Bile salt-stimulated lipase in non-primate milk: longitudinal variation and lipase characteristics in cat and dog milk

We report the presence of bile salt-stimulated lipase in milk collected from dog and cat. This enzyme has previously been found only in the milk of human and gorilla. Bile salt-stimulated lipase activity in individual dog milk specimens (range: 4.8-107.4 U/ml; 1 U = 1 mumol [3H]oleic acid released/min) was similar, while that in cat milk specimens (range: 2.2-16.9 U/ml) was lower than in human milk (range: 10-80 U/ml). Longitudinal patterns for bile salt-stimulated lipase activity differed depending upon the enzyme source: in dog milk, lipase activity was lowest in colostrum, while in cat milk, lipase activity was highest in colostrum and decreased at mid-lactation. In human milk, bile salt-stimulated lipase activity levels remain fairly constant throughout the first 3 months of lactation. Dog, cat and human milk bile salt-stimulated lipase activity had a neutral-to-alkaline pH optimum of 7.3-8.5, was stable at low pH (above 3.0 for at least 1 h), and was inhibited 95-100% by eserine (at concentrations greater than 0.6 mM). The lipase in the milk of the three species studied had an absolute requirement for primary bile salts (tauro- and glycocholate), and was inhibited by secondary bile salts (tauro- and glycodeoxycholate). These data are the first to report bile salt-stimulated lipase activity in milk from mammals other than the highest primates. Presence of this lipase in non-primate milk will permit the study of the factors that regulate the ontogeny, synthesis and secretion of the enzyme during pregnancy and lactation as well as its function in neonatal fat digestion.     

Human milk lipases. I. Serum-stimulated lipase

Lipase activity has previously been demonstrated in human milk. This study shows that there are two separate triglyceride lipases in human milk. One is mainly in the skim milk and is stimulated by bile salts; the other is mainly in the cream and is inhibited by bile salts but stimulated by serum. The serum-stimulated lipase was purified by affinity chromatography on heparin-substituted Sepharose 4B. This gave a 9500-fold purification over whole milk. Although polyacrylamide gel electrophoresis showed that the enzyme was not purified to homogeneity, it had the highest specific activity so far reported for a human serum-stimulated lipase. The purified enzyme was free from bile salt-stimulated lipase activity and had the characteristics of other serum-stimulated or so-called lipoprotein lipases. Thus, it was almost completely inhibited by 1 M NaCl. The purified enzyme was active against tributyrylglycerol also in the absence of exogenous serum factors.     

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.     

Synthesis of Extracellular Proteinase by Pseudomonas fluorescens Under Conditions of Limiting Carbon, Nitrogen, and Phosphate

The influence of carbon, nitrogen, and phosphate concentrations on growth and proteinase production by Pseudomonas fluorescens 32A was examined. In mineral salts medium containing dialyzed skim milk supernatant as an inducer, maximum growth was obtained at 1.0 and 2.5 mM orthophosphate at 20 and 5°C, respectively. At both temperatures, 5 mM orthophosphate was required for maximum proteinase production, whereas significant inhibition was found at 10 mM. Orthophosphate was the only phosphate compound able to support growth. With sodium pyruvate as the carbon source, maximum enzyme synthesis was at 100 mM carbon at both temperatures. At both 20 and 5°C maximum growth and enzyme production was found with 10 mM NH₄Cl. A bioassay for available phosphate based on the growth of P. fluorescens 32A in phosphate-limited mineral salts medium showed that skim milk and skim milk supernatant contained 50 and 10 mM orthophosphate, respectively. Proteinase production in skim milk was 2.6- and 12-fold greater than that in optimal mineral salts medium at 20 and 5°C, respectively. These results suggest that proteinase production in milk does not occur as a result of nutrient limitation and may be regulated in part by milk phosphates.     

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with α-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 × 10⁴-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 × 10⁴-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H₂O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

Identification and Characterization of a Bile Salt Hydrolase from Lactobacillus salivarius for Development of Novel Alternatives to Antibiotic Growth Promoters

Antibiotic growth promoters (AGPs) have been used as feed additives to improve average body weight gain and feed efficiency in food animals for more than 5 decades. However, there is a worldwide trend to limit AGP use to protect food safety and public health, which raises an urgent need to discover effective alternatives to AGPs. The growth-promoting effect of AGPs has been shown to be highly correlated with the decreased activity of intestinal bile salt hydrolase (BSH), an enzyme that is produced by various gut microflora and involved in host lipid metabolism. Thus, BSH inhibitors are likely promising feed additives to AGPs to improve animal growth performance. In this study, the genome of Lactobacillus salivarius NRRL B-30514, a BSH-producing strain isolated from chicken, was sequenced by a 454 GS FLX sequencer. A BSH gene identified by genome analysis was cloned and expressed in an Escherichia coli expression system for enzymatic analyses. The BSH displayed efficient hydrolysis activity for both glycoconjugated and tauroconjugated bile salts, with slightly higher catalytic efficiencies (k_cat/K_m) on glycoconjugated bile salts. The optimal pH and temperature for the BSH activity were 5.5 and 41°C, respectively. Examination of a panel of dietary compounds using the purified BSH identified some potent BSH inhibitors, in which copper and zinc have been recently demonstrated to promote feed digestion and body weight gain in different food animals. In sum, this study identified and characterized a BSH with broad substrate specificity from a chicken L. salivarius strain and established a solid platform for us to discover novel BSH inhibitors, the promising feed additives to replace AGPs for enhancing the productivity and sustainability of food animals.     

Effect of Various Inhibitors on Lipase Action of Thermophilic Fungus Humicola lanuginosa S-38

The hydrolysis of olive oil by the Humicola lipase was inhibited by the addition of n-alcohols, fatty acids and surface active agents. The inhibition of n-alcohols was overcomed by the addition of more substrate but not by the addition of more enzyme. The inhibition of fatty acids and bile salts was eliminated by adding calcium ion. It was concluded that the inhibition of the Humicola lipase by n-alcohols, fatty acids and bile salts was not due to inactivation of the enzyme directly but due to the displacing of the substrate from the oil/water interface, thus blocking the enzyme from the substrate.     

Lipase from new isolate Bacillus cereus ATA179: optimization of production conditions,partial purification,characterization and its potential in the detergent industry

In this study, 341 Bacillus sp. strains were isolated from agricultural soils of Turkey. The potent extracellular lipase producer was selected. It was identified by 16S rRNA, named as Bacillus cereus ATA179. Optimal nutritional and physical parameters for lipase production were determined. Sucrose as the carbon source, (NH₄)₂HPO₄ as the nitrogen source, CaCl₂ as the metal ion were obtained. The best results of physical parameters were stated at 45°C, pH 7.0, shaking rate 50 rpm, inoculation amount 7% and inoculum age 24 h. ATA179 strain showed a 51% increase in enzyme production in the modified medium created by optimizing nutritional and physical conditions. Optimum pH value and temperature were found as 6.0 and 55 °C, respectively. CaCl₂, Tween 20, Triton X-100 had an activating effect on enzyme activity. V_max and K_m kinetic values were found as 18.28 U/mL and 0.11 mM, respectively. The molecular weight was determined as 47 kDa. Lipase was found to be stable up to 75 days at -20 ºC. The potential of the enzyme in detergent industry was also investigated. It was not affected by detergent additives, but was found to be effective in removing oils from contaminated fabrics. This new lipase may have potential to be used in detergent industry.     

A highly stable Yarrowia lipolytica lipase formulation for the treatment of pancreatic exocrine insufficiency

Yarrowia lipolytica lipase has been assumed to be a good candidate for the treatment of fat malabsorption in patients with pancreatic insufficiency. Nevertheless, no systematic studies on its stability under physiological conditions pertaining to the human GI (gastrointestinal) tract have been published. Stability of various Y. lipolytica lipase powder formulations at various physiological pH values as well as the effect of digestive proteases and bile salts on enzyme activity were investigated. Results were compared with those obtained from another competing fungal lipase sourced from Candida rugosa. Among the studied formulations, Y. lipolytica lipase stabilized with gum arabic and skimmed milk powder was the most promising powder formulation. Under acidic conditions (pH 3-5), this formulation showed higher stability than those observed with the other Y. lipolytica lipase formulations and C. rugosa lipase. In addition, in the presence of gum arabic and skimmed milk powder as additives, Y. lipolytica lipase exhibited markedly higher resistance to pepsin, trypsin and chymotrypsin actions. Resistance to proteolytic degradation by digestive proteases was also by far higher than that observed with C. rugosa lipase. Similar behaviour was, however, observed when these two fungal lipases were incubated with increased concentrations of bile salts. Residual lipase activity of both fungal lipases showed a slight decrease in NaTDC (sodium taurodeoxycholate) concentration above 4 mM. Consequently, Y. lipolytica lipase formulated with gum arabic and milk powder seemed to have great potential for use as a therapeutic tool for patients with pancreatic insufficiency.     

Studies on the effect of bile salt and colipase on enzymatic lipolysis. Improved method for the determination of pancreatic lipase and colipase.

The rate of hydrolysis of long chain triglycerides by pure bovine pancreatic lipase has been determined in the presence of variable amounts of bile salts and colipase. Cofactor-free lipase is strongly inhibited by sodium taurodesoxycholate and by mixed bovine bile salts at concentrations higher than the critical micellar concentration. Bile salt inhibited lipase is reactivated by the addition of bovine colipase. Gel filtration of pancreatic juice from several species (Cow, dog, pig) on Sephadex G 100 allows the separation of lipase from colipase. It is found that the enzyme catalyzed hydrolysis of long chain triglycerides by pancreatic lipase from one species is activated by the addition of colipase from other species. Studies on the activation of pancreatic lipase by colipase in the presence of bile salts allowed the re-evaluation of optimal conditions for the determination of lipase and the development of a procedure to assay colipase.     

Production and Properties of Alkaline Lipase from Alcaligenes sp. Strain No. 679

For the production of extracellular lipase by Alcaligenes species No. 679, NaNO₃, polyoxyethylene alkyl ether, Fe⁺⁺, sodium citrate and fructose were found to be effective. The enzyme was prepared by acetone precipitation from the filtrate of the culture broth of this strain. The enzyme was most active at pH 9.0 and 50°C, while 35% of its activity was lost on heat treatment at 60°C for 10 min. Sodium salts of bile acids stimulated the enzyme activity. This lipase could hydrolyse natural fats and oils as well as olive oil. During the hydrolysis of olive oil, monoglyceride was found to accumulate up to 70 mol percent. This lipase possesses special properties similar to those of pancreatic lipase as shown in the comparative experiments.     

Intestinal Mucus Protects Giardia lamblia from Killing by Human Milk1

We have previously shown that nonimmune human milk kills Giardia lamblia trophozoites in vitro. Killing requires a bile salt and the activity of the milk bile salt-stimulated lipase. We now show that human small-intestinal mucus protects trophozoites from killing by milk. Parasite survival increased with mucus concentration, but protection was overcome during longer incubation times or with greater milk concentrations. Trophozoites preincubated with mucus and then washed were not protected. Protective activity was associated with non-mucin CsCl density gradient fractions. Moreover, it was heat-stable, non-dialyzable, and non-lipid. Whereas whole mucus inhibited milk lipolytic activity, protective mucus fractions did not inhibit the enzyme. Furthermore, mucus partially protected G. lamblia trophozoites against the toxicity of oleic acid, a fatty acid which is released from milk triglycerides by lipase. These studies show that mucus protects G. lamblia both by inhibiting lipase activity and by decreasing the toxicity of products of lipolysis. The ability of mucus to protect G. lamblia from toxic lipolytic products may help to promote intestinal colonization by this parasite.     

Low-Temperature Lipase from Psychrotrophic Pseudomonas sp. Strain KB700A

We have previously reported that a psychrotrophic bacterium, Pseudomonas sp. strain KB700A, which displays sigmoidal growth even at −5°C, produced a lipase. A genomic DNA library of strain KB700A was introduced into Escherichia coli TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open reading frame (KB-lip) consisting of 1,422 nucleotides that encoded a protein (KB-Lip) of 474 amino acids with a molecular mass of 49,924 Da. KB-Lip showed 90% identity with the lipase from Pseudomonas fluorescens and was found to be a member of Subfamily I.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activity in the presence of Ca²⁺. Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca²⁺-dependent lipase. Addition of Mn²⁺ and Sr²⁺ also led to enhancement of lipase activity but to a much lower extent than that produced by Ca²⁺. The optimal pH of KB-Lip was 8 to 8.5. The addition of detergents enhanced the enzyme activity. When p-nitrophenyl esters and triglyceride substrates of various chain-lengths were examined, the lipase displayed highest activity towards C₁₀ acyl groups. We also determined the positional specificity and found that the activity was 20-fold higher toward the 1(3) position than toward the 2 position. The optimal temperature for KB-Lip was 35°C, lower than that for any previously reported Subfamily I.3 lipase. The enzyme was also thermolabile compared to these lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily I.3, indicating that KB-Lip has evolved to function in cold environments, in accordance with the temperature range for growth of its psychrotrophic host, strain KB700A.     

Pyruvatephosphate dikinase from Bacteroides symbiosus

1. An improved method is given for preparation of pyruvate,phosphate dikinase from Bacteroides symbiosus. 2. The bacterial enzyme is stable, free from interfering enzyme activities, and does not require thiol compounds to maintain stability during storage or assay. 3. New direct assays of enzyme activity are based on acid evolution or consumption as measured at constant pH in a pH-stat. 4. The optimum rate of reaction in the direction of pyruvate formation occurs at about pH6.4; in the direction of phosphoenolpyruvate formation, it is at pH7.2–7.8. 5. Newly determined substrate K_m values for the enzyme are: AMP, 3.5×10⁻⁶m; ATP, 1×10⁻⁴m; pyruvate, 8×10⁻⁵m; P_i, 6×10⁻⁴m. 6. K⁺ may substitute for NH₄⁺ in activating the reaction catalysed by the B. symbiosus enzyme. 7. In the direction of pyruvate formation the bivalent metal ion requirement of the enzyme is fulfilled by salts of nickel, manganese, magnesium and cobalt. In the other direction only magnesium salts were effective. 8. The nucleotide specificity of the enzyme is strictly limited to the adenine nucleotides. CTP and ITP strongly inhibit the reaction in the direction of phosphoenolpyruvate formation.     

Conversion of a Rhizopus chinensis lipase into an esterase by lid swapping

In an effort to explore the feasibility of converting a lipase into an esterase by modifying the lid region, we designed and characterized two novel Rhizopus chinensis lipase variants by lid swapping. The substrate specificity of an R. chinensis lipase was successfully modified toward water-soluble substrates, that is, turned into an esterase, by replacing the hydrophobic lid with a hydrophilic lid from ferulic acid esterase from Aspergillus niger. Meanwhile, as a comparison, the lid of R. chinensis lipase was replaced by a hydrophobic lid from Rhizomucor miehei lipase, which did not alter its substrate specificity but led to a 5.4-fold higher catalytic efficiency (k*_cat/K*_m) toward p-nitrophenyl laurate. Based on the analysis of structure-function relationships, it suggests that the amphipathic nature of the lid is very important for the substrate specificity. This study provides new insight into the structural basis of lipase specificities and a way to tune the substrate preference of lipases.     

Importance of microbial defence systems to bile salts and mechanisms of serum cholesterol reduction

An important feature of the intestinal microbiota, particularly in the case of administered probiotic microorganisms, is their resistance to conditions in the gastrointestinal tract, particularly tolerance to and growth in the presence of bile salts. Bacteria can use several defence mechanisms against bile, including special transport mechanisms, the synthesis of various types of surface proteins and fatty acids or the production of exopolysaccharides. The ability to enzymatically hydrolyse bile salts occurs in a variety of bacteria. Choloylglycine hydrolase (EC 3.5.1.24), a bile salt hydrolase, is a constitutive intracellular enzyme responsible for the hydrolysis of an amide bond between glycine or taurine and the steroid nucleus of bile acids. Its presence was demonstrated in specific microorganisms from several bacterial genera (Lactobacillus spp., Bifidobacterium spp., Clostridium spp., Bacteroides spp.). Occurrence and gene arrangement encoding this enzyme are highly variable in probiotic microorganisms. Bile salt hydrolase activity may provide the possibility to use the released amino acids by bacteria as sources of carbon and nitrogen, to facilitate detoxification of bile or to support the incorporation of cholesterol into the cell wall. Deconjugation of bile salts may be directly related to a lowering of serum cholesterol levels, from which conjugated bile salts are synthesized de novo. Furthermore, the ability of microorganisms to assimilate or to bind ingested cholesterol to the cell wall or to eliminate it by co-precipitation with released cholic acid was also documented. Some intestinal microflora produce cholesterol reductase that catalyses the conversion of cholesterol to insoluble coprostanol, which is subsequently excreted in faeces, thereby also reducing the amount of exogenous cholesterol.     

A Cold-Adapted Lipase of an Alaskan Psychrotroph, Pseudomonas sp. Strain B11-1: Gene Cloning and Enzyme Purification and Characterization

A psychrotrophic bacterium producing a cold-adapted lipase upon growth at low temperatures was isolated from Alaskan soil and identified as a Pseudomonas strain. The lipase gene (lipP) was cloned from the strain and sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (924 bp) corresponded to a protein of 308 amino acid residues with a molecular weight of 33,714. LipP also has consensus motifs conserved in other cold-adapted lipases, i.e., Lipase 2 from Antarctic Moraxella TA144 (G. Feller, M. Thiry, J. L. Arpigny, and C. Gerday, DNA Cell Biol. 10:381–388, 1991) and the mammalian hormone-sensitive lipase (D. Langin, H. Laurell, L. S. Holst, P. Belfrage, and C. Holm, Proc. Natl. Acad. Sci. USA 90:4897–4901, 1993): a pentapeptide, GDSAG, containing the putative active-site serine and an HG dipeptide. LipP was purified from an extract of recombinant Escherichia coli C600 cells harboring a plasmid coding for the lipP gene. The enzyme showed a 1,3-positional specificity toward triolein. p-Nitrophenyl esters of fatty acids with short to medium chains (C₄ and C₆) served as good substrates. The enzyme was stable between pH 6 and 9, and the optimal pH for the enzymatic hydrolysis of tributyrin was around 8. The activation energies for the hydrolysis of p-nitrophenyl butyrate and p-nitrophenyl laurate were determined to be 11.2 and 7.7 kcal/mol, respectively, in the temperature range 5 to 35°C. The enzyme was unstable at temperatures higher than 45°C. The K_m of the enzyme for p-nitrophenyl butyrate increased with increases in the assay temperature. The enzyme was strongly inhibited by Zn²⁺, Cu²⁺, Fe³⁺, and Hg²⁺ but was not affected by phenylmethylsulfonyl fluoride and bis-nitrophenyl phosphate. Various water-miscible organic solvents, such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30% (vol/vol) activated the enzyme.     

Engineering of Yarrowia lipolytica lipase Lip8p by circular permutation to alter substrate and temperature characteristics

Applications of lipases are mainly based on their catalytic efficiency and substrate specificity. In this study, circular permutation (CP), an unconventional protein engineering technique, was employed to acquire active mutants of Yarrowia lipolytica lipase Lip8p. A total of 21 mutant lipases exhibited significant shifts in substrate specificity. Cp128, the most active enzyme mutant, showed higher catalytic activity (14.5-fold) and higher affinity (4.6-fold) (decreased K _m) to p-nitrophenyl-myristate (pNP-C₁₄) than wild type (WT). Based on the three-dimensional (3D) structure model of the Lip8p, we found that most of the functional mutation occurred in the surface-exposed loop region in close proximity to the lid domain (S112–F122), which implies the steric effect of the lid on lipase activity and substrate specificity. The temperature properties of Cp128 were also investigated. In contrast to the optimal temperature of 45 °C for the WT enzyme, Cp128 exhibited the maximal activity at 37 °C. But it is noteworthy that there is no change in thermostability.     

Engineering a Disulfide Bond in the Lid Hinge Region of Rhizopus chinensis Lipase: Increased Thermostability and Altered Acyl Chain Length Specificity

The key to enzyme function is the maintenance of an appropriate balance between molecular stability and structural flexibility. The lid domain which is very important for “interfacial activation” is the most flexible part in the lipase structure. In this work, rational design was applied to explore the relationship between lid rigidity and lipase activity by introducing a disulfide bond in the hinge region of the lid, in the hope of improving the thermostability of R. chinensis lipase through stabilization of the lid domain without interfering with its catalytic performance. A disulfide bridge between F95C and F214C was introduced into the lipase from R. chinensis in the hinge region of the lid according to the prediction of the “Disulfide by Design” algorithm. The disulfide variant showed substantially improved thermostability with an eleven-fold increase in the t _1/2 value at 60°C and a 7°C increase of T _m compared with the parent enzyme, probably contributed by the stabilization of the geometric structure of the lid region. The additional disulfide bond did not interfere with the catalytic rate (k _cat) and the catalytic efficiency towards the short-chain fatty acid substrate, however, the catalytic efficiency of the disulfide variant towards pNPP decreased by 1.5-fold probably due to the block of the hydrophobic substrate channel by the disulfide bond. Furthermore, in the synthesis of fatty acid methyl esters, the maximum conversion rate by RCLCYS reached 95% which was 9% higher than that by RCL. This is the first report on improving the thermostability of the lipase from R. chinensis by introduction of a disulfide bond in the lid hinge region without compromising the catalytic rate.     

Role of 57-72 loop in the allosteric action of bile salts on pancreatic IB phospholipase A2: Regulation of fat and cholesterol homeostasis

Mono- and biphasic kinetic effects of bile salts on the pancreatic IB phospholipase A2 (PLA2) catalyzed interfacial hydrolysis are characterized. This novel phenomenon is modeled as allosteric action of bile salts with PLA2 at the interface. The results and controls also show that these kinetic effects are not due to surface dilution or solubilization or disruption of the bilayer interface where in the mixed-micelles substrate replenishment becomes the rate-limiting step. The PLA2-catalyzed rate of hydrolysis of zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles depends on the concentration and structure of the bile salt. The sigmoidal rate increase with cholate saturates at 0.06 mole fraction and changes little at the higher mole fractions. Also, with the rate-lowering bile salts (B), such as taurochenodeoxycholate (TCDOC), the initial sigmoidal rate increase at lower mole fraction is followed by nearly complete reversal to the rate at the pre-activation level at higher mole fractions. The rate-lowering effect of TCDOC is not observed with the (62-66)-loop deleted ΔPLA2, or with the Naja venom PLA2 that is evolutionarily devoid of the loop. The rate increase is modeled with the assumption that the binding of PLA2 to DMPC interface is cooperatively promoted by bile salt followed by allosteric k_cat^?-activation of the bound enzyme by the anionic interface. The rate-lowering effect of bile salts is attributed to the formation of a specific catalytically inert E^?B complex in the interface, which is noticeably different than the 1:1 EB complex in the aqueous phase. The cholate-activated rate of hydrolysis is lowered by hypolidemic ezetimibe and guggul extract which are not interfacial competitive inhibitors of PLA2. We propose that the biphasic modulation of the pancreatic PLA2 activity by bile salts regulates gastrointestinal fat metabolism and cholesterol homeostasis.