Isolation of two distinct activator proteins for lipoprotein lipase from ovine plasma

Two distinct activator proteins for lipoprotein lipase (LPL) have been isolated in approximately equal amounts from ovine plasma. These low molecular weight proteins were readily separated from each other on the basis of size and charge. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated proteins of Mr about 8000 and 5000, with pI in urea-containing gels of about 5.1 and 4.8 respectively. Each of the ovine activator proteins was as effective as human apolipoprotein C-II (apo C-II) in activating LPL, with 1 microgram/ml giving near to maximum activation, and in lowering the apparent Km of LPL for triolein substrate. As the ratio of activator to triolein increased from 0.16 to 5.2 (micrograms/mg) the apparent Km fell from about 0.5 to 0.18 mM. Whereas human apo C-II and the two ovine activators were equally effective in stimulating the hydrolysis of triolein, differences were observed between the human and ovine activators when p-nitrophenylbutyrate was used as substrate. Unlike human apo C-II, which produced significant inhibition of p-nitrophenylbutyrate hydrolysis, the ovine activators were without effect. This suggests that the interaction between the ovine activators and LPL is different from that of human apo C-II.     

Hepatic lipase. Purification and characterization

Hepatic lipase has been purified to homogeneity from rat liver homogenates. The purified enzyme exhibits a single band on SDS-polyacrylamide gel electrophoresis. The molecular size of the native hepatic lipase is 200 000, while on SDS-polyacrylamide gel electrophoresis the apparent minimum molecular weight of the enzyme is 53 000, suggesting that the active enzyme is composed of four subunits. The relationship between triacylglycerol, monoacylglycerol and phospholipid hydrolyzing activities of the purified rat liver enzyme was studied. All three activities had a pH optimum of 8.5. The maximal reaction rates obtained with triolein, monoolein and dipalmitoylphosphatidylcholine were 55 000, 66 000 and 2600 mumol fatty acid/mg per h with apparent Michaelis constant (Km) values of 0.4, 0.25 and 1.0 mM, respectively. Hydrolysis of triolein and monoolein probably takes place at the same site on the enzyme molecule, since competitive inhibition between these two substrates was observed, and a similar loss of hydrolytic activity occurred in the presence of diisopropylfluorophosphate. Addition of apolipoproteins C-II and C-I had no effect on the hydrolytic activity of the enzyme with the three substrates tested. However, the triacylglycerol hydrolyzing activity was inhibited by the addition of apolipoprotein C-III. Monospecific antiserum to the pure hepatic lipase has been raised in a rabbit.     

Purification and properties of human placental acid lipase

Two peaks of lysosomal acid lipase activity were purified from normal human placenta. Acid lipase I, with an estimated molecular weight of 102 500, was purified 1016-fold while acid lipase II, with an estimated molecular weight of 30 600, was purified 3031-fold. The final yields of enzyme activity for acid lipase I and II were 0.9% and 2.2% respectively. The purity of the final preparations was documented by demonstration of a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both preparations of the purified enzyme demonstrated activity towards triolein, cholesteryl oleate and the artificial substrate 4-methylumbelliferyl oleate. Examination of Km values, thermal stability, pH optima, and electrophoretic mobility revealed similar properties for the two enzyme peaks. The response of the two enzyme preparations to inhibitors was similar with both being significantly inhibited by 0.2 M NaCl, 0.2 M KCl, 5 mM HgCl2 and 5 mM p-chloromercuribenzoate. The activity of the two preparations as assayed with either triolein or cholesterol oleate was not significantly affected by the addition of bovine serum albumin. In contrast, the 4-methylumbelliferyl oleate activity of both preparations was significantly inhibitred by albumin. These findings support the hypothesis that the same enzyme or enzymes are responsible for the intralysosomal hydrolysis of triacylglycerols and cholesterol esters in human tissues.     

Kinetics of bovine milk lipoprotein lipase and the mechanism of enzyme activation by apolipoprotein C-II

The kinetics of bovine milk lipoprotein lipase (LPL) were studied in order to determine the reaction mechanism of this enzyme. Reaction velocities were determined at varying concentrations of emulsified trioleoylglycerol (TG) and different fixed concentrations of apolipoprotein C-II (C-II) or at varying C-II concentrations and different fixed concentrations of TG. Neither the apparent Km(TG) nor the apparent Km(C-II) was affected by varying the concentrations of C-II or TG, respectively. However, C-II increased the apparent Vmax for the enzyme about 20-fold. The following kinetic parameters were calculated from Lineweaver-Burk plots: Km(C-II) = 2.5 X 10(-8) M and Km (TG) = 2.5 X 10(-3) M. The dissociation constant (KS) of the enzyme-TG binary complex was determined from Scatchard plots to be 7.6 X 10(-8) M. Heparin was found to be a competitive dead-end inhibitor against both TG and C-II. Tricapryloylglycerol represented a competitive inhibitor against TG but a noncompetitive inhibitor against C-II. C-II was shown to interact with dansylated bovine milk LPL, increasing its fluorescent emission by inducing a conformational change in the enzyme. Based on these studies, it was concluded that the LPL-catalyzed reaction follows a random, bireactant, rapid-equilibrium mechanism and the role of C-II in the activation process involves an increase in the catalytic rate constant (Kp) resulting from conformational changes of LPL induced by C-II.     

The kinetics of heparin inhibition of the esterase and basal lipase activities of lipoprotein lipase

The kinetics of inhibition of the esterase and lipase activities of bovine milk lipoprotein lipase (LPL) were compared. The esterase LPL activity against emulsified tributyrylglycerol was not affected by the enzyme activator apolipoprotein C-II (C-II) and amounted to about 15% of the "plus activator" lipase enzyme activity. Heparin at concentrations of 20 micrograms/ml inhibited 25% of the esterase activity. The reaction followed Henri-Michaelis-Menten kinetics and the inhibition by heparin followed a linear, intersecting, noncompetitive kinetic model. On the other hand, the basal lipase activity of LPL against emulsified trioleoylglycerol (TG) was very sensitive to inhibition by heparin: 1 microgram/ml inhibited about 80% of the reaction and 3 micrograms/ml drove the reaction to zero. The velocity curve for the uninhibited basal LPL activity was sigmoidal with an apparent nH(TG) of 2.94. Heparin inhibited the lipase activity competitively: heparin decreased nH(TG) and increased[TG]0.5 6.4-fold, while TG decreased the nH(Heparin) from 2.14 to 0.95 and caused a 3-fold increase in [Heparin]0.5. C-II, at concentrations lower than 2.5 X 10(-8) M (i.e., lower than KA), countered the inhibitory effects of heparin: at constant inhibitor concentrations, C-II increased nH(TG) from 1.78 to 2.52 and decreased [TG]0.5 about 10-fold; it also increased the apparent Vmax. At the lower C-II concentrations, nH(C-II) was approximately equal to 1.0 and increasing the TG concentrations decreased [C-II]0.5 from 3.8 X 10(-8) to 8.5 X 10(-9) M, with no effect on the nH(C-II). At the higher C-II concentrations, nH(C-II) was 2.5 and TG decreased [C-II]0.5 about 2-fold with no effect on the nH(C-II). In the absence of heparin, C-II had no effect on nH(TG) nor on [TG]0.5, but it increased the apparent Vmax. On the other hand, TG had no effect on nH(C-II) nor on [C-II]0.5, but at any given C-II concentration, the reaction velocity increased with increasing TG concentrations. It is concluded that TG and heparin as well as C-II and heparin are mutually exclusive and that lipoprotein lipase is a multisite enzyme, possibly a tetramer, with three high-affinity catalytic sites, and an equal number of sites for C-II and heparin per oligomer. However, LPL differs from classical allosteric enzymes in that its activator has no effect on substrate cooperativity nor on [S]0.5; its only effect is to increase Vmax by increasing the catalytic rate constant kp by inducing conformational changes in the enzyme.     

Mechanism of action of lipoprotein lipase on triolein particles: effect of apolipoprotein C-II

Triolein particles stabilized by a phosphatidylcholine monolayer were used to study the lipoprotein lipase (LpL) reaction. They were prepared in two different sizes and with triolein and phosphatidylcholine in the molar ratios of 0.9-1.2 : 1 (small particles) and 8-17 : 1 (large particles). The rate of hydrolysis by LpL of phosphatidylcholine on the surface of both lipid particles was only 1/20 as much as that of triolein, even if it was activated to the maximum by apolipoprotein C-II (apoC-II). Thus, the phospholipase activity of LpL was low enough to measure the initial rate of hydrolysis of triolein without causing a gross change of the surface of the lipid particle. When the hydrolysis of triolein by LpL was monitored, fatty acid was released at a constant rate until all of the triolein molecules were hydrolyzed. The enzyme required 220 +/- 17 and 66 +/- 9 nM apoC-II for its half-maximal activity (Km (apoC-II] with small and large particles as a substrate (1.15 mM triolein for small and 2.13 mM triolein for large particles), respectively, using various concentrations of LpL. The Km(apoC-II) values for these two substrates became similar when LpL activity was analyzed with respect to the density of apoC-II on the phosphatidylcholine monolayer at the surface of the particles (bound apoC-II/phosphatidylcholine). The concentration of substrate particles did not affect the Km(apoC-II) values. The presence of an adequate amount of apoC-II increased the maximal activity of LpL (Vmax(triolein)) from 0.48 +/- 0.21 to 6.81 +/- 0.45 and from 0.32 +/- 0.04 to 7.13 +/- 0.64 mmol/h/mg with a slight decrease in the apparent Michaelis constant (Km(triolein)) for small (from 90 to 54 microM triolein) and large (from 1.00 to 0.65 mM triolein) particles, respectively. Although the apparent Km for triolein in large particles was about ten times greater than that in small particles, the values became similar when they were corrected for the concentration of phosphatidylcholine (50-100 microM phosphatidylcholine), which corresponded to the surface area of the substrate particles. It was suggested that bound apoC-II molecules were transferred relatively slowly to other lipid particles while LpL molecules moved rapidly among the lipid particles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on the degradation of human very low density lipoproteins by human milk lipoprotein lipase

Human milk lipoprotein lipase (LPL) was purified by heparin-Sepharose 4B affinity chromatography. The time required for the purification was approximately 2 h. The acetone-diethyl ether powder of milk cream was extracted by a 0.1% Triton X-100 buffer solution and the extract was applied to the heparin-Sepharose 4B column. The partially purified LPL eluted by heparin had a specific activity of 5120 units/mg which represented a 2500-fold purification of the enzyme. The LPL was found to be stable in the heparin solution for at least 2 days at 4 °C. This enzyme preparation was found to be free of the bile salt-activated lipase activity, esterase activity, and cholesterol esterase activity. The LPL had no demonstrable basal activity with emulsified triolein in the absence of a serum cofactor. The enzyme was activated by serum and by apolipoprotein C-II. The application of milk LPL to studies on the in vitro degradation of human very low density lipoproteins can result in a 90–97% triglyceride hydrolysis. The LPL degraded very low density lipoprotein triglyceride and phospholipid without any effect on cholesterol esters. Of the partial glycerides potentially generated by lipolysis with milk LPL, only monoglycerides were present in measurable amounts after 60 min of lipolysis. These results show that the partially purified human milk LPL with its high specific activity and ease of purification represents a very suitable enzyme preparation for studying the kinetics and reaction mechanisms involved in the lipolytic degradation of human triglyceride-rich lipoproteins.     

Activation and inhibition of lipoprotein lipase. Studies with artificial lipoproteins.

Human plasma very low density apolipoproteins C-I, C-II and C-III were recombined in vitro with triolein. The lipid-protein complexes were analyzed by ultracentrifugal flotation, agarose gel electrophoresis, immunoelectrophoresis and electron microscopy. Maximal protein/triolein ratios for apoprotein C-I, C-II, C-III-1 and C-III-2 were 50, 45, 95 and 55 microgram/mg, respectively. Electron micrographs exhibited spherical particles with diameters ranging from 200--2000 A comparable to native VLDL and chylomicrons. On agarose gel electrophoresis these complexes showed alpha-mobility. Kinetics of triolein hydrolysis by purified human plasma lipoprotein lipase were studied using these artificial lipoprotein substrates with different apoprotein/triolein ratios. The reaction followed the Michaelis-Menten equation. With increasing amounts of apo C-II, the apparent Km decreased from 0.60 to 0.11 mM. Incubation of the substrate with either rabbit anti-apo C-II gamma-globulins or digestion with trypsin prior to hydrolysis reversed this lowering effect on apparent Km. V was not altered significantly. Increasing amounts of apo C-I, apo C-III-1 or apo C-III-2 without apo C-II caused inhibition of triolein hydrolysis. In the presence of apo C-II, however, similar kinetic parameters were obtained as described above.     

Chimeras of hepatic lipase and lipoprotein lipase. Domain localization of enzyme-specific properties.

Chimeric molecules between human lipoprotein lipase (LPL) and rat hepatic lipase (HL) were used to identify structural elements responsible for functional differences. Based on the close sequence homology with pancreatic lipase, both LPL and HL are believed to have a two-domain structure composed of an amino-terminal (NH2-terminal) domain containing the catalytic Ser-His-Asp triad and a smaller carboxyl-terminal (COOH-terminal) domain. Experiments with chimeric lipases containing the HL NH2-terminal domain and the LPL COOH-terminal domain (HL/LPL) or the reverse chimera (LPL/HL) showed that the NH2-terminal domain is responsible for the catalytic efficiency (Vmax/Km) of these enzymes. Furthermore, it was demonstrated that the stimulation of LPL activity by apolipoprotein C-II and the inhibition of activity by 1 M NaCl originate in structural features within the NH2-terminal domain. HL and LPL bind to vascular endothelium, presumably by interaction with cell surface heparan sulfate proteoglycans. However, the two enzymes differ significantly in their heparin affinity. Experiments with the chimeric lipases indicated that heparin binding avidity was primarily associated with the COOH-terminal domain. Specifically, both HL and the LPL/HL chimera were eluted from immobilized heparin by 0.75 M NaCl, whereas 1.1 M NaCl was required to elute LPL and the HL/LPL chimera. Finally, HL is more active than LPL in the hydrolysis of phospholipid substrates. However, the ratio of phospholipase to neutral lipase activity in both chimeric lipases was enhanced by the presence of the heterologous COOH-terminal domain, demonstrating that this domain strongly influences substrate specificity. The NH2-terminal domain thus controls the kinetic parameters of these lipases, whereas the COOH-terminal domain modulates substrate specificity and heparin binding.     

Immunological studies on bovine milk lipoprotein lipase. Effects of Fab fragments on enzyme activity

Rabbit antiserum was prepared against purified bovine mild lipoprotein lipase. Immunoelectrophoresis of lipoprotein lipase gave a single precipitin line against the antibody which was coincident with enzyme activity. The gamma-globulin fraction inhibited heparin-releasable lipoprotein lipase activity of bovine arterial intima, heart muscle and adipose tissue. The antibody also inhibited the lipoprotein lipase activity from adipose tissue of human and pig, but not that of rat and dog. Fab fragments were prepared by papain digestion of the gamma-globulin fraction. Fab fragments inhibited the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine vesicles and trioleoylglycerol emulsions to the same extent. The Fab fragments also inhibited the lipolysis of human plasma very low density lipoproteins. The change of the kinetic parameters for the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol by the Fab fragments was accompanied with a 3-fold increase in Km and a 10-fold decrease in Vmax. Preincubation of lipoprotein lipase with apolipoprotein C-II, the activator protein for lipoprotein lipase, did not prevent inhibition of enzyme activity by the Fab fragments. However, preincubation with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol or Triton X-100-emulsified trioleoylglycerol had a protective effect (remaining activity 7.0 or 25.8%, respectively, compared to 1.0 or 0.4% with no preincubation). The addition of both apolipoprotein C-II and substrate prior to the incubation with the Fab fragments was associated with an increased protective effect against inhibition of enzyme activity; remaining activity with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol was 40.6% and with Triton X-100-emulsified trioleoylglycerol, 45.4%. Human plasma very low density lipoproteins also protected against the inhibition of enzyme activity by the Fab fragments. These immunological studies suggest that the interaction of lipoprotein lipase with apolipoprotein C-II in the presence of lipids is associated with a conformational change in the structure of the enzyme such that the Fab fragments are less inhibitory. The consequence of a conformational change in lipoprotein lipase may be to facilitate the formation of an enzyme-triacylglycerol complex so as to enhance the rate of the lipoprotein lipase-catalyzed turnover of substrate to products.     

Kinetics of product inhibition and mechanisms of lipoprotein lipase activation by apolipoprotein C-II

The kinetics of product inhibition of bovine milk lipoprotein lipase (LPL) were studied in a system of emulsified trioleoylglycerol (TG) at different fixed initial concentrations of oleic acid [( OA]0) without a fatty acid (FA) acceptor. In the absence of apolipoprotein C-II (C-II), the apparent Vmax and the nH(TG) (the slope of the corresponding Hill plot for TG) of 1.82 decreased by about 52% and [TG]0.5 increased 13-fold by raising the [OA]0 to 0.3 mM. At low [OA]0, product inhibition was competitive with respect to TG: the nH(OA) averaged 1.1, and [OA]0.5 was increased about 2-fold by TG. At the higher [OA]0, nH(OA) was 3.5, and TG had no effect on [OA]0.5. In the presence of 3 micrograms/mL C-II, the apparent Vmax was 4.3-7.1-fold higher than in its absence, and the nH(TG) was 2.45. Both parameters decreased by only 20-25%, and [TG]0.5 increased only 3-fold at an [OA]0 of 0.3 mM. Conversely, nH(OA) decreased by 35% and [OA]0.5 increased 6-fold by increasing TG concentrations. Similar kinetics were observed with very low density lipoproteins (VLDL). At saturating TG and varying C-II concentrations, nH(C-II) was 1.78, and product inhibition was found to be competitive with respect to C-II. At the [OA]0 employed, the FA had no effect on enzyme binding to TG emulsions, and there was no evidence that LPL catalyzes the reverse reaction. It is concluded that (a) the LPL kinetics are those of a multisite enzyme that probably has three high-affinity binding sites for TG, two for C-II, and four for OA.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Relationship of lipoprotein lipase and hepatic triacylglycerol lipase activity to serum adiponectin levels in Japanese hyperlipidemic men.

OBJECTIVE: The aim of this study was to determine how lipoprotein lipase (LPL) and hepatic triacylglycerol lipase (HTGL) activity relate to serum adiponectin levels. RESEARCH DESIGN AND METHODS: Fifty-five hyperlipidemic Japanese men were recruited for this study. LPL and HTGL activity in post-heparin plasma (PHP) was measured using Triton X-100 emulsified-[14C] triolein. The remaining activity in the presence of 1M NaCl was defined as HTGL activity. Serum adiponectin levels were determined by an enzyme-linked immunosorbent assay system. RESULT: LPL activity had a positive relationship with HDL2, but had no relation with HDL3, while HTGL had positive relationship with HDL3, but had no relationship with HDL2. LPL activity showed a positive relationship [r = 0.345, p = 0.010] to serum adiponectin levels, while and HTGL activity showed an inverse relationship [r = - 0.365 p = 0.006]. Multiple regression analysis with LPL and HTGL as dependent variables and age, BMI, serum adiponectin and the homeostasis model assessment of insulin resistance (HOMA-IR) as independent variables showed LPL and HTGL's association to adiponectin did not persist after adjustments for these covariants. However, the association of LPL activity to HOMA-IR was found to persist after adjustments of age, BMI, and serum adiponectin. CONCLUSIONS: There was a co-linearity between insulin sensitivity and adiponectin as well as insulin sensitivity and LPL/HTGL activity.     

Purification and characterization of a second form of acid lipase in human liver.

We describe here the purification and characterization of a form of acid lipase from human liver (designated ALII), which differed from the more abundant Mr-29000 form (ALI). ALII was solubilized from frozen human liver with Triton X-100 and purified 8500-fold by chromatography over concanavalin A-sepharose, CM-cellulose and finally h.p.l.c. over a Mono S column. ALII migrated as a single band on polyacrylamide-gel electrophoresis in both the presence and the absence of SDS. The Mr of ALII was estimated to be 58,500 by SDS/polyacrylamide-gel electrophoresis. Gel filtration on Sephacryl S-200 gave an apparent Mr of 69,000. 4-Methylumbelliferyl (4MU) palmitate, cholesterol oleate and triolein were substrates for ALII, with apparent Vmax values of 5000, 1100 and 2500 nmol/min per mg respectively and Km values of 1.0, 1.5 and 1.8 mM respectively. Cholesterol oleate and triolein were hydrolysed optimally by ALII at pH 4.5, whereas 4MU palmitate was hydrolysed optimally at pH 5.3. Antisera were raised against ALI and ALII and, on immunoblot analysis, no antigenic similarity was observed between ALI and ALII. Cellulose acetate electrophoresis followed by reaction with 4MU palmitate revealed two forms of lipase, corresponding to ALI and ALII. The two enzymes were also separated by hydrophobic chromatography. The activity of ALII was stimulated by several proteins and was partially inhibited by millimolar concentrations of NaCl, CaCl2 and MgSO4.     

Conformational changes in human urokinase induced by a specific reduction of disulfide bond in Cys-194-Cys-222 associated with exhibition of enzymatic activity

The mechanism of action of hepatic triacylglycerol lipase (EC 3.1.1.3) was examined by comparing the hydrolysis of a water-soluble substrate, tributyrin, with that of triolein by hepatic triacylglycerol lipase purified from human post-heparin plasma. The hydrolyzing activities toward tributyrin and triolein were coeluted from heparin-Sepharose at an NaCl concentration of 0.7 M. The maximal velocity of hepatic triacylglycerol lipase (Vmax) for tributyrin was 17.9 mumol/mg protein per h and the Michaelis constant (Km) value was 0.12 mM, whereas the Vmax for triolein was 76 mumol/mg per h and the Km value was 2.5 mM. The hydrolyses of tributyrin and triolein by hepatic triacylglycerol lipase were inhibited to similar extends by procainamide, NaF, Zn2+, Cu2+, Mn2+, SDS and sodium deoxycholate. Triolein hydrolysis was inhibited by the addition of tributyrin. Triolein hydrolysis was also inhibited by the addition of dipalmitoylphosphaidylcholine vesicles. In contrast, the additions of triolein emulsified with Triton X-100 and dipalmitoylphosphatidylcholine vesicles enhanced the rate of tributyrin hydrolysis by hepatic triacylglycerol lipase. In the presence of dipalmitoylphosphatidylcholine, the Vmax and Km values of hepatic triacylglycerol lipase for tributyrin were 41 mumol/mg protein per h and 0.12 mM, respectively, indicating that the enhancement of hepatic triacylglycerol lipase activity for tributyrin by dipalmitoylphosphatidycholine vesicles was mainly due to increase in the Vmax. The enhancement of hepatic triacylglycerol lipase activity for tributyrin by phospholipid was not correlated with the amount of tributyrin associated with the phospholipid vesicles. On Bio-Gel A5m column chromatography, glycerol tri[1-14C]butyrate was not coeluted with triolein emulsion, and hepatic triacylglycerol lipase activity was associated with triolein emulsion even in the presence of 2 mM tributyrin. These results suggest that hepatic triacylglycerol lipase has a catalytic site for esterase activity and a separate site for lipid interface recognition, and that on binding to a lipid interface the conformation of the enzyme changes, resulting in enhancement of the esterase activity.     

Characterization of the lipolytic activity of endothelial lipase

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family previously reported to have phospholipase activity. Using radiolabeled lipid substrates, we characterized the lipolytic activity of this enzyme in comparison to lipoprotein lipase (LPL) and hepatic lipase (HL) using conditioned medium from cells infected with recombinant adenoviruses encoding each of the enzymes. In the absence of serum, EL had clearly detectable triglyceride lipase activity. Both the triglyceride lipase and phospholipase activities of EL were inhibited in a dose-dependent fashion by the addition of serum. The ratio of triglyceride lipase to phospholipase activity of EL was 0.65, compared with ratios of 24.1 for HL and 139.9 for LPL, placing EL at the opposite end of the lipolytic spectrum from LPL. Neither lipase activity of EL was influenced by the addition of apolipoprotein C-II (apoC-II), indicating that EL, like HL, does not require apoC-II for activation. Like LPL but not HL, both lipase activities of EL were inhibited by 1 M NaCl. The relative ability of EL, versus HL and LPL, to hydrolyze lipids in isolated lipoprotein fractions was also examined using generation of FFAs as an end point. As expected, based on the relative triglyceride lipase activities of the three enzymes, the triglyceride-rich lipoproteins, chylomicrons, VLDL, and IDL, were efficiently hydrolyzed by LPL and HL. EL hydrolyzed HDL more efficiently than the other lipoprotein fractions, and LDL was a poor substrate for all of the enzymes.     

Coexistence of abnormalities of hepatic lipase and lipoprotein lipase in a large family.

A large family is reported with familial hepatic triglyceride lipase (HTGL) deficiency and with the coexistence of reduced lipoprotein lipase (LPL) similar to the heterozygote state of LPL deficiency. The proband was initially detected because of hypertriglyceridemia and chylomicronemia. He was later demonstrated to have beta-VLDL despite an apo E3/E3 phenotype and the lack of stigmata of type III hyperlipoproteinemia. The proband had no HTGL activity in postheparin plasma. Two of his half-sisters had very low HTGL activity (39 and 31 nmol free fatty acids/min/ml; normal adult female greater than 44). His son and daughters had decreased HTGL activity (normal male and preadolescent female greater than 102), which would be expected in obligate heterozygotes for HTGL deficiency. Low HTGL activity was associated with LDL particles which were larger and more buoyant. Several family members, including the proband, had reduced LPL activity and mass less than that circumscribed by the 95% confidence-interval ellipse for normal subjects and had hyperlipidemia similar to that described in heterozygote relatives of patients with LPL deficiency. All the sibs with hyperlipidemia had a reduced LPL activity and mass, while subjects with isolated reduced HTGL (with normal LPL activity) had normal lipid phenotypes. Analysis of genomic DNA from these subjects by restriction-enzyme digestion revealed no major abnormalities in the structure of either the HTGL or the LPL gene. Compound heterozygotes for HTGL and LPL deficiency show lipoprotein physiological characteristics typical for HTGL deficiency, while their variable lipid phenotype is typical for LPL deficiency.     

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.     

Partial purification and characterization of a triglyceride lipase from pig adipose tissue.

A triglyceride lipase was extracted from defatted pig adipose tissue powder with dilute ammonia and purified about 230-fold by a combination of ammonium sulfate fractionation, heparin-Sepharose 4B, DEAE-cellulose, and Sephadex G-150 column chromatographies and isoelectrofocusing electrophoresis. The enzyme was distinguishable in physical and kinetic properties from the two previously defined lipases in adipose tissue, lipoprotein lipase, and hormone-sensitive lipase. The purified enzyme was fully active in the absence of serum lipoprotein and was not stimulated by adenosine 3':5'-monophosphate-dependent protein kinase. In marked contrast to the already defined lipases, the enzyme was strongly inhibited by serum albumin. The enzyme had a molecular weigt of about 43,000, a pI of 5.2, and pH optimum of 7.0. The enzyme hydrolyzed triolein to oleic acid and glycerol, and did not exhibit esterase activity. The apparent Km for triolein was 0.05 mM. Physiological roles of this new species of lipase remained to be explored.     

Triglyceridase and phospholipase A1 activities of rat-heart lipoprotein lipase. Influence of apolipoproteins C-II and C-III.

The influence of purified human apolipoprotein C-II on phospholipase A1 and triglyceridase activities of lipoprotein lipase were compared. Lipoprotein lipase was obtained from rat hearts by perfusion with a medium containing heparin and purified on a heparin Sepharose 4-B column. Using phosphatidyl-ethanolamine-coated triglyceride particles as substrate it was found that the phospholipase A1 and triglyceridase activities of lipoprotein lipase similarly depend on the presence of apolipoprotein C-II. Apolipoprotein C-III cannot replace apolipoprotein C-II. However, addition of apolipoprotein C-III in the presence of C-II affects both lipase activities. While strong inhibition of triglyceridase activity was observed under these conditions, phospholipase A1 activity was slightly stimulated. On the basis of these findings a model was constructed for the role of apolipoprotein C-II in lipoprotein lipase action.