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PubMed1994

Evidence for a pancreatic lipase subfamily with new kinetic properties.

Several new members of the pancreatic lipase family have been reported recently, and amino acid sequence comparison reveals that this family can now be divided into three subgroups: (1) "classical" pancreatic lipases, (2) related proteins 1 (RP1), and (3) related proteins 2 (RP2) (Giller, T., et al. (1992) J. Biol. Chem. 267(23), 16509-16516). Whereas "classical" pancreatic lipases are well characterized with respect to kinetic properties, i.e., interfacial activation and dependence on colipase in the presence of bile salts, the two latter subfamilies have been poorly investigated so far. The kinetic behavior of a lipase from guinea pig pancreas differs, however, from that of "classical" lipases (Hjorth, A., et al. (1993) Biochemistry 32, 4702-4707). This enzyme is highly homologous to RP2 lipases with the exception of a deletion in the so-called lid domain that regulates access to the active center of pancreatic lipases. We have now characterized a novel lipase from coypu (Myocastor coypus) pancreas. This enzyme, also belonging to the RP2 subfamily, possesses a full-length lid domain, but its kinetic properties are very similar to those of the guinea pig enzyme: (1) a high phospholipase activity, (2) the absence of interfacial activation, and (3) the absence of a colipase effect at high bile salt concentrations. Since both guinea pig and coypu pancreas produce a classical pancreatic lipase and no measurable phospholipase A2 activity, it is suggested that RP2 enzymes act as real phospholipases under physiological conditions. In fact, all RP2 lipases from other species might share phospholipase activity and fulfill new biological functions.

Amino Acid Sequence↗

PubMed1983

Pancreatic hydrolases in cold-induced hyperphagia of rats fed a low or high-fat diet.

Rats fed either a low (2p. 100) or high (40 p. 100)-fat diet were exposed to 22 or 5 degrees C. The resulting hyperphagia adequately compensated energy losses as judged from body weight. The cold-induced hyperphagia was accompanied by a non-parallel increase in pancreatic hydrolases. Amylase and lipase were not increased above the adaptive levels they had respectively reached in the heat with a high-starch or high-lipid diet. Chymotrypsinogen, on the contrary, responded to increased intake of both diets. It also responded to the higher protein concentration in the high-fat diet caused by isocaloric replacement of starch by fat. Colipase varied independently of lipase and was increased additively by fat and protein intakes. Consequently, although limiting for lipase in the warm, colipase rose to a 1:1 ratio in the cold. Increased intake had a consistent pleiotropic effect evidenced by an increase of amylase with the high-fat diet and of lipase with the low-fat diet. The net effect was a significant increase in the lipid-digesting potential of the organism of lipid-fed animals upon exposure to cold, while the starch-digesting potential remained unaffected in starch-fed animals.

Animals↗

PubMed1985

Effects of dietary fibers and cholestyramine on the activity of pancreatic lipase in vitro.

Most experiments were conducted in the presence of human gallbladder bile; colipase and pancreatic lipase were purified using porcine pancreas. The adsorption of bile salts, phospholipids and cholesterol from the bile, together with that of pancreatic lipase was measured on wheat bran, cellulose, hemicellulose (xylan), slightly methylated pectin (42%) and cholestyramine. In contrast to cholestyramine which intensively binds biliary lipids (61.7-81.7%) and pancreatic lipase (47.5%), the fibers studied only had a low adsorbent power. The direct influence of these fibers and of cholestyramine at concentrations ranging from 0-5% on lipase activity was measured at constant pH, using two conventional assay systems, long chain triglycerides and tributyrin. In the presence of human bile and colipase, a drastic reduction in triglyceride hydrolysis by lipase was observed with cholestyramine (loss of 66-82%) and wheat bran (loss of 77-94%) at 1% concentration. The other fibers did not have any marked effects on enzyme activity. The use of a radio labeled lipase made it possible to demonstrate that the inhibitory effect of bran on enzyme activity was independent of adsorption phenomena on bran. The fraction of bran that can be solubilized in the aqueous phase, in fact, induced this reduction in activity. The presence of protein inhibitor in bran may be responsible for the reduction in pancreatic lipase activity.

Animals↗

PubMed2004

The enzyme levels in blood are not affected by oral administration of a pancreatic enzyme preparation (Creon 10,000) in pancreas-insufficient pigs.

After oral intake, small amounts of intact protein may be absorbed into the blood circulation. The current study investigated whether orally administered pancreatic enzymes were absorbed from the intestine. The study included 28 pigs; 3 control pigs with intact pancreatic function and 25 pigs that were made exocrine pancreas insufficient by duct ligation (20 pigs) or total pancreatectomy (5 pigs). The pigs received a pancreatic enzyme preparation (0, 2, 4, or 8 g of Creon 10,000) together with the feed. The blood plasma was analyzed for pancreatic lipase activity with a [3H]-triolein substrate assay, while (pro)colipase and cationic trypsin(ogen) levels were measured with enzyme-linked immunosorbent assay (ELISA). Administration of Creon (0-8 g) caused no significant changes in plasma (pro)colipase or cationic trypsin(ogen) levels. Lipase activity peaks in plasma samples were found, but they did not correspond to the administration of Creon. The potential source of these plasma lipase activity peaks is discussed. The results showed no absorption into blood of pancreatic enzymes after oral administration (0, 2, 4, or 8 g of Creon mixed with 100 g of feed) to pancreas-insufficient pigs.

Administration, Oral↗

PubMed1982

Snake venom: protein CM-2 from Bitis arietans (puff adder) venom.

Protein CM-2 from Bitis arietans venom was purified by chromatographic procedures involving Sephadex G-50 and CM-cellulose. The purified protein comprises 82 amino acids including 14 half-cystine residues and its primary structure has been elucidated. Protein CM-2 is not toxic. Although the protein clears a suspension of egg yolk, structural features and the inability to hydrolyse L-alpha-lecithin reveal that it cannot be a phospholipase A2. In spite of its sequence showing some homology with that of porcine colipase, protein CM-2 is not a colipase.

Amino Acid Sequence↗

PubMed1993

Purification of lipase and other lipolytic enzymes from the human pancreas.

A pure lipase has been isolated from extracts of the human pancreas. The purification process includes centrifugation, two ion-exchange chromatography steps, and one gel filtration step. Compared with other reports, a high recovery, large amounts, and a high specific activity were obtained. Lipase is present at 1-2 mg/g in the pancreatic gland. In the absence of colipase and bile salts with tributyrine as substrate, the specific activity at room temperature and at pH 7.0 is 4000 mumol/min/mg. It increases to 8000-10,000 in the presence of colipase and bile salts at a temperature of 37 degrees C. The fate of the other human lipolytic proteins during the different purification steps is also indicated. Lipase purified by this method has been used for crystallization.

Chromatography, Agarose↗

PubMed1983

Effects of age and weaning on enzyme activities of abomasum and pancreas of the lamb.

The effect of age and weaning on enzyme activities of abomasum and pancreas of the lamb was studied. Sixty-nine lambs were either maintained at the preruminant stage until 42 days of age or weaned between 20 and 30 days. They were slaughtered between birth and 100 days, and the mucosa of their abomasum and their pancreas were collected. At 2 days of age there was a threshold of glandular growth and of development of enzyme secretory potentialities. Pancreas showed an extensive hyperplasia without growth until day 2; its weight increased after day 7 but only by hypertrophy. Quantities of gastric enzymes in relation to empty live weight increased between birth and 2 days, but that of chymosin then decreased, whereas pepsin did not change significantly; effects of weaning were similar. The evolution of pancreatic enzyme activity was usually the reverse of that of chymosin; however, trypsin activity was low at birth and that of colipase did not increase at weaning. The ratio of colipase to lipase was always higher than 1, showing that lipase always was saturated in the lamb. The pancreas seemed to take over the secretion of abomasal proteolytic enzymes. Potentialities of enzyme digestion appeared to be influenced mainly by the age or weight of the animal; these potentialities were minimal for at least several days during the first neonatal week and, if there was enzymatic adaptation to the amounts of the ingested substrate, it did not occur rapidly.

Abomasum↗

PubMed2001

Intact intestinal mRNAs and intestinal epithelial cell esterase, but not Cryptosporidium parvum, reach mesenteric lymph nodes of infected mice.

Dendritic cells from the mesenteric lymph nodes (MLN) contain dense esterase-positive inclusions that may originate in effete intestinal epithelial cells and reach MLN without degradation. The MLN esterases have the electrophoretic mobilities of both intestinal and mononuclear cells. Cryptosporidium parvum (CP)-infected mice have CP Ag-positive cells in MLN and also increased numbers of dense esterase-positive cells, but the CP Ag-positive cells do not stain for esterase. To characterize the handling of epithelial cell products by dendritic cells, we analyzed mRNAs in the MLN of control and CP-infected recombination-activating gene(-/-)DO11.10 mice by oligoarrays. mRNAs for 115 proteins were increased in MLN after CP infection, of which the principal increases in trypsin and chymotrypsin approximated to 250-fold. Colipase, reg-1, C-reactive protein-ductin, and amyloid were also up-regulated >10-fold and all returned to baseline by 28 days after infection. mRNAs for the same proteins were detected in intestinal epithelial cells of infected mice by oligoarrays and RT-PCR after infection. mRNA for CP beta-tubulin was detectable in intestinal epithelial cells between 5 and 18 days after infection but was not detected in the MLN throughout the observation period. It appears that host response to CP infection includes expression of mRNA for some pancreatic enzymes by intestinal epithelial cells and their subsequent transport to the MLN. The esterase and trypsin, and mRNAs for chymotrypsin, colipase, and others that may derive from uninfected epithelial cells, appear to be transported to the MLN intact, while mRNA for CP beta-tubulin that is derived from infected cells is degraded.

Animals↗

PubMed2001

Specificity of the lipid-binding domain of apoC-II for the substrates and products of lipolysis.

Functional similarities between colipase and apolipoprotein C-II (apoC-II) in activating lipases suggest that apoC-II may, like colipase, preferentially interact with interfaces containing the substrates and products of lipolysis. To test this hypothesis, the binding of a peptide comprising residues of the cofactor implicated in lipid binding, apolipoprotein C-II(13-56), and, to a lesser extent, apoC-II, to monomolecular lipid films was characterized. The lipids used were a diacylphosphatidylcholine, a diacylglycerol, and a fatty acid. The peptide had an affinity for the argon-buffer interface and for all lipids consistent with a dissociation constant of <10 nM. Changes in surface pressure accompanying peptide binding were comparable to those reported for native apoC-II and indicate peptide miscibility with each of the lipids tested. The capacity of the surfaces to accommodate the peptide decreased with increasing lipid concentration in the interface, indicating competition between lipid and peptide for interfacial occupancy. At a lipid acyl chain density of 470 pmol/cm2, or 35 A2 per acyl chain, a lower limit of peptide adsorption was reached with all lipids. The limiting level of adsorption to phosphatidylcholine was only 1 pmol/cm2 compared with 6;-7 pmol/cm2 for fatty acid and diacylglycerol. Similar results were obtained with apoC-II. The difference in the extent of protein adsorption to lipid classes suggests that the distribution of apoC-II among lipoproteins will depend on their lipid composition and surface pressure.

Adsorption↗

PubMed1984

Studies on the inhibition of pancreatic and microbial lipases by soybean proteins.

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.

Amino Acids↗

PubMed1997

Enterostatin--a peptide regulating fat intake.

A high fat intake, together with an inability to match lipid oxidation to fat intake, has been found to be correlated with obesity in humans. This review describes our current understanding of enterostatin, a peptide that selectively reduces fat intake. Enterostatin is formed in the intestine by the cleavage of secreted pancreatic procolipase, the remaining colipase serving as an obligatory cofactor for pancreatic lipase during fat digestion. Enterostatin is also produced in the gastric mucosa and the mucosal epithelia of the small intestine. Procolipase gene transcription and enterostatin release into the gastrointestinal lumen are increased by high-fat diets. After feeding, enterostatin appears in the lymph and circulation. Enterostatin will selectively inhibit fat intake during normal feeding and in experimental paradigms that involve dietary choice. Its anorectic effect has been demonstrated in a number of species. Both peripheral and central sites of action have been proposed. The peripheral mechanism involves an afferent vagal signaling pathway to hypothalamic centers. The central responses are mediated through a pathway that includes both serotonergic and opioidergic components. Chronically, enterostatin reduces fat intake, bodyweight, and body fat. This response may involve multiple metabolic effects of enterostatin, which include a reduction of insulin secretion, an increase in sympathetic drive to brown adipose tissue, and the stimulation of adrenal corticosteroid secretion. A possible pathophysiological role is suggested by studies that have linked low enterostatin production and/or responsiveness to strains of rat that become obese and prefer dietary fat. Humans with obesity also exhibit a lower secretion of pancreatic procolipase after a test meal, compared with persons of normal weight.

Animals↗

PubMed1994

Human pancreatic procolipase expressed in insect cells: purification and characterization.

A cDNA clone encoding human pancreatic procolipase was incorporated into a recombinant baculovirus. Spodoptera frugiperda insect cells infected with the recombinant baculovirus secreted procolipase into the medium, which could be isolated in a single step by immunoaffinity chromatography. The highly purified protein reactivated human pancreatic lipase in a concentration-dependent fashion and was efficiently converted to colipase by limited trypsin digestion. This expression system is suitable for producing amounts of procolipase adequate for biophysical studies.

Animals↗

PubMed1998

Purification and characterization of human procolipase expressed in yeast cells.

We report the successful, efficient, and large-scale expression of recombinant human procolipase in yeast. Using the full-length cDNA of human procolipase, constructs were made using either the native human procolipase signal peptide sequence or the signal peptide sequence of yeast. These constructs were used to transform yeast cells, and expression was followed. Only minimal expression was seen with the procolipase using the native human signal peptide. Robust secretion of the procolipase occurred when the yeast signal peptide was exchanged for the native signal peptide. Expression yielded more than 30 mg/liter. The recombinant protein was purified from the medium by immunoaffinity chromatography. The highly purified procolipase was free of proteolytic degradation and displayed activity and binding characteristics that were indistinguishable from those of tissue-purified human pancreatic colipase. Expression in yeast cells provides a useful tool for expressing intact, unprocessed recombinant wild-type and mutated procolipase.

Chromatography, Affinity↗

PubMed1978

Adsorption and activation of pancreatic lipase at interfaces.

The first step of the lipase-catalyzed hydrolysis of insoluble long chain triglycerides is the adsorption of the enzyme to the interface. This adsorption, which is spontaneous when the interface is hydrophobic, is hindered by bile salts. Under these conditions, a small protein cofactor designated colipase adsorbs first and then anchors lipase at the interface. Interfacial adsorption enhances lipase activity, due, at least in part, to an acceleration of the rate-limiting deacylation step of the reaction. In this respect, lipase appears to be a most interesting model of an enzyme being activated by the presence of a lipid. The 3 steps of the heterogeneous catalysis induced by lipase, interfacial adsorption, interfacial activation and catalysis proper are under the control, respectively, of a serine hydroxyl group, a carboxyl and a histidine imidazole.

Adsorption↗

PubMed1990

Lipolytic enzymes of the human pancreas. III. Auxiliary function of lipase in the cholesterol-esterase-dependent oral test on exocrine pancreatic output.

In the oral exocrine pancreatic function test using fluorescein dilaurate, this synthetic substrate attaches primarily to the triglyceride surfaces of the neutral lipids administered as part of the breakfast: these fluorescein dilaurate molecules cannot be attacked by cholesterol esterase. In the course of triglyceride saponification by lipase and colipase, however, the fluorescein dilaurate is liberated and hydrolyzed by cholesterol esterase. The pancreatic function test, therefore, measures the lipolytic activities not merely of cholesterol esterase, but indirectly of lipase, as well.

Bile Acids and Salts↗

PubMed1997

The effect of selective sympathetic denervation on pancreatic exocrine secretion.

The purpose of this study was to investigate the influence of selective sympathetic denervation of the rat pancreas on exocrine secretion and to study whether the observed effects were due to pancreatic trophism. Sprague-Dawley rats were divided into two groups. One group underwent selective sympathetic denervation by skeletonizing the superior and inferior pancreaticoduodenal and splenic arteries. The other group underwent simple laparotomy and served as controls. One week after the operation a catheter was introduced into the bile-pancreatic duct and pancreatic juice was collected at 30-min intervals for 4 h. The output of bicarbonate, total protein, amylase, trypsin, chymotrypsin, lipase, colipase and carboxyesterlipase were determined. Following denervation secretion of pancreatic enzymes was significantly enhanced compared with sham-operated animals. We did not find any signs of pancreatic trophism 1 week after denervation.

Amylases↗

PubMed1980

The influence of bile salts and bile lipoprotein complex on pancreatic lipase hydrolysis of monomolecular films.

We report a new technique which allows us to follow the lipolysis of monomolecular films in the presence of bile salts by using a 'zero-order' trough (Verger, R. and de Haas, G.H. (1973) Chem. Phys. Lipids 10, 127). The effects of bile salts, the bile lipoprotein complex and colipase on pancreatic lipase hydrolysis of rac-1,2-didodecanoylglycerol films were studied at different surface pressures. Taking into account previous studies, lipase activity was interpreted as a function of its degree of binding to the bile lipoprotein complex.

Animals↗

PubMed1984

Substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas. I. Studies on neutral glycerides.

The substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas has been tested towards various neutral glycerides. Triolein hydrolysis proceeded in the absence of di- and monoolein accumulation. Optimal conditions for di- and monoolein hydrolysis included an alkaline pH (9-10), a substrate concentration of 10 mM, and the presence of sodium deoxycholate (12 and 24 mM, respectively). Pancreatic colipase (bovine) had no effect on the activity of the two lipases. The comparison between the rates of hydrolysis of various substrates revealed the following order of decreasing enzyme activity: diolein greater than 1(3)-monoolein greater than tributyrin = triacetin greater than or equal to triolein = 2-monoolein. No hydrolysis of p-nitrophenylacetate and cholesteryloleate could be detected. Using 1-[3H]palmitoyl-2-[14C]linoleoyl-sn-glycerol, both enzymes displayed a strong preference for the 1-position, leading to the accumulation of 2-[14C]linoleoyl-sn-glycerol. Identical activities were found for the two lipases. It is concluded that the two cationic lipases from guinea pig pancreas represent a unique group of lipolytic enzymes different from other previously described enzymes, including classical pancreatic lipase, gastric and lingual enzymes, mold lipases and carboxylesterhydrolase.

Animals↗