Characterization of two triacylglycerol lipase activities in pig post-heparin plasma.

Two triacylglycerol lipase activities were characterized after partial purification from pig post-heparin plasma. These two lipase activities were eluted sequentially with a NaCl gradient from columns containing Sepharose with covalently linked heparin. The first lipase activity, which was eluted at 0.75M-NaCl, was not inhibited at 28 degrees C in the presence of 1M-NaCl and was not further activated by plasma apolipoproteins. The absence of this lipase activity from post-heparin plasma from hepatectomized pigs indicates that the liver plays a role in the synthesis of this enzyme. A second lipase activity, which was eluted at 1.2M-NaCl, was inhibited when assayed in the presence of 1.0M-NaCl and was activated 14-fold by an apolipoprotein isolated from human very-low-density lipoprotein. The characteristics are identical with those of lipoprotein lipase purified from pig adipose tissue.     

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.     

Effects of threonine-poor apolipoproteins on post-heparin plasma hepatic triacylglycerol lipase and lipoprotein lipase

Whole-irradiated rabbit pre-heparin plasma had an important inhibitory effect on hepatic triacylglycerol lipase and lipoprotein lipase activities, whereas control rabbit pre-heparin plasma slightly inhibited hepatic triacylglycerol lipase activity at a high concentration and enhanced lipoprotein lipase activity. As some apolipoproteins were known to modulate these two lipolytic enzymes, the inhibitory effects of irradiated rabbit plasma were investigated in apolipoproteins. Three apolipoproteins, with isoelectric points of about 6.58, 6.44 and 6.12, characterized by their low content in threonine (threonine-poor apolipoproteins) were produced in high concentrations in rabbit VLDL and HDL after irradiation. The effects of these apolipoproteins on control rabbit post-heparin plasma hepatic triacylglycerol lipase and extrahepatic lipoprotein lipase were studied. Threonine-poor apolipoproteins substantially inhibited the hepatic triacylglycerol lipase activity and enhanced the apolipoprotein C-II-stimulated activity of lipoprotein lipase. The amounts of these apolipoproteins in triacylglycerol-rich lipoprotein particles may determine the lipolytic activity of lipoprotein lipase and hepatic triacylglycerol lipase in triacylglycerol hydrolysis. The existence of another inhibitor of lipoprotein lipase remains to be determined.     

Measurement of lipoprotein lipase and hepatic triacylglycerol lipase in post-heparin plasma of the cynomolgus monkey

Conditions for measurement of the lipolytic activities, lipoprotein lipase and hepatic triacylglycerol lipase in cynomolgus monkey postheparin plasma are described. The two activities are separable by heparin-Sepharose chromatography. Goat anti-human hepatic triacylglycerol lipase serum inhibits monkey hepatic triacylglycerol lipase activity and allows direct measurement of lipoprotein lipase in post-heparin plasma. While both human and homologous serum can be used as a source of activator apolipoprotein, homologous serum produces a much greater activation.     

Properties of salt-resistant lipase and lipoprotein lipase purified from human post-heparin plasma.

Lipoprotein lipase and salt-resistant lipase were isolated from human post-heparin plasma. The proteins of human post-plasma lipoprotein lipase and salt-resistant lipase were identified and demonstrated to be immunologically different. Significant differences between the two enzymes in their relative amino acid composition were demonstrated, which indicates that the two enzymes are different proteins. When analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the enzymes seemed to have monomer molecular weights similar to that of lipoprotein lipase purified from bovine milk.     

Effect of pantethine on post-heparin plasma lipolytic activities and adipose tissue lipoprotein lipase in rats

The lipid-lowering effect of pantethine, a new drug affecting lipid metabolism, had been evaluated in carbohydrate-induced hyperlipidemic rats. Administration of the drug raised post-heparin lipolytic activities, the change being due to an increase in lipoprotein lipase activity, whereas hepatic lipase activity remained virtually unchanged. Total lipoprotein lipase activity per g of adipose tissue increased in pantethine-treated rats compared with controls. Furthermore, the soluble lipoprotein lipase of fat-pads was fractionated by heparin-Sepharose affinity chromatography. The first active peak, originated from the microsomal fractions, significantly increased after the drug treatment, while the second one, originated from the plasma membranes, remained unchanged. The increase in the microsomal lipoprotein lipase activity may be due to an increase in intracellular synthesis of lipoprotein lipase enzyme proteins. The heterogeneity of lipoprotein lipase of rat adipose tissues was ensured using affinity chromatography on heparin-Sepharose.     

Isolation of apolipoprotein-free lipoprotein lipase from human post-heparin plasma

Lipoprotein lipase from human post-heparin plasma was purified at least 10,000-fold using the Fielding procedure. When the purified lipoprotein lipase was subjected to polyacrylamide electrophoresis, a single band with lipolytic activity and four additional bands were observed. These four bands are identical in their electrophoretic and immunochemical properties to the polypeptides of apolipoprotein C. Evidence is presented which suggests that one or more of these polypeptides may serve as a partial activator of this enzyme.     

Lipoprotein lipase in rat heart--I. Characterization of tri-, di- and monoacylglycerol lipase activities in post-heparin effluents

1. The lipolytic activities that sequentially hydrolyze tri-, di- and monoacylglycerol in rat post-heparin heart effluents were examined. 2. Properties of triacylglycerol lipase (TAGL) activity were typical of lipoprotein lipase. Diacylglycerol lipase (DAGL) behaved similarly to TAGL, suggesting that both activities refer to the same catalytic entity. 3. Differences, particularly in thermal stability, between TAGL and DAGL activities on one hand, and monoacylglycerol lipase (MAGL) activity on the other, may reflect different intrinsic molecular properties. 4. TAGL, DAGL and MAGL activities could not be separated by physical means and appeared to belong to a single unit at the same site on the capillary wall.     

The contribution of hepatic triglyceride lipase to plasma post-heparin lipolytic activity in the rat.

Plasma post-heparin lipolytic activity (PHLA) was reduced by 50% in functionally hepatectomised compared with shamoperated rats. Inhibition of PHLA by protamine sulphate was significantly increased in functionally hepatectomised compared with shamoperated rats. Hepatic triglyceride lipase contributes significantly to total plasma PHLA. Its function is not yet clear.     

Evidence for separate monoglyceride hydrolase and triglyceride lipase in post-heparin human plasma

Post-heparin plasma contains an enzyme or enzymes with both triglyceride lipase (TGL) and monoglyceride hydrolase (MGH) activities. A simple and reproducible radioactive assay for measurement of MGH activity was developed and used, with a previously reported assay for TGL, to study lipolysis in plasma. After the injection of heparin, enzymatic activity against both tri- and monoglycerides appeared and disappeared from plasma at approximately the same rates. However, in contrast to TGL activity, MGH activity was: (a) much greater, (b) considerably less heat-sensitive, (c) unaffected by three inhibitors (NaCl, protamine, and pyrophosphate), (d) not influenced by radical changes in fat and carbohydrate content of the diet, and (e) normal in familial Type I hyperlipoproteinemia. The dichotomy between MGH and TGL activities in patients with genetic deficiency of TGL constitutes strong evidence that these are two different enzymes. The findings further indicate that when post-heparin lipolytic activity is measured for the purpose of detecting TGL deficiency, it may be necessary to perform the assay with a substrate free from partial glycerides.     

Differentiation of microsomal from lysosomal triacylglycerol lipase activities in rat liver

The F1-ATPase from Micrococcus lysodeikticus has been purified to 95% protein homogeneity in this laboratory and as all other bacterial F1S, possesses five distinct subunits with molecular weights ranging from 60 000 to 10 000 (Huberman, M. and Salton, M.R.J. (1979) Biochim. Biophys. Acta 547, 230-240). In this communication, we demonstrate the immunochemical reactivities of antibodies to native and SDS-dissociated subunits with the native and dissociated F1-ATPase and show that: (1) the antibodies generated to the native or SDS-dissociated subunits react with the native molecule; (2) all of the subunits comprising the F1 are antigenically unique as determined by crossed immunoelectrophoresis and the Ouchterlony double-diffusion techniques; (3) antibodies to the SDS-denatured individual delta- and epsilon-subunits can be used to destabilize the interaction of these specific subunits with the rest of the native F1; and (4) all subunit antibodies as well as anti-native F1 were found to inhibit ATPase activity to varying degrees, the strongest inhibition being seen with antibodies to the total F1 and anti-alpha- and anti-beta-subunit antibodies. The interaction of specific subunit antibodies may provide a new and novel way to study further and characterize the catalytic portions of F1-ATPases and in general may offer an additional method for the examination of multimeric proteins.     

Lipoprotein lipase in rat heart--III. Effects of sex steroid hormones on tri-, di- and monoacylglycerol lipase activities in post-heparin effluents

The in vivo administration of ethinylestradiol to female rats causes dose-dependent decreases in tri-, di- and monoacylglycerol lipase activities in post-heparin heart effluents; monoacylglycerol lipase is less sensitive to estrogen than the other two.     

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.     

Studies on the effect of hepatectomy on pig post-heparin plasma lipases

The effect of different amounts of heparin injected intravenously in swine on lipoprotein lipase and hepatic lipase activities in post-heparin plasma was studied using an immunochemical method. After the injection of 50 I.U. of heparin/kg body weight the apparent half-life of lipoprotein lipase and hepatic lipase activity measurable in post-heparin plasma was 15 min. This was prolonged to more than 60 min after the injection of 1000 I.U./kg body weight. It is concluded that the higher the heparin dose injected the longer can lipolytic activities be measured in plasma. A possible explanation for these findings is that the amount of circulating heparin governs the distribution of lipoprotein lipase and hepatic lipase between an endothelial-bound form and a circulating form and thus determines the apparent ‘half-life’ of lipase activity measurable in plasma. The apparent half-life of radioactively labelled heparin in normal swine was not different from that observed in hepatectomized animals. After hepatectomy no immunoreactive hepatic lipase activity could be demonstrated in post-heparin plasma confirming our previous findings that the liver is the only source of hepatic lipase.To study the role of the liver in the clearance of plasma lipoprotein lipase activity after the administration of heparin normal and hepatectomized pigs were given 200 I.U./kg body weight followed by a heparin infusion of 100 I.U./ h per kg body weight. In the control pigs the heparin injection caused a rapid release of lipoprotein lipase and hepatic lipase activities. These activities were maintained in the circulation during the 3-h infusion at a level of about 60% of the levels measurable 30 min after the injection. In hepatectomized pigs the lipoprotein lipase activity rose during the infusion to about six times the activity recorded 30 min after heparin administration. From these experiments we conclude that after heparin injection the liver is involved in the clearance of post-heparin plasma lipolytic activity.