Kinetics of acylglycerol sequential hydrolysis by human milk bile salt activated lipase and effect of taurocholate as fatty acid acceptor

The simplest reaction scheme for the conversion of trioleoylglycerol to glycerol catalyzed by human milk bile salt activated lipase can be described by consecutive first-order reactions: triacylglycerol k1----diacylglycerol k2----monoacylglycerol k3----glycerol. In these equations, k1, k2, and k3 represent the pseudo-first-order rate constants for the indicated reactions. The results from this study show that although the relative ratio of k2/k1 or k3/k1 may change somewhat, depending on the reaction conditions, the enzyme has a reactivity with the order of dioleoylglycerol greater than trioleoylglycerol greater than monooleoylglycerol. The incomplete equilibration of the intermediary diacylglycerol and monoacylglycerol with the bulk of the substrate during sequential lipolysis of triacylglycerol provides a means for their efficient lipolysis and minimizes the effect of partial acylglycerol as competitive substrates for intact triacylglycerol lipolysis. Taurocholate functions both as an activator of the enzyme and also as fatty acid acceptor to relieve product inhibition. In the presence of sufficient taurocholate, bovine serum albumin is no longer required as a fatty acid acceptor for the in vitro lipolysis.     

Lipoprotein lipase hydrolysis of trioleoylglycerol in a phospholipid interface. Effect of cholesteryl oleate on catalysis

The effect of cholesteryl oleate on the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol was determined in monolayers of egg phosphatidylcholine at a constant surface pressure of 24 mN m-1. The phospholipid monolayers contained 1.0 to 7.5 mol % trioleoylglycerol and various amounts (0 to 20 mol %) of cholesteryl oleate. The initial rates of trioleoylglycerol hydrolysis were determined with lipoprotein lipase purified from bovine milk. In phospholipid monolayers containing 5.0 or 7.5 mol % trioleoylglycerol, the further addition of cholesteryl oleate caused a decrease in lipoprotein lipase activity. In contrast, addition of cholesteryl oleate to phospholipid monolayers containing 1.0 or 2.5 mol % trioleoylglycerol enhanced enzyme activity; a 3-fold enhancement was observed with 5.0-7.5 mol % cholesteryl oleate. Based on force-area measurements, the cholesteryl ester-mediated decrease in lipoprotein lipase activity observed at high substrate concentrations may be explained by displacement of trioleoylglycerol from the interface, thereby reducing the interfacial trioleoylglycerol concentration available for enzyme catalysis. One explanation for the cholesteryl oleate-mediated enhancement of lipoprotein lipase activity at low trioleoylglycerol concentrations is that the additional spreading of cholesteryl oleate disrupts microemulsions of trioleoylglycerol, thereby increasing the effective monomer substrate concentration available for enzyme catalysis. Based on these monolayer studies with model systems, we suggest that the relative amount of cholesteryl esters in plasma triacylglycerol-rich lipoproteins plays a regulatory role in determining the rate at which triacylglycerols are cleared from the circulation.     

Interaction of synthetic peptides of apolipoprotein C-II and lipoprotein lipase at monomolecular lipid films

The triacylglycerol hydrolyase and phospholipase A1 activities of bovine milk lipoprotein lipase toward long-chain fatty acyl ester substrates were investigated with monomolecular lipid films containing trioleoylglycerol and phosphatidylcholine. In a monolayer of egg phosphatidylcholine containing 3 mol% [14C]trioleoylglycerol, and in the presence of apolipoprotein C-II, a 79 amino acid activator protein for lipoprotein lipase, enzyme activity was maximal at a surface pressure of 21-22 mN X m-1 (37 mumol oleic acid released/h per mg enzyme); enzyme activity was enhanced 9-fold by apolipoprotein C-II. At surface pressures between 22 and 30 mN X m-1, lipoprotein lipase activity decreased over a broad range and was nearly zero at 30 mN X m-1. Apolipoprotein C-II and the synthetic fragments of the activator protein containing residues 56-79, 51-79 and 44-79 were equally effective at 20 mN X m-1 in enhancing lipoprotein lipase catalysis. However, at surface pressures between 25 and 29 mN X m-1, only apolipoprotein C-II and the phospholipid-associating fragment containing residues 44-79 enhanced enzyme catalysis. The effect of apolipoprotein C-II and synthetic peptides on the phospholipase A1 activity of lipoprotein lipase was examined in sphingomyelin:cholesterol (2:1) monolayers containing 5 mol% di[14C]myristoylphosphatidylcholine. At 22 mN X m-1, apolipoprotein C-II and the synthetic fragments containing residues 44-79 or 56-79 enhanced lipoprotein lipase activity (70-80 nmol/h per mg enzyme). In contrast to trioleoylglycerol hydrolysis, the synthetic fragments were not as effective as apolipoprotein C-II enhancing enzyme activity towards di[14C]myristoylphosphatidylcholine at higher surface pressures. We conclude that the minimal amino acid sequence of apolipoprotein C-II required for activation of lipoprotein lipase is dependent both on the lipid substrate and the packing density of the monolayer.     

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.     

Hydrolysis of tri- and monoacylglycerol by lipoprotein lipase: evidence for a common active site.

The relationship between triacylglycerol and monoacylglycerol hydrolyzing activities of purified rat heart lipoprotein lipase was studied using emulsified trioleoylglycerol and micellar or albumin-bound monooleoylglycerol as substrates. The maximal reaction rates obtained with the two substrates were similar (650 and 550 nmol of fatty acid released per min per mg of protein, respectively). Addition of apolipoprotein C-II or serum increased the maximal reaction rate for the trioleolyglycerol hydrolyzing activity about four-fold, but had no effect on the monooleolyglycerol hydrolyzing activity. Hydolysis of the two substrates apparently takes place at the same active site of the enzyme since (1) mutual competitive inhibition between the substrates could be demonstrated; (2) the rate of inactivation of enzymatic activity with the two substrates in 1.2 M NaCl was the same; (3) similar losses of hydrolytic activity with tri- and monooleoylglycerol were observed in the presence of low concentrations of n-butyl (p-nitrophenyl) carbamide; (4) inhibition of both hydrolytic activities by this compound could be prevented by prior exposure of lipoprotein lipase to either substrate.     

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.     

Enzymatic production of linalool esters in organic and solvent-free system

This work investigated the influence of temperature, enzyme concentration, substrates molar ratio, in the absence and presence of organic solvent, at two molar ratios of the substrates on the enzymatic production of linalil esters using the immobilized lipase Novozym 435 as catalyst, different acids and linalool and Ho-Sho essential oil as substrates. The best reaction conversion was obtained at the highest temperature (70 °C), for both solvent free (3.81%) and with solvent addition (2.25%), for a solvent to substrates molar ratio of 2:1, enzyme concentration of 5 wt% and acid to alcohol molar ratio of 1:1. The reaction kinetics revealed that Ho-Sho essential oil afforded the greatest conversions when compared with pure linalool. Higher linalil esters production were achieved after 10 h reaction (5.58%) in 2:1 solvent to substrates molar ratio, with enzyme concentration of 5 wt%, at 70 °C and anhydride to alcohol molar ratio of 1:1 using Ho-Sho essential oil as substrate.     

Fate of lipoprotein lipase taken up by the rat liver. Evidence for a conformational change with loss of catalytic activity

When isolated rat livers were perfused with medium containing lipoprotein lipase, 40-60% was taken up during a single passage. This value was similar for lipoprotein lipase derived from culture medium of rat preadipocytes, and for lipoprotein lipase purified from bovine milk. It was also, similar, irrespective of the lipoprotein lipase concentration, at least up to 1 microgram/ml. Immediately following its uptake by the liver, a large fraction of the lipoprotein lipase could be released by heparin, but the magnitude of this fraction decreased with time. The enzyme lost its catalytic activity rather rapidly, but its degradation to acid-soluble products, or to larger fragments, was much slower. On heparin-agarose chromatography, the enzyme taken up by the liver eluted at a lower salt concentration than the original lipoprotein lipase preparation. This change in affinity for heparin suggests that the originally dimeric lipoprotein lipase had dissociated into monomers, in analogy to the findings in model experiments. It is suggested that the initial uptake of lipoprotein lipase occurs by binding to a polyanion at the liver cell surface. This is followed by endocytosis and dissociation of the enzyme from its heparan sulfate-like binding site. Acidification of the endosome may cause a conformational change in the lipase molecule with dissociation to inactive monomers, preceding ultimate proteolytic degradation.     

Fatty acid alcohol ester-synthesizing activity of lipoprotein lipase

The fatty acid alcohol ester-synthesizing activity of lipoprotein lipase (LPL) was characterized using bovine milk LPL. Synthesizing activities were determined in an aqueous medium using oleic acid or trioleylglycerol as the acyl donor and equimolar amounts of long-chain alcohols as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with LPL, palmityl oleate was synthesized, in a time- and dose-dependent manner. Apo-very low density lipoprotein (apoVLDL) stimulated LPL-catalyzed palmityl oleate synthesis. The apparent equilibrium ratio of fatty acid alcohol ester/oleic acid was estimated using a high concentration of LPL and a long (20 h) incubation period. The equilibrium ratio was affected by the incubation pH and the alcohol chain length. When the incubation pH was below pH 7.0 and long chain fatty acyl alcohols were used as substrates, the fatty acid alcohol ester/free fatty acid equilibrium ratio favored ester formation, with an apparent equilibrium ratio of fatty acid alcohol ester/fatty acid of about 0.9/0.1. The equilibrium ratio decreased sharply at alkaline pH (above pH 8.0). The ratio also decreased when fatty alcohols with acyl chains shorter than dodecanol were used. When a trioleoylglycerol/fatty acyl alcohol emulsion was incubated with LPL, fatty acid alcohol esters were synthesized in a dose- and time-dependent fashion. Fatty acid alcohol esters were easily synthesized from trioleoylglycerol when fatty alcohols with acyl chains longer than dodecanol were used, but synthesis was decreased with fatty alcohols with acyl chain lengths shorter than decanol, and little synthesizing activity was detected with shorter-chain fatty alcohols such as butanol or ethanol.     

Ultrasound-assisted enzymatic transesterification of methyl benzoate and glycerol to 1-glyceryl benzoate in organic solvent

The aim of this work is to report the enzymatic transesterification production of 1-glyceryl benzoate under ultrasound irradiation, using a commercial immobilized lipase, Novozym 435. Firstly, a preliminary evaluation was carried out at 2, 4 and 6h, at constant temperature of 50 °C, methyl benzoate to glycerol molar ratio of 1:1 and 5.5 wt% of enzyme concentration. After analyzing the results obtained, the experimental design technique was used to evaluate the effects of temperature, substrates molar ratio, enzyme concentration, solvent volume and ultrasonic power on the 1-glyceryl benzoate production. The highest conversion, around 16%, was obtained at 65 °C, 1:1 of methyl benzoate to glycerol molar ratio, 15 wt% of enzyme concentration, 7 mL of solvent and 40% ultrasonic power in 4h of reaction. A preliminary kinetic experiment carried out varying the enzyme concentration (15 and 20 wt%) keeping fixed the temperature at 35 °C, 1:1 of substrates molar ratio, 3 mL of solvent and 40% of maximum ultrasonic power led to lower (around 15% after 12 h of reaction) conversions compared to that achieved in the experimental design.     

Preparation of Triacylglycerol Molecular Species by Interesterification Using Endocellular Lipase in n-Hexane

The interesterification of triacylglycerol with fatty acid was done to prepare triacylglycerol molecular species. Optimum operating conditions for the interesterification using a 1,3-positional specific endocellular lipase from Rhizopus japonicus NR400 in a batch system were investigated. The reaction was done at 40°C for 5 hr in the following system: Trioleoylglycerol-palmitic acid = 1:3.5 (mol/mol), 10 ml n-hexane/g trioleoylglycerol, and 2500 units of enzyme/g trioleoylglycerol. Under these conditions, the content of palmitoyl groups in 1,3-positions of triacylglycerol was about 60 mol%. Additional interesterification (2-cycle reaction) using palmitic acid and the novel triacylglycerol prepared by one-step interesterification (1-cycle reaction) resulted in a preparation of highly pure 1,3-dipalmitoyl-2-oleoylglycerol.     

Human monocytes in culture synthesize and secrete lipoprotein lipase

Within the first day in culture, human monocytes begin to synthesize and secrete a triglyceride lipase. The designation of this activity as lipoprotein lipase is based upon: 1) a requirement of serum or apolipoprotein C-II for full activity; 2) inhibition by 1M NaCl or apolipoprotein C-III₂; 3) a pH optimum of 8; and 4) binding to endothelial cells that is releasable by heparin. The enzyme also exhibits immunological cross reactivity with antibody to purified bovine milk lipoprotein lipase as does human postheparin plasma lipoprotein lipase. Lymphocytes and polymorphonuclear leukocytes do not appear to contain this enzyme.     

Hydrolysis of chylomicron polyenoic fatty acid esters with lipoprotein lipase and hepatic lipase

The lipolysis of rat chylomicron polyenoic fatty acid esters with bovine milk lipoprotein lipase and human hepatic lipase was examined in vitro. Chylomicrons obtained after feeding fish oil or soy bean oil emulsions were used as substrates. The lipolysis was followed by gas chromatography or by using chylomicrons containing radioactive fatty acids. Lipoprotein lipase hydrolyzed eicosapentaenoic (20:5) and arachidonic acid (20:4) esters at a slower rate than the C14-C18 acid esters. More 20:5 and 20:4 thus accumulated in remaining tri- and diacylglycerols. Eicosatrienoic, docosatrienoic and docosahexanoic acids exhibited an intermediate lipolysis pattern. When added together with lipoprotein lipase, hepatic lipase increased the rate of lipolysis of 20:5 and 20:4 esters of both tri- and diacylglycerols. Addition of NaCl (final concentration 1 M) during the course of lipolysis inhibited lipoprotein lipase as well as the enhancing effect of hepatic lipase on triacylglycerol lipolysis. Hepatic lipase however, hydrolyzed diacylglycerol that had already been formed. Chylomicron 20:4 and 20:5 esters thus exhibit a relative resistance to lipoprotein lipase. It is suggested that the tri- and diacylglycerol species containing these fatty acids may accumulate at the surface of the remnant particles and act as substrate for hepatic lipase during a concerted action of this enzyme and lipoprotein lipase.     

Fatty acyl chain specificity of phosphatidylcholine hydrolysis catalyzed by lipoprotein lipase. Effect of apolipoprotein C-II and its (56-79) synthetic fragment

Mixed acyl chain phosphatidylcholine molecules in Triton N-101 micelles were employed as substrates for lipoprotein lipase to test which substrate acyl chain has the greatest effect on activation of the enzyme by apolipoprotein C-II. The phospholipase A1 activity of lipoprotein lipase was measured by pH-stat. The activation factor (lipoprotein lipase activity plus apolipoprotein C-II/activity minus apolipoprotein C-II) increased monotonically with apolipoprotein C-II concentration up to 1 microM apolipoprotein C-II at an enzyme concentration of 0.01 microM. The maximal activation factor for phosphatidylcholine substrate molecules with sn-2 acyl chain lengths of 14 averages 14.8. By contrast, for sn-2 acyl chain lengths of 16 the activation factor was 29.2. Varying the sn-1 acyl chain length had no significant effect on the activation factor. The chain-length dependence of the activation factor is similar with the apolipoprotein C-II peptide fragment comprising residues 56-79, which does not include the lipid-binding region of apolipoprotein C-II. These data are consistent with a model for activation of lipoprotein lipase in which residues 56-79 bind to lipoprotein lipase and alter the interaction of the sn-2 acyl chain of the phosphatidylcholine (PC) substrate or the lysoPC product within the activated state complex.     

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.     

A stable, radioactive substrate emulsion for assay of lipoprotein lipase.

A method is described for the assay of lipoprotein lipase, using a stable, radioactive substrate emulsion. Fatty acid-labeled trioleoylglycerol was emulsified by homogenization in glycerol with lecithin as detergent. This anhydrous emulsion was stable for at least six weeks. Substrate solutions for enzyme assay were prepared by diluting the emulsion with buffer containing serum and albumin. The fatty acid produced on hydrolysis was isolated in a one-step liquid-liquid partition system. Incubations with extracts of acetone powders from adipose tissue displayed characteristics of lipoprotein lipase activity, i.e., serum dependence and inhibition by NaCl and protamine. The method is rapid (less than 1 hour), sensitive and reproducible, and suitable for routine use.     

Secretion and degradation of lipoprotein lipase in cultured adipocytes. Binding of lipoprotein lipase to membrane heparan sulfate proteoglycans is necessary for degradation

Equilibrium-binding data of highly purified 125I-labeled avian lipoprotein lipase to cultured avian adipocytes demonstrate the presence of a class of high affinity binding sites. Analysis of the binding function yielded an association constant of 0.62 x 10(8)M-1 and a maximum binding capacity of 2.1 micrograms/60-mm dish. From a time course of dissociation of 125I-lipoprotein lipase from adipocytes at 4 degrees C, a dissociation rate constant of 6.1 x 10(-5)s-1 was obtained. Pretreatment of cells with heparinase and heparitinase resulted in a quantitative suppression of the high affinity binding component, establishing that lipoprotein lipase is bound to cell surface heparan sulfate proteoglycans. At 37 degrees C, cell surface-bound 125I-lipoprotein lipase is internalized and either degraded or recycled to the medium. The degradation rate constant for 125I-lipoprotein lipase was estimated to be 0.78 h-1. The degradation rate constant was reduced 6-fold when cells were exposed to 100 microM chloroquine, indicating that most of the degradation occurs within the lysosomal compartment. By using cells that had been pulsed with Trans35S-label for 1 h, it was demonstrated that acute treatment with endoglycosidases for up to 1 h resulted in a new lipoprotein lipase secretion rate which was 6-fold higher than that of control cells. Degradation of newly synthesized lipoprotein lipase was essentially blocked 30 min after the initiation of the chase. In other studies it was observed that there were no additive effects of chloroquine and either endoglycosidase or heparin treatment on total lipoprotein lipase levels (intracellular, cell surface, and medium) in adipocyte cultures. These experiments support the hypothesis that the release of lipoprotein lipase from its receptor prevents its internalization and degradation and enhances enzyme efflux from the adipocyte. A new model of lipoprotein lipase secretion in cultured adipocytes is proposed: Newly synthesized lipoprotein lipase is transported to the cell surface where it binds to specific heparan sulfate proteoglycan receptors. The enzyme is either released to the medium or internalized via the receptor, in which case the enzyme is degraded or recycled to the cell surface. Major determinants of enzyme efflux from the cell surface include the number and integrity of receptors, the association constant of the enzyme-receptor complex, and the presence in the medium of competing molecules with high affinity for lipoprotein lipase. In this model, modulation of lipoprotein lipase degradation rate may be a significant mechanism for acute regulation of enzyme efflux independent of changes in the rate of enzyme synthesis.     

Separation and characterization of the acid lipase and neutral esterases from human liver.

Electrophoresis of human liver homogenates followed by reaction with 4-methylumbelliferyl palmitate reveals the presence of two major electrophoretic forms with esterase (lipase) activity toward this substrate. The two enzymes were isolated and partially purified based on their solubility differences and their relative affinities for the lectin column concanavalin A-Sepharose 4B. Lipase A was particulate with an acidic pH optimum (5.2) and could be solubilized with the non-ionic surfactant Triton X-100. Lipase B was soluble and had a more neutral pH optimum (6.3--6.6). Both forms bound to immobilized concanavalin A and could be specifically eluted. Buffers containing alpha-methylmannoside eluted lipase B, and buffers with alpha-methylmannoside and Triton X-100 eluted lipase A, giving a 22- and 257-fold purification, respectively, over whole-tissue homogenates. Cholesterol oleate, trioleoylglycerol, and 4-methylumbelliferyl palmitate were substrates for solubilized lipase A. Lipase B hydrolyzed 4-methylum-belliferyl palmitate but not trioleoylglycerol or cholesterol oleate. Lipase B was more thermolabile than lipase A, and it was selectively inhibited by diethyl-p-nitrophenyl phosphate at low concentrations. We conclude that lipase A and B are distinctly different enzymes and that they are probably not related polymorphic forms of one another.     

The site of cartilage matrix degradation.

1. The metabolism of VLD lipoproteins (very-low-density lipoproteins) was studied in intact isolated beating-heart cells and isolated perfused rat heart from starved animals by using [14C]triacylglycerol fatty acid-labelled VLD lipoprotein prepared from rats previously injected with [1-14C]palmitate. 2. 14C-labelled VLD lipoprotein was metabolized by the isolated perfused heart, but was only minimally metabolized by the heart cells unless an exogenous source of lipoprotein lipase was added. 3. Measurements of lipoprotein lipase at pH 7.4 with the natural substrate 14C-labelled VLD lipoprotein indicated that during collagenase perfusion of the heart the enzyme was released into the perfusate, the activity released being proportional to the concentration of collagenase used. Lipoprotein lipase activity in homogenates of hearts that had been perfused with collagenase showed a corresponding loss of activity. 4. At high perfusate concentrations of collagenase, inactivation of the released lipoprotein lipase occurred. 5. Lipoprotein lipase activity was largely undetectable in the homogenate of the isolated heart cells. 6. It is concluded that the lipoprotein lipase responsible for the hydrolysis of VLD lipoprotein triacylglycerol is predominantly located externally to the heart muscle cells and that its release can be facilitated by perfusion of the heart with bacterial collagenase.     

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.