Search

A protein, molecular weight 70,000 that inhibits pancreatic lipase has been isolated from soybean seeds. Inhibition is not reversed by colipase unless bile salts are added to the assay system. Inhibitory properties of the purified protein are very similar to those of serum albumin or alpha-lactoglobulin. It has been confirmed that, during intestinal lipolysis of dietary fats, bile salts play an essential role for the activation of the lipase-colipase system in the presence of inhibitory proteins. The purified soybean lipase inhibitory protein was shown to be highly surface-active and able to penetrate monomolecular films of various glycerides and phospholipids at high surface pressure. Inhibition of pancreatic lipase by proteins is related to their capacity to interact with lipids and to modify the quality of the substrate-water interface. The protein isolated from soybeans inhibits pancreatic and Rh. delemar lipase in contrast to the Rh. arrhizus enzyme.

The adsorption of pancreatic phospholipase was studied in vitro in the presence of egg yolk lipoprotein emulsion, Intralipid emulsion, and milk fat globules. When the emulsions are incubated with bile salts, the latter dissociate a considerable fraction of the phospholipids initially associated with the emulsions, leading to the coexistence of an emulsified phase and a phase of mixed micelles. After the addition of pancreatic phospholipase A2, gel filtration shows that the enzyme was more than 90% bound to mixed micelles, regardless of the type of emulsion used. Comparable results were obtained by replacing the bile salts with human gallbladder bile. In parallel, pancreatic zymogen was never found to be bound to any of the lipid structures present (emulsion or mixed micelles). When the catalytic site of pancreatic phospholipase A2 was blocked with 4-bromophenacylbromide, there was no fixation on mixed micelles. Fixation was restored by the presence of lysolecithins and fatty acids in the incubation mixtures. The partial transformation of all emulsified substrates to mixed micelles by bile salts in vivo would thus lead to optimum activity of pancreatic phospholipase A2.

Pancreatic lipase requires colipase, a protein cofactor, to counteract the in vitro inhibition by bile salt. Lipase activity is inhibited by nonsteroidic detergents regardless of their charge and structure. Detergent-inhibited lipase is reactivated by colipase but in all cases activation is limited to a narrow range of detergent concentration. Complementary studies on the bile salt and detergent effect on lipase activity and on interfacial tension at the substrate-water interface show that inhibition is not related to the interfacial surface tension. It is hypothesized that absorption of amphiphilic compounds to the substrate surface modifies the distribution of the enzyme between the lipid surface and the aqueous phase. The activity of detergent-inhibited lipase is fully restored by adding bile salt to the reaction system. Bile salt might play a critical role during in vivo lipolysis by desorbing surface-active substances from the lipid-water interface thus allowing lipase and colipase to interact with substrate.

Hormone-sensitive lipase (HSL) contributes importantly to the hydrolysis of cholesteryl ester in steroidogenic tissues, releasing the cholesterol required for adrenal steroidogenesis. HSL has broad substrate specificity, because it hydrolyzes triacylglycerols (TAGs), diacylglycerols, monoacylglycerols, and cholesteryl esters. In this study, we developed a specific cholesterol esterase assay using cholesterol oleate (CO) dispersed in phosphatidylcholine and gum arabic by sonication. To continuously monitor the hydrolysis of CO by HSL, we used the pH-stat technique. For the sake of comparison, the hydrolysis of CO dispersion was also tested using other cholesteryl ester-hydrolyzing enzymes. The specific activities measured on CO were found to be 18, 100, 27, and 3 micromol/min/mg for HSL, cholesterol esterase from Pseudomonas species, Candida rugosa lipase-3, and cholesterol esterase from bovine pancreas, respectively. The activity of HSL on CO is approximately 4- to 5-fold higher than on long-chain TAGs. In contrast, with all other enzymes tested, the rates of TAG hydrolysis were higher than those of CO hydrolysis. The relatively higher turnover of HSL on CO observed in vitro adds further molecular insight on the physiological importance of HSL in cholesteryl ester catabolism in vivo. Thus, HSL could be considered more as a cholesteryl ester hydrolase than as a TAG lipase.

The aim of this study was to design a convenient, specific, sensitive, and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs were used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent because more than half of the fatty acids from Parinari oil are known to contain 9,11,13, 15-octadecatetraenoic acid (parinaric acid) in its esterified form. The presence of detergents (sodium taurodeoxycholate, CHAPS, Sulfobetaine SB12, Tween 20, Brij 35, Dobanol, n-dodecylglucoside) above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase in the fluorescence intensity is linear with time and proportional to the amount of lipase added. This new method, performed under non-oxidative conditions, was applied successfully to detecting low lipase levels in crude protein extracts from plant seeds and could be scaled down to microtiterplate measurements. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8). Lipase activity can also be assayed in acidic media (pH 5) using human gastric lipase. This simple and continuous assay is compatible with a high sample throughput and might be applied to detecting true lipase activities in various biological samples.

The adsorption of pancreatic phospholipase was studied in vitro in the presence of egg yolk lipoprotein emulsion, Intralipid emulsion, and milk fat globules. When the emulsions are incubated with bile salts, the latter dissociate a considerable fraction of the phospholipids initially associated with the emulsions, leading to the coexistence of an emulsified phase and a phase of mixed micelles. After the addition of pancreatic phospholipase A2, gel filtration shows that the enzyme was more than 90% bound to mixed micelles, regardless of the type of emulsion used. Comparable results were obtained by replacing the bile salts with human gallbladder bile. In parallel, pancreatic zymogen was never found to be bound to any of the lipid structures present (emulsion or mixed micelles). When the catalytic site of pancreatic phospholipase A2 was blocked with 4-bromophenacylbromide, there was no fixation on mixed micelles. Fixation was restored by the presence of lysolecithins and fatty acids in the incubation mixtures. The partial transformation of all emulsified substrates to mixed micelles by bile salts in vivo would thus lead to optimum activity of pancreatic phospholipase A2.

Pancreatic lipase requires colipase, a protein cofactor, to counteract the in vitro inhibition by bile salt. Lipase activity is inhibited by nonsteroidic detergents regardless of their charge and structure. Detergent-inhibited lipase is reactivated by colipase but in all cases activation is limited to a narrow range of detergent concentration. Complementary studies on the bile salt and detergent effect on lipase activity and on interfacial tension at the substrate-water interface show that inhibition is not related to the interfacial surface tension. It is hypothesized that absorption of amphiphilic compounds to the substrate surface modifies the distribution of the enzyme between the lipid surface and the aqueous phase. The activity of detergent-inhibited lipase is fully restored by adding bile salt to the reaction system. Bile salt might play a critical role during in vivo lipolysis by desorbing surface-active substances from the lipid-water interface thus allowing lipase and colipase to interact with substrate.

A protein, molecular weight 70,000 that inhibits pancreatic lipase has been isolated from soybean seeds. Inhibition is not reversed by colipase unless bile salts are added to the assay system. Inhibitory properties of the purified protein are very similar to those of serum albumin or alpha-lactoglobulin. It has been confirmed that, during intestinal lipolysis of dietary fats, bile salts play an essential role for the activation of the lipase-colipase system in the presence of inhibitory proteins. The purified soybean lipase inhibitory protein was shown to be highly surface-active and able to penetrate monomolecular films of various glycerides and phospholipids at high surface pressure. Inhibition of pancreatic lipase by proteins is related to their capacity to interact with lipids and to modify the quality of the substrate-water interface. The protein isolated from soybeans inhibits pancreatic and Rh. delemar lipase in contrast to the Rh. arrhizus enzyme.