Kinetic properties of human milk bile salt-activated lipase: studies using long chain triacylglycerol as substrate

Studies on the hydrodynamic properties of human milk bile salt-activated lipase (BAL) indicated that it is a monomer with molecular weight of 107,000. The presence of taurocholate (1 mM) did not lead to an association of the enzyme. The enzyme had a basal activity with trioctanoylglycerol and with shorter chain, but not with longer chain, monoacid triacylglycerols. Based on kinetic analyses, we suggest that the BAL-catalyzed lipolysis of long-chain triacylglycerol can be described to follow a compulsory sequential mechanism. The initial interaction of BAL with the activator (taurocholate) leads to a conformational change of the enzyme which facilitates the further interaction with the long chain triacylglycerol substrate in forming the enzyme-bile salt-substrate ternary complex. We also suggest that the binding of BAL with substrate involves direct interaction of the active site with the fatty acyl-chain of the triacylglycerol rather than with nonspecific hydrophobic interactions at the emulsion interface.     

Purification of carboxyl ester lipase from human pancreas and the amino acid sequence of the N-terminal region

A procedure for the purification of carboxyl ester lipase from human pancreas has been developed. The determined N-terminal 10 amino acid residues of the purified enzyme, NH2-Ala-Lys-Leu-Gly-Ala-Val-Tyr-Thr-Glu-Gly, was identical to the terminal of human milk bile salt-activated lipase. The human pancreatic carboxyl ester lipase has an apparent molecular weight slightly smaller than that of human milk bile salt-activated lipase (105,000 vs 125,000) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, it is possible that the human pancreatic carboxyl ester lipase and human milk bile salt-activated lipase could be produced by the same gene by a different splice or post-translational modification. Alternatively, they could simply be the products of two closely related but separate genes.     

Purification of human milk bile salt-activated lipase by cholic acid-coupled sepharose 4B affinity chromatography

Sequential chromatography of human milk whey on concanavalin A—Sepharose 4B followed by cholate—Sepharose 4B yielded a bile salt-activated lipase with 150-fold purification. The lipase was not retained by concanavalin A—Sepharose 4B but was retained by the cholate—Sepharose 4B, from which it was eluted with 2% sodium cholate. The affinity chromatography procedure on cholate—Sepharose 4B was based on the specific structural requirement of the enzyme for a 7-hydroxyl group of bile salt. Sodium deoxycholate, which lacks the 7-hydroxyl group, was effective in removing the nonspecifically bound proteins without affecting the binding of the enzyme. Bile salt-activated lipase showed a single band on urea-sodium dodecyl sulfate—polyacrylamide gel electrophoresis with an apparent molecular weight of 125,000, and based on densitometric measurement accounted for 0.5–1.0% of the protein mass of human whole milk. A rabbit antiserum to the purified bile salt-activated lipase caused no inhibition of human milk lipoprotein lipase activity but completely inhibited bile salt-activated lipase activity.     

Purification and characterization of bovine pancreatic bile salt-activated lipase

An enzyme with lipase and esterase activity was purified from bovine pancreas. Furthermore, a non-radioactive lipase assay was developed which is 100 times more sensitive than the conventional methods and allowed the characterization of the lipase activity of the enzyme. The lipase activity increased 42 times in the presence of 10 mM sodium taurocholate, which for the first time provides direct evidence that a bile salt-activated lipase (bp-BAL) was isolated from bovine pancreas. This conclusion is further supported by the fact that the N-terminal amino acid sequence of this lipase/esterase is 88% homologous to human milk BAL and human pancreatic BAL. Staining with various lectins showed that bp-BAL is a glycoprotein which contains fucose residues. Previously from bovine pancreas a lysophospholipase has been purified and a gene was cloned and sequenced encoding an enzyme with cholesterol esterase/lysophospholipase activity. Comparison of the N-terminal amino acid sequence of bp-BAL with the deduced amino acid sequence of the latter revealed that they are identical. Furthermore, the molecular weight of the purified bp-BAL of 63,000, as estimated by SDS-PAGE, is very similar to that of the purified lysophospholipase (65,000) and to the theoretical molecular weight of 65,147 of the cholesterol esterase/lysophospholipase. These data suggest that these three enzymes are one and the same.     

Pancreatic cholesterol esterases. 3. Kinetic characterization of cholesterol ester resynthesis by the pancreatic cholesterol esterases

The ability of cholesterol esterase to catalyze the synthesis of cholesterol esters has been considered to be of limited physiological significance because of its bile salt requirements for activity, though detailed kinetic studies have not been reported. This study was performed to determine the taurocholate, pH, and substrate requirements for optimal cholesterol ester synthesis catalyzed by various pancreatic lipolytic enzymes, including the bovine 67- and 72-kDa cholesterol esterases, human 100-kDa cholesterol esterase, and human 52-kDa triglyceride lipase. In contrast to current beliefs, cholesterol esterase exhibits a bile salt independent as well as a bile salt dependent synthetic pathway. For the bovine pancreatic 67- and 72-kDa cholesterol esterases, the bile salt independent pathway is optimal at pH 6.0-6.5 and is stimulated by micromolar concentrations of taurocholate. For the bile salt dependent synthetic reaction for the 67-kDa enzyme, increasing the taurocholate concentration from 0 to 1.0 mM results in a progressive shift in the pH optimum from pH 6.0-6.5 to pH 4.5 or lower. In contrast, cholesterol ester hydrolysis by the 67-, 72-, and 100-kDa enzymes was characterized by pH optima from 5.5 to 6.5 at all taurocholate concentrations. Optimum hydrolytic activity for these three enzyme forms occurred with 10 mM taurocholate. Since hydrolysis is minimal at low taurocholate concentrations, the rate of synthesis actually exceeds hydrolysis when the taurocholate concentration is less than 1.0 mM. The 52-kDa enzyme exhibits very low cholesterol ester synthetic and hydrolytic activities, and for this enzyme both activities are bile salt independent. Thus, our data show that cholesterol esterase has both bile salt independent and bile salt dependent cholesterol ester synthetic activities and that it may catalyze the net synthesis of cholesterol esters under physiological conditions.     

Author index to volume 155

Bile salt-stimulated lipase is a milk enzyme unique to the higher primates. Its molecular and kinetic characteristics differ greatly from other lipolytic enzymes; e.g., pancreatic lipase and lipoprotein lipase. It has a much higher app. M_r, 310 000 on gel filtration and 100 000 after denaturation. It requires primary bile salts for optimal activity and bile salts also protect the enzyme from proteolytic and heat inactivation. It may, due to its low substrate specificity, contribute to the utilization of a variety of milk lipids. Since it lacks positional specificity, digestion of milk triglycerides should be complete, which may explain why fat absorption is more efficient in breast-fed than in formula-fed infants.     

Purification and properties of a cholesteryl ester hydrolase from rat liver microsomes

This report describes a purification procedure for a cholesteryl ester hydrolase (CEH) from female rat liver microsomes, and some structural, immunological, kinetic, and regulatory properties of the enzyme that distinguish the microsomal CEH from other hepatic cholesteryl ester-splitting enzymes. CEH was purified 12.4-fold from reisolated microsomes using sequential solubilization by sonication, polyethylene glycol precipitation, fractionation with hydroxyapatite, anion exchange chromatography, and chromatography on hydroxyapatite, with an overall yield of 3.2%. CEH activity was purified 141-fold over nonspecific esterase activity and 56-fold over triacylglycerol lipase activity. In sharp contrast with most esterases and lipases, CEH did not bind to concanavalin A-Sepharose and heparin-Sepharose. After polyacrylamide gel electrophoresis, the purified enzyme exhibited two silver-stained bands, but only the protein electroeluted from the low mobility band had CEH activity. Affinity-purified polyclonal antibodies raised to electroeluted CEH inhibited 90% of the activity of liver microsomal CEH and reacted with a 106 kDa protein band on Western blot analysis. This 106 kDa CEH contains a unique N-terminal amino acid sequence. The purified enzyme had optimal activity at pH 6 and no taurocholate requirements, and was inhibited by the serine active site inhibitor phenylmethylsulfonyl fluoride and by free sulfhydryl specific reagents. It hydrolyzed cholesteryl oleate much more efficiently than trioleine, and hydrolytic activity with p-nitrophenyl acetate was higher than with p-nitrophenyl butyrate. These results indicate that rat liver microsomes contain a bile salt-independent catalytic protein that is relatively specific for cholesteryl ester hydrolysis.     

Inactivation of gastric and pancreatic lipases by diethyl p-nitrophenyl phosphate

Reacting gastric and pancreatic lipases with mixed diethyl p-nitrophenyl phosphate/bile salt micelles resulted in a stoichiometric inactivation of these enzymes as tested on emulsified tributyroylglycerol and trioleoylglycerol as substrates. Diethyl p-nitrophenyl phosphate treated gastric lipases were also inactive on water-soluble p-nitrophenyl acetate, whereas the modified pancreatic lipase was still able to hydrolyze this water-soluble substrate. The binding of diethyl p-nitrophenyl phosphate modified pancreatic and gastric lipases to tributyroylglycerol/water interface was comparable to that of native lipases. The essential free sulfhydryl group of gastric lipases underwent no chemical changes due to the reaction with micellar diethyl p-nitrophenyl phosphate. All in all, these results indicate that, in both gastric and pancreatic lipases, the essential serine residue which was stoichiometrically labeled by this organophosphorus reagent is involved in catalysis and not in lipid binding.     

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.     

A quick method for purifying bile salt-activated lipases

Summary A simple, one step chromatography procedure of general utility for purifying bile salt-activated lipases (BAL; cholesterol esterases) using a cholate affinity column is described. A key feature of the method is the use of a taurocholate concentration gradient in the eluent. The method has been used successfully to purify bovine and porcine BAL from commercial crude sources and BAL from human milk whey.     

Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: comparison with non-lipolytic and lipolytic carboxylesterases

We have studied the kinetics of hydrolysis of triacylglycerols, vinyl esters and p-nitrophenyl butyrate by four carboxylesterases of the HSL family, namely recombinant human hormone-sensitive lipase (HSL), EST2 from Alicyclobacillus acidocaldarius, AFEST from Archeoglobus fulgidus, and protein RV1399C from Mycobacterium tuberculosis. The kinetic properties of enzymes of the HSL family have been compared to those of a series of lipolytic and non-lipolytic carboxylesterases including human pancreatic lipase, guinea pig pancreatic lipase related protein 2, lipases from Mucor miehei and Thermomyces lanuginosus, cutinase from Fusarium solani, LipA from Bacillus subtilis, porcine liver esterase and Esterase A from Aspergilus niger. Results indicate that human HSL, together with other lipolytic carboxylesterases, are active on short chain esters and hydrolyze water insoluble trioctanoin, vinyl laurate and olive oil, whereas the action of EST2, AFEST, protein RV1399C and non-lipolytic carboxylesterases is restricted to solutions of short chain substrates. Lipolytic and non-lipolytic carboxylesterases can be differentiated by their respective value of K(0.5) (apparent K(m)) for the hydrolysis of short chain esters. Among lipolytic enzymes, those possessing a lid domain display higher activity on tributyrin, trioctanoin and olive oil suggesting, then, that the lid structure contributes to enzyme binding to triacylglycerols. Progress reaction curves of the hydrolysis of p-nitrophenyl butyrate by lipolytic carboxylesterases with lid domain show a latency phase which is not observed with human HSL, non-lipolytic carboxylesterases, and lipolytic enzymes devoid of a lid structure as cutinase.     

Ontogeny of lipase expression in winter flounder

The partial sequencing of two lipases from winter flounder Pseudopleuronectes americanus , one most closely related to gastric, lingual and lysosomal acid lipase from other vertebrates and one most closely related to bile salt-activated lipase, is reported. Biochemical analyses of enzymatic activity demonstrated the greater contribution made by bile salt-activated lipase relative to neutral bile salt-independent lipase. Using molecular techniques, the tissue-specific expression of bile salt-activated lipase in pancreatic tissue and acid triacylglycerol lipase in a wide variety of organs was demonstrated. Furthermore, the developmental expression of these types of lipase in larval fish was established.     

Sequence of horse pancreatic lipase as determined by protein and cDNA sequencing. Implications for p-nitrophenyl acetate hydrolysis by pancreatic lipases.

The complete sequence of the horse pancreatic lipase was elucidated by combining polypeptide chain and cDNA sequencing. Among the structural features of horse lipase, it is worth mentioning that Lys373 is not conserved. This residue, which is present in human, porcine and canine lipases, has been assumed to be involved in p-nitrophenyl acetate hydrolysis by pancreatic lipases. Kinetic investigation of the p-nitrophenyl acetate hydrolysis by the various pancreatic lipases and by the C-terminal domain (336-449) of human lipase reveals that this hydrolysis is the result of the superimposition of independent events; a specific linear hydrolysis occurring at the active site of lipase, a fast acylation depending on the presence of Lys373 and a non-specific hydrolysis most likely occurring in the C-terminal domain of the enzyme. This finding definitely proves that pancreatic lipase bears only one active site and raises the question of a covalent catalysis by pancreatic lipases. Moreover, based on sequence comparison with the above-mentioned pancreatic lipases, three residues located in the C-terminal domain, Lys349, Lys398 and Lys419, are proposed as possible candidates for lipase/colipase binding.     

Purification of pancreatic carboxylic-ester hydrolase by immunoaffinity and its application to the human bile-salt-stimulated lipase

A column of immobilized antibodies directed against pure human pancreatic carboxylic (cholesterol) ester hydrolase was used to purify in a single step the enzyme from human pancreatic juice as well as carboxylic-ester hydrolases from other species (rat, dog). This immunoaffinity method was also used for the purification of the related bile-salt-stimulated lipase from the human skim milk. The enzymes were homogeneous on SDS-PAGE. The yields obtained were always higher than those previously observed using either conventional or affinity columns. The human and dog carboxylic-ester hydrolases as well as the bile-salt-stimulated lipase, in contrast to the rat enzyme, are glycoproteins. From our results, it can be speculated that these enzymes, which differ in their molecular weight but not in their N-terminal sequences or amino-acid compositions, might have a similar proteic core with a molecular mass between 65 and 75 kDa. The difference in their respective molecular masses might result from a different level of glycosylation of pancreatic carboxylic-ester hydrolases (and milk bile-salt-stimulated lipase).     

Lipolytic and Esterolytic Activities in Posterior Lingual Glands of Rat: A Histochemical Study

Catalytic activities of lingual lipase were investigated by enzyme histochemistry in post-mortem tongues from male rats. Sections of fresh-frozen or formalin-calcium fixed tissue were incubated with naphthol-AS-nonanoate and α-naphthyl acetate substrate mixtures. The effects of pH level, sodium taurocholate activator and E600 inhibitor were also examined. The use of cryostat sections of tissues fixed in formalin-calcium and of nonanoate substrate within the range of pH 4.4–6.4, were optimal for localizing maximum reaction product, captured by Fast Blue BB, in acini and demilunes of the posterior deep and superficial lingual glands respectively. The reaction product corresponded with the distribution of secretory granules and failed to develop when taurocholate was omitted from the incubation medium. Similarly localized E600-resistant reaction product occurred with the acetate substrate and hexazotized New Fuchsin at pH 7.4, in the absence of taurocholate. Lipase and conventional esterase activities appear to be superimposed in posterior lingual glands of rat. The ability of their acini and demilunes to hydrolyse nonanoate substrate at an acidic pH optimum, when activated by sodium taurocholate, seems attributable to lipase destined for secretion into saliva – hence convenient for routine histochemical identification of the enzyme.     

Lipolytic and esterolytic activities in posterior lingual glands of rat: a histochemical study

Catalytic activities of lingual lipase were investigated by enzyme histochemistry in post-mortem tongues from male rats. Sections of fresh-frozen or formalin-calcium fixed tissue were incubated with naphthol-AS-nonanoate and alpha-naphthyl acetate substrate mixtures. The effects of pH level, sodium taurocholate activator and E600 inhibitor were also examined. The use of cryostat sections of tissues fixed in formalin-calcium and of nonanoate substrate within the range of pH 4.4-6.4, were optimal for localizing maximum reaction product, captured by Fast Blue BB, in acini and demilunes of the posterior deep and superficial lingual glands respectively. The reaction product corresponded with the distribution of secretory granules and failed to develop when taurocholate was omitted from the incubation medium. Similarly localized E600-resistant reaction product occurred with the acetate substrate and hexazotized New Fuchsin at pH 7.4, in the absence of taurocholate. Lipase and conventional esterase activities appear to be superimposed in posterior lingual glands of rat. The ability of their acini and demilunes to hydrolyse nonanoate substrate at an acidic pH optimum, when activated by sodium taurocholate, seems attributable to lipase destined for secretion into saliva--hence convenient for routine histochemical identification of the enzyme.     

Interfacial reaction dynamics and acyl-enzyme mechanism for lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters

The fatty acyl (lipid) p-nitrophenyl esters p-nitrophenyl caprylate, p-nitrophenyl laurate and p-nitrophenyl palmitate that are incorporated at a few mol % into mixed micelles with Triton X-100 are substrates for bovine milk lipoprotein lipase. When the concentration of components of the mixed micelles is approximately equal to or greater than the critical micelle concentration, time courses for lipoprotein lipase-catalyzed hydrolysis of the esters are described by the integrated form of the Michaelis-Menten equation. Least square fitting to the integrated equation therefore allows calculation of the interfacial kinetic parameters Km and Vmax from single runs. The computational methodology used to determine the interfacial kinetic parameters is described in this paper and is used to determine the intrinsic substrate fatty acyl specificity of lipoprotein lipase catalysis, which is reflected in the magnitude of kcat/Km and kcat. The results for interfacial lipoprotein lipase catalysis, along with previously determined kinetic parameters for the water-soluble esters p-nitrophenyl acetate and p-nitrophenyl butyrate, indicate that lipoprotein lipase has highest specificity for the substrates that have fatty acyl chains of intermediate length (i.e. p-nitrophenyl butyrate and p-nitrophenyl caprylate). The fatty acid products do not cause product inhibition during lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles. The effects of the nucleophiles hydroxylamine, hydrazine, and ethylenediamine on Km and Vmax for lipoprotein lipase catalyzed hydrolysis of p-nitrophenyl laurate are consistent with trapping of a lauryl-lipoprotein lipase intermediate. This mechanism is confirmed by analysis of the product lauryl hydroxamate when hydroxylamine is the nucleophile. Hence, lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles occurs via an interfacial acyl-lipoprotein lipase mechanism that is rate-limited by hydrolysis of the acyl-enzyme intermediate.     

Esterastin: a potent inhibitor of lysosomal acid lipase

The effect of a fungal metabolite, esterastin, on lysosomal acid lipase purified from rabbit liver was studied. Esterastin inhibited the enzyme activity very strongly (IC50, about 80 nM). The inhibition of acid lipase by esterastin was competitive with respect to the substrate and the inhibition constant for esterastin was 90 nM. Esterastin was less inhibitory to other lipolytic enzymes, such as pancreatic lipase and carboxylesterase. Thus esterastin is a potent new inhibitor of lysosomal acid lipase.     

Giardia lamblia: the role of conjugated and unconjugated bile salts in killing by human milk

Killing of Giardia lamblia trophozoites by nonimmune human milk in vitro is dependent upon the presence of cholate which activates the milk bile salt-stimulated lipase to cleave fatty acids from milk triglycerides. In the present studies, conjugated bile salts, which predominate in vivo, displayed striking differences from unconjugated bile salts in ability to support killing by milk. Human milk killed greater than 99% of the parasites in the presence of cholate, but not glycocholate or taurocholate. In contrast, after brief sonication which disrupts milk fat globules, milk killed G. lamblia after addition of either conjugated or unconjugated bile salts. Whereas cholate stimulated milk lipase to cleave triglycerides of either unsonicated or sonicated human milk, glycocholate or taurocholate stimulated lipolysis only in sonicated milk. Since the concentration of bile salts in the small intestine fluctuates, the effect of this variable on killing was examined. Each bile salt at and above its critical micellar concentration increased Giardia survival of human milk probably because it sequestered released fatty acids in micelles. This partial protection could be overcome by increasing the milk concentration. Human hepatic and gall bladder bile and artificial bile also activated human milk to kill at low concentrations but partly protected the parasite at higher concentrations. These studies show that conjugated bile salts can activate the bile salt-stimulated lipase of sonicated human milk to release fatty acids; and kill G. lamblia. Conversely, bile salts in concentrations above their critical micellar concentration sequester fatty acids and interfere with killing. Thus, nonimmune host secretions such as milk and bile may affect the course of infection by G. lamblia.     

Inhibition of pancreatic lipase B activity by taurodeoxycholate and its reversal by colipase.

In our two-phase reaction system taurodexycholate prevents the adsorption of pancreatic lipase B to the nonaqueous phase. Our data are consistent with a mechanism for this reaction which involves the cooperative formation of an enzyme-(bile salt)4 complex in solution with a dissociation constant of 1.4 X 10(-15)M4. Whereas the free enzyme is readily adsorbed to a bile salt-substrate-covered surface, the complex is not. Thus, the "inhibition" of substrate hydrolysis occurs because enzyme and substrate are separated physically. The protein cofactor, colipase, reverses the inhibitory effects of bile salt by providing a high affinity binding site at the interface for the lipase-(bile salt)4 complex. Steady state and presteady state kinetic data are consistent with the formation of a complex with a 1/1, lipase/colipase, ratio, and a dissociation constant of 0.4 to 2.8 X 10(-9)M. The rate of adsorption of lipase to adsorbed colipase appears to be controlled by diffusion through the unstirred layer with a second order rate constant of 1.3 X 10(6)M-1S-1.