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PubMed1994

Rat liver contains a limited number of binding sites for hepatic lipase.

The binding of hepatic lipase to rat liver was studied in an ex vivo perfusion model. The livers were perfused with media containing partially purified rat hepatic lipase or bovine milk lipoprotein lipase. The activity of the enzymes was determined in the perfusion media before and after passage through the liver. During perfusion with a hepatic-lipase-containing medium the lipase activity in the medium did not change, indicating that there was no net binding of lipase by the liver. In contrast, more than 80% of the lipoprotein lipase was removed from the medium. This lipoprotein lipase activity could be recovered into the perfusion medium completely by heparin perfusion of the liver. If livers, first depleted of hepatic lipase by heparin, were subsequent perfused with a hepatic-lipase-containing medium, 90 +/- 24 m-units of the lipase activity was bound per g of liver (up to 1000 m-units/total liver). However, heparin treatment of the liver decreases the ability of the liver to re-bind hepatic lipase by 80%. Perfusion of rat livers with 0.3 M NaCl released 60% of the lipase activity into the medium. Upon subsequent perfusion of these livers with hepatic-lipase-containing media, 541 +/- 164 m-units of hepatic lipase could be bound per g of liver (up to 5000 m-units/total liver). The binding of hepatic lipase was also studied in livers of corticotropin (ACTH)-pre-treated rats. In these rats also, hepatic lipase bound only to livers which had been pre-perfused with heparin or 0.3 M NaCl. After heparin pre-perfusion, 88 +/- 12 m-units of hepatic lipase could be bound per g of liver, similar to that with livers of control rats not treated with ACTH. After prior salt perfusion, however, the capacity of the livers of ACTH-pre-treated rats to bind hepatic lipase was 212 +/- 60 m-units/g of liver. This is less than in livers of control rats (541 +/- 164 m-units/g of liver). These results indicate that in rat liver the binding of hepatic lipase is heterogeneous in character and consists of heparin-resistant and heparin-sensitive components. The hepatic-lipase binding capacity of the liver is saturable and fully utilized under various conditions. The heparin-sensitive binding capacity is lowered in ACTH-treated rats, whereas the heparin-resistant binding is unaffected. We postulate that the functional hepatic lipase activity can be regulated by changes in the binding capacity of the liver.

Adrenocorticotropic Hormone↗
PubMed1994

Bacterial lipases.

Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, meaning a sharp increase in lipase activity observed when the substrate starts to form an emulsion, thereby presenting to the enzyme an interfacial area. As a consequence, the kinetics of a lipase reaction do not follow the classical Michaelis-Menten model. With only a few exceptions, bacterial lipases are able to completely hydrolyze a triacylglycerol substrate although a certain preference for primary ester bonds has been observed. Numerous lipase assay methods are available using coloured or fluorescent substrates which allow spectroscopic and fluorimetric detection of lipase activity. Another important assay is based on titration of fatty acids released from the substrate. Newly developed methods allow to exactly determine lipase activity via controlled surface pressure or by means of a computer-controlled oil drop tensiometer. The synthesis and secretion of lipases by bacteria is influenced by a variety of environmental factors like ions, carbon sources, or presence of non-metabolizable polysaccharides. The secretion pathway is known for Pseudomonas lipases with P. aeruginosa lipase using a two-step mechanism and P. fluorescens lipase using a one-step mechanism. Additionally, some Pseudomonas lipases need specific chaperone-like proteins assisting their correct folding in the periplasm. These lipase-specific foldases (Lif-proteins) which show a high degree of amino acid sequence homology among different Pseudomonas species are coded for by genes located immediately downstream the lipase structural genes. A comparison of different bacterial lipases on the basis of primary structure revealed only very limited sequence homology. However, determination of the three-dimensional structure of the P. glumae lipase indicated that at least some of the bacterial lipases will presumably reveal a conserved folding pattern called the alpha/beta-hydrolase fold, which has been described for other microbial and human lipases. The catalytic site of lipases is buried inside the protein and contains a serine-protease-like catalytic triad consisting of the amino acids serine, histidine, and aspartate (or glutamate). The Ser-residue is located in a strictly conserved beta-epsilon Ser-alpha motif. The active site is covered by a lid-like alpha-helical structure which moves away upon contact of the lipase with its substrate, thereby exposing hydrophobic residues at the protein's surface mediating the contact between protein and substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

Comparison of lipases from different strains of the fungus Geotrichum candidum.

The type A lipase and the cis-9 18:1 specific type B lipase of different strains of Geotrichum candidum were compared. Comparing the enzyme activity of crude lipase preparation and purified type A and type B lipases and the protein pattern of these preparations in denaturing polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the specific activity for cis-9 18:1 fatty acids was related to the content of the type B lipase. Tandem-crossed immunoelectrophoresis was used to demonstrate immunological identity between type A and type B lipase of G. candidum ATCC 66592. Partial immunological identity was observed between type B lipase of this strain and type A lipase of G. candidum ATCC 34614 and two commercial crude G. candidum lipase preparations (Amano and Biocatalyst), i.e., the type B lipase of G. candidum ATCC 66592 had immunogenic epitopes which are not present on the other lipases. Enzymatic deglycosylation of the lipases did not alter this pattern. After partial proteolysis of purified type A and type B lipases of G. candidum ATCC 66592, Amano and Biocatalyst, no difference between the type A lipase of the three strains was observed in SDS-PAGE. For all strains the type B lipase exhibited a distinctly different peptide pattern to that of the type A lipase. In addition, the type B lipase of G. candidum ATCC 66592 differed from the type B lipase of Amano and Biocatalyst by having an additional peptide band. The results indicate that the G. candidum ATCC 66592 should be considered a distinct strain regarding the protein chemical characteristics of its type B lipase, whereas the two commercial lipase preparations appear to be very similar.

Geotrichum↗
PubMed2000

Properties and function of pancreatic lipase related protein 2.

The lipase gene family includes pancreatic triglyceride lipase and two pancreatic proteins, pancreatic lipase related proteins 1 and 2, with strong nucleotide and amino acid sequence homology to pancreatic triglyceride lipase. All three proteins have virtually identical three-dimensional structures. Of the pancreatic triglyceride lipase homologues, only pancreatic lipase related protein 2 has lipase activity. Like pancreatic triglyceride lipase, related protein 2 cleaves triglycerides, but it has broader substrate specificity. Pancreatic lipase related protein 2 also hydrolyzes phospholipids and galactolipids, two fats that are not substrates for pancreatic triglyceride lipase. The rat-related protein 2 also differs from pancreatic triglyceride lipase in sensitivity to bile salts and in response to colipase. Although the pancreas expresses both lipases, their temporal pattern of expression differs. Pancreatic lipase-related protein 2 mRNA appears before birth and persists into adulthood, whereas PTL mRNA first appears at the suckling-weanling transition. Additionally, intestinal enterocytes, paneth cells and cultured cytotoxic T-cells express mRNA encoding pancreatic lipase related protein 2. A physiological function for pancreatic lipase related protein 2 was demonstrated in mice that did not express this protein. Pancreatic lipase related protein 2 deficient mice malabsorbed fat in the suckling period, but not after weaning. They also had a defect in T-cell mediated cytotoxicity. Thus, pancreatic lipase related protein 2 is a lipase that participates in the cytotoxic activity of T-cells and plays a critical role in the digestion of breast milk fats.

Animals↗
PubMed1989

Two different mechanisms are involved in nutritional regulation of lipoprotein lipase in guinea-pig adipose tissue.

Lipoprotein lipase activity in adipose tissue responds rapidly to changes in the physiological state. To study what mechanisms are involved in the regulation, guinea pigs were fasted and the decrease in adipose-tissue lipoprotein lipase activity was compared with the decreases in mRNA and lipase synthesis. The mRNA pattern (three species) did not change. There was a close parallelism between the abundance of lipase mRNA and relative lipase synthesis (immunoprecipitable 35S-labelled lipoprotein lipase as fraction of total [35S]protein after pulse-labelling with [35S]methionine). Total protein synthesis decreased on fasting, compounding the decrease in relative lipase synthesis. Lipoprotein lipase mRNA changed similarly in fat-pads and in isolated adipocytes, whereas lipase activity changed more in the pads, indicating disproportionally large changes in extracellularly located lipase. In old guinea pigs the decreases in lipoprotein lipase activity and lipase synthesis were comparable, but in young animals the change in lipase activity was substantially larger than the change in lipase synthesis. Refeeding of fasted young guinea pigs with glucose resulted in a rapid increase in lipoprotein lipase activity, but there was only a small change in lipase mRNA. Old animals responded slowly to refeeding. The results indicate that in older animals the major mechanism for regulation of adipose lipoprotein lipase activity is a relatively slow change in lipase mRNA, whereas in younger animals an additional, more rapid, regulation is exerted on the transport and turnover of the enzyme.

Adipose Tissue↗
PubMed1998

Anatomy of lipase binding sites: the scissile fatty acid binding site.

Shape and physico-chemical properties of the scissile fatty acid binding sites of six lipases and two serine esterases were analyzed and compared in order to understand the molecular basis of substrate specificity. All eight serine esterases and lipases have similar architecture and catalytic mechanism of ester hydrolysis, but different substrate specificities for the acyl moiety. Lipases and esterases differ in the geometry of their binding sites, lipases have a large, hydrophobic scissile fatty acid binding site, esterases like acetylcholinesterase and bromoperoxidase have a small acyl binding pocket, which fits exactly to their favorite substrates. The lipases were subdivided into three sub-groups: (1) lipases with a hydrophobic, crevice-like binding site located near the protein surface (lipases from Rhizomucor and Rhizopus); (2) lipases with a funnel-like binding site (lipases from Candida antarctica, Pseudomonas and mammalian pancreas and cutinase); and (3) lipases with a tunnel-like binding site (lipase from Candida rugosa). The length of the scissile fatty acid binding site varies considerably among the lipases between 7.8 A in cutinase and 22 A in Candida rugosa and Rhizomucor miehei lipase. Location and properties of the scissile fatty acid binding sites of all lipases of known structure were characterized. Our model also identifies the residues which mediate chain length specificity and thus may guide protein engineering of lipases for changed chain length specificity. The model was supported by published experimental data on the chain length specificity profile of various lipases and on mutants of fungal lipases with changed fatty acid chain length specificity.

Animals↗
PubMed1992

Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols.

Diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) activities were investigated in subcellular fractions from neonatal and adult rat liver in order to determine whether one or more different lipases might provide the substrate for the developmentally expressed, activity monoacylglycerol acyltransferase. The assay for diacylglycerol lipase examined the hydrolysis of sn-1-stearoyl,2- [14C]oleoylglycerol to labeled monoacylglycerol and fatty acid. Highest specific activities were found in lysosomes (pH 4.8) and cytosol and microsomes (pH 8). The specific activity from plasma membrane from adult liver was 5.8-fold higher than the corresponding activity in the neonate. In other fractions, however, no developmental differences were observed in activity or distribution. In both lysosomes and cytosol, 75 to 90% of the labeled product was monoacylglycerol, suggesting that these fractions contained relatively little monoacylglycerol lipase activity. In contrast, 80% of the labeled product from microsomes was fatty acid, suggesting the presence of monoacylglycerol lipase in this fraction. Analysis of the reaction products strongly suggested that the lysosomal and cytosolic diacylglycerol lipase activities hydrolyzed the acyl-group at the sn-1 position. The effects of serum and NaCl on diacylglycerol lipase from each of the subcellular fractions differed from those effects routinely observed on lipoprotein lipase and hepatic lipase, suggesting that the hepatic diacylglycerol lipase activities were not second functions of these triacylglycerol lipases. Cytosolic diacylglycerol lipase activity from neonatal liver and adult liver was characterized. The apparent Km for 1-stearoyl,2-oleoylglycerol was 115 microM. There was no preference for a diacylglycerol with arachidonate in the sn-2 position. Bovine serum albumin stimulated the activity, whereas dithiothreitol, N-ethylmaleimide, and ATP inhibited the activity. Both sn-1(3)- and 2-monooleylglycerol ethers stimulated cytosolic diacylglycerol lipase activity 2-3-fold. The corresponding amide analogs stimulated 28 to 85%, monooleoylglycerol itself had little effect, and 1-alkyl- or 1-acyl-lysophosphatidylcholine inhibited the activity. These data provide the first characterization of hepatic subcellular lipase activities from neonatal and adult rat liver and suggest that independent diacylglycerol and monoacylglycerol lipase activities are present in microsomal membranes and that the microsomal and cytosolic diacylglycerol lipase activities may describe an ambipathic enzyme. The data also suggest possible cellular regulation by monoalkylglycerols.

Aging↗
PubMed1985

Importance of the different steps of glycosylation for the activity and secretion of lipoprotein lipase in rat preadipocytes studied with monensin and tunicamycin.

Lipoprotein lipase synthesized by cultured rat preadipocytes is present in three compartments: an intracellular, a surface-related 3-min heparin-releasable, and that secreted into the culture medium. 30 min after addition of 6 microM monensin, the lipoprotein lipase activity in the heparin-releasable compartment starts to decrease; by 4 h of monensin treatment the lipoprotein lipase activity in the heparin-releasable pool and in the culture medium is about 10% of that found in control dishes. The intracellular activity, which had been identified as lipoprotein lipase by an antiserum to lipoprotein lipase, increases slowly and doubles by 24 h. However, since the cellular compartment accounts for 10-25% of total activity, this increase does not account for the missing enzyme activity. To determine whether this enzyme molecule is synthesized but is not active, incorporation of labeled leucine, mannose and galactose into immunoadsorbable lipoprotein lipase was studied in control, monensin- or tunicamycin-treated cells. Addition of tunicamycin (5 micrograms/ml) for 24 h caused a 30-50% reduction in immunoadsorbable lipoprotein lipase, but the enzyme activity was reduced by 90%. On the other hand, 4 h monensin treatment reduced both incorporation of [3H]leucine into immunoadsorbable lipoprotein lipase and heparin-releasable and medium lipoprotein lipase activity by 57 to 77%. The immunoadsorbable lipoprotein lipase in the intracellular compartment has a [14C]mannose to [3H]galactose ratio of 0.15 and this ratio increased 6-fold in monensin-treated cells. The intracellular lipoprotein lipase in monensin-treated cells had the same affinity for both the native and synthetic substrate as the lipoprotein lipase in control cells, yet its spontaneous secretion into the culture medium and its release by 3 min heparin treatment was markedly decreased. The present results indicate that: the presence of asparagine-linked oligosaccharide (formation of which is inhibited by tunicamycin) is mandatory for the expression of lipoprotein lipase activity; lipoprotein lipase is active also in a high mannose form; and terminal glycosylation and oligosaccharide processing, which is inhibited by monensin, may be important for the appearance of heparin-releasable lipoprotein lipase and secretion of lipoprotein lipase into the medium.

Adipose Tissue↗
PubMed1986

Production and use of an inhibitory monoclonal antibody to human lipoprotein lipase.

Studies were performed to produce a monoclonal antibody to human lipoprotein lipase, verify the specificity of the antibody for lipoprotein lipase, and use this antibody for detection of lipoprotein lipase protein in human post-heparin plasma. Partially purified lipoprotein lipase from human milk was used as an antigen for the production of anti-lipoprotein lipase antibodies in mice. The spleen was removed from the animal having the highest titer of inhibitory antibodies to lipoprotein lipase and the cells were fused mouse myeloma cells. Culture media from the resulting hybridomas were screened for their ability to inhibit lipoprotein lipase catalytic activity. This screening procedure thus identified only those hybridomas which produced antibodies directed against lipoprotein lipase. One monoclonal antibody, from one clone, was selected for detailed study. The specificity of this antibody for lipoprotein lipase protein was established by three methods. First, post-heparin plasma lipoprotein lipase activity and immunoreactivity detected by an enzyme-linked immunosorbent assay (ELISA) co-eluted during heparin-agarose and phenyl-Sepharose chromatography. Second, the antibody detected a protein which was released into the circulation after intravenous injection of heparin into humans. Third, both immunoreactive lipoprotein lipase protein and lipoprotein lipase enzymatic activity were lost by heat-inactivation of lipoprotein lipase. The use of active enzyme as an antigen and the procedure used to screen the monoclonal antibody-producing hybridomas allowed the production of an inhibitory anti-human lipoprotein lipase monoclonal antibody. This antibody is useful for detection of lipoprotein lipase protein in plasma and should allow for immunohistochemical staining of active lipoprotein lipase enzyme in tissues. Moreover, the methods described for screening hybridomas may be modified and used to produce specific antibodies against other partially purified enzymes.

Adult↗
PubMed2004

Changes in lipid fractions and sensory properties of Idiazabal cheese induced by lipase addition.

This work studied the addition of an adequate lipase to enhance lipolysis reactions and the development of piquant flavour and sharp odour in Idiazabal cheese, as an alternative to the use of lamb rennet paste. Cheeses were manufactured from bulk raw ewes' milk in 50 l vats with commercial bovine rennet and 80 lipase units of pregastric or 180 lipase units of fungal lipase and ripened for 180 days. A higher lipolytic activity was induced by lipase addition promoting strong changes in odour and flavour attributes. Both fungal and pregastric lipases increased the content of total free fatty acids (FFA), but the fungal lipase released mainly medium- and long-chain FFA. In contrast, the pregastric lipase preferably released short-chain FFA. Diglyceride (DG) content was considerably higher in cheeses made with added pregastric lipase compared with those made with fungal lipase or with no lipase. Monoglycerides (MG) were detected only in cheeses made with either lipase added, reaching comparable concentrations after ripening for 180 days. The cheeses made with pregastric lipase had the highest scores for odour and flavour intensity, and sharp and rennet odours, desirable attributes for the Idiazabal cheese made with lamb rennet paste. None of the texture attributes were significantly influenced by the concentrations of MG and DG in the cheeses made with either lipase. Thus, the pregastric lipase was more appropriate than the fungal lipase to develop a more traditionally-flavoured Idiazabal cheese.

Animals↗
PubMed1996

Chinese hamster ovary cells expressing a cell surface-anchored form of hepatic lipase. Characterization of low density lipoprotein and chylomicron remnant uptake and selective uptake of high density lipoprotein-cholesteryl ester.

The enzyme hepatic lipase may play several roles in lipoprotein metabolism. Recent investigation has suggested a role for the enzyme in lipoprotein and/or lipoprotein lipid uptake. To study this, a simple isolated system that mimics the in vivo system would be desirable. The enzyme is secreted by the hepatic parenchymal cell but exists, and presumably exerts its effects, while bound to capillary endothelial cells in the liver, adrenal gland, and the ovary. We constructed a cDNA that encodes the expression of a chimeric protein composed of rat hepatic lipase and the signal sequence for the addition of the glycophosphatidylinositol (GPI) anchor from human decay-accelerating factor. When transfected into Chinese hamster ovary (CHO) cells this gave rise to a cell population that had immunoreactive hepatic lipase on the cell surface. Cloning of the transfected cells produced several cell lines that expressed the chimeric protein bound to the cell surface by a GPI anchor. This was documented by demonstrating incorporation of [3H]ethanolamine into anti-hepatic lipase immunoprecipitable material; in addition, hepatic lipase was released from the cells by phosphatidylinositol-specific phospholipase C but not by heparin. Phosphatidylinositol-phospholipase C treatment of cells expressing the anchored lipase released material that comigrated with hepatic lipase on SDS-polyacrylamide gel electrophoresis and was immunoreactive with antibody to the cross-reacting determinant of GPI anchors. Cell lysates containing the anchored protein contained salt-resistant lipase activity, a known feature of the secreted hepatic lipase; thus it appears that these cells have a surface-anchored hepatic lipase molecule. Although it was not possible to demonstrate lipolysis by the enzyme while it was on the cell surface for technical reasons, the protein produced by these cells was active when studied in cell membranes. The ability of the cells to take up lipoproteins was studied. The cells demonstrated an increased affinity for low density lipoprotein (LDL) receptor mediated uptake of LDL. They did not, however, demonstrate any enhanced binding or removal of chylomicron remnants. With respect to LDL and remnants, the cells expressing anchored lipase behaved similarly to CHO cell that expressed secreted hepatic lipase. The cells expressing anchored hepatic lipase had a marked increase in the uptake of high density lipoprotein and high density lipoprotein cholesteryl ester when compared to that seen with CHO cells secreting hepatic lipase. This increase occurred primarily via the selective pathway, and was not reduced by addition of anti-LDL receptor or anti-hepatic lipase antibodies or the receptor-associated protein. Together the results suggest that hepatic lipase, when bound to the cell surface by a GPI anchor, plays a role in enhancing lipoprotein uptake. For LDL this may involve the provision of a second foot for particle binding, thus enhancing affinity for the LDL receptor. For chylomicron remnants an additional molecule or molecules are necessary to mediate this effect. For HDL, the enzyme facilitates uptake of cholesteryl ester primarily by the selective pathway.

Amino Acid Sequence↗
PubMed1991

Structural and functional roles of highly conserved serines in human lipoprotein lipase. Evidence that serine 132 is essential for enzyme catalysis.

The structure of human lipoprotein lipase was recently deduced from its cDNA sequence. It contains 8 serine residues (residues 45, 132, 143, 172, 193, 244, 251, and 363) that are absolutely conserved in both lipoprotein lipase and hepatic lipase across all species studied. The high homology between lipoprotein lipase, hepatic lipase, and pancreatic lipase suggests that the catalytic functions of these enzymes share a common mechanism and that one of the 8 conserved serines in human lipoprotein lipase must play a catalytic role as does serine 152 in the case of pancreatic lipase (Winkler, F. K., D'Arcy, A., and Hunziker, W. Nature 343, 771-774). We expressed wild-type and site-specific mutants of human lipoprotein lipase in COS cells in vitro. We produced two to four substitution mutants involving each of the 8 serines and assayed a total of 22 mutants for both enzyme activity and the amount of immunoreactive enzyme mass produced. Immunoreactive lipase was detected in all cases. With the exception of Ser132, for each of the 8 serine mutants we studied, at least one of several mutants at each position showed detectable enzyme activity. All three substitution mutants at Ser132, Ser----Thr, Ser----Ala, and Ser----Asp, were totally inactive. Ser132 occurs in the consensus sequence Gly-Xaa-Ser-Xaa-Gly present in all serine proteinases and in human pancreatic lipase. The x-ray crystallography structure of human pancreatic lipase suggests that the analogous serine residue in human pancreatic lipase, Ser152, is the nucleophilic residue essential for catalysis. Our biochemical data strongly support the conclusion that Ser132 in human lipoprotein lipase is the crucial residue required for enzyme catalysis. The observed specific activities of the variants involving the other seven highly conserved serines in human lipoprotein lipase are consistent with the interpretation that this enzyme has a three-dimensional structure very similar to that of human pancreatic lipase.

Amino Acid Sequence↗