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PubMed1992

Inhibition of rat alveolar macrophage phagocytic function by a Pseudomonas cepacia lipase.

The effects of purified Pseudomonas cepacia lipase on rat pulmonary alveolar function and morphology were examined. Lipase (2.5-20 micrograms/ml) adversely effected the phagocytic function of rat pulmonary alveolar macrophages in a dose-dependent manner. The lipase itself was not directly cytotoxic to these cells. Alveolar macrophages, in the absence of lipase, phagocytosed c. 35% of a given population of opsonised P. cepacia in 30 min when the ratio of bacteria:phagocyte was 10:1. Phagocytosis of P. cepacia by rat pulmonary alveolar macrophages was significantly reduced when the cells were either pre-incubated with the lipase or when phagocytosis occurred in the presence of the lipase. This was confirmed by transmission electronmicroscopy. These functional changes were associated with marked alterations of the macrophage morphology. Scanning electronmicroscopy showed that macrophages exposed to the P. cepacia lipase had fewer specialised surface structures and did not spread on plastic surfaces as well as untreated macrophages. The effects of the lipase were lost after heat inactivation, which indicates that the effects of the P. cepacia lipase were due to its enzymic activity. These results suggest that, if sufficient quantities of the enzyme are produced in vivo, lipase may be an important virulence factor for P. cepacia, allowing the organism to evade phagocytic cells.

Animals↗
PubMed1994

Polymorphism in the lipase genes of Geotrichum candidum strains.

The fungus Geotrichum candidum produces extracellular lipases. Purification and characterization of different lipase isoforms from various G. candidum strains is difficult due to the close physical and biochemical properties of the isoforms. Consequently, the characterization of these enzymes and their substrate specificities has been difficult. We have determined the lipase genes present in four strains of G. candidum (ATCC 34614, NRCC 205002, NRRL Y-552 and NRRL Y-553) by molecular cloning and DNA sequencing. Each strain contains two genes similar to the previously identified lipase I and lipase II cDNAs. Our data suggest that no other related lipase genes are present in these strains. Each lipase-gene family shows sequence variation (polymorphism) that is confirmed by Southern-blot analysis. This polymorphism and the sequence differences between lipase I and lipase II have been localized within the previously determined three-dimensional structure of lipase II. Although most of the amino acid substitutions are located on the protein surface, some are present in structural features possibly involved in determining substrate specificity.

Amino Acid Sequence↗
PubMed1993

Characterization of a triacylglycerol lipase that liberates arachidonic acid from bovine chromaffin cells during secretion.

Primary cultures of chromaffin cells from bovine adrenal medullae were used as a model to study lipolytic events during stimulus-secretion coupling. It has been shown that chromaffin cells liberate arachidonic acid in addition to their main secretion product, the catecholamines. To understand more about the mechanism of arachidonic acid liberation, chromaffin cells were labeled with radioactive arachidonic acid, stimulated, and then analyzed for changes in lipid composition. After stimulation with 10(-4) M acetylcholine, the radioactivity of triacylglycerols decreased to the same extent that the free arachidonic acid level rose. This finding suggests that in bovine chromaffin cells a stimulation-dependent triacylglycerol lipase (triacylglycerol hydrolase; EC 3.1.1.3) is involved in arachidonic acid liberation. Further work was performed on detection, characterization, and isolation of this enzyme. Triacylglycerol lipase activity was found in whole cell homogenates and in plasma membrane fractions isolated from adrenal medullary tissue. The plasma membrane lipase showed a pH optimum of 4.3. The apparent Michaelis constant was determined as 3.3 x 10(-4) mol/L. Ca2+ did not influence the enzymatic activity. To differentiate the plasma membrane triacylglycerol lipase from the previously described plasma membrane diacylglycerol lipase of chromaffin cells, the influence of RG 80267, a specific diacylglycerol lipase inhibitor, was examined. RG 80267 (50 microM) inhibited the triacylglycerol lipase by only 24%, although diacylglycerol lipase was totally inhibited with only 20 microM RG 80267. The pH optimum of homogenate lipase was broad, lying between 4 and 7. Starting from the soluble fraction of whole cell homogenates, the triacylglycerol lipase was partially purified by ultracentrifugation and size-exclusion chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Genetic and biochemical characterization of a new extracellular lipase from Streptomyces cinnamomeus.

Streptomyces cinnamomeus Tü89 secretes a 30-kDa esterase and a 50-kDa lipase. The lipase-encoding gene, lipA, was cloned from genomic DNA into Streptomyces lividans TK23 with plasmid vector pIJ702. Two lipase-positive clones were identified; each recombinant plasmid had a 5.2-kb MboI insert that contained the complete lipA gene. The two plasmids differed in the orientation of the insert and the degree of lipolytic activity produced. The lipA gene was sequenced; lipA encodes a proprotein of 275 amino acids (29,213 Da) with a pI of 5.35. The LipA signal peptide is 30 amino acids long, and the mature lipase sequence is 245 amino acids long (26.2 kDa) and contains six cysteine residues. The conserved catalytic serine residue of LipA is in position 125. Sequence similarity of the mature lipases (29% identity, 60% similarity) was observed mainly in the N-terminal 104 amino acids with the group II Pseudomonas lipases; no similarity to the two Streptomyces lipase sequences was found. lipA was also expressed in Escherichia coli under the control of lacZ promoter. In the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), growth of the E. coli clone was severely affected, and the cells lysed in liquid medium. Lipase activity in the E. coli clone was found mainly in the pellet fraction. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, three additional protein bands of 50, 29, and 27 kDa were visible. The 27-kDa protein showed lipolytic activity and represents the mature lipase; the 29- and 50-kDa forms showed no activity and very probably represent the unprocessed form and a dimeric misfolded form, respectively. For higher expression of lipA in S. lividans, the gene was cloned next to the strong aphII promoter. In contrast to the lipA-expressing E. coli clone, S. cinnamomeus and the corresponding S. lividans clone secreted only an active protein of 50 kDa. The lipase showed highest activity with C6 and C18 triglycerides; no activity was observed with phospholipids, Tween 20, or p-nitrophenylesters. Upstream of lipA and in the same orientation, an open reading frame, orfA, is found whose deduced protein sequence (519 amino acids) shows similarity to various membrane-localized transporters. Downstream of lipA and in the opposite orientation, an open reading frame, orfB (encoding a 199-amino-acid protein) is found, which shows no conspicuous sequence similarity to known proteins, other than an NAD and flavin adenine dinucleotide binding-site sequence.

Amino Acid Sequence↗
PubMed2001

Disulfide bond in Pseudomonas aeruginosa lipase stabilizes the structure but is not required for interaction with its foldase.

Pseudomonas aeruginosa secretes a 29-kDa lipase which is dependent for folding on the presence of the lipase-specific foldase Lif. The lipase contains two cysteine residues which form an intramolecular disulfide bond. Variant lipases with either one or both cysteines replaced by serines showed severely reduced levels of extracellular lipase activity, indicating the importance of the disulfide bond for secretion of lipase through the outer membrane. Wild-type and variant lipase genes fused to the signal sequence of pectate lyase from Erwinia carotovora were expressed in Escherichia coli, denatured by treatment with urea, and subsequently refolded in vitro. Enzymatically active lipase was obtained irrespective of the presence or absence of the disulfide bond, suggesting that the disulfide bond is required neither for correct folding nor for the interaction with the lipase-specific foldase. However, cysteine-to-serine variants were more readily denatured by treatment at elevated temperatures and more susceptible to proteolytic degradation by cell lysates of P. aeruginosa. These results indicate a stabilizing function of the disulfide bond for the active conformation of lipase. This conclusion was supported by the finding that the disulfide bond function could partly be substituted by a salt bridge constructed by changing the two cysteine residues to arginine and aspartate, respectively.

Biological Transport↗
PubMed1999

Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases.

Bacteria produce and secrete lipases, which can catalyze both the hydrolysis and the synthesis of long-chain acylglycerols. These reactions usually proceed with high regioselectivity and enantioselectivity, and, therefore, lipases have become very important stereoselective biocatalysts used in organic chemistry. High-level production of these biocatalysts requires the understanding of the mechanisms underlying gene expression, folding, and secretion. Transcription of lipase genes may be regulated by quorum sensing and two-component systems; secretion can proceed either via the Sec-dependent general secretory pathway or via ABC transporters. In addition, some lipases need folding catalysts such as the lipase-specific foldases and disulfide-bond-forming proteins to achieve a secretion-competent conformation. Three-dimensional structures of bacterial lipases were solved to understand the catalytic mechanism of lipase reactions. Structural characteristics include an alpha/beta hydrolase fold, a catalytic triad consisting of a nucleophilic serine located in a highly conserved Gly-X-Ser-X-Gly pentapeptide, and an aspartate or glutamate residue that is hydrogen bonded to a histidine. Four substrate binding pockets were identified for triglycerides: an oxyanion hole and three pockets accommodating the fatty acids bound at position sn-1, sn-2, and sn-3. The differences in size and the hydrophilicity/hydrophobicity of these pockets determine the enantiopreference of a lipase. The understanding of structure-function relationships will enable researchers to tailor new lipases for biotechnological applications. At the same time, directed evolution in combination with appropriate screening systems will be used extensively as a novel approach to develop lipases with high stability and enantioselectivity.

Bacteria↗
PubMed1988

Inactivation of human lipase by proteases.

To determine if human lipase is inactivated by trypsin and chymotrypsin, we intubated 22 human subjects with an oroduodenal tube and stimulated pancreatic secretion with cholecystokinin octapeptide. The duodenal aspirate from each subject was divided into a control and a test sample and incubated in a 37 degrees C water bath for 2 h. An inhibitor of trypsin or chymotrypsin or more of one of these enzymes was added to the test sample. We found that the loss of lipase activity was partly prevented by inhibiting trypsin with aprotinin (910 KU/ml; P = 0.03) and was accelerated by adding bovine trypsin (2.5 mg/ml; P = 0.01). Inhibiting chymotrypsin with turkey egg white (2.5 mg/ml) totally abolished the loss of lipase activity (P = 0.01), and addition of bovine chymotrypsin (5 mg/ml) accelerated the loss of lipase activity more than adding trypsin (P = 0.01). After inhibiting chymotrypsin (to maintain lipase activity), increasing trypsin activity by adding a single or repeated doses of trypsin did not decrease lipase activity. Conversely, the addition of a single dose of chymotrypsin after inhibiting trypsin activity markedly decreased lipase activity (P less than 0.004). In conclusion, chymotrypsin is a more potent inactivator of human lipase than trypsin; chymotrypsin inactivates lipase in the absence of trypsin, but trypsin inactivation of lipase requires chymotrypsin.

Adult↗
PubMed1982

Lipoprotein metabolism during acute inhibition of hepatic triglyceride lipase in the cynomolgus monkey.

The role of the enzyme hepatic triglyceride lipase was investigated in a primate model, the cynomolgus monkey. Antisera produced against human postheparin hepatic lipase fully inhibited cynomolgus monkey posttheparin plasma hepatic triglyceride lipase activity. Lipoprotein lipase activity was not inhibited by this antisera. Hepatic triglyceride lipase activity in liver biopsies was decreased by 65-90% after intravenous infusion of this antisera into the cynomolgus monkey. After a 3-h infusion of the antisera, analytic ultracentrifugation revealed an increase in mass of very low density lipoproteins (S(f) 20-400). Very low density lipoprotein triglyceride isolated by isopycnic ultracentrifugation increased by 60-300%. Analytic ultracentrifugation revealed an increase in mass of lipoproteins with flotation greater than S(f) 9 (n = 4). The total mass of intermediate density lipoproteins (S(f) 12-20) approximately doubled during the 3 h of in vivo enzyme inhibition. While more rapidly floating low density lipoproteins (S(f) 9-12) increased, the total mass of low density lipoproteins decreased after infusion of the antibodies. The changes in high density lipoproteins did not differ from those in control experiments. In order to determine whether the increases of plasma concentrations of very low density lipoproteins were due to an increase in the rate of synthesis or a decrease in the rate of clearance of these particles, the metabolism of radiolabeled homologous very low density lipoproteins was studied during intravenous infusion of immunoglobulin G prepared from the antisera against hepatic triglyceride lipase (n = 3) or preimmune goat sera (n = 3). Studies performed in the same animals during saline infusion were used as controls for each immunoglobulin infusion. There was a twofold increase in the apparent half-life of the very low density lipoprotein apolipoprotein-B tracer in animals receiving the antibody, consistent with a decreased catabolism of very low density lipoproteins. Concomitantly, the rise in low density lipoprotein apoprotein-B specific activity was markedly delayed. None of these changes were observed during infusion of preimmune immunoglobulin G.Hepatic triglyceride lipase participates with lipoprotein lipase in the hydrolysis of the lipid in very low density lipoproteins, intermediate density lipoproteins, and the larger low density lipoproteins (S(f) 9-12). Thus, hepatic triglyceride lipase appears to function in a parallel role with lipoprotein lipase in the conversion of very low density and intermediate density lipoproteins to low density lipoproteins (S(f) 0-9).

Animals↗
PubMed1985

Bovine milk lipoprotein lipase transfers tocopherol to human fibroblasts during triglyceride hydrolysis in vitro.

Lipoprotein lipase appears to function as the mechanism by which dietary vitamin E (tocopherol) is transferred from chylomicrons to tissues. In patients with lipoprotein lipase deficiency, more than 85% of both the circulating triglyceride and tocopherol is contained in the chylomicron fraction. The studies presented here show that the in vitro addition of bovine milk lipoprotein lipase (lipase) to chylomicrons in the presence of human erythrocytes or fibroblasts (and bovine serum albumin [BSA]) resulted in the hydrolysis of the triglyceride and the transfer of both fatty acids and tocopherol to the cells; in the absence of lipase, no increase in cellular tocopherol was detectable. The incubation system was simplified to include only fibroblasts, BSA, and Intralipid (an artificial lipid emulsion containing 10% soybean oil, which has gamma but not alpha tocopherol). The addition of lipase to this system also resulted in the transfer of tocopherol (gamma) to the fibroblasts. Addition of both lipase and its activator, apolipoprotein CII, resulted in a further increase in the cellular tocopherol content, but apolipoprotein CII alone had no effect. Heparin, which is known to prevent the binding of lipoprotein lipase to the cell surface membrane, abrogated the transfer of tocopherol to fibroblasts without altering the rate of triglyceride hydrolysis. Thus, in vitro tocopherol is transferred to cells during hydrolysis of triglyceride by the action of lipase, and for this transfer of tocopherol to occur, the lipase itself must bind to the cell membrane.

Animals↗
PubMed2006

The main triglyceride-lipase from the insect fat body is an active phospholipase A(1): identification and characterization.

The main triglyceride-lipase (TG-lipase) from the fat body of Manduca sexta has been identified as the homolog of Drosophila melanogaster CG8552. This protein is conserved among insects and also shares significant sequence similarity with vertebrate phospholipases (PLs) from the phosphatidic acid preferring-phospholipase A1 (PA-PLA(1)) family. It is shown here that the TG-lipase is also a PL. TG-lipase and PL activities copurify and are inhibited by, or resistant to, the same lipase inhibitors, indicating that both activities are catalyzed by the same enzyme and active site. The PL activity of TG-lipase corresponded to PL type A(1). The concentration dependence of lipase activity with TG and PL micellar substrates showed saturation kinetics, with apparent K(m) values of 152 +/- 11 and 7.8 +/- 1.1 muM, respectively. TG-lipase was able to hydrolyze the major phospholipid components of the lipid droplets, phosphatidylcholine and phosphatidylethanolamine. The enzyme hydrolyzes 77 molecules of TG for every molecule of PL contained in the lipid droplets. It was observed that the activation of lipolysis in vivo is accompanied by activation of the hydrolysis of phospholipids of the lipid droplets. These results suggest that the PL activity of the insect TG-lipase could be required to allow access of the lipase to TG molecules contained in the core of the lipid droplets.

Amino Acid Sequence↗
PubMed1994

Purification, characterization, and crystallization of two types of lipase from Rhizopus niveus.

The purification and some properties of two types of lipase (Lipase I and Lipase II) from Rhizopus niveus are described. The enzymes were purified to homogeneity by column chromatographies on DEAE-Toyopearl (1 pass) and CM-Toyopearl (2 passes). Lipase I consists of two polypeptide chains [a small peptide with sugar moiety (A-chain) and a large peptide of molecular weight 34,000 (B-chain)]. Lipase II has a molecular weight of 30,000 consisting of a single polypeptide chain. Lipase I appeared to be converted to Lipase II by limited proteolysis by a specific protease a small amount of which is in the culture supernatant from Rh. niveus, because one of the peptides formed has the same N-terminal sequence and C-terminal amino acid as Lipase II, as well as the molecular mass estimated by SDS-PAGE. Lipase I had a pH optimum of 6.0-6.5 and a temperature optimum of 35 degrees C, while, for Lipase II these values were pH 6.0 and 40 degrees C. Both enzymes were obtained in the crystalline state using the hanging drop method of vapor diffusion and PEG as the precipitating agents.

Amino Acid Sequence↗
PubMed2006

Ontogeny and chain-length specificity of gastrointestinal lipases affect medium-chain triacylglycerol utilization by newborn pigs.

Ontogeny and fatty acid chain-length specificity of gastrointestinal lipases in neonatal piglets were examined to explore the basis for variations in postnatal use of medium-chain triacylglycerols (MCT). Twenty-four newborn pigs were studied at 4 ages: 0, 6, 18, and 48 h postpartum (n = 6 pigs/age). Piglets were gastrically intubated and given 3.0 mmol/kg of BW(0.75) each of emulsified tri-C6:0 and tri-C8:0. One hour after intubation, the plasma concentration of C6:0 was 7.5-fold greater than that of C8:0 (P < 0.001), with total plasma medium-chain fatty acid concentrations 3.7-fold greater at 48 h than at 6 h of age (P < 0.05). Pancreatic, gastric, and lingual tissues were analyzed for lipase activity using an equimolar mixture of tri-C6:0 and tri-C8:0 as substrate. Pancreatic lipase activity averaged 7.0 +/- 0.8 micromol of fatty acid released/min per mg of protein for the medium-chain fatty acid substrates. Hexanoate (C6:0) release was greater at 0 h than at 6, 18, or 48 h (P < 0.05); however, age did not affect C8:0 release (P > 0.05). The lowest lipase activity was observed at 18 h for both tri-C6:0 and tri-C8:0. Chain-length specificity of pancreatic lipase was measured with tri-C4:0, tri-C6:0, tri-C8:0, and tri-C10:0 as combined or separate substrates. As separate substrates, the lipase activity decreased progressively as chain length increased from tri-C4:0 to tri-C10:0. As combined substrates, tri-C6:0 was hydrolyzed fastest (P < 0.05), followed by C4:0, C8:0, and C10:0. Gastric and lingual lipase activities averaged 2.7 nmol/min per mg of protein for the medium-chain fatty acid substrates, with hydrolysis of C6:0 being 7-fold greater than that of C8:0. In conclusion, pancreatic lipase dominates the preduodenal lipases in the neonatal pig, and greater activity of the gastrointestinal lipases toward tri-C6:0 underlies its increased rate of use.

Animals↗
PubMed2003

Purification and partial characterization of psychrotrophic Serratia marcescens lipase.

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6 h at 30 degrees C and after 12 h at 6 degrees C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.

Animals↗
PubMed2003

Inhibition of lipase activities by citrus pectin.

The oral administration of pectin to rats reduced and delayed the peak plasma triacylglycerol concentration. Pectin inhibited the hydrolysis of trioleoylglycerol emulsified with soybean phosphatidylcholine by pancreatic, carboxylester, and lingual lipases in a concentration-dependent manner. However, the effective concentration of pectin for lingual lipase was 100 times lower than that for pancreatic lipase. Pectin did not inhibit the tributyrin- and p-nitrophenylbutyrate-hydrolyzing activities by pancreatic and carboxylester lipase. When low molecular weight pectin was assayed, pectin at a molecular weight of 90,000 (MW 90) most strongly inhibited three lipase activities. When the effect of pH on pectin inhibition was analyzed using pancreatic lipase, strong inhibition was observed at an acidic pH (below pH 7.0). In the assay system, the pancreatic lipase protein levels in the supernatant and fat layer were estimated by Western blotting with an anti-pancreatic lipase antibody. Pectin reduced the amount of pancreatic lipase protein in the fat layer in a concentration-dependent manner and concomitantly increased that in the supernatant. These results suggest that pectin may interact with emulsified substrates and inhibit the adsorption of lipase to the surface of substrate emulsion.

Adipose Tissue↗
PubMed1992

The admission serum lipase:amylase ratio differentiates alcoholic from nonalcoholic acute pancreatitis.

To determine whether the lipase:amylase ratio differentiates alcoholic from nonalcoholic pancreatitis, we conducted a retrospective review of charts with the diagnosis of acute pancreatitis at the George Washington University Medical Center between January 1988 and July 1990. A total of 446 charts were reviewed. For a patient to be included in the subsequent analysis, the following criteria were met: 1) the patient had typical symptoms of pancreatitis, 2) serum amylase and lipase were analyzed on admission, and 3) a computerized tomographic (CT) scan or ultrasound of the abdomen was obtained within 72 h of admission. Forty-seven charts satisfied the requirements for inclusion in the study. Data collected from the charts included history of alcohol consumption, age, sex, race, admission serum amylase and serum lipase (from this the amylase:lipase ratio was calculated), peak serum amylase and serum lipase, and number of days of abdominal pain before admission. Patients with alcoholic pancreatitis had significantly lower serum amylase levels and significantly higher lipase:amylase ratios than those with nonalcoholic pancreatitis (p < 0.01). There was no difference in the serum lipase between the groups. The higher the lipase:amylase ratio, the greater the specificity of alcohol as the etiology of acute pancreatitis. Only patients with alcoholic acute pancreatitis had lipase:amylase ratios > 5.0 (sensitivity 31%, specificity 100%). Our data point to the clinical utility of the lipase:amylase ratio in differentiating alcoholic from nonalcoholic acute pancreatitis. Prospective studies will be needed to confirm the clinical utility of this ratio.

Acute Disease↗
PubMed2005

Does the pancreas really produce much more lipase than required for fat digestion?

Thirty years ago, it was reported that a linear relationship does not exist between the amounts of human pancreatic lipase secreted in chronic pancreatitis and the degree of steatorrhea, which was considered to appear only after more than 90% of the pancreatic secretory capacity had been lost. From these observations, it was generally thought that the lipolytic potential of the pancreas is much higher than required. In recent years, however, it has been noted that: 1) the level of inhibition of digestive lipases and gastrointestinal lipolysis by the lipase inhibitor orlistat were almost linearly correlated with the amount of excreted fat; 2) in minipigs with experimentally-induced pancreatic exocrine insufficiency, the amounts of enteric-coated pancreatic extracts needed for restoring fat digestion to normal levels were estimated to be much higher than those usually administered; 3) human pancreatic lipase specific activity on meal triglycerides is 3 orders of magnitude lower than the very high specific activity usually measured under experimental in vitro conditions which are far from physiological conditions; 4) in patients with reduced human pancreatic lipase secretion, gastric lipase plays a significant role in fat digestion. This last observation might explain the absence of a linear relationship between human pancreatic lipase secretion in chronic pancreatitis and steatorrhea. From the low specific activity displayed by human pancreatic lipase on meal triglycerides, one can better understand why more lipase than expected is needed, why fat digestion lasts for more than a few minutes and, finally, why there is not such an excess secretory capacity for lipase as had been previously thought.

Animals↗
PubMed1991

Identification of two separate allelic mutations in the lipoprotein lipase gene of a patient with the familial hyperchylomicronemia syndrome.

The molecular defects resulting in a deficiency of lipoprotein lipase activity in a patient with the familial hyperchylomicronemia syndrome have been identified. Increased lipoprotein lipase mass but undetectable lipoprotein lipase activity in the patient's post-heparin plasma indicate the presence of an inactive enzyme. No major gene rearrangements were identified by Southern blot analysis of the patient's lipoprotein lipase gene and Northern blot hybridization revealed an lipoprotein lipase mRNA of normal size. Sequence analysis of polymerase chain reaction-amplified lipoprotein lipase cDNA identified two separate allelic mutations. A T to C transition at nucleotide 836 results in the substitution of Ile194, located near the putative interfacial recognition site of lipoprotein lipase, to a Thr. A G to A mutation at base 983 leads to the substitution of a His for Arg243 and the loss of a HhaI restriction enzyme site. Arg243 is near His241, which has been postulated to be part of the catalytic triad of lipoprotein lipase. Direct sequencing of amplified cDNA and digestion with HhaI established that the proband is a compound heterozygote for each base substitution. Transient expression of each of the mutant lipoprotein lipase cDNAs in human embryonal kidney-293 cells resulted in the synthesis of enzymically inactive proteins, establishing the functional significance of the mutations. We conclude that the Ile194 to Thr194 and Arg243 to His243 substitutions occur in lipoprotein lipase regions essential for normal enzyme activity and each mutation results in the expression of a nonfunctional enzyme leading to the hyperchylomicronemia syndrome manifested in the proband.

Adult↗
PubMed1989

The relation between glycosylation and activity of guinea pig lipoprotein lipase.

Previous studies have indicated that the processing of oligosaccharide chains is necessary for lipoprotein lipase to become catalytically active and may be involved in the regulation of lipase release. Guinea pig adipocytes and perfused guinea pig hearts were labeled with [35S]methionine, and lipoprotein lipase was immunoprecipitated. Digestion with endo-beta-N-acetylglucosaminidase H (Endo H) showed that the mature enzyme contains one high mannose and two complex oligosaccharide chains. Limited proteolysis indicated where in the molecule the chains are attached. Pulse-chase experiments showed that some lipase molecules were rapidly processed and appeared in the medium within 40 min. Other lipase molecules remained fully Endo H-sensitive for more than 2 h, and this form of the lipase did not appear in the medium. Both forms co-eluted with the sole lipoprotein lipase activity peak from heparin-Sepharose; this indicates that both were dimeric. Separation of the two forms was achieved by lectin chromatography and demonstrated that both were catalytically active. Cells treated with methyl-deoxynojirimycin or with deoxymannojirimycin produced and released active lipoprotein lipase which was fully Endo H-sensitive. These studies demonstrate that the trimming and processing of the oligosaccharide chains is not necessary for lipoprotein lipase to become catalytically active and be secreted, and they suggest that a comparatively large fraction of the lipase molecules is retained in the endoplasmic reticulum. Whether they ever reach the processing apparatus in the Golgi or are degraded is not clear.

1-Deoxynojirimycin↗