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.     

Preparation of a homogeneous and stable form of bovine milk lipoprotein lipase

Lipoprotein lipase was purified to homogeneity from bovine skim milk by a two-step procedure using chromatography on heparin-Sepharose. As determined by gradient-polyacrylamide gel electrophoresis in sodium dodecyl sulfate, purified lipoprotein lipase showed a single band with an apparent molecular weight of 55,000. The use of Triton N-101 in the washing buffers was the major improvement from previously reported purification procedures that resulted in a stable homogeneous preparation of the enzyme.     

Preparation of a Homogenous and Stable form of Bovine Milk Lipoprotein Lipase

Abstract

Lipoprotein lipase was purified to homogeneity from bovine skim milk by a two-step procedure using chromatography on heparin-Sepharose. As determined by gradient-polyacrylamide gel electrophoresis in sodium dodecyl sulfate, purified lipoprotein lipase showed a single band with an apparent molecular weight of 55,000. The use of Triton N-101 in the washing buffers was the major improvement from previously reported purification procedures that resulted in a stable homogeneous preparation of the enzyme.     

Synthesis and secretion of lipoprotein lipase in 3T3-L1 adipocytes. Demonstration of inactive forms of lipase in cells

3T3-L1 adipocytes in culture incorporated [35S]methionine into a protein which could be immunoprecipitated with chicken antiserum to bovine lipoprotein lipase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed this protein had an Mr of 55,000, similar to that of bovine lipoprotein lipase, and accounted for 0.1-0.5% of total protein synthesis in the adipocytes. Lipoprotein lipase protein was present in small amounts in confluent 3T3-L1 fibroblasts, and the amount increased many-fold as the cells differentiated into adipocytes. This increase was accompanied by parallel increases in cellular lipase activity and secretion. When cells were grown with [35S]methionine, the amount of label incorporated into lipoprotein lipase increased for 2 h and then leveled off. Pulse-chase experiments showed that half-life of newly synthesized lipase was about 1 h. Turnover of lipoprotein lipase in control cells involved both release to the medium and intracellular degradation. When N-linked glycosylation was blocked by tunicamycin, the cells synthesized a form of lipase that had a smaller Mr (48,000), was catalytically inactive, and was not released to the medium. Radioimmunoassay demonstrated that 3T3-L1 adipocytes contained an unexpectedly large amount of lipoprotein lipase protein. 55% of the enzyme protein in acetone/ether powder of the cells was insoluble in 50 mM NH3/NH4Cl at pH 8.1, a solution commonly used to extract lipoprotein lipase; 27% of the lipase protein was soluble but did not bind to heparin-Sepharose and had very low lipase activity; and the remaining 13% was soluble, bound to heparin-Sepharose, and had high lipolytic activity. About one-half of the lipase released spontaneously to the medium was inactive, and lipase inactivation proceeded in the medium with little loss of enzyme protein. Lipoprotein lipase released heparin, in contrast, was fully active and more stable. When protein synthesis was blocked by cycloheximide, the level of lipoprotein lipase activity in adipocytes decreased more rapidly than the amount of lipase protein in the cells. Most of the inactive lipoprotein lipase in adipocytes probably results from dissociation of active dimeric lipase, but some could be a precursor of active enzyme.     

Characterization of rat adipose tissue lipoprotein lipase using a monospecific antibody

An antibody to a highly pure enzyme preparation was developed to facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation. Lipoprotein lipase was purified by heparin-Sepharose affinity chromatography followed by preparative isoelectric focusing. The enzyme migrated as a single broad band on SDS disc gel and two-dimensional gel electrophoresis with an apparent molecular mass of 67 000 and 62 000 Da, respectively. The amino acid composition of the purified rat enzyme was virtually identical to that of bovine milk. A major protein component with no lipase activity co-eluted with the enzyme from the affinity column, but was separated by the isoelectric focusing step. The molecular mass was slightly lower (58 000 Da) but the amino acid composition of this protein was similar to that of the enzyme. An antibody raised against the purified rat enzyme was highly potent and was effective in inhibiting rat heart lipoprotein lipase, but not the salt-resistant hepatic lipase. Analysis of crude acetone-ether adipose tissue preparation on SDS slab polyacrylamide gel coupled to Western blotting revealed five protein bands = (62 000, 56 000, 41 700, 22 500, 20 000 Da). Similarly, following affinity purification by immunoadsorption, the purified antibody reacted with five equivalent protein bands. Fluorescent concanavalin A binding data indicated that the 56 kDa band is a glycosylated form of lipoprotein lipase. Pretreatment of adipose tissue with proteinase inhibitors revealed that the lower molecular mass proteins (41 700 and 20 000 Da) were degradation products of lipoprotein lipase, and the 22 500 Da band could be accounted for by non-specific binding.     

Purification from human plasma of a heparin-released lipase with activity against triglyceride and phospholipids.

A triglyceride lipase different from lipoprotein lipase, but measurable only after intravenous heparin injection, has been isolated from human plasma by sequential use of heparin-Sepharose and concanavalin A-Sepharose affinity chromatography. Using these procedures, phospholipase A1 activity was found to chromatograph identically with the triglyceride lipase. The constancy of the ratio of activities after isoelectric focusing (pI 4.1) and during thermal deactivation indicates that this enzyme has hydrolase activity against both triglycerides and phospholipids. This conclusion was supported further by the homogeneity of the protein as indicated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Cell-free translation of avian adipose tissue lipoprotein lipase messenger RNA

Poly(A)+ mRNA isolated from chicken adipose tissue directed cell-free translation in a rabbit reticulocyte lysate system. Immunoadsorption with polyclonal antibodies against lipoprotein lipase detected a protein of 56 +/- 2 kDa. Immunodetection of this protein was prevented by inclusion of purified lipoprotein lipase in the assay mixture. Identification of the 56 kDa protein as lipoprotein lipase was confirmed by immunoadsorption to the monoclonal antibody CAL 1-11. Inclusion of dog pancreatic microsomal membranes in the translation system resulted in isolation of an additional protein of 62 kDa. Treatment of the 62 kDa protein with endo-beta-N-acetylglycosaminidase H or endo-beta-N-acetylglucosaminidase F decreased the observed molecular mass to that of the primary translation product, indicating that the increase in molecular mass resulted from the addition of N-linked oligosaccharides. Starving and refeeding chickens prior to poly(A)+ mRNA isolation resulted in a 3-fold increase in the amount of immunodetectable lipoprotein lipase synthesized.     

Avian adipose lipoprotein lipase: cDNA sequence and reciprocal regulation of mRNA levels in adipose and heart

cDNA clones for chicken adipose lipoprotein lipase were isolated from an expression library in lambda gt11 by antibody screening and characterized by hybridization selection and nucleotide sequencing. Based on the cDNA sequence and on N-terminal sequence analysis of the purified enzyme, chicken adipose lipoprotein lipase is a mature protein of 465 amino acids with a signal peptide of 19 or 25 amino acids, depending on which of two methionine residues is used for translation initiation. The predicted amino-acid sequence was found to be 73-77% identical to the four known mammalian adipose lipoprotein lipase sequences, with conservation of position of cysteine residues and putative functional domains, and number of potential N-glycosylation sites. Chicken lipoprotein lipase differs from mammalian lipoprotein lipases with respect to the position of one N-glycosylation site and the presence of an additional 15-17 C-terminal amino acids. 32P-labeled cDNA clones hybridized to mRNA species of 3.7 and 4.0 kb in Northern blots of heart and adipose, but not of liver RNA. In chickens that were fasted for 48 h and then refed, lipoprotein lipase mRNA levels in adipose increased to a maximal level of 350% that of controls at 10 h, whereas heart lipoprotein lipase mRNA levels fell to 40% of controls at 14 h. Concomitantly, no changes in total RNA were observed. Thus, avian lipoprotein lipase is subject to reciprocal pretranslational regulation in adipose and heart.     

Purification and characterization of rat adipose tissue lipoprotein lipase.

Lipoprotein lipase (EC 3.1.1.34) extracted from adipose tissue of glucose-fed rats with 5 mM-sodium barbital, pH 7.5, containing 20% (v/v) glycerol and 0.1% (v/v) Triton X-100, was partially purified by affinity chromatography on heparin linked to Sepharose 4B. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the partially purified enzyme preparation revealed the presence of two major Coomassie-staining bands (mol.wts. 62 000 and 56 000) as well as a number of minor bands. Treatment of partially purified enzyme with [1,3-3H]di-isopropyl fluorophosphate resulted in the incorporation of radiolabel into the band of mol.wt. 56 000, but not into the band of mol.wt. 62 000. Both the amount of the 56 000-mol.wt. polypeptide and the incorporation of [1,3-3H]di-isopropyl fluorophosphate into this band were greatly reduced in the enzyme preparations isolated from adipose tissue of 48 h-starved rats. whereas the amount of the 62 000-mol.wt. polypeptide was unaffected by starvation. Purification of lipoprotein lipase from adipose tissue of glucose-fed rats was also carried out using affinity chromatography on Sepharose 4B linked to heparin with low affinity for antithrombin-III. This procedure resulted in the presence of a single band of mol.wt. 56 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These results suggest that the polypeptide of mol.wt. 56 000 corresponds to the subunit of lipoprotein lipase, whereas the 62 000-mol.wt. polypeptide probably represents antithrombin-III.     

Monoclonal antibodies to bovine milk lipoprotein lipase. Evidence for proteolytic degradation of the native enzyme

Lipoprotein lipase was purified from bovine skim milk by chromatography on heparin-Sepharose. Polyacrylamide gel electrophoresis showed a single protein with an apparent molecular weight of 55,000 in the trailing edge of the elution profile; fractions in the leading edge contained additional proteins with molecular weights of 36,000 and 18,000-22,000. Nine monoclonal antibodies were prepared against the 55,000-dalton protein. By immunoblotting, we show that the Mr = 18,000-22,000 components share common antigen determinants with the 55,000-dalton protein, suggesting that they represent proteolytic degradation products. Incubation of partially purified lipoprotein lipase for 24 h at 37 degrees C results in breakdown of the 55,000-dalton protein with concomitant enrichment in lower Mr components; the proteolytic activity is prevented by incubating the milk with phenylmethane, sulfonyl fluoride prior to chromatography on heparin-Sepharose. This study shows the presence of milk proteases which co-purify and degrade lipoprotein lipase. We suggest that this degradation could account for part of the known instability of the enzyme.     

Purification and characterization of lipoprotein lipase from pig myocardium.

1. Lipoprotein lipase was purified from pig myocardium by a two-step purification procedure involving (a) the formation of an enzyme-substrate complex and (b) affinity chromatography on Sepharose which contained covalently linked heparin. The purified enzyme gave in sodium dodecyl sulphate-polyacrylamide-gel electrophoresis one main band with an apparent molecular weight of 73 000. The enzyme, which was purified 70 000-fold, had a specific activity of 860 mumol of unesterified fatty acid liberated/h per mg of protein. 2. The purified enzyme hydrolysed [14C]triolein emulsions in the absence of added cofactors but its activity was increased fivefold by adding normal human serum. Of the low-density lipoprotein apoproteins only apolipoprotein CII could be substituted for serum in activating the enzyme. This lipase had maximum activity at 0.05-0.15 M-NaCl. Heparin increased the activity of the purified enzyme twofold at low concentrations, but high concentrations inhibited. The triglyceride lipase of pig myocardium thus resembles lipoprotein lipase purified from adipose tissue and from plasma, but is clearly different from pig hepatic triglyceride lipase.     

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.     

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.     

Structural Properties of the Heparan Sulfate Proteoglycans of Brain

The heparan sulfate proteoglycans present in a deoxycholate extract of rat brain were purified by ion exchange chromatography, affinity chromatography on lipoprotein lipase agarose, and gel filtration. Heparitinase treatment of the heparan sulfate proteoglycan fraction (containing 86% heparan sulfate and 10% chondroitin sulfate) that was eluted from the lipoprotein lipase affinity column with 1 M NaCl led to the appearance of a major protein core with a molecular size of 55,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the effects of heparinase and heparitinase treatment revealed that the heparan sulfate proteoglycans of brain contain a significant proportion of relatively short N-sulfoglucosaminyl 6-O-sulfate [or N-sulfoglucosaminyl](alpha 1-4)iduronosyl 2-O-sulfate(alpha 1-4) repeating units and that the portions of the heparan sulfate chains in the vicinity of the carbohydrate-protein linkage region are characterized by the presence of D-glucuronic acid rather than L-iduronic acid. After chondroitinase treatment of a proteoglycan fraction that contained 62% chondroitin sulfate and 21% heparan sulfate (eluted from lipoprotein lipase with 0.4 M NaCl), the charge and density of a portion of the heparan sulfate-containing proteoglycans decreased significantly. These results indicate that a population of "hybrid" brain proteoglycans exists that contain both chondroitin sulfate and heparan sulfate chains covalently linked to a common protein core.     

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.     

The characterization of lipoprotein lipase isolated from the post-heparin plasma of the rainbow trout, Salmo gairdneri Richardson.

1. Intravenous injection of heparin into the trout resulted in the appearance in the plasma of a lipase with the properties of lipoprotein lipase. 2. The enzyme was purified to apparent electrophoretic homogeneity by means of heparin-Sepharose affinity chromatography. The enzyme was eluted with 1.5 M-NaCl and had a specific activity approx. 450-fold that of the post-heparin plasma. 3. The activity of the purified enzyme was inhibited by 1.0 M-NaCl and protamine sulphate and was stimulated between 3- and 8.8-fold by the addition of trout plasma. 4. The activity was strongly stimulated by trout very low density lipoproteins and to a lesser extent by high density lipoproteins. 5. The isolated enzyme fraction gave a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had an apparent subunit M4 of 63 000. 6. These results suggest that the uptake of lipid by the tissues in the trout can occur by a process similar to that in mammals.     

Characterization of serum-stimulated lipoprotein lipase from bovine heart

1. A triglyceride (TG) lipase is present in whole homogenate and tissue extracts of beef myocardium with characteristics of lipoprotein lipase (LPL); i.e., activity is stimulated by serum, inhibited by NaCl and protamine sulfate, the protein binds to heparin-Sepharose, and the enzyme has an alkaline pH optimum. 2. This TG lipase, eluted from heparin-Sepharose at 0.9-1.0 M NaCl, has an apparent mol. wt of 64 K daltons. Its primary mRNA is 3.7 kb. 3. Expression of LPL mRNA and enzyme activities are in the ratio of approximately 20:8:1 for hearts of mouse, rat and beef, respectively and correlate with r = +0.99.     

Activation of myocardial neutral triglyceride lipase and neutral cholesterol esterase by cAMP-dependent protein kinase

Lipolysis of intracellular triglycerides in the heart has been shown to be regulated by hormones. However, activation of myocardial triglyceride lipase in a cell-free system has not been directly demonstrated. In the present studies, initial attempts to demonstrate cAMP-dependent activation of triglyceride lipase using the 1,000 X g supernatant fraction (S1) of mouse heart homogenate were unsuccessful, presumably due to the masking effects of high levels of lipoprotein lipase activity even when assayed at pH 7.4 and in the absence of apolipoprotein C-II. Myocardial lipoprotein lipase in the 40,000 X g supernatant fraction was then removed by heparin-Sepharose affinity chromatography. The lipoprotein lipase-free fractions were shown to contain neutral triglyceride lipase and neutral cholesterol esterase of about equal activities. The triglyceride lipase and cholesterol esterase activities fell progressively during preincubation in the presence of 5 mM Mg2+. Additions of cAMP and ATP resulted in 40-70% activation of both triglyceride lipase and cholesterol esterase. The activation was blocked by protein kinase inhibitor and was restored by the addition of exogenous cAMP-dependent protein kinase. Since lipoprotein lipase has no activity toward cholesteryl oleate, activation of cholesterol esterase in untreated S1 was readily demonstrable. Both triglyceride lipase and cholesterol esterase activities were present in homogenates prepared from isolated rat heart myocytes. We conclude that the myocardium contains a hormone-sensitive lipase that is regulated in a fashion similar to that of the adipose tissue enzyme.     

Enhanced release and synthesis of lipoprotein lipase in rat heart cell cultures exposed to high concentrations of Hepes

While attempting to optimize conditions for synthesis of lipoprotein lipase by cultured heart cells, we encountered an unexpected rise in enzyme activity when media were supplemented inadvertently with 100 mM Hepes buffer (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid). This finding was further investigated and optimal results were obtained at pH 7.0-7.2. The increase in lipoprotein lipase activity was time dependent; after 3-6 h there was a rise in medium activity but cellular activity increased only after 24 h. The increased enzyme activity was defined as lipoprotein lipase by inhibition with antiserum to rat adipose tissue lipoprotein lipase. A 72-h exposure to Hepes resulted in a 30% increase in the incorporation of [35S]methionine into cellular proteins and a 2-fold increase into heparin-releasable proteins. Using heparin Sepharose chromatography and stepwise elution, a lipoprotein lipase enriched fraction was recovered with 2 M NaCl. The amount of [35S]methionine and [3H]galactose incorporated into protein of this fraction derived from Hepes-treated cells was 2-6-fold that of controls. A 4-fold increase in cellular lipoprotein lipase mass in Hepes-treated cells was shown by immunoblotting. Results obtained with Hepes-conditioned medium suggest the presence of cell-derived compounds that enhance release and subsequent synthesis of lipoprotein lipase. The effect of Hepes-conditioned medium on lipoprotein lipase resembled to some extent that of the addition of heparin. Therefore, it appears that when Hepes is first added to the culture medium, it might promote a release of heparan sulfate or related compounds, possibly by virtue of its negatively charged sulfonic acid residue. The accumulated heparan sulfate could then promote a sustained release of lipoprotein lipase into the culture medium which in turn leads to increased enzyme synthesis.