3.1.22) enzymatic reactions are not stereospecific, and that the CDP-diglyceride synthase (EC 2.7.7.41) and phosphatidic acid phosphatase (EC 3.1.3.4) metabolic steps are stereospecific in S. mutans BHT. The synthesis of phosphatidic acid and lysophosphatidic acid from sn-3-DDG provides a unique method for synthesizing these glycerol lipids with the uncommon stereochemical configuration in which the phosphate moiety is in the sn-1 position.

Role of small intestine in pathogenesis of hyperlipidemia in diabetic rats.

The small intestine can utilize endogenous substrates for triglyceride synthesis. In diabetes mellitus, potential endogenous substrates are elevated. This study was designed to investigate whether intestinal triglyceride production utilizing endogenous substrates contributes to the pathogenesis of hyperlipidemia in diabetes. Intestinal fatty acid esterification as well as activities of acyl-CoA synthetase and acyl-CoA monoglyceride acyltransferase are the same in diabetic and control rats when the results are expressed per milligram protein. However, due to marked intestinal hypertrophy these activities are increased when the results are expressed as per centimeter gut length. In the mesenteric lymph fistula rat model, we found that during fasting diabetic rats have a greater than twofold increase in triglyceride output that is carried mainly by very low-density lipoproteins (VLDL). During lipid infusion, total triglyceride fatty acid output was not different between diabetic and control rats, although there were significant differences in the patterns of partition of endogenous and exogenous triglyceride into chylomicrons and VLDL. Endogenous triglyceride production did not increase in diabetic rats during lipid infusion. In contrast, there was a substantial increase in endogenous triglyceride production in the control group to a level comparable with that of the diabetic rats. There was a significant reduction in incorporation of exogenous triglyceride into chylomicrons in diabetic rats.

Triacylglycerol biosynthesis in human small intestinal mucosa. Acyl-CoA: monoglyceride acyltransferase.

Acyl-CoA: monoglyceride acyltransferase (MGAT; EC 2.3.1.22) has been studied in human small intestinal mucosa by means of a spectrophotometric method based on the detection of liberated CoA employing 5,5'-dithiobis-(2-nitrobenzoic acid). With optimal assay conditions available the pH optimum was spread between 7.0 and 7.7 with a maximum at a pH of 7.4. Dependent on its concentration one of the substrates, palmitoyl-CoA, caused severe inhibition which was largely prevented by the addition of albumin. Using palmitoyl-CoA and 1-monooleoylglycerol as substrates specific activities were 23.0 +/- 8.4 in total homogenates compared with 92.9 +/- 28.3 nmol CoA released/min/mg protein in microsomal fractions from jejunal mucosa. Concerning the substrate specificity, a broad acyl-donor pattern exists with maximal activities for C10:0 to C16:0, the highest for C16:0. A preferential esterification of acyl-acceptors was shown for 2-monoacylglycerols as compared with the 1-isomers, and for monoacylglycerols with unsaturated versus saturated fatty acids. MGAT was mainly localized in the microsomal fraction. The properties of MGAT from human small intestine are discussed with respect to the intestinal enzyme from other species.

Subcellular localization of monoglyceride acyltransferase, xanthine oxidation, NADP: Isocitrate dehydrogenase and alkaline phosphatase in the mucosa of the guinea-pig small intestine

1. 1. Rate dependent and isopycnic banding in a zonal rotor were used to analyse the subcellular sites of enzymes in homogenates of guinea-pig small intestinal mucosa. 2. 2. The results demonstrate the following localizations: monoglyceride acyltransferase—microsomal; xanthine oxidase and dehydrogenase—soluble phase, and NADP: isocitrate dehydrogenase—soluble phase and mitochondrial. 3. 3. Alkaline phosphatase is confined to brush borders and is absent from the basolateral plasma membrane. A variable proportion of the activity, up to 40% is on brush borders which during homogenization break up into particles of reduced density and slow sedimentation rate.

Topographic distribution of enzymes involved in glycerolipid synthesis in rat small intestinal epithelium

1. 1. The enzymes involved in glycerolphosphate and monoacylglycerol acylation of rat small intestine were more active in villi than in crypts. Monoglyceride acyltransferase (EC 2.3.1.22) was found to be absent from crypts. 2. 2. In the villi, the enzymes are mainly localized in microsomes, although low activities of palmitoyl-CoA synthetase (EC 6.2.1.3), glycerolphosphate acyltransferase (EC 2.3.1.15) and cholinephosphotransferase (EC 2.7.8.2) are found in mitochondria. Mitochondria lack monoglyceride acyltransferase and lysolecithin acyltransferase (EC 2.3.1.23), both of which are involved in the reacylation of alimentary partial glycerides. Therefore, this process is confined to microsomes. 3. 3. The monoacylglycerol and lysolecithin acyltransferases, as well as cholinephosphotransferase, are probably localized within the endoplasmic reticulum, since these enzymes are relatively Nagerse resistant (subtilisin; EC 3.4.2.1, compared with palmitoyl-CoA synthetase and glycerolphosphate acyltransferase, which are highly Nagarse-sensitive and therefore probably localized on the outside of the microsomes (and mitochondria). 4. 4. The physical separation of alimentary product reacylation from de novo synthetic processes provides the basis of metabolic compartmentation observed by other workers. 5. 5. The use of sucrose instead of a salt medium for the isolation and homogenization of small intestinal epithelial cells allowed the separation of mitochondria and microsomes by differential centrifugation without mutual contamination. 6. 6. Phospholipids were found to stimulate glycerolphosphate acylation in vitro. 7. 7. The glycerolphosphate and monoacylglycerol acylation pathways are not competitive.

Fatty acid binding protein. Role in esterification of absorbed long chain fatty acid in rat intestine.

Fatty acid binding protein (FABP) is a protein of 12,000 mol wt found in cytosol of intestinal mucosa and other tissues, which exhibits high affinity for long chain fatty acids. It has been suggested that FABP (which may comprise a group of closely related proteins of 12,000 mol wt) participates in cellular fatty acid transport and metabolism. Although earlier findings were consistent with this concept, the present studies were designed to examine its physiological function more directly. Everted jejunal sacs were incubated in mixed fatty acid-monoglyceride-bile acid micelles, in the presence or absence of equimolar concentrations of either of two compounds which inhibit oleate binding to FABP:flavaspidic acid-N-methyl-glucaminate and alpha-bromopalmitate. Oleate uptake, mucosal morphology, and oxidation of [14C]acetate remained unaffected by these agents, but oleate incorporation into triglyceride was inhibited by 62-64% after 4 min. The inhibition by flavaspidic acid was reversible with higher oleate concentrations. The effect of these compounds on enzymes of triglyceride biosynthesis was examined in intestinal microsomes. Neither flavaspidic acid nor alpha-bromopalmitate inhibited acyl CoA:monoglyceride acyl-transferase. Fatty acid:coenzyme A ligase activity was significantly enhanced in the presence of partially purified FABP, probably reflecting a physical effect on the fatty acid substrate or on the formation of the enzyme-substrate complex. Activity of the enzyme in the presence of 0.1 mM oleate was only modestly inhibited by equimolar flavaspidic acid and alpha-bromopalmitate, and this effect was blunted or prevented by FABP. We conclude that in everted gut sacs, inhibition of triglyceride synthesis by flavaspidic acid and alpha-bromopalmitate could not be explained as an effect on fatty acid uptake or on esterifying enzymes in the endoplasmic reticulum but rather can be interpreted as reflecting inhibition of fatty acid binding to FABP. These findings lend further support to the concept that FABP participates in cellular fatty acid transport and metabolism. It is also possible that FABP, by effecting an intracellular compartmentalization of fatty acids and acyl CoA, may play a broader role in cellular lipid metabolism.

Uptake, activation, and esterification of fatty acids in the small intestine of suckling rat.

The small intestinal mucosal phase of fatty acid absorption was studied in suckling and adult rats. Fatty acid binding protein (FABP) is present in the cytosol of jejunal mucosa of 6-day-old rats in amounts equivalent to that found in mucosal cytosol of adult rats (16.4% and 15.0%, respectively). The percentage of oleic acid binding to FABP is the same in 6-day-old and adult rats (13.9% and 10.2%, respectively). The specific activity of jejunal microsomal oleoyl-CoA synthetase is high in the fetus, falls abruptly after birth, but increased by the third day of life to remain constant thereafter into adult life. In contrast the specific activity of acyl-CoA:monoglyceride acyltransferase is low in the fetal jejunum, gradually increases, and is significantly higher in the 6- and 12-day-old rat than in the adult. Uptake of oleic acid by jejunal slices of 6- and 11-day-old animals is three- to fivefold higher than uptake by jejunal slices prepared from adult rats. The rate of esterification of oleic acid is higher in jejunal slices from 6- and 11-day-old rats, reflecting the enhanced uptake of oleic acid.

The effect of acute ethanol treatment on lipid-reesterifying enzymes of the rat small bowel.

The acute effects of intraduodenal administration of ethanol, 5 g/kg body weight, on intestinal activities of lipid-reesterifying and disaccharidase enzymes of the small bowel were studied. Results were compared to those produced in controls receiving isocaloric amounts of glucose by the same route. Acyl-CoA:monoglyceride acyltransferase, acyl-CoA synthetase (acid:CoA ligase (AMP) EC 6.2.1.3), sucrase, and lactase assays were performed on jejunal samples; acyl-CoA synthetase assay was performed on ileal samples. Ethanol produced greater activities of the lipid-reesterifying enzymes in the jejunum than did glucose. Ileal specific activity of acyl-CoA synthetase was also increased in the experimental group. No effect of ethanol on jejunal disaccharidase enzyme activities was noted. It is concluded that ethanol given acutely has a specific stimulating effect on intestinal enzymes involved in lipid absorption.

Fatty acid uptake and esterification by proximal and distal intestine in bile fistula rats

The uptake and reesterification of 3H-labelled oleic acid was studied in intestinal sacs from the duodeno-jejunum and ileum of control and 48 h bile-diverted rats. In addition, the levels of two of the enzymes involved in the reesterification of absorbed fatty acid, acyl-CoA: monoglyceride acyl transferase and acyl-CoA synthetase, were measured in proximal and distal intestine from control and bile diverted animals. Ileal sacs from bile diverted rats absorbed more and reesterified more labelled fatty acid than sacs from control animals. The increased ideal uptake was associated with an increase in the level of acyl-CoA synthetase and a decrease in the level of acyl-CoA: monoglyceride acyl transferase in the distal intestine of bile-diverted compared with control animals. These changes in ileal enzyme activity may result from changes in the composition of the lipid mixture absorbed by the distal ileum when bile is diverted. The increased enzyme activity may in turn promote uptake by increasing the rate of handling of absorbed lipid.

A new assay procedure for monoglyceride acyltransferase.

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 V(max.) 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.

PERINATAL CHANGES IN THE ACTIVATION AND ESTERIFICATION OF FATTY ACIDS IN THE RAT INTESTINAL MUCOSA

The high dietary triglyceride intake in the perinatal period of all mammals including man occurs when the lipolytic activity in the gastrointestinal tract is low. In previous experiments the activity of one of the enzymes of the re-esterification pathway, acyl-CoA:monoglyceride acyltransferase was found to be higher in microsomes prepared from suckling rat jejunal mucosa than in adults(Experientia 29, 405, 1973). To evaluate conclusively the esterification processes during the suckling period, the activity of the fatty acid: CoA ligase in jejunal microsomes and the in-vitro esterification of 1-14C-oleic acid by jejunal segments was determined. The activity of the fatty acid:CoA ligase in newborn, 5, 12 and 90 day old rats was 239±15, 154±24, 149±10 and 195±21 nmoles/min/mg protein, respectively. The results of these enzymological studies were confirmed by the finding that the esterification rate of oleic acid occurs as rapidly in jejunal mucosa of suckling rats as in adults. Thus these studies indicate that, of the phases of fat absorption, the re-esterification processes are not a limiting factor. The apparent discrepancy of low lipolytic and high esterification activity during the suckling period remains to be explained.

Activities of lipid reesterifying enzymes in jejunal microsomes of bile fistula rats attempts to correlate enzyme activities with microsomal phospholipid content

1. 1. Microsomes from control and bile fistula rat jejuna were assayed for specific and total activities of acyl-CoA synthetase (acid:CoA ligase (AMP) EC 6.2.1.3) and acyl-CoA:monoglyceride acyltransferase. In addition, measurements of microsomal phospholipid content were performed. In certain groups of rats the classes of micro-somal phospholipid were quantitated. 2. 2. Specific and total enzyme activities were found to be reduced in bile fistula rats as previously observed. Enzyme activities were restored to normal levels in bile fistula rats by infusing sodium taurocholate and lecithin at concentrations of 7.5 and 2 mM, respectively. Infusion of 7.5 mM sodium taurocholate alone resulted in some increase in enzyme specific activities toward the normal range. Values for total enzyme activities were actually normal under these conditions. Infusion of greater concentrations of bile salts and/or lecithin was not as effective in maintaining normal enzyme activities. 3. 3. Phospholipid content of microsomes was decreased in bile fistula rats. This change was found to be the result of reduced recoveries of lecithin. Intraduodenal infusion of moderate amounts of sodium taurocholate (7.5 mM) and lecithin (2 mM) restored microsomal phospholipid content to normal. It was also observed, however, that this could be accomplished by infusing 7.5 mM sodium taurocholate without lecithin into bile fistula rats. It is concluded, therefore, that under the conditions of this study the presence of lecithin in the intestinal lumen is not essential for the maintenance of normal amounts of phospholipid of jejunal microsomes. 4. 4. Microsomal enzyme activity was generally normal when microsomal phospholipid content was found to be normal and was abnormal when phospholipid levels were depressed. Statistical analysis showed a correlation existed between enzyme specific activities and phospholipid content. It was not definitely established, however, whether changes in microsomal phospholipid composition directly influenced microsomal enzyme activities or whether changes in enzyme activities and phospholipid levels merely vary in the same direction in response to changes in the amount of fat being absorbed by the intestine under different experimental conditions.

Utilization of Neutral Glycerides and Phosphatidylethanolamine by the Phospholipase A1 of the Plasma Membranes of Rat Liver

Abstract The phospholipase A1 solubilized by heparin from the plasma membranes of rat liver can catalyze the hydrolysis of monoglyceride, diglyceride, and monoacylglycerophosphorylethanolamine in addition to phosphatidylethanolamine. Furthermore, the enzyme catalyzes the transacylation of the acyl group from position 1 of these glycerides to an acceptor monoglyceride to form diglyceride. Triton X-100 which disperses the lipids reduces transacylation and favors the utilization of water as the acyl acceptor (hydrolysis). Since free fatty acid is not incorporated, the transacylation appears to be direct rather than the combination of hydrolysis and reacylation. The influence of Ca2+ on transacylation and hydrolysis was dependent upon the mixtures of lipids used as substrates. Co-elution of the hydrolytic and transacylation activities from a column of Sephadex G-200 and heat inactivation of all activities indicate that a single enzyme catalyzes both reactions. It is tentatively suggested this enzyme be more appropriately named monoglyceride acyltransferase since monoglyceride is the preferred substrate and transacylation is the predominating reaction.

Fat absorption in essential fatty acid deficiency: a model experimental approach to studies of the mechanism of fat malabsorption of unknown etiology

Male rats were made deficient in essential fatty acids by feeding them a fat-free diet supplemented with 4% tripalmitin for 8-12 wk from the time of weaning. After feeding 0.5 ml of [(14)C]triolein or [(3)H]oleic acid, 72-hr stool recoveries of radioactivity were significantly greater in deficient rats than in chow-fed controls. Essential fatty acid deficiency did not reduce the absorptive capacities for triolein or for a medium-chain fat, trioctanoin, measured after 3 and 2 hr of maximal-rate duodenal infusion. In everted jejunal slices from essential fatty acid-deficient rats, uptake of micellar [(14)C]oleic acid at 0-1 degrees C was similar to that of controls, but the rate of incorporation of fatty acid into triglyceride after rewarming to 37 degrees C was significantly reduced. The specific activities of the microsomal esterifying enzymes, acyl CoA:monoglyceride acyltransferase and fatty acid CoA ligase in jejunal mucosa were 30% lower in essential fatty acid-deficient rats. However, the total microsomal enzyme activity adjusted to constant weight did not differ significantly in deficient rats compared with controls. After intraduodenal perfusion of triolein, accumulation of lipid in the intestinal wall was increased in the deficient rats. Because over 90% of the absorbed mucosal lipid was present as triglyceride, essential fatty acid deficiency appears to affect the synthesis or release of chylomicron lipid from the intestine. Analysis of regions of intestine showed that this delay in transport was most marked in the midportion of the small intestine.

Effects of Bile Diversion on the Lipid-Reesterifying Capacity of the Rat Small Bowel

Intestinal absorption of lipid by the small bowel proceeds via enzymatic processes by which the products of lipid digestion are reesterified to triglyceride. This study was done to determine whether bile diversion which causes steatorrhea due to impairment of micelle formation also has an adverse effect on the small bowel itself by producing a reduction in its lipid-reesterifying capacity. Two methods were employed to investigate this question. The first utilized the everted gut sac technique incubating jejunal sacs in a micellar solution of radioactive free fatty acid. Rates of esterification of the fatty acid were determined in both control and experimental sacs incubated for brief periods in solutions of varying concentrations of free fatty acid. Morphological studies were also performed on these sacs both before and after incubation. The other method used to determine the reesterifying capacity of the intestine was the direct enzymatic assays of acyl-coenzyme A ligase and acyl-coenzyme A: monoglyceride acyltransferase. All studies were performed 72 hr after surgery. Maximal rates of esterification of oleic acid by sacs were sustained for only brief periods. Histological examination demonstrated slight but definite abnormalities of the epithelial cells after only 5 min and partial villous sloughing after 15 min of incubation. Using brief periods of incubation, studies were performed on sacs from both nonfasting and fasting control and bile fistula rats. Rates of esterification were greater in sacs from either nonfasting or fasting control rats compared with sacs from experimental rats under comparable conditions of nonfasting and fasting. The activities of the mucosal lipid-reesterifying enzymes were also found to be greater in the control rats. Enzymatic activities were determined to be greater in either the nonfasting or fasting control groups compared with the respective nonfasting and fasting experimental groups. Similar enzyme assays were performed in rats infused intraduodenally with a synthetic diet at a constant rate. Under these conditions, enzymatic activities were still greater in control rats. Additional studies in experimental rats demonstrated that these enzymatic activities could be maintained in the normal range by infusing artificial bile intraduodenally. It is concluded that, in addition to causing lipid malabsorption by impairing micelle formation, bile diversion also has an adverse effect on the intracellular process of lipid absorption by producing a reduction in the lipid-reesterifying capacity of the small bowel.

Lipid-metabolizing enzymes in the ovary of the laying hen

The activities of cholesteryl ester hydrolase, di- and monoglyceride acyltransferase, glycerokinase, triglyceride lipase and lipoprotein lipase in the ovary of the laying hen were assayed by standard methods in vitro. Except for lipoprotein lipase, all enzymes showed significant activities, which, however, were incompatible with the hypothesis that the lipid moieties of plasma lipoproteins undergo extensive transformation during transfer into the egg yolk. It is suggested that these enzymes function exclusively in the metabolism of the follicular cells.

Acyl-CoA synthetase for long-chain fatty acids in rat small bowel and the influence of diets containing different compositions of fatty acids on intestinal lipid reesterifying enzyme activities

1. 1. Kinetic studies of microsomal long chain acyl-CoA synthetase (acid:CoA ligase (AMP), EC 6.2.1.3) from rat jejunum using palmitic acid, oleic acid, and linoleic acid as substrates were performed. Kinetic studies were also done using whole jejunal mucosal homogenates. The enzyme assay employed was the hydroxamate-trapping method. 2. 2. Palmitic acid was found to mildly inhibit the enzyme reaction while oleic acid was shown to be a strong inhibitor. This inhibition by oleate could be partially overcome by the addition of large amounts of albumin. Plant lecithin, however, was found to be much more effective in reversing the inhibitory effects of oleic acid. 3. 3. Under ideal conditions for the enzyme assay specific activity of acyl-CoA synthetase for jejunal microsomes was 6–7 times greater than for jejunal homogenate. Recovery of total enzymatic activity in this subcellular fraction was about one-third of the amount calculated to be present in the whole jejunal homogenate. 4. 4. The effect of the long chain fatty acid composition of the diet on lipid reesterifying enzyme activities in the small bowel including palmitoyl-CoA synthetase, oleoyl-CoA synthetase and acyl-CoA :monoglyceride acyltransferase were also determined. No difference in specific or total small intestinal enzymatic activities were observed between two groups of rats, one on a diet for 3 weeks with a fatty acid composition of 13.8% saturated and 86.2% unsaturated and the other on a diet for a similar period containing 43.6% saturated and 56.4% unsaturated fatty acids. It was concluded that both saturated and unsaturated long chain fatty acids in the diet stimulate intestinal lipid reesterifying enzymatic activities equally well.

Preservation of lipid reesterifying enzymes in the microsomal fraction of the rat jejunum

Studies were performed on methods of storage of rat jejunal tissue that would preserve activities of the lipid reesterifying enzymes, acyl CoA:monoglyceride acyltransferase and fatty acid CoA ligase. Storage at -80 degrees C of microsomes prepared from jejunal mucosa or storage of lyophilized microsomes at -20 degrees C was shown to preserve acyl CoA:monoglyceride acyltransferase very well for a matter of weeks. Preservation of fatty acid CoA ligase activity was adequate with either method, but results were not as good as for the transacylase enzyme.

Localization of lipid reesterifying enzymes of the rat small intestine effects of jejunal removal on ileal enzyme activities

1. 1. The difference between proximal and distal small bowel regarding lipid absorption was studied. Uptake and esterification of [1- 14C]oleic acid by everted sacs from the jejunum and terminal ileum were determined. Assays were also done for two of the lipid reesterifying enzymes at the various levels of the small bowel. Fatty acid-CoA ligase was determined spectrophotometrically by employing hydroxylamine which assays thioester bonds. Acyl-CoA: monoglyceride acyltransferase was determined by continuous recording spectro photometry using 5,5'-dithiobis-(2-nitrobenzoic acid) which reacts with sulfhydryl groups on liberated CoA. 2. 2. Uptake and esterification of [ 1–14C] oleic acid was greater in sacs prepared from the proximal compared to the distal small bowel. Activities for fatty acid-CoA ligase and acyl-CoA: monoglyceride acyltransferase were significantly greater in the proximal small bowel. 3. 3. An attempt to induce greater enzyme activities in the ileum was done by surgically removing or bypassing the jejunum. Enzyme activities were elevated in the ileum in experimental rats compared to controls when assays were done 1 month af ter sargey. Total enzyme activities were more increased compared to the specific activities because of the hypertrophic effects produced in the remaining ileum following removal of the jejunum. 4. 4. These findings support the concept that the proximal small bowel is the major site of lipid absorption under normal conditions but that the ileum has the potential to absorb lipid if the jejunum is removed.