In vitro studies on interaction of rat pancreatic lipase and colipase with biliary lipids

Lipase and colipase were prepared separately from rat pancreatic juice, and their respective interaction with biliary lipids was investigated by gel filtration on agarose in the presence of a micellar solution of sodium taurocholate. It was found that the cofactor can associate with the biliary lipids, whereas the enzyme forms a high mol wt complex only in the presence of colipase. It is suggested that biliary phospholipids might participate in the in vivo formation of the enzyme-cofactor substrate complex at the triglyceride-water interface in the presence of bile salts.     

Uncoupling of catalysis and colipase binding in pancreatic lipase by limited proteolysis

In the intestine, the hydrolysis of triglycerides by pancreaticlipase is performed only in the presence of colipase, whosefunction is to anchor lipase to the bile-salt-coated lipid interface.Biochemical and crystallographk data on porcine and human Upaseshave shown that the molecule is made of two well-delimited domains.In order to get more information on the role of the domainsin catalysis and colipase binding, we performed limited proteolysison lipase from various species and obtained different patternsof cleavage. In the case of porcine and human Upases, only theC-terminal domain (12 kDa) could be obtained after chymotrypticattack, whereas in the horse enzyme the cleavage of the Leu410-Thr411bond gave rise to a large N-terminal (45 kDa) and a small C-terminal(4 kDa) fragment. The isolated porcine and human C-terminaldomains were completely inactive towards emulsified tributyrin,though were able to bind colipase. Conversely, the horse 45kDa fragment retained the lipase activity but failed to correctlybind colipase. This work definitely proves that catalysis andcoUpase binding are separate events involving topographicallydistinct regions of the molecule and focuses attention on therole of the C-terminal domain in colipase binding     

Recombinant C-terminal domain of pancreatic lipase retains full ability to bind colipase

The organization of the pancreatic lipase in two well defineddomains has been correlated to a specific function for eachdomain, catalytic activity for the N-terminal domain and colipasebinding for the C-terminal domain. In order to see if such anorganization implies that the two domains can behave as separateentities, we expressed the N- and C-terminal domains in insectcells. The recombinant proteins secreted in the cell supernatantspresent the expected molecular properties. However, whereasthe C-terminal domain retains its function of colipase binding,the N-terminal domain appears to be unable to ensure catalysis.The lack of activity of the recombinant N-terminal domain couldresult either from a (partially) incorrect folding or from anincapacity to function by itself. These results suggest that,although both are structurally well defined, the two domainsof the pancreatic lipase behave differently when they are expressedas separate entities.     

A colorimetric assay of pancreatic lipase: Rapid detection of lipase and colipase separated by gel filtration

A rapid assay for pancreatic lipase (E.C., glycerol-ester hydrolase 3.1.1.3) is described. The assay is based on the color change of a pH indicator as butyric acid is released from the substrate tributyrin. A mixture made with tributyrin and the water soluble components of the assay is ideally suited for use as a rapid test as, for example, when assaying chromatography fractions. Quantitative data can be obtained by measuring the disappearance of absorbance at 557 nm versus a blank reaction. The assay has been used in the rapid preparation of colipase-free lipase and colipase.     

Effect of biliary obstruction on canine plasma and biliary lipids

The common bile duct was obstructed in 17 dogs. Reciprocal changes were noted for the plasma and biliary lipid concentrations of each after obstruction. As the plasma lecithin and free cholesterol concentrations increased, the biliary lipid concentrations declined. After biliary obstruction the reflux of biliary lecithin into the plasma of these animals was demonstrated with both labeled and unlabeled lecithin. The plasma lipid abnormalities seen after acute biliary obstruction were closely simulated by the reflux of lecithin alone from the biliary tree. The isolated reflux of biliary tract taurocholate produced a distinct lowering of plasma phospholipid and cholesterol concentrations, quite different from the plasma lipid alteration noted with acute biliary obstruction. Similar to observations in human obstruction, some of the plasma lipid was in mesophase form after these animals were obstructed.     

Effect of platelet-activating factor on lipoprotein lipase and blood lipids

We investigated the effect of platelet-activating factor (PAF) and of the PAF specific antagonist CV-6209 on plasma lipid metabolism, and particularly on post-heparin plasma lipolytic activity in male Wistar rats. Lipoprotein lipase (LPL) activity was enhanced by intravenous injection of PAF before intravenous injection of heparin when the PAF dose was low (0.2 μg/kg). PAF activated hepatic triacylglycerol lipase (HTGL) activity dose-dependently. Plasma triacylglycerols (TG) significantly decreased with the activation of LPL and/or HTGL. Plasma total cholesterol (TC) and phospholipid (PL) levels decreased at a low dose of PAF (0.2 μg/kg), but increased when higher doses were used. The PAF antagonist CV-6209 partially reversed the PAF induced effects on HTGL, TC and PL. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Glycodihydrofusidate: Biliary excretion and its effect on biliary secretion of the rat

Glycodihydrofusidate, which has the same detergent properties as bile salts, is excreted almost exclusively by the bile duct after intravenous injection in the rat. As with bile salts, it leads to a significant (P≤0.05) increase in excretion of lecithins and cholesterol (0.15 μmol lecithin and 0.026 μmol cholesterol per 1 μmol of glycodihydrofusidate excreted). In addition, this drug simultaneously inhibits excretion of both endogenous bile salts and bile pigments.     

Lipolysis of trivernolin by pancreatic lipase

Vernolic acid (cis-12,13-epoxy-cis-9-oetadece-noic acid) occurs as the triglyceride in the seed ofVernonia anthelmintica. Incubation of the seed produces a 1,3-divernolin. To determine whether the structure of trivernolin is responsible for the apparent secondary ester position specificity of the natural enzyme, trivernolin and tri-olein, were incubated with pancreatic lipase and the free fatty acids and monoglycerides were determined after 5 and 15 min digestion periods. The preponderance of 2-monoglyceride produced by the action of pancreatic lipase was interpreted to indicate that the structure of trivernolin was not solely responsible for the secondary position specificity of theV. anthelmintica lipase toward trivernolin.     

Effect of lipids and surfactants on extracellular lipase production by Yarrowia lipolytica

The production of extracellular lipase in submerged cultures of Yarrowia lipolytica CECT 1240 has been investigated. Several compounds have been added to the culture medium, in order to assess their efficiency as inducers of lipase production. First, the effect of triglycerides (olive oil, sunflower oil, tributyrin) and fatty acids (oleic acid) has been studied. The highest activity level was obtained with sunflower oil (58 U cm^?3), followed by olive oil (49 U cm^?3). The cultures with tributyrin and oleic acid attained similar activities (33 U cm^?3). Then, several surfactants (Tween 80, Triton X‐100, gum arabic, polyethylene glycol 200) were added to the cultures with sunflower oil, in an attempt to increase the levels of extracellular lipase activity. The obtained activities were slightly lower than those achieved without surfactants. The assay of a wide range of surfactant concentrations in the case of PEG‐200 (with which the highest activity levels had been attained) did not improve the results. This strain secreted lipase concentrations two‐fold higher and showed significantly different behaviour towards the presence of surfactants in the culture medium, compared with other wild‐type Yarrowia lipolytica strains. Copyright © 2003 Society of Chemical Industry     

Dietary fat effect on incorporation and release of lipids and cholesterol by rat intestinal slices

The effect of saturated and unsaturated fats on in vitro formation and release of lipids and cholesterol from¹⁴C acetate by rat intestinal tissue was investigated. The rats were fed a basal diet enriched with either 25% corn oil or lard and then sacrificed after a 10- or 25-day feeding period. It was observed that a similar¹⁴C lipid content but a greater¹⁴C cholesterol content was found in the intestinal tissue of rats fed corn oil than in rats fed lard for 10 days. After a longer period of feeding of 25 days, the intestinal tissue¹⁴C cholesterol level was decreased in the corn oil fed rats without any significant effect on other lipids. These data suggest that corn oil in some way influences cholesterol biosynthesis depending upon its degree of unsaturation and the period of time for which it is fed. The decrease at the later time might involve some mechanism which aids in getting rid of accumulated tissue cholesterol. Less¹⁴C lipid and¹⁴C cholesterol were released by the intestinal tissue of rats fed the unsaturated fat as compared with those fed the saturated fat, suggesting a possible role in vivo in reducing blood lipids and blood cholesterol levels. Robert A. Welch Foundation.     

Digestion of butyrate glycerides by pancreatic lipase

The racemic triglycerides, glyceryl-1-palmitate-2,3-dibutyrate (PBB), glyceryl-1-butyrate-2,3-dipalmitate (PPB), glyceryl-2-butyrate-1,3-dipalmitate (PBP), and the diglyceride, racemic glyceryl-1-palmitate-3-butyrate (P-B) were synthesized and digested with pancreatic lipase. Each triglyceride was mixed with equimolar amounts of triolein (OOO) prior to incubation. The following order of digestion rates was observed: PBB>PPB>PBP>P-B. There was no evidence for short-chain fatty acid specificity; however the triglycerides containing butyric acid were hydrolyzed more rapidly than OOO. Based upon the fatty acid composition of partial glycerides, digestion of butyrate glycerides was not a simple phenomenon. For example, in the digestion of PBB, butyric acid accumulated faster than palmitic acid in the diglycerides, and monobutyrin was found to accumulate when the diglyceride, P-B, was digested. As evidenced by the fatty acid composition of the monoglycerides, positional specificity of pancreatic lipase was always maintained. Scientific contribution No. 245, Agricultural Experiment Station, University of Connecticut, Storrs. Presented in part at the AOCS Meeting, Philadelphia, October 1967.     

The effect of lipids on taurocholate absorption from intestinal loops in the rat

The rates of uptake and serosal trans-fer of [¹⁴C]-labelled taurocholate (7.77 mM in bicarbonate buffer, pH 6.5) were determined in situ in ligated segments of rat intestine in the presence of lipids. Oleic acid, monoolein, lecithin, and lysolecithin enhanced taurocholate uptake and transfer in the jejunum, each lipid exhibiting and optimal concentration at which the bile acid fluxes were maximal. The maximal rates of bile acid uptake observed with the various lipids were close to four times the uptake rates found with the lipid-free taurocholate medium, whereas serosal transfer rates under optimal conditions were enhanced about six-fold. The optimal concentrations differed widely among the various lipids, being inversely related to the lipids’ polarity. Simultaneous measurement of taurocholate and [³H]-labelled oleic acid showed that under optimal conditions, when the molar concentration of oleic acid was about equal to that of the bile acid, the fatty aicd and bile acid also exhibited closely similar rates of absorption. At other fatty acid concentrations, the fractional rate of absorption of the bile acid was much lower than that of the fatty acid. The rates of uptake and serosal transfer of pure taurocholate by the ileum exceeded those of the jejunum by factors of about 7 and 15, respectively, but in the presence of lipids this difference in absorptive capacity for bile acid between the distal and proximal segment largely disappeared.     

Effect of fructose on rat lipids

Young and mature albino rats were fed Purina chow with and without fructose in the drinking water, and the lipid contents in the serum and livers were determined. Fructose elevated the serum triglyceride and caused an accumulation after 24 hr of liver triglyceride in fed but not fasted mature rats. In young male rats, the liver triglyceride was increased initially but was not found after 10 days. Serum phospholipids were increased in young and mature rats; the content and specific activity of the high density (HD)-lipoproteins being increased in young rats. In vivo incorporation of labeled acetate into liver cholesterol was reduced. Results suggest that fructose or triglyceride metabolism, or both, in rats differ with age.     

Hydrolysis of synthetic triacylglycerols by pancreatic and lipoprotein lipase

The stereochemical course of the hydrolysis of synthetic sn-glycerol-1-palmitate-2-oleate-3-linoleate, sn-glycerol-1,2-dipalmitate-3-oleate and their antipodes by pancreatic and milk lipoprotein lipase was investigated by thin layer and gas liquid chromatographies of the diacylglycerol intermediates. The enzymic hydrolyses were made with bile salts or lysolecithin in a 1∶1 molar ratio to the substrate as emulsifiers and were limited to short time intervals which minimized isomerization and the reversal of lipolysis. In all instances, the products of hydrolysis by lipoprotein lipase contained a marked preponderance of the 2,3-diacylglycerols, while the composition of the diacylglycerol intermediates in the products of pancreatic lipase varied with the nature of the fatty acid in the 1 and 3 positions of the triacylglycerol molecule. Pancreatic lipase, but not lipoprotein lipase, gave a preferential release of unsaturated fatty acids. The above results are similar to those obtained with radioactive trioleoylglycerol and conventional stereospecific analyses and suggest that lipoprotein lipase may favor attack on the sn-1 position. It is hypothesized that the small amounts of the 1,2-diacylglycerols present may have arisen from a reversal of lipolysis also catalyzed by this enzyme. Presented in part at the AOCS Fall Meeting, Ottawa, September 1972.     

Analytical supercritical fluid extraction with lipase catalysis: Conversion of different lipids to methyl esters and effect of moisture

The fat content of lipid-containing samples has been determined by extraction of the fat with supercritical carbon dioxide, followed by enzyme-catalyzed methylation of the fat under supercritical conditions, prior to gas chromatography (GC) analysis. This study was initiated to determine the effect of moisture content on the extraction and conversion of lipids in oilseed and meat samples to their fatty acid methyl ester (FAME) derivatives. These samples were freeze-dried or mixed with Hydromatrix and compared with untreated control samples by employing the above-described supercritical fluid extraction-reaction sequence. Particular attention was focused on minor constituents, such as phospholipids and cholesteryl esters, to see if they could be extracted and derivatized by the above technique. Recoveries and reaction conversions of the lipid species were determined with the aid of GC, high-performance liquid chromatography, and supercritical fluid chromatography for analyses of the extracted lipids. Total fat values were higher from the freeze-dried meat and oilseed samples than from samples mixed with Hydromatrix or left untreated. Extraction of cholesteryl esters was better than 90%, and conversion of the cholesteryl esters to FAME was 93% or higher. Extraction of phosphatidic acid was only 88% compared to more than 90% recoveries for the other phospholipid species. FAME conversion was better than 96% for all phospholipid samples in the study.     

Effect of magnesium deficiency on post-heparin lipase activity and tissue lipoprotein lipase in the rat

Previous studies have provided evidence that Mg deficiency affects lipid metabolism. The present experiments were designed to assess whether the hypertriglyceridemia associated with Mg deficiency was related to alterations in post-heparin lipase activity (PHLA). Mg-deficient and control diets were pair-fed to weanling Wistar rats for eight days and plasma lipoproteins were separated into various density classes by sequential preparative ultracentrifugation. Triglycerides were significantly increased in chylomicrons and in the very low density lipoprotein, low density lipoprotein and high density lipoprotein (HDL) fractions. Cholesterol and phospholipid levels were significantly lower in the HDL fraction. PHLA in deficient rat was substantially lower than in control rats. The inverse correlation between plasma triglyceride concentration and PHLA strongly suggests that hypertriglyceridemia is the result of defective lipolysis of plasma triglycerides in Mg-deficient rats. Further examination of the PHLA was carried out by salt-mediated inhibition of lipoprotein lipase (LPL) and by heparin sepharose affinity chromatography and purified rat LPL antiserum. The results indicate that hepatic lipase is significantly decreased in Mg-deficient rats but the low PHLA is due mainly to a decline in LPL. However, total LPL activity, that is, both the intracellular and the extracellular oools of LPL in adipose tissue, heart and diaphragm, were unaffected by Mg deficiency. The results suggest that the decrease of LPL activity in the plasma of Mg-deficient rats may be due to a selective decrease in the heparin-releasable pool of enzyme.     

The incorporation of fatty acids of different chain length into liver and biliary lipids in the perfused rat liver

In an attempt to correlate the incorporation of fatty acids (FA) of different chain length into liver and biliary lipids’ isolated rat livers were perfused for 2 h with Krebs-Ringer bicarbonate containing 1% albumin and 10 μmol of [1-¹⁴C]-labeled FA: C₂’ C₈’ C₁₀’ C₁₂’ C₁₆’ and C_18∶1. One to 1.36 μmol of medium-chain fatty acids (MCFA’ C₈’ C₁₀’ and C₁₂) and 6.6 μmol of long-chain FA (LCFA) were incorporated into liver lipids’ 40% of the latter into phosphatidylcholine (PC). ¹⁴C-acetate (13 nmol) was incorporated into biliary cholesterol; ¹⁴C-MCFA contributed only 3.2–5 nmol; LCFA did not lead to newly synthesized cholesterol. Newly synthesized liver PC (2.75 to 3.25%) and newly synthesized liver cholesterol (6.5 to 10%) were secreted into bile. The specific radioactivity of biliary PC after infusion of all-saturated FA was 3.8–6.8 times higher than that of liver PC; for C_18∶1 it was only 1.7-fold. The specific radioactivity of biliary cholesterol’ as compared to liver cholesterol’ was 12 times higher for C₂ and five times higher for MCFA. This indicates that a considerable proportion of the newly synthesized lipids was secreted into bile prior to significant mixing with preexisting liver PC and cholesterol pools. liver PC contained 8% of unchanged ¹⁴C−C₁₂; while ¹⁴C−C₁₀ was not detected. Biliary PC’ in contrast’ contained 18% of unchanged ¹⁴C−C₁₂ and 3% ¹⁴C−C₁₀. These results suggest that after prolonged infusion of medium-chain triacylglycerols/longchain triacylglycerols to patients’ biliary PC may become enriched with MCFA. In addition’ the oxidation of these FA may provide C-2 units which increase cholesterol synthesis.     

The effect of sonication on lipase activity

Studies were made to evaluate the effect of sonication on activity of porcine pancreatic lipase and on activity of this lipase when in contact with substrate. Lipase was rapidly inactivated by sonication at 50 C, but was stable at 30 C or lower temperatures. Sonication for 4.5 min of lipase and olive oil at 38 C produced 2.7 times as much free fatty acid as without sonication. Hydrolysis of tripalmitin was achieved by sonication of lipase and tripalmitin dissolved in methyl myristate. Journal Paper No. 4181 of the Michigan Agricultural Experiment Station.     

Pancreatic and microbial lipases: A comparison of the interaction of pancreatic colipase with lipases of various origins

Conjugated bile salts inhibit the hydrolysis of triglycerides (TG) by the lipases fromRhizopus arrhizus andGeotrichum candidum. This occurs for detergent concentrations similar to those which suppress the action of mammalian pancreatic lipases upon the same substrates. However, in opposition with what is observed with the latter enzymes, the activity is not restored by the addition of pancreatic colipase. Both pancreatic andR. arrhizus lipases are inactivated at tributyrin/water interface, but only the first enzyme is protected against this surface denaturation by the pancreatic cofactor. These observations suggest that colipases synthesized in mammalian pancreas display specific interaction towards the lipases made by the same organ.     

Interaction of porcine pancreatic colipase with a nonionic detergent,triton X-100: Spectrophotometric studies

Strong perturbation of the ultraviolet spectrum of the tyrosines of porcine pancreatic colipase A is observed in the presence of Triton X-100 at concentration above the critical micellar concentration. Spectrophotometric titration of the phenolic groups of the protein shows that the apparent pKa value for two tyrosines is about 10.3, while the third tyrosine has a higher pKa value above 11.6. This residue is still protonated at pH 13 in the presence of Triton X-100. All perturbations induced by the nonionic detergent can be interpreted as resulting from interactions between colipase and Triton X-100 molecules at a hydrophobic site of the protein that includes the tyrosine residues. Results obtained in studies with Triton X-100 are similar to those already reported by Sari et al. (Eur. J. Biochem. 58∶561 (1975) on the interaction of colipase with taurodeoxycholate. It is likely that the binding of both types of detergent occurs at the same specific site on the protein molecule. Data presented in this communication give further support to the hypothesis that a hydrophobic domain (residues 49–57), including all three tyrosines of the colipase molecule, participate to the well characterized interaction of the lipase cofactor with triglycerides at lipid-water interfaces.