Regulation of fatty acid oxygen-18 exchange catalyzed by pancreatic carboxylester lipase. 1. Mechanism and kinetic properties

Muderhwa, J.M.; Schmid, Patricia C.; Brockman, Howard L.

doi:10.1021/bi00116a021

Cited by 22 publications

(20 citation statements)

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“…When the enzyme was incubated with fatty acid and ethanol, an acyl-enzyme intermediate was formed and fatty acid ethyl ester was synthesized though nucleophilic displacement by ethanol. Exchange of fatty acid carboxyl oxygens with water oxygens was demonstrated by Muderhwa et al (18) using 18 O water. They suggested that fatty acids are good "substrates" for lipase(s).…”

Section: Discussionmentioning

confidence: 95%

Coenzyme A-independent Monoacylglycerol Acyltransferase from Rat Intestinal Mucosa

Tsujita¹,

Miyazaki

Tabei

et al. 1996

Journal of Biological Chemistry

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Rat intestinal mucosa contains high diacylglycerolsynthesizing activity (monoacylglycerol acyltransferase (MGAT) activity) due to monoacylglycerol and fatty acid, independently of coenzyme A and ATP. MGAT activity was purified from rat intestinal mucosa by successive chromatography separations on DEAEcellulose, CM-Sephadex, and anti-IgG-Sepharose against rat pancreatic lipase. The enzyme was electrophoretically homogeneous, and its molecular weight was 49,000, which is identical with that of rat pancreatic lipase. Immunoblotting analysis with antibody against rat pancreatic lipase showed one immunoreactive protein with an estimated molecular weight of 49,000. The activity of the purified enzyme was completely inhibited by addition of the antibody. Using immunocytochemical techniques, it was found that immunoreactive protein against rat pancreatic lipase was uniformly distributed within the absorptive cells of the intestine but was absent from the microvillar membrane. The MGAT activity of intestinal mucosal homogenate was inhibited by about 65% by addition of antibody against rat pancreatic lipase. Trioleoylglycerol-and dioleoylglycerol-hydrolyzing activities of the purified enzyme and pancreatic lipase were inhibited by addition of intestinal mucosa extract.These results suggest that pancreatic lipase is present in intestinal absorptive cells and that it may contribute to resynthesis of diacylglycerol from monoacylglycerol and fatty acids in these cells.Dietary fat (triacylglycerol) is digested by pancreatic lipase to 2-monoacylglycerols and free fatty acids prior to its absorption in the intestinal lumen (1). Unlike esterases, pancreatic lipase develops its full activity only with emulsified substrates; i.e. it is activated at lipid-water interfaces (2). The adsorbed 2-monoacylglycerols and free fatty acids are resynthesized to triacylglycerol by enterocytes. Triacylglycerols are synthesized by two pathways, the glycerol 3-phosphate pathway and the monoacylglycerol pathway (3). During fat absorption in monogastric animals, the monoacylglycerol pathway is the major route of triacylglycerol synthesis (4). This pathway involves the stepwise acylation of 2-monoacylglycerols absorbed from the intestinal lumen. Monoacylglycerol acyltransferase (MGAT) 1 is a microsomal enzyme that catalyzes the synthesis of 1,2-diacylglycerols from 2-monoacylglycerols and long chain fatty acyl coenzyme A (5, 6). Fatty acyl coenzyme A is synthesized from free fatty acid and coenzyme A by fatty acyl-CoA synthetase, a reaction which requires energy (ATP). Because the main purpose of eating fat diets is to gain energy, it is comprehensible that ATP is required in the fat adsorption process. Therefore, we examined a newly discovered pathway which does not require energy.Previously, we reported that, under physiological conditions, lipases/esterases catalyze the synthesis of acyl-alcohol ester from free fatty acids and alcohols (7-9). These reactions do not require ATP or coenzyme A. Pancreatic cholesterol esterase, which is known to hyd...

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Section: Discussionmentioning

confidence: 95%

Coenzyme A-independent Monoacylglycerol Acyltransferase from Rat Intestinal Mucosa

Tsujita¹,

Miyazaki

Tabei

et al. 1996

Journal of Biological Chemistry

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“…31, No. 1, 1992 Muderhwa and Brockman oxygens of 13,16-cis,cis-docosadienoic acid (DA) in monolayers, the accessibility of the fatty acid by the enzyme can be monitored (Muderhwa et al, 1992). With monolayers of pure DA and a CEL concentration of 30 nM, exchange is essentially complete in 10 min.…”

Section: Resultsmentioning

confidence: 99%

“…Most reagents and procedures for CEL adsorption and l 8 0 exchange experiments were as described in the preceding paper (Muderhwa et al, 1992). All experiments were performed at pH 6.6 on a subphase containing 0.01 M potassium phosphate buffer and 0.1 M NaCl at 24 OC.…”

Section: Methodsmentioning

confidence: 99%

“…In the preceding paper in this issue, the mechanism for oxygen exchange occurring in pure DA films was characterized by plotting the ratio of the surface concentrations of the singly and doubly 180-labeled species determined experimentally, r18,lbDA/r18,18-DA, against the corresponding ratio calculated for a randomly labeled sample with the same total content of '*O (Muderhwa et al, 1992). To examine the dependence of the mechanism on other parameters like lipid composition, we define the mechanism parameter R m = (r18,16-DA/r18,18-DA)cxp/(rl8,~~-DA/r18,18-DA)~l~ (3)…”

Section: K ' = Kik[eo]mentioning

confidence: 99%

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Regulation of fatty acid oxygen-18 exchange catalyzed by pancreatic carboxylester lipase. 2. Effects of lateral lipid distribution in mixtures with phosphatidylcholine

Muderhwa

Brockman²

1992

Biochemistry

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The lipase-catalyzed exchange of the carboxyl oxygens of 13,16-cis,cis-docosadienoic acid (DA) was studied in the presence of a nonsubstrate matrix lipid, 1-palmitoyl-2-oleoylphosphatidylcholine. For mixed lipid films at the argon-water interface exposed to pancreatic carboxylester lipase (EC 1.1.1.13), the extent of oxygen exchange showed an abrupt increase as the abundance of DA in the interface was increased from 0.5 to 0.6 mole fraction. This compositional range was independent of the level of enzyme used and of the surface pressure, i.e., lipid packing density, of the film. Concomitant with the transition was a change in the apparent mechanism of exchange from coupled to random sequential. Like the extent of oxygen exchanged, the shift in mechanism was independent of all variables except the lipid composition of the interface. The absence of any chemical or physical change accompanying the exchange reaction precludes mechanistic explanations based on the generation of reaction products by the enzyme. Instead, the results suggest that the lateral distribution of DA in phosphatidylcholine-DA interfaces regulates the expression of carboxylester lipase activity and its apparent mechanism. Preliminary measurements give an average cluster size of 1825 molecules of DA when its mole fraction is 0.35. As the DA content of the interface reaches 0.5-0.6, there appears to be a lipid head-group based percolative transition in which DA becomes the continuum. Because this transition involves the lateral organization of the lipids themselves, other interfacially active enzymes may be regulated similarly.

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“…Wang et al (1988) used the kinetic equations for sequential reactions to investigate the effect of taurocholate on human milk bile-salt-activated lipase. Muderhwa et al (1992) studied the exchange of "0 between water and fatty acids catalyzed by carboxylester lipase. We therefore believe that kinetic equations for sequential reactions are useful tools in studying lipolytic reactions.…”

Section: Discussionmentioning

confidence: 99%

Kinetics of the two‐step hydrolysis of triacylglycerol by pancreatic lipases

Lykidis¹,

Mougios²,

Arzoglou³

1995

European Journal of Biochemistry

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Pancreatic lipases catalyze the hydrolysis of triacylglycerol in a sequential manner. First, triacylglycerol is hydrolyzed to 1,2-diacylglycerol, which is subsequently converted to 2-monoacylglycerol. We studied the kinetics of trioleoylglycerol hydrolysis by rabbit and human pancreatic lipases. The products (acylglycerols and fatty acid) were analyzed by extraction from the reaction mixture, separation by thinlayer chromatography, and quantification by capillary gas chromatography. The first-order rate constants of trioleoylglycerol and dioleoylglycerol hydrolysis were calculated showing that both enzymes hydrolyze dioleoylglycerol faster than trioleoylglycerol. Using rabbit pancreatic lipase, we found that deoxycholate enhanced dioleoylglycerol hydrolysis to a higher degree than trioleoylglycerol hydrolysis. Colipase increased both rate constants similarly at high deoxycholate concentrations (35 mM), while at low concentrations (5 mM) a selectivity toward trioleoylglycerol was observed. From the variation of the rate constants with respect to temperature, we calculated the apparent activation energies of trioleoylglycerol and dioleoylglycerol hydrolysis to be 59.8 kJ . mol-' and 53.5 kJ . mol-', respectively. Upon storage, both rabbit and human pancreatic lipases showed a greater loss of activity toward dioleoylglycerol as compared to trioleoylglycerol, suggesting that different conformational elements of the enzyme molecule are responsible for the interaction with each substrate.Keywords. Pancreatic lipase ; kinetics ; rate constants ; triacylglycerol ; diacylglycerol.Pancreatic lipases catalyze the hydrolysis of triacylglycerol producing 1,2-diacyIglycerol, 2-monoacylglycerol, and fatty acids. Lipases are active at the oil/water interface in heterogeneous reaction systems (Brockman, 1984).One of the main characteristics of lipolysis by pancreatic lipases is that it takes place in a sequential manner. First, a molecule of triacylglycerol is hydrolyzed yielding one molecule of fatty acid and one molecule of 1,2-diacylglycerol, which is subsequently hydrolyzed to 2-monoacylglycerol and fatty acid. Therefore, 1,2-diacylglycerol constitutes both the initial product of lipolysis and the substrate for the second reaction. Using porcine pancreatic lipase, Constantin et al. (1960) reported a transient accumulation of 1 ,2-diacylglycerol and a late production of glycerol resulting from the cleavage of 1-monoacylglycerol after isomerization of 2-monoacylglycerol.A matter of great interest for understanding the whole mechanism of lipolysis, is the kinetics of the hydrolysis of diacylglycerol subsequent to the degradation of triacylglycerol. Despite intense research in the general field of lipases in recent years, the kinetics of this reaction remain unclear. Using trioctanoylglycerol and 1,2-dioctanoylglycero1 monolayers, Lagocki et al.(1 973) reported that the hydrolysis of the diester to 2-monooctanoylglycerol by porcine pancreatic lipase was slower than hydrolysis of the triester. The latter study was made in the abs...

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Regulation of fatty acid oxygen-18 exchange catalyzed by pancreatic carboxylester lipase. 1. Mechanism and kinetic properties

Cited by 22 publications

References 45 publications

Coenzyme A-independent Monoacylglycerol Acyltransferase from Rat Intestinal Mucosa

Coenzyme A-independent Monoacylglycerol Acyltransferase from Rat Intestinal Mucosa

Regulation of fatty acid oxygen-18 exchange catalyzed by pancreatic carboxylester lipase. 2. Effects of lateral lipid distribution in mixtures with phosphatidylcholine

Kinetics of the two‐step hydrolysis of triacylglycerol by pancreatic lipases

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