Inhibition of lipases by ∊-polylysine

Oral administration of ∊-polylysine to rats reduced the peak plasma triacylglycerol concentration. In vitro, ∊-polylysine and polylysine strongly inhibited the hydrolysis, by either pancreatic lipase or carboxylester lipase, of trioleoylglycerol (TO) emulsified with phosphatidylcholine (PC) and taurocholate. The ∊-polylysine concentration required for complete inhibition of pancreatic lipase, 10 μg/ml, is 1,000 times lower than that of BSA required for the same effect. Inhibition requires the presence of bile salt and, unlike inhibition of lipase by other proteins, is not reversed by supramicellar concentrations of bile salt. Inhibition increases with the degree of polylysine polymerization, is independent of lipase concentration, is independent of pH between 5.0 and 9.5, and is accompanied by an inhibition of lipase binding to TO-PC emulsion particles. However, ∊-polylysine did not inhibit the hydrolysis by pancreatic lipase of TO emulsions prepared using anionic surfactants, TO hydrolysis catalyzed by lingual lipase, or the hydrolysis of a water-soluble substrate. In the presence of taurocholate, ∊-polylysine becomes surface active and adsorbs to TO-PC monomolecular films.These results are consistent with ∊-polylysine and taurocholate forming a surface-active complex that binds to emulsion particles, thereby retarding lipase adsorption and triacylglycerol hydrolysis both in vivo and in vitro. Oral administration of ∊-polylysine to rats reduced the peak plasma triacylglycerol concentration. In vitro, ∊-polylysine and polylysine strongly inhibited the hydrolysis, by either pancreatic lipase or carboxylester lipase, of trioleoylglycerol (TO) emulsified with phosphatidylcholine (PC) and taurocholate. The ∊-polylysine concentration required for complete inhibition of pancreatic lipase, 10 μg/ml, is 1,000 times lower than that of BSA required for the same effect. Inhibition requires the presence of bile salt and, unlike inhibition of lipase by other proteins, is not reversed by supramicellar concentrations of bile salt. Inhibition increases with the degree of polylysine polymerization, is independent of lipase concentration, is independent of pH between 5.0 and 9.5, and is accompanied by an inhibition of lipase binding to TO-PC emulsion particles. However, ∊-polylysine did not inhibit the hydrolysis by pancreatic lipase of TO emulsions prepared using anionic surfactants, TO hydrolysis catalyzed by lingual lipase, or the hydrolysis of a water-soluble substrate. In the presence of taurocholate, ∊-polylysine becomes surface active and adsorbs to TO-PC monomolecular films. These results are consistent with ∊-polylysine and taurocholate forming a surface-active complex that binds to emulsion particles, thereby retarding lipase adsorption and triacylglycerol hydrolysis both in vivo and in vitro. In mammals, dietary neutral lipid digestion is commonly assumed to be mediated by three main enzymes: preduodenal (lingual or gastric) lipase, carboxylester lipase (cholesterol esterase), and pancreatic lipase (1Phan C.T. Tso P. Intestinal lipid absorption and transport.Front. Biosci. 2001; 6: D299-D319Crossref PubMed Google Scholar). Typical substrates for these enzymes are water-insoluble long-chain triacylglycerols. In contrast to their lipid substrates, these lipases are water soluble. Thus, for catalysis to occur, these enzymes must be adsorbed to the lipid surfaces and, therefore, the quality of the surface of substrate lipids is an important factor for lipase activity. Enzymes are sometimes activated or denatured by surface adsorption (2Rietsch J. Pattus F. Desnuelle P. Verger R. Further studies of mode of action of lipolytic enzymes.J. Biol. Chem. 1977; 252: 4313-4318Abstract Full Text PDF PubMed Google Scholar). As a consequence of these properties, amphiphilic substances, acting as emulsifiers, would be expected to influence the lipase reaction rate. It is well known that bile salts and synthetic detergents behave as inhibitors of lipolysis (3Borgström B. Erlanson C. Pancreatic lipase and co-lipase. Interactions and effects of bile salts and other detergents.Eur. J. Biochem. 1973; 37: 60-68Crossref PubMed Scopus (208) Google Scholar, 4Gargouri Y. Julien R. Bois A.G. Verger R. Sarda L. Studies on the detergent inhibition of pancreatic lipase activity.J. Lipid Res. 1983; 24: 1336-1342Abstract Full Text PDF PubMed Google Scholar). Amphiphilic proteins, such as BSA and β-lactoglobulin, have been shown to inhibit lipase activity toward its triglyceride substrate (5Brockerhoff H. On the function of bile salts and proteins as cofactors of lipase.J. Biol. Chem. 1971; 246: 5828-5831Abstract Full Text PDF PubMed Google Scholar, 6Gargouri Y. Julien R. Sugihara A. Verger R. Sarda L. Inhibition of pancreatic and microbial lipases by proteins.Biochim. Biophys. Acta. 1984; 795: 326-331Crossref PubMed Scopus (92) Google Scholar). Previously, we demonstrated that a basic protein, protamine, strongly inhibited the hydrolysis of trioleoylglycerol (TO) emulsified with phosphatidylcholine (PC) (7Tsujita T. Matsuura Y. Okuda H. Studies on the inhibition of pancreatic and carboxylester lipases by protamine.J. Lipid Res. 1996; 37: 1481-1487Abstract Full Text PDF PubMed Google Scholar). Polylysine, another basic protein, has been suggested as a food antiseptic. Polylysine binds to some proteins, nucleic acids, viruses, or bacteria through electrostatic or hydrophobic interaction and inhibits their functions. Another form of polylysine, ∊-polylysine, is synthesized by linking the α-carboxyl groups of lysine with its ∊-amino groups. As a consequence of this linkage, it is not hydrolyzed by proteases, such as trypsin, but retains its basic character. Therefore, ∊-polylysine is a candidate for an agent that inhibits intestinal lipid absorption while resisting proteolysis. The experiments presented in this report were undertaken to evaluate this possibility and elucidate its mode of action. The enzyme substrates and reagents used were obtained as follows. TO, taurocholate, deoxycholate, colipase, and β-lactoglobulin were from Sigma (St. Louis, MO). PC (from soybean) was from Nippon Shoji (Tokyo, Japan). Phosphatidylethanolamine (PE, from egg), phosphatidylserine (PS, from bovine brain), phosphatidic acid (PA, from egg PC), and PC (from egg) were from Serdary Research Laboratories (London, Canada). ∊-Polylysine (average molecular weight 5,000) was from Chisso Co. (Tokyo, Japan) and polylysine (average molecular weight 1,000–4,000) was from Wako Pure Chemical Industries (Osaka, Japan). The defined length polylysine polypeptides were synthesized using a peptide synthesizer (ABI 432A Synergy, Applied Biosystems Japan, Tokyo, Japan) following the manufacturer's recommendations. BSA was from Wako Pure Chemical Industries and was extracted by the method of Chen (8Chen R.F. Removal of fatty acids from serum albumin by charcoal treatment.J. Biol. Chem. 1967; 242: 173-181Abstract Full Text PDF PubMed Google Scholar) to remove free fatty acid. Pancreatic lipase for emulsion studies was purified from rat pancreas by the procedure of Gidez (9Gidez L.I. Purification of rat pancreatic lipase.J. Lipid Res. 1968; 9: 794-798Abstract Full Text PDF PubMed Google Scholar) with some modifications. Activity during purification was monitored using the soybean PC-TO assay described below under “Enzyme activity assays.” The purified enzyme (3,200 U/mg protein, at pH 6.8) gave a single band on SDS-PAGE from which its molecular weight was estimated to be 49,000. Carboxylester lipase was purified from porcine pancreas by the procedure of Rudd, Mizuno, and Brockman (10Rudd E.A. Mizuno N.K. Brockman H.L. Isolation of two forms of carboxylester lipase (cholesterol esterase) from porcine pancreas.Biochim. Biophys. Acta. 1987; 918: 106-114Crossref PubMed Scopus (59) Google Scholar) with some modifications (11Tsujita T. Okuda H. Effect of bile salts on the interfacial inactivation of pancreatic carboxylester lipase.J. Lipid Res. 1990; 31: 831-838Abstract Full Text PDF PubMed Google Scholar). The purified enzyme preparations were found to have specific activities of 700–800 μmol p-nitrophenol released/mg protein/min with p-nitrophenyl butyrate as the substrate. Lingual lipase fraction was prepared from rat tongues. The entire lingual serous glandular region was homogenized in cold 25 mM potassium phosphate buffer (pH 6.3) containing 0.9% NaCl. The was at for and the which was used as the enzyme was at form of pancreatic lipase in which the active was by was by reaction using the as described for and are for digestion in the presence of bile salt Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The was in and purified as described for Y. pancreatic triglyceride lipase in purification and PubMed Scopus Google Scholar). and ∊-polylysine were by as described for porcine A. Brockman H.L. The of and for are by J. 1996; Full Text PDF PubMed Scopus Google Scholar). The of reagents were to for the of free groups in the three of was with μmol of and of in and μmol of ∊-polylysine was with μmol of and of in was using The specific of and were and pancreatic lipase activity was by the of of acid from of μmol TO, μmol soybean and μmol taurocholate in mM (pH containing was for The assay was of the following in a of 25 enzyme colipase, μmol TO, μmol taurocholate μmol 10 μmol and μmol NaCl. was at pH and for The of acid was by the method of J. Effect of of rat on effects and binding of and Further for the action of through the J. Biochem. PubMed Scopus Google Scholar) with a T. Okuda H. in rat and and their of serum and J. Biochem. 1983; PubMed Scopus Google Scholar). using this assay were a function of lipase activity but with As a in experiments enzyme the of inhibition be Carboxylester lipase activity was using the same assay In some rat pancreatic lipase activity TO was using the assay described but with the μmol of soybean PC by of activity by pancreatic lipase was as described T. A. Y. Okuda H. activity of triglyceride lipase from rat Biol. Chem. 1984; Full Text PDF PubMed Google Scholar). activity of carboxylester lipase was by the of acid from as described (7Tsujita T. Matsuura Y. Okuda H. Studies on the inhibition of pancreatic and carboxylester lipases by protamine.J. Lipid Res. 1996; 37: 1481-1487Abstract Full Text PDF PubMed Google Scholar). Lingual lipase activity was using soybean TO as described for the pancreatic lipase assay but bile salt and using buffer (pH NaCl. lipase was in using purified rat pancreatic lipase T. T. R. Okuda H. from rat intestinal Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Pancreatic lipase was with the assay emulsion containing of 10 the and lipid were by at for 10 and were in buffer containing and of was to the proteins were to a which was with and with the C. A. H. T. Okuda H. of the action of acid on lipolysis in rat Lipid Res. Full Text Full Text PDF PubMed Google Scholar). was with and and the was using a of and in water was for and two were and was to rat a of this containing of peptide or or free and the a containing ∊-polylysine were from the or and at for triacylglycerol concentrations were using Pure Chemical The for the of the adsorption of proteins to lipid have been described Brockman H.L. acid adsorption of pancreatic lipase to 37: PubMed Scopus Google Scholar, Brockman H.L. of the of for the substrates and of Lipid Res. 2001; Full Text Full Text PDF PubMed Google Scholar). a was with a buffer of mM and mM (pH was monitored using the method and was at Lipid were from a the surface was the lipid to was the from a through a in the of the an of was in the same was monitored the was on of a hydrophobic and the of adsorption was As was used to the adsorption for the of in the that to the Brockman H.L. of monomolecular in studies of PubMed Scopus Google Scholar). the of the plasma triacylglycerol concentration emulsion with or ∊-polylysine was to ∊-polylysine the plasma triacylglycerol concentration was as with but were and the peak plasma triacylglycerol concentration was reduced by ∊-polylysine of the free or plasma triacylglycerol not the on triglyceride in the serum be to triglyceride hydrolysis in the the activity of rat pancreatic lipase toward TO emulsified with taurocholate and soybean PC was in the presence of concentrations of ∊-polylysine ∊-Polylysine inhibited TO hydrolysis activity by between and and hydrolysis was inhibited at On a weight this concentration is 1,000 times lower than that of β-lactoglobulin or BSA required to the same effect. was using of of on a weight concentration that the the of polymerization, the is the free to did not TO hydrolysis, and at it inhibited lipase activity by These that the concentration of that of free groups is for the inhibition of pancreatic the is of polylysine length on the of hydrolysis by rat pancreatic lipase of TO emulsified with were in the presence of mM taurocholate and concentrations of ∊-polylysine polylysine polylysine polylysine or polylysine the of three The action of ∊-polylysine was using three concentrations of pancreatic lipase a not to the activity in the of the of lipase inactivation by ∊-polylysine concentration was independent of the enzyme concentration. that either the is not acting on lipase or it the is at lipase concentrations of the known of lipolysis to the of the the results suggested that ∊-polylysine its on the the of ∊-polylysine on TO hydrolysis by pancreatic lipase was using other In the presence of taurocholate, ∊-polylysine strongly inhibited hydrolysis of TO-PC and but the TO was emulsified with either or the TO hydrolysis TO was emulsified with of soybean the activity was reduced to is to its surface activity as by A. F. Verger R. of on lipase interfacial binding and Biochem. 2001; PubMed Scopus Google Scholar). However, ∊-polylysine did not inhibit TO ∊-polylysine, to did not the hydrolysis of the water-soluble butyrate not These results are consistent with the of ∊-polylysine interfacial and that inhibition is reduced or by the presence of anionic lipids or an anionic in the of on the activity of pancreatic phosphatidic (TO) was emulsified with or as described in and and activity was in the presence and of ∊-polylysine The results are as of in a phosphatidic (TO) was emulsified with or as described in and and activity was in the presence and of ∊-polylysine The results are as of The hydrolysis of TO by the other intestinal lipase, carboxylester lipase, was inhibited by ∊-polylysine, the concentration required was as BSA did not carboxylester lipase activity to but at 10 it inhibited it not The activity of lingual lipase was not inhibited by ∊-polylysine but was As lingual lipase activity was at pH the effects of pH on the inhibition of pancreatic lipase activity were as a function of ∊-Polylysine inhibited activity of pancreatic lipase at pH between 5.0 and not it be that of activity pH or pH be the of the of pancreatic lipase Brockman H.L. of and on the and of pancreatic lipase at an Biol. Chem. Full Text PDF PubMed Google Scholar). The concentration of ∊-polylysine required to inhibit the activity of carboxylester lipase was times than that required to inhibit its activity these results are consistent with ∊-polylysine lipolysis at the interfacial as other proteins Y. Julien R. Sugihara A. Verger R. Sarda L. Inhibition of pancreatic and microbial lipases by proteins.Biochim. Biophys. Acta. 1984; 795: 326-331Crossref PubMed Scopus (92) Google but with It the possibility that the be with lipase and carboxylester lipase in of concentrations of ∊-polylysine on the of hydrolysis by carboxylester lipase of emulsified with soybean the of in which a water-soluble lipase be inhibited by proteins at the interfacial is to its interaction with the Y. Julien R. Sugihara A. Verger R. Sarda L. Inhibition of pancreatic and microbial lipases by proteins.Biochim. Biophys. Acta. 1984; 795: 326-331Crossref PubMed Scopus (92) Google Scholar). this adsorption the interaction of an substrate with the active of the this was in the pancreatic lipase was with TO-PC emulsion containing of a 10 the and lipid were by and the pancreatic lipase in the and were estimated by with an lipase of pancreatic lipase in the and ∊-Polylysine reduced the of pancreatic lipase in the in a and that in the inhibition the same of concentrations that TO hydrolysis Thus, as for proteins, the of inhibition to a of enzyme adsorption to the In the the concentration of taurocholate was below its concentration. concentration was used that two were in the assay the emulsion and the but the was to with bile salt A. H. P. A. Verger R. of bile salts and at and 2001; PubMed Scopus Google Scholar). pancreatic lipase inhibition by other proteins, bile salt to supramicellar concentration inhibition Y. Julien R. Sugihara A. Verger R. Sarda L. Inhibition of pancreatic and microbial lipases by proteins.Biochim. Biophys. Acta. 1984; 795: 326-331Crossref PubMed Scopus (92) Google by the from the to the of pancreatic lipase inhibition to bile salt concentration, the hydrolysis of the PC-TO emulsion containing was in the presence and of ∊-polylysine The results that in the of ∊-polylysine, the enzyme of its activity to a taurocholate concentration of but that activity was inhibited at mM in to the mM in the substrate activity was to mM by at The taurocholate was emulsions prepared in its lipase activities of it was The concentration with bile salt is at or its concentration under of and bile effects of and 31: PubMed Scopus Google Scholar, and surface of by surface and Scopus Google Scholar). concentrations of taurocholate and the presence of ∊-polylysine complete inhibition of TO In the presence of mM taurocholate or mM deoxycholate, the concentrations of bile salts that did not inhibit lipolysis in the of ∊-polylysine concentrations as as complete inhibition of lipolysis The of concentrations of bile salts either to the inhibition of pancreatic lipase by ∊-polylysine or to its some of this with other using substrate emulsions prepared in the of bile inhibition by ∊-polylysine was activity was at and at mM The results that ∊-polylysine inhibits lipolysis by enzyme binding in a bile However, not emulsion surfaces were of in the of the to the TO and other ∊-Polylysine was a bile salt was not but the of the Therefore, we used monomolecular lipid at the to the inhibition of pancreatic lipase by ∊-polylysine in the presence of bile The of this is that the interfacial surface is and independent of the lipids the lipid or the in the The surface activity of in the of an lipid at the was taurocholate in the the surface of the to buffer was of ∊-polylysine to the a in the surface that not a of such the of the surface on the concentration of ∊-polylysine in the is shown in of the surface for the was and the surface concentration of was The of the surface and the surface concentration of were The binding by both surface and adsorption was using the adsorption for that the in surface is to adsorbed Brockman H.L. with 1983; PubMed Scopus Google Scholar). The of and the in the surface concentration of was to be taurocholate was from the the surface did not following of not the binding of independent of its concentration at a surface concentration than that obtained with taurocholate taurocholate the surface activity and interfacial binding of the of a surface-active complex between taurocholate and that to the this is the that in the concentration of ∊-polylysine which of the inhibition the bile salt is in the of on the anionic taurocholate, to free groups on the from to the of this complex with the would in the presence of the lipid used to the emulsions for experiments described binding were with taurocholate was under lipid of or a at an surface of adsorbed to a surface concentration of with a of an surface of the was to However, the surface concentration of from the binding was at with TO adsorbed to an at to a surface of with a of However, the taurocholate concentration in the was to the surface concentration of to that with mM taurocholate at and with taurocholate adsorbed to or These that binding of to lipid on the presence of taurocholate, the of a surface-active complex at the the binding was at the concentration at which lipase binding and activity toward emulsions is in the emulsion In the lipase experiments using emulsion ∊-polylysine lipase of was as a function of the surface using on a a concentration of its adsorption to the lipid to 10 to the taurocholate was not is a that expected for of a complete However, the to binding by not In the presence of ∊-polylysine binding was reduced by at 10 and at not that under ∊-polylysine increases surface by the that the of ∊-polylysine to surface to the inhibition of lipase adsorption at 10 However, the presence of was to the effects of both surface and ∊-polylysine at surface of 10 and but not at The for this are not but the that surface not the of ∊-polylysine on lipids are water-insoluble and are emulsified by bile concentrations of are in and the bile is of the In the presence of PC and bile acids, the lipid surface at which the enzyme is neutral lipids are to be by three main enzymes: and pancreatic lipases (1Phan C.T. Tso P. Intestinal lipid absorption and transport.Front. Biosci. 2001; 6: D299-D319Crossref PubMed Google Scholar). of these are water-soluble enzymes and have to the lipid at the surface of the substrate lipid Therefore, the lipase reaction at the substrate surface and is surface adsorption of the amphiphilic proteins are adsorbed to the and lipase activity (5Brockerhoff H. On the function of bile salts and proteins as cofactors of lipase.J. Biol. Chem. 1971; 246: 5828-5831Abstract Full Text PDF PubMed Google Scholar, 6Gargouri Y. Julien R. Sugihara A. Verger R. Sarda L. Inhibition of pancreatic and microbial lipases by proteins.Biochim. Biophys. Acta. 1984; 795: 326-331Crossref PubMed Scopus (92) Google Scholar). albumin has been shown to lipolysis in two it the lipase from and at it inhibits activity by the substrate Y. Julien R. Sugihara A. Verger R. Sarda L. Inhibition of pancreatic and microbial lipases by proteins.Biochim. Biophys. Acta. 1984; 795: 326-331Crossref PubMed Scopus (92) Google Scholar, Y. C. Sugihara A. Sarda L. Verger R. Inhibition of lipases by with Biol. Chem. Full Text PDF PubMed Google Scholar) that albumin and other proteins, β-lactoglobulin and inhibited pancreatic lipase and their effects be the of lipase from its substrate to a in interfacial salts were to this by these proteins from the In this we that polylysine and ∊-polylysine inhibit the hydrolysis of TO emulsified with PC with a that is two to three of than that of proteins used in The for the inhibition to be the same as that by which other amphiphilic proteins inhibit by lipase adsorption to the inhibition by ∊-polylysine on substrate was presented to the it with TO-PC and and not or The between the is that PC and are the are to the emulsion in these was taurocholate, an anionic bile salt. taurocholate in lipase inhibition the concentration of ∊-polylysine by a the of of a of taurocholate or the concentration of to inhibit emulsion experiments that bile salt is required for inhibition to be is in contrast to for other proteins, albumin and β-lactoglobulin, that of inhibition by supramicellar concentrations of bile salts Y. Julien R. Sugihara A. Verger R. Sarda L. Inhibition of pancreatic and microbial lipases by proteins.Biochim. Biophys. Acta. 1984; 795: 326-331Crossref PubMed Scopus (92) Google Scholar). Y. Julien R. Sugihara A. Verger R. Sarda L. Inhibition of pancreatic and microbial lipases by proteins.Biochim. Biophys. Acta. 1984; 795: 326-331Crossref PubMed Scopus (92) Google Scholar) that proteins inhibit pancreatic lipase, of their at the assay In the the that is not with to the but with to the of polylysine The for this is suggested by the experiments with and taurocholate These that the surface activity of the as both by surface and by increases in the presence of bile salt and in the at which the of taurocholate the on It be that the procedure used not the of the ∊-polylysine, and the of of groups was the results that inhibition from the of a surface-active complex of ∊-polylysine and taurocholate. a complex to taurocholate The of between and bile salts is well known and the for and The inhibition requires bile salt and to be with of a surface-active salt complex that the and thereby lipase Inhibition of the binding of to at be reversed by colipase, a surface-active Thus, as Y. C. Sugihara A. Sarda L. Verger R. Inhibition of lipases by with Biol. Chem. Full Text PDF PubMed Google the of proteins in this an salt to to the and surface a for the has suggested that the of of proteins with the not in this is important Y. C. Sugihara A. Sarda L. Verger R. Inhibition of lipases by with Biol. Chem. Full Text PDF PubMed Google Scholar). In this inhibition by ∊-polylysine be that of other the of inhibition of lipase to be a consequence of the of bile salt in that ∊-polylysine was in its presence not In this lingual lipase is the lipase from that is not inhibited by proteins under in which pancreatic lipase is inhibited Y. Julien R. Sugihara A. Verger R. Sarda L. Inhibition of pancreatic and microbial lipases by proteins.Biochim. Biophys. Acta. 1984; 795: 326-331Crossref PubMed Scopus (92) Google Scholar). has been as that inhibition of lipolysis by proteins not by a of the substrate by the as a consequence of its binding to the Y. C. Sarda L. Verger R. Inhibition of lipases by a binding using PubMed Scopus Google Scholar). The of an salt complex in the of inhibition is by the that the presence of anionic at a than that of taurocholate in the assay or the in the of anionic complex for not to inhibit possibility is of the complex on the substrate the of the surface to lipase However, this have TO the interfacial of the is not demonstrated in this is the possibility be that the complex that adsorbs to the by PC be in it lipase and its binding to the the results that the in triacylglycerol absorption in vivo results from the inhibition of lipolysis demonstrated in possibility is that the reaction thereby their these but the the of concentrations of bile salt to the are consistent with ∊-polylysine to as an interfacial lipase in the Previously, we demonstrated that the basic protein, protamine, strongly inhibited hydrolysis of TO emulsified with PC (7Tsujita T. Matsuura Y. Okuda H. Studies on the inhibition of pancreatic and carboxylester lipases by protamine.J. Lipid Res. 1996; 37: 1481-1487Abstract Full Text PDF PubMed Google Scholar). a fraction of basic acid for of the and its is pH that to the PC on the TO, thereby lipase but did not the of bile salt. The of of and its that inhibit the lipase activity by the same as that for polylysine and These results that as a lipase in the presence of bile salts in the ∊-Polylysine be to polylysine or by of its to proteolysis. However, it to be some to such inhibitors their was by and from the of and by the The for the of phosphatidic acid phosphatidylcholine phosphatidylserine trioleoylglycerol

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