Validation of a "clearing" assay for milk lipoprotein lipase in agarose gel

We have developed a simplified method for the quantitative assay of lipoprotein lipase in cow's milk based on the hydrolysis of a glyceride emulsion in semisolid agarose gel. The area of clearing produced thereby is a function of enzyme concentration. Absolute molar rates for unknown samples may be calculated if standards of known activity are used concurrently. The precision of the simplified assay compared favorably with a method based on titrimetric determination of the rate of free fatty acid release. A modified assay has been used to assess the potency of lipoproteins in lipoprotein lipase activation.     

Macrophages take up triacylglycerol-rich emulsions at a faster rate upon co-incubation with native and modified LDL: An investigation on the role of natural chylomicrons in atherosclerosis.

Chylomicrons play a role in atherosclerosis, however, because the mechanisms involved in the cell uptake of these particles are not fully understood, investigations were carried out using a radioactively labeled protein-free triacylglycerol-rich emulsion incubated with peritoneal macrophages obtained from normal and apoE-knockout mice. Experiments were done in the presence of substances that inhibit several endocytic processes: EDTA for low density lipoprotein receptor, fucoidan for scavenger receptor, cytochalasin B for phagocytosis, and a lipopolysaccharide for lipoprotein lipase. In addition, triacylglycerol-rich emulsions were also prepared in the presence of native or modified radioactively labeled low density lipoprotein particles that are known to accumulate in the arterial intima. Probucol was also used to prevent the possible role played by an antioxidant in triacylglycerol-rich emulsion uptake. We have shown that triacylglycerol-rich emulsion alone is taken up by a coated-pit-dependent mechanism, mediated by macrophage secretion of apolipoprotein E. Furthermore, native, aggregated, acetylated, and moderately macrophage-oxidized low density lipoprotein stimulate the uptake of a triacylglycerol-rich emulsion through several mechanisms such as an actin-dependent pathway, scavenger receptors, and lipolysis mediated by lipoprotein lipase. On the other hand, in spite of the interaction of low density lipoprotein forms with a triacylglycerol-rich emulsion, the cellular triacylglycerol-rich emulsion uptake is impaired by copper-oxidized low density lipoprotein, possibly due to its diminished affinity towards lipoprotein lipase. We have also shown that macrophages take up aggregated low density lipoprotein better than the acetylated or oxidized forms of low density lipoprotein.     

Lipoprotein lipase activity in bovine aorta.

A lipoprotein lipase in the bovine arterial wall has been identified and partially characterized. The enzyme has a Km apparent of 1 mM for triolein in a phosphatidylcholine stabilized emulsion. The lipase was stimulated 20- to 30-fold by the addition of heated rat plasma to the assay medium. The activity exhibited a pH optimum at 8.6. Protamine sulfate (1.0 mg/ml) inhibited the activity by 50%, whereas 1.4 M sodium chloride inhibited by 85%. Sodium fluoride, an inhibitor of the hormone-sensitive lipase, had no effect on the activity. Additions of low concentrations of heparin or Ca-2+ to the enzyme caused a slight stimulation of the lipolytic activity. A crude sectioning of the aorta revealed specific activity of lipoprotein lipase to be highest at the endothelial side of the artery.     

Studies of lipoprotein lipase during the adipose conversion of 3T3 cells.

Lipoprotein lipase activity is negligible in exponentially growing 3T3-L1 cells and 3T3-F442A cells, but develops in both lines when they reach a confluent state and undergo adipose conversion. 3T3-C2 cells, which undergo adipose conversion with extremely low frequency, do not develop the enzyme. The lipase activity of 3T3-L1 and 3T3-F442A is greatly enhanced by insulin and increases 80–180 fold during the adipose conversion. The lipase has the following characteristics in common with lipoprotein lipase from adipose and other tissues: it is dependent upon serum, is inhibited by 0.5–1.0 M sodium chloride, is recovered from acetone powders, has an alkaline pH optimum and is released from the cells by heparin. Like the lipoprotein lipase of tissue adipose cells, the enzyme of 3T3-L1 decays in the presence of cycloheximide with a half-time of about 25 min at 37°C.The ability of 3T3-F442A and 3T3-L1 to take up triglyceride from the medium depends almost completely upon lipoprotein lipase. They incorporate the fatty acids of a large fraction of a triglyceride emulsion added to the medium, and this utilization is stimulated by heparin. Very little of the glycerol portion of the triglyceride is incorporated. 3T3-C2, which lacks lipoprotein lipase, utilizes very little of either the fatty acid or the glycerol portion of triglyceride.The relevance of external lipid or lipoprotein to both the adipose conversion and the appearance of lipoprotein lipase was tested using confluent cultures in medium depleted of these components. In the presence of serum whose lipoproteins have been removed by flotation, lines 3T3-F442A and 3T3-L1 undergo adipose conversion as completely as in the presence of untreated serum, and lipoprotein lipase activity appears at essentially the same rate. In medium whose serum supplement has been extracted with acetone:ethanol, 3T3-F442A cells undergo adipose conversion to nearly the same extent as in untreated serum, and develop nearly the same increase in lipoprotein lipase activity.Unless even very low concentrations of lipids or lipoprotein are saturating it can be concluded that the adipose conversion does not depend upon external lipids or lipoproteins for its induction; rather the differentiation program is built into the cell type and comes into operation when growth is arrested even in their absence. The source of fatty acids utilized for triglyceride synthesis, however, may be affected by the amount of lipid provided to the cells.     

A modified assay system for enzymes involved in N-acetyl group transfer reactions: its use to study enzymes involved in ornithine biosynthesis in plants

An enzymatic method for the determination of the amount of free fatty acids released from triglyceride by lipoprotein lipase is described. The quantity of free fatty acids present in media before and after incubation is measured spectrophotometrically by the oxidation of NADH in the final reaction of a series of coupled enzymatic reactions. This assay for lipoprotein lipase is unlike previously described assays in that radioactive substrates or titration procedures are not used in the free fatty acid determination. In addition, another method for assay of lipoprotein lipase activity that involves the separation of free fatty acids from triglycerides by adsorption chromatography with Florisil as a stationary phase is described.     

The effect of human arginine rich apoprotein on rat adipose lipoprotein lipase.

Heterologous human arginine rich apoprotein purified by heparin affinity chromatography from very low density lipoproteins produces a pronounced inhibition of the activity of lipoprotein lipase obtained from rat adipose tissue when the apoprotein is added directly to the assay medium. If, on the other hand, only the triglyceride emulsion bound arginine rich apoprotein is presented to the enzyme, a two-fold increment in the activity of the enzyme is noted. The ratio of the substrate bound arginine rich apoprotein to the free apoprotein importantly influences the effect of this apoprotein on the lipoprotein lipase reaction. These findings suggest a potential receptor role for the protein in this enzyme-substrate interaction.     

A simple lipase assay using trichloroacetic acid

An extremely rapid and sensitive assay for lipoprotein lipase activity, suitable for routine determinations, is described. The substrate for the assay is emulsified [2-(3)H]glyceryl trioleate, activated by serum. The method is based on trichloroacetic acid precipitation of unreacted substrate and measurement of (3)H-labeled glycerol.     

Inhibition of human and rat lipoprotein lipase by high-density lipoprotein

The hydrolysis in vitro of preactivated Intralipid (an artificial triacylglycerol-phospholipid emulsion) by rat adipose tissue lipoprotein lipase is inhibited by rat high-density lipoprotein (HDL). The aim of this work was to investigate whether human lipoprotein lipase was also inhibited, the mechanism of inhibition of the rat enzyme by HDL, and the role of the various individual apolipoproteins. Both human and rat lipoprotein lipase from post-heparin plasma are inhibited by HDL. This inhibition is considerably decreased if the HDL is first made 'apolipoprotein poor' by removal of some transferable apolipoproteins. In contrast, both native and apolipoprotein poor HDL inhibit the hydrolysis of Intralipid by rat hepatic lipase. Apolipoproteins C and E, either free in solution or attached to lipid vesicles, inhibit the hydrolysis of activated Intralipid by rat lipoprotein lipase to a maximum of 85% and 50%, respectively. Apolipoprotein A attached to vesicles gives little inhibition. HDL apolipoprotein and apolipoprotein C compete with the substrate for binding to lipoprotein lipase with apolipoprotein C having a higher affinity for the enzyme than HDL apolipoprotein. The inhibition of lipoprotein lipase by HDL can be explained by the association of the constituent apolipoproteins, in particular apolipoprotein C, with the enzyme so that there is less enzyme available to act on substrate.     

Studies on triglyceride lipases from rat adipose tissue.

A new assay procedure for triglyceride lipase [EC 3.1.1.3] was developed in which radioactive triolein was dissolved in ethanol and directly added to the reaction mixture in the absence of serum and albumin. In the rat adipose tissue there appeared to be a triglyceride lipase measurable with this assay in addition to the two previously defined lipases, lipoprotein lipase [EC 3.1.1.34] and hormone-sensitive lipase. The enzyme was active in the absence of serum and was strongly inhibited by albumin. The molecular weight was estimated to be about 42,000. Adenosine 3',5'-monophosphate-dependent protein kinase [EC 2.7.1.27] was unable to activate the enzyme. The three species of lipases mentioned above behaved differently upon chromatography on a Sepharose 4B column, and were distinguishable from each other in their physical and kinetic properties. The physiological roles of the new species of lipase remain to be explored.     

Studies on the Lipoprotein Lipases of Microorganisms

About 2000 strains of microorganisms were examined for lipoprotein lipase producibilities and some microorganisms were found to produce lipases similar to animal lipoprotein lipases.

Microorganisms were cultured on solid media containing a serum-activated olive oil emulsion, and strains which formed a clear zone around the colony were collected. The collected microorganisms were cultured on liquid media containing 0.5% of olive oil by shaking and the culture filtrates were tested for lipoprotein lipase activity by a turbidity method. The superior lipoprotein lipase producers obtained belonged to genera of Serratia, Pseudomonas, Mucor, and Streptomyces.     

The lipoprotein lipase of white adipose tissue. Studies on the intracellular distribution of the adipocyte-associated enzyme.

The separation of rat epididymal adipocytes into plasma-membrane, mitochondrial, microsomal and cytosol fractions is described. The fractions, which were characterized by marker-enzyme analysis and electron-micrographic observation, from the cells of fed and 24 h-starved animals were used to prepare acetone/diethyl ether-dried powders for the measurement of lipoprotein lipase activities. The highest specific activities and proportion of recovered lipoprotein lipase activity were found in the plasma-membrane and microsomal fractions. The two fractions from the cells of fed rats showed similar activities and enrichments of the enzyme, these activities being higher than the plasma-membrane and lower than the microsomal activities recovered from the cells of starved animals. Chicken and guinea-pig anti-(rat lipoprotein lipase) sera were prepared, and an indirect labelled-second-antibody cellular immunoassay, using 125I-labelled rabbit anti-(chicken IgG) or 125I-labelled sheep anti-(guinea-pig IgG) antibodies respectively, for the detection of cell-surface enzyme was devised and optimized. The amount of immunodetectable cell-surface lipoprotein lipase was higher for cells isolated from fed animals than for cells from 24 h-starved animals, when either anti-(lipoprotein lipase) serum was used in the assay. The amount of immunodetectable cell-surface lipoprotein lipase fell further when starvation was extended to 48 h. The lipoprotein lipase of plasma-membrane vesicles was shown to be a patent activity and to be immunodetectable in a modification of the cellular immunoassay. Although the functional significance of the adipocyte surface lipoprotein lipase is not known, the possibility of it forming a pool of enzyme en route to the capillary endothelium is advanced.     

Monoclonal antibodies against bovine milk lipoprotein lipase. Characterization of an antibody specific for the apolipoprotein C-II binding site

Ten murine monoclonal antibodies have been produced that are specific for bovine milk lipoprotein lipase. One monoclonal antibody, bLPL-mAb-7, inhibited completely the apolipoprotein C-II (apo-C-II)-dependent enzymic hydrolysis of trioleoylglycerol in a phospholipid-stabilized emulsion, but had no effect on the hydrolysis of the water-soluble substrate p-nitro-phenylacetate. Four times more bLPL-mAb-7 was required to achieve 50% inactivation of lipoprotein lipase activity when the enzyme was preincubated with excess apo-C-II. Disruption of the binding of a dansyl-labeled apo-C-II peptide to lipoprotein lipase by bLPL-mAb-7 was demonstrated by resonance energy transfer, both in the presence and absence of lipid. This antibody thus appears to recognize the apo-C-II binding site of lipoprotein lipase. In addition, bLPL-mAb-7 also inhibited the lipoprotein lipase activity of human post-heparin plasma.     

Triacylglycerol lipase activities of cultured rat L6 myoblasts

The utilization of exogenous triacylglycerol by fusing and non-fusing rat L6 myoblasts grown in culture was investigated. Although small quantities of triacylglycerol were accumulated by both cell lines during an incubation of 2 h, no evidence could be found for the presence of lipoprotein lipase, either in the cells or released into the medium. Cell homogenate studies confirmed the absence of lipoprotein lipase but revealed the presence of an acid lipase having a pH optimum at 4.6. Acid lipase activity was mainly associated with a 15 000 g pellet and was capable of hydrolysing triolein at maximum velocity in the millimolar range. Unlike lipoprotein lipase, acid lipase was strongly inhibited by serum and preliminary investigations suggest that the inhibitory component of serum is located amongst the higher density lipoproteins. It is likely that the acid lipase is of lysosomal origin and is responsible for the hydrolysis of internalized triacylglycerol for subsequent utilization by the cell.     

Selective release of extrahepatic lipoprotein lipase by polymetaphosphate

We have studied the lipase released into the circulation by polymetaphosphate injection into rats. Lipase release was in proportion to the dose injected. The post-polymetaphosphate lipase was almost completely inhibited by high salt concentrations or by addition of protamine sulfate to the assay system suggesting that this compound released lipoprotein lipase and not hepatic triglyceride lipase. The lipases released by polymetaphosphate and by heparin were compared using a heparin-sepharose affinity column technique which separates lipoprotein lipase from hepatic triglyceride lipase. While heparin released both lipoprotein lipase and hepatic triglyceride lipase, polymetaphosphate released almost exclusively lipoprotein lipase. Other experiments showed that neither polymetaphosphate nor heparin inhibited the hepatic lipase when added to the assay. These results suggest that lipoprotein lipase may be released by the negative charge on these high-charge polymers while hepatic triglyceride lipase release may require the specific sugar configuration of heparin.     

Medium-chain versus long-chain triacylglycerol emulsion hydrolysis by lipoprotein lipase and hepatic lipase: implications for the mechanisms of lipase action

To explore how enzyme affinities and enzyme activities regulate hydrolysis of water-insoluble substrates, we compared hydrolysis of phospholipid-stabilized emulsions of medium-chain (MCT) versus long-chain triacylglycerols (LCT). Because substrate solubility at the emulsion surface might modulate rates of hydrolysis, the ability of egg yolk phosphatidylcholine to solubilize MCT was examined by NMR spectroscopy. Chemical shift measurements showed that 11 mol % of [13C]carbonyl enriched trioctanoin was incorporated into phospholipid vesicles as a surface component. Similar methods with [13C]triolein showed a maximum solubility in phospholipid bilayers of 3 mol % (Hamilton & Small, 1981). Line widths of trioctanoin surface peaks were half that of LCT, and relaxation times, T1, were also shorter for trioctanoin, showing greater mobility for MCT in phospholipid. In assessing the effects of these differences in solubility on lipolysis, we found that both purified bovine milk lipoprotein lipase and human hepatic lipase hydrolyzed MCT at rates at least 2-fold higher than for LCT. With increasing concentrations of MCT, saturation was not reached, indicating low affinities of lipase for MCT emulsions, but with LCT emulsion incubated with lipoprotein lipase, saturation was reached at relatively low concentration, demonstrating higher affinity of lipase for LCT emulsions. Differences in affinity were also demonstrated in mixed incubations where increasing amounts of LCT emulsion resulted in decreased hydrolysis of MCT emulsions. Increasing MCT emulsion amounts had little or no effect on LCT emulsion hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ingestion of unsaturated fat on lipolytic activity of rat tissues

Homogenates of some rat tissues, incubated in Tris-maleate buffer containing bovine serum albumin, olive oil emulsion, heparin, and serum, liberated free fatty acids. The total lipolytic activity in tissues of rats fed a low fat, 20% lard, or 20% corn oil diet for 6 wk was measured. Similar activities were found in all the livers, but there was a significant increase in the total lipolytic activity of the mucosa, epididymal fat, and mesenteric tissues after ingestion of an unsaturated fat diet as compared with that containing a more saturated fat. From measurements of the lipolytic activity in the presence of 1 M NaCl or 0.2 M NaF and in the absence and presence of heparin and serum, the conclusion is drawn that more lipoprotein lipase was present in adipose tissue of rats on unsaturated fat diets. An increase in available lipoprotein lipase after unsaturated fat diets may aid in clearing lipids from the blood of these rats and thus in producing the lower blood lipid levels obtained.     

Lipoprotein lipase in heart and myocytes: characteristics with intralipid as substrate.

1. Intralipid is a suitable substrate for measuring lipoprotein lipase activity in the presence of other triacylglycerol lipases in heart and myocytes. 2. Triacylglycerol lipase activity in heart and myocytes was increased 10-fold in the presence of serum at pH 7.4 and 8.1. The serum-stimulated activity in myocytes was 95% inhibited by saturating concentrations of antiserum to lipoprotein lipase. 3. Both heparin-releasable and non-releasable lipoprotein lipase fractions had similar Km values for Intralipid and a similar pattern of inhibition by high density lipoprotein but different responses to heparin. 4. Isoproterenol did not alter lipoprotein lipase activity in cardiac myocytes.     

Combined lipase deficiency (cld/cld) in mice. Demonstration that an inactive form of lipoprotein lipase is synthesized

Combined lipase deficiency, cld, is a recessive mutation within the T/t complex of mouse chromosome 17. Mice homozygous for this defect display severe functional deficiencies of lipoprotein lipase and the related hepatic lipase. They develop massive hyperchylomicronemia and die within 3 days when allowed to suckle. Heart, diaphragm muscle, and brown adipose tissue of 1-day-old cld/cld and unaffected mice incorporated in vivo [35S]methionine into a protein that could be immunoprecipitated by antilipoprotein lipase serum. The immunoprecipitated protein in all tissues had the same Mr as bovine lipoprotein lipase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proportion of radioactivity in the lipoprotein lipase band to that in total protein was 0.02% in tissues of cld/cld mice and 0.01% in tissues of unaffected mice. There was 2-6 times more lipoprotein lipase-like protein (determined by immunoassay) in tissues of defective mice than in those of unaffected mice. These findings indicate that the cld mutation did not cause deletion of the structural gene for lipoprotein lipase. Lipoprotein lipase activity in heart, diaphragm muscle, brown adipose tissue, and lung of cld/cld mice was less than 5% of that in tissues of unaffected mice. This low activity could be inhibited more than 85% by antilipoprotein lipase serum, but not by nonimmune serum. It is concluded that tissues in cld/cld mice synthesize a lipoprotein lipase-like protein which has subnormal catalytic activity.     

The functional status of lipoprotein lipase in rat liver

1. Acetone-dried powders of liver and heart tissues from rats given a high-carbohydrate diet or a fat meal were assayed for lipoprotein lipase activity. Heart tissue showed typical lipoprotein lipase activity, whereas none was detected in liver by the usual assay procedures. 2. When mixed acetone-dried powders were prepared from heart plus liver, there was a marked suppression of the expected activity, indicating that an inhibitor was present in the liver. This inhibition was partially overcome in the presence of relatively large amounts of heparin. 3. Lipoprotein lipase was also detected in liver alone when large quantities of heparin were added to the assay system. 4. No increase in lipoprotein lipase activity in either liver or heart was detected when rats were given a fat meal. 5. It is concluded that the liver of the rat contains lipoprotein lipase that is normally present in an inactive state. The results imply that a heparinase is the agent responsible for the inactivation. 6. The significance of the non-functional status of lipoprotein lipase in the liver is discussed. The results support the view that direct hydrolysis of plasma triglycerides by the liver is not a significant physiological process.     

Lipoprotein lipase and acid lipase activity in rabbit brain microvessels.

A preparation of cerebral microvessels was used to demonstrate the presence of lipoprotein lipase and acid lipase activity in the microvasculature of rabbit brain. Microvessels, consisting predominantly of capillaries, small arterioles, and venules, were islated from rabbit brain. Homogenates were assayed for lipolytic activity using a glycerol-stabilized trioleoylglycerol-phospholipid emulsion as substrate. Lipoprotein lipase activity was characterized with this substrate by previously established criteria including an alkaline pH optimum, increased activity in the presence of heparin and heat-inactivated plasma, and reduced activity in the presence of NaCl and protamine sulfate. A different substrate, containing trioleoylglycerol incorporated into phospholipid vesicles, was used to reveal acid lipase activity that was not affected by heparin, plasma, NaCl, or protamine sulfate. Lipoprotein lipase did not show activity with the vesicle preparation as substrate. Intact microvessels, when incubated in the presence of heparin, release lipoprotein lipase into the incubation solution. In contrast, release of acid lipase activity from intact microvessels was not dependent on heparin. The data show the presence of both lipoprotein lipase and acid lipase in brain microvessels and suggest that lipoproteins are metabolized within the cerebral vasculature.