Differential characteristics of purified hepatic triglyceride lipase and lipoprotein lipase from human postheparin plasma.

Evidence is presented that hepatic triglyceride lipase (H-TGL) and lipoprotein lipase (LPL), purified from human postheparin plasma, can each hydrolyze both glyceryl trioleate and palmitoyl-CoA. The average ratio of glyceryl trioleate/palmitoyl-CoA hydrolase activities, obtained with enzyme preparations from 15 human postheparin plasma samples was 1.30 (1.18-1.52) for H-TGL and 8.75 (7.45-10.25) for LPL. Albumin was identified as the serum cofactor required for the hydrolysis of palmitoyl-CoA by H-TGL. It protected this enzyme from inactivation by this substrate. In contrast, palmitoyl-CoA activated and protected LPL from denaturation by dilution and incubation at 25 degrees C. The effects of other detergents were investigated on glyceryl trioleate hydrolase activities of both enzymes. Sodium dodecyl sulfate (0.4 mM) and Trisoleate (0.4 mM), which also effectively activated and protected LPL against inactivation, had only moderate protective effect on H-TGL. Sodium dodecyl sulfate at a higher concentration (1 mM) produced little or no inhibition of LPL, while completely inactivating H-TGL. Conversely, sodium taurodeoxycholate (0.4 mM) protected and activated H-TGL, but had only moderate protective effect on LPL. Triton X-100 (0.1-0.8 mM) and egg lysolecithin (0.05-2 mM) also protected H-TGL, but not LPL. The very dissimilar effects of detergents on preparations on H-TGL and LPL may form the basis for the direct assay of each enzyme in the presence of the other.     

Purification and characterization of human lipoprotein lipase and hepatic triglyceride lipase. Reactivity with monoclonal antibodies to hepatic triglyceride lipase

Human lipoprotein lipase and hepatic triglyceride lipase were purified to homogeneity from post-heparin plasma. These enzymes were purified 250,000- and 100,000-fold with yields of 27 +/- 15 and 19 +/- 6%, respectively. Molecular weight determination by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents yielded Mr of 60,500 +/- 1,800 and 65,200 +/- 400, respectively, for lipoprotein lipase and hepatic triglyceride lipase. These lipase preparations were shown to be free of detectable antithrombin by measuring its activity and by probing of Western blots of lipases with a monospecific antibody against antithrombin. In additions, probing of Western blots with concanavalin A revealed no glycoproteins corresponding to the molecular weight of antithrombin. Four stable hybridoma-producing distinct monoclonal antibodies (mAb) to hepatic triglyceride lipase were isolated. The specificity of one mAb, HL3-5, was established by its ability to immunoprecipitate hepatic triglyceride lipase catalytic activity. Interaction of HL3-5 with this lipase did not inhibit catalytic activity. The three other mAb interacted with hepatic triglyceride lipase only after denaturation of the enzyme with detergents. The relatedness of these two enzymes was examined by comparing under the same conditions the thermal inactivation, the sensitivity to sulfhydryl and reducing agents, amino acid composition, and the mobility of peptide fragments generated by cyanogen bromide cleavage. The results of these studies strongly support the view that the two enzymes are different proteins. Immunological studies confirm this conclusion. Four mAb to hepatic triglyceride lipase did not interact with lipoprotein lipase in Western blots, enzyme-linked immunosorbent assay, and immunoprecipitation experiments. These immunological studies demonstrate that several epitopes of the hepatic triglyceride lipase protein moiety are not present in the lipoprotein lipase molecule.     

Isolation and cDNA sequence of human postheparin plasma hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was isolated from human postheparin plasma by column chromatography on heparin-Sepharose and phenyl-Sepharose and immunoaffinity chromatography with monoclonal antibodies. The purified enzyme had an apparent molecular weight of 65,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an amino-terminal sequence of Leu-Gly-Gln-Ser-Leu-Lys-Pro-Glu. Partial amino acid sequences of seven cyanogen bromide peptides were obtained. A human hepatoma cDNA library was screened with synthetic oligonucleotides derived from the partial protein sequence. The cloned H-TGL cDNA of 1569 nucleotides predicts a mature protein of 477 amino acids plus a leader sequence of 22 amino acids. Blot hybridization analysis of poly(A)+ mRNA with a putative H-TGL cDNA clone gave a single hybridizing band of 1.7 kilobases. The protein contains four consensus N-glycosylation sequences based on the cDNA sequence. Comparison of the enzyme sequence with that of other lipases reveals highly conserved sequences in regions of putative lipid and heparin binding. The carboxyl terminus of H-TGL contains a highly basic sequence which is not reported to be present in rat H-TGL or other members of the lipase gene family.     

Apo A-I and apo A-II inhibit hepatic triglyceride lipase from human postheparin plasma

Like α-amanitin, tRNA binds reversibly to yeast RNA polymerases A or B, in a 1:1 stoechiometry and inhibits enzyme B preferentially. Kinetic studies showed that the binding sites for the two inhibitors are completely independent. The dissociation constant of the enzyme-inhibitor complexes was determined. Only in the case of tRNA, it varied with the nature and concentration of the template. The stimulation factor P₃₇ does not interfere with the binding of both inhibitors.     

A sandwich-enzyme immunoassay for the quantification of lipoprotein lipase and hepatic triglyceride lipase in human postheparin plasma using monoclonal antibodies to the corresponding enzymes

We have developed a sandwich-enzyme immunoassay (EIA) for the quantification of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) in human postheparin plasma (PHP) using monoclonal antibodies (MAbs) directed against the corresponding enzymes purified from human PHP. The sandwich-EIA for LPL was performed by using the combination of two distinct types of anti-LPL MAbs that recognize different epitopes on the LPL molecule. The immunoreactive mass of LPL was specifically measured using a beta-galactosidase-labeled anti-LPL MAb as an enzyme-linked MAb, an anti-LPL MAb linked with the bacterial cell wall as an insolubilized MAb, and purified human PHP-LPL as a standard. The sandwich-EIA for HTGL was carried out by using two distinct anti-HTGL MAbs that recognize different epitopes on HTGL. The limit of detection was 20 ng/ml for LPL and 60 ng/ml for HTGL. Each method yielded a coefficient of variation of less than 6% in intra- and inter-assays, and a high concentration of triglyceride did not interfere with the assays. The average recovery of purified human PHP-LPL and -HTGL added to human PHP samples was 98.8% and 97.5%, respectively. The immunoreactive masses of LPL and HTGL in PHP samples, obtained at a heparin dose of 30 IU/kg, from 34 normolipidemic and 20 hypertriglyceridemic subjects were quantified by the sandwich-EIA. To assess the reliability of the measured mass values, they were compared with the corresponding enzyme activities measured by selective immunoinactivation assay using rabbit anti-human PHP-LPL and -HTGL polyclonal antisera. Both assay methods yielded a highly significant correlation in either normolipidemic (r = 0.945 for LPL; r = 0.932 for HTGL) or hypertriglyceridemic subjects (r = 0.989 for LPL; r = 0.954 for HTGL). The normal mean (+/- SD) level of lipoprotein lipase mass and activity in postheparin plasma was 223 +/- 66 ng/ml and 10.1 +/- 2.9 mumol/h per ml, and that of hepatic triglyceride lipase mass and activity was 1456 +/- 469 ng/ml and 26.4 +/- 8.7 mumol/h per ml, respectively. The present sandwich-enzyme immunoassay methods make it possible to study the molecular nature of LPL and HTGL in PHP from patients with either primary or secondary hyperlipoproteinemia.     

Mechanism of inhibition of hepatic triglyceride lipase from human postheparin plasma by apolipoproteins A-I and A-II

The present data describe the mechanism of the inhibitory effects of human plasma apolipoproteins A-I and A-II on hydrolysis of triglyceride catalyzed by hepatic triglyceride lipase using a substrate of triolein particles stabilized with gum arabic in vitro. The experimental data could well be described by a model in which apolipoproteins bound to the surface of lipid substrate particles inhibited the enzyme reaction. The values of Km obtained were similar with or without inhibitors and the calculated saturation levels of apolipoprotein binding to the lipid were in good agreement with those obtained in independent binding experiments.     

A novel method for measuring human lipoprotein lipase and hepatic lipase activities in postheparin plasma

The objective of this study was to establish a new lipoprotein lipase (LPL) and hepatic lipase (HL) activity assay method. Seventy normal volunteers were recruited. Lipase activities were assayed by measuring the increase in absorbance at 546 nm due to the quinoneine dye. Reaction mixture-1 (R-1) contained dioleoylglycerol solubilized with lauryldimethylaminobetaine, monoacylglycerol-specific lipase, glycerolkinase, glycerol-3-phosphate oxidase, peroxidase, ascorbic acid oxidase, and apolipoprotein C-II (apoC-II). R-2 contained Tris-HCl (pH 8.7) and 4-aminoantipyrine. Automated assay of lipase activities was performed with an automatic clinical analyzer. In the assay for HL + LPL activity, 160 microl R-1 was incubated at 37 degrees C with 2 microl of sample for 5 min, and 80 microl R-2 was added. HL activities were measured under the same conditions without apoC-II. HL and LPL activities were also measured by the conventional isotope method and for HL mass by ELISA. Lipase activity detected in a 1.6 M NaCl-eluted fraction from a heparin-Sepharose column was enhanced by adding purified apoC-II in a dose-dependent manner, whereas that eluted by 0.8 M NaCl was not. Postheparin plasma-LPL and HL activities measured in the present automated method had high correlations with those measured by conventional activity and mass methods. This automated assay method for LPL and HL activities is simple and reliable and can be applied to an automatic clinical analyzer.     

A comparison of molecular properties of hepatic triglyceride lipase and lipoprotein lipase from human post-heparin plasma

Hepatic triglyceride lipase was isolated from human post-heparin plasma by the method of Ehnholm et al. using modifications which increased the specific activity 12-fold to approximately 3,000 mumol of free fatty acid/h/mg of protein. Lipoprotein lipase with similar specific activity was prepared from the same plasma samples using heparin and concanavalin A affinity chromatography. The molecular weight of hepatic triglyceride lipase (69,000) was slightly greater than that of lipoprotein lipase (67,000) as determined by polyacrylamide electrophoresis in sodium dodecyl sulfate-containing buffers. These proteins had identical amino acid compositions, terminal amino acid residues, and tryptic peptide maps. However, the differences previously described regarding optima of pH and ionic strength and the requirement for apolipoprotein CII (only for lipoprotein lipase) were maintained in the highly purified state. It was found that both proteins contain approximately 8% carbohydrate. Antisera prepared in goats selectively precipitated each activity. Other antisera prepared in chickens reacted with both enzymes, suggesting a common antigenic determinant.     

Monoclonal antibodies that distinguish between active and inactive forms of human postheparin plasma hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was purified from human postheparin plasma. Specific monoclonal antibodies (MAbs) were produced that discriminate between active (native) and inactive (denatured) forms of the enzyme. Mice immunized with native H-TGL resulted in MAbs that recognized only the native protein. The antibodies did not react with H-TGL treated with 1% sodium dodecyl sulfate or heated at 60 degrees C. The loss of immunoreactivity with heating correlated directly with the loss of enzyme activity and there was a corresponding increase in immunoreactivity with the MAbs prepared against the denatured enzyme. Western blot analysis of postheparin plasma with the MAbs against denatured H-TGL gave a single protein band of 65 kD; preheparin plasma showed no detectable immunoreactivity with either MAb. These immunochemical studies suggest that there are no circulating active or inactive forms of H-TGL in man. Furthermore, the MAbs provide the necessary reagents for development of immunoassays for H-TGL.     

A new method for the measurement of lipoprotein lipase in postheparin plasma using sodium dodecyl sulfate for the inactivation of hepatic triglyceride lipase.

Lipoprotein lipase (LPL) and hepatic triglyceride lipase (H-TGL) are lipolytic activities found in postheparin plasma. A simple and precise method for the direct determination of LPL in postheparin plasma is described. Pre-incubations of this plasma (45--60 min at 26 degrees C) with sodium dodecyl sulfate (35--50 mM) in 0.2 M Tris-HCl buffer, pH 8.2, results in the inactivation of H-TGL, while leaving LPL fully active. Direct determination of H-TGL is done in a separate aliquot of the same postheparin plasma sample using previously reported assay conditons that do not measure LPL. The sodium dodecyl sulfate-resistant lipolytic activity has the characteristics of LPL as judged by a) its activation by serum and by apolipoprotein C-II; b) its inactivation (over 90%) by 0.75 M NaCl; and c) its inactivation by a specific antiserum. No sodium dodecyl sulfate-resistant activity was found in postheparin plasma from a patient with LPL deficiency (primary type I hyperlipoproteinemia). An excellent correlation of values was obtained (r = 0.99) for 30 samples assayed after sodium dodecyl sulfate treatment and after immuno-inactivation of H-TGL. The intra-assay coefficient of variation was +/- 11% and 4% before and after normalization of values, respectively.     

Effect of acute ethanol load on postheparin plasma lipoprotein lipase and hepatic lipase activities and intravenous fat tolerance

This study aimed to examine the possibility that ethanol-induced rise of serum triglyceride concentration in man is partly due to an impaired removal of triglycerides from the circulation. Acute ethanol loads given to normal human subjects after an overnight fast reduced the postheparin plasma lipoprotein lipase activity by an average of 25% but did not influence the postheparin plasma hepatic lipase activity or fractional removal of Intralipid triglyceride. When alcolhol was administered to fed subjects in the evening the postheparin plasma hepatic lipase was significantly decreased in the next morning as compared to corresponding control value but the lipoprotein lipase and Intralipid clearance were not changed. It is concluded that the slight decrease of lipoprotein lipase during alcohol intoxication may contribute to the hyperlipemic effect of ethanol.     

Purification, stabilization, and characterization of rat hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was purified to near homogeneity from heparin-containing rat liver perfusates with the following column chromatography steps: heparin-Sepharose affinity chromatography, anion-exchange chromatography on DEAE-Sephacel, and gel filtration on Ultrogel AcA 34. A final specific activity of 45,000 μmol fatty acid/mg/h was obtained with an overall 31% recovery of catalytic activity. The heparin-Sepharose step resulted in a 20-fold purification, while the DEAE and gel filtration steps led to further purification with complete recovery of activity. An extensive survey of various detergents as potential stabilizers of H-TGL activity led to the selection of Triton N-101 for use in the column buffers of the DEAE and gel filtration steps. Relative to initial H-TGL activity upon dilution in buffer without detergent, recoveries between 90 and 100% were consistently obtained with Triton N-101-containing buffers following a 24-h incubation at 20°C. In contrast after a 24-h incubation at 20°C those control samples lacking detergent were at least 95% inactivated. The highly purified H-TGL exhibited a single major band by sodium dodecyl sulfate-electrophoresis. The use of DEAE chromatography and stabilization of H-TGL with Triton N-101 are the improvements in purification that resulted in an 8-fold enhancement in specific activity relative to the highest previous report of purification from rat liver perfusates.     

Lipoprotein substrates of lipoprotein lipase and hepatic triacylglycerol lipase from human post-heparin plasma.

The number and the substrate specificities of glutathione thiol esterases of human red blood cells have been investigated by gel electrophoresis and isoelectric focusing and staining methods devised for the location of these enzymes on gels. Several glutathione thiol esterase forms, both unspecific (with respect to the S-acyl group of the substrate) and specific were found. Electrophoresis on both polyacrylamide and agarose gels resolved three enzyme components with apparently similar substrate specificity. Isoelectric focusing in liquid column separated two unspecific thiol esterase components with S-lactoylglutathione (pI = 8.4) and S-propionylglutathione (pI = 8.1) as the best substrates, respectively, and two specific enzymes, S-formylglutathione hydrolase (pI = 5.2) and S-succinylglutathione hydrolase (pI = 9.0). Isoelectric focusing on polyacrylamide gel resolved nine unspecific glutathione thiol esterase bands (between pH values 7.0 and 8.4). Partially purified glyoxalase II (S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) from erythrocytes or liver still gave three components on electrophoresis and several activity bands on gel electrofocusing. These results indicate that human red cells contain at least four separate glutathione thiol esterases. Glyoxalase II, one of these enzymes, apparently occurs in multiple forms. These were neither influenced by preptreatment of the samples with neuraminidase or thiols nor were interconvertible during the fractionations.     

Human hepatoma (HepG2) cells secrete a single 65 K dalton triglyceride lipase immunologically identical to postheparin plasma hepatic lipase

A triacylglycerol lipase was isolated from the culture medium of HepG2 human hepatoma cells and its properties were compared to hepatic triglyceride lipase (H-TGL) from human postheparin plasma. The HepG2 cell enzyme bound to heparin-Sepharose, was eluted with 1 M NaCl and was not inhibited by 1 M salt. Western-blotting of the fractions from the heparin-Sepharose column with a monoclonal antibody prepared against postheparin plasma H-TGL and which binds to an epitope in the carboxyl-terminus of H-TGL gave a single immunoreactive protein band of 65 kDa. This finding of immunochemical identity was confirmed with polyclonal antibodies prepared against synthetic peptides of H-TGL corresponding to amino acid residues 82-94 near the amino-terminus and residues 468-477, the carboxyl-terminus of the enzyme. We conclude that HepG2 cells secrete a single triacylglycerol lipase with molecular weight properties and immunological characteristics identical to post-heparin plasma H-TGL.     

Function of hepatic triglyceride lipase in lipoprotein metabolism

Rat hepatic triglyceride lipase (H-TGL) was purified from liver tissue extracts by affinity chromatography on Sepharose 4B with covalently linked heparin. The purified rat H-TGL exhibited the properties previously described for this enzyme. Enzyme protein was injected into rabbits for anti-H-TGL antibody production. Antirat-H-TGL did not react against rat lipoprotein lipase (LPL) but inhibited H-TGL-activity both in vitro and in vivo greater than 90%. These antibodies were injected into rats and lipoprotein analyses were performed over a 36-hr period. It could be shown that inactivation of H-TGL by anti-H-TGL gamma-globulins in vivo led to an increase in total triglyceride concentration from 70 mg/dl to 230 mg/dl due to an increase in very low density lipoprotein (VLDL) and low density lipoprotein (LDL) triglycerides 4 hr after antibody injection; a marked increase in high density lipoprotein (HDL) phospholipid concentration was observed with almost no change in HDL-cholesterol and HDL-triglycerides. This study documents the ability of antirat-H-TGL-gamma-globulins to inhibit H-TGL in vitro and in vivo. Furthermore, the inhibition of triglyceride removal in vivo demonstrated that this enzyme together with LPL is responsible for the catabolism of VLDL-triglyceride.     

A novel method for measuring human hepatic lipase activity in postheparin plasma

The objective of this study was to establish a hepatic lipase (HL) assay method that can be applied to automatic clinical analyzers. Seventy-four hyperlipidemic subjects (men/women 45/29) were recruited. Lipase activity was assayed measuring the increase in absorbance at 546 nm due to quinonediimine dye production. Reaction mixture R-1 contained 50 mM Tris-HCl (pH 9.5), 0.5 mM glycerol-1,2-dioleate, 0.4% (unless otherwise noted) polyoxyethylene-nonylphenylether, 3 mM ATP, 3 mM MgCl(2), 1.5 mM CaCl(2), monoacylglycerol-specific lipase, glycerol kinase, glycerol-3-phosphate oxidase, 0.075% N,N-bis-(4-sulfobutyl)-3-methylaniline-2 Na, peroxidase, ascorbic acid oxidase. Reaction mixture R-2 contained 50 mM Tris-HCl (pH9.5), 0.15% 4-aminoantypirine. Automated assay for activity was performed with a Model 7080 Hitachi analyzer. In the lipase assay, 160 microl of R-1 was incubated at 37 degrees C with 3 microl of samples for 5 min, and 80 microl of R-2 was added. Within-run coefficient of variations was 0.9-1.0%. Calibration curve of lipase activity was linear (r = 0.999) between 0 and 320 U/l. Analytical recoveries of purified HL added to plasma were 96.6-99.8%. HL activity in postheparin plasma measured in this method had a closer correlation with HL mass by a sandwich ELISA (r = 0.888, P < 0.0001) than those in the conventional method using [(14)C-]triolein (r = 0.730, P < 0.0001). This assay method for HL activity can be applied to an automatic clinical analyzer.