Development of an indirect method for measuring porcine pancreatic lipase in human duodenal fluid.

Patients with exocrine pancreatic insufficiency are usually treated with porcine pancreatic enzymes but the bioavailability of these enzymes in the gut remains a matter of discussion. In order to determine the duodenal availability of porcine pancreatic lipase (PPL) present in pancreatic extracts (PE) taken orally, we developed a method for quantifying PPL in samples containing both PPL and human pancreatic lipase (HPL). Total pancreatic lipase activity measurements using the pH-stat technique and tributyrin as substrate were combined with an HPL-specific ELISA. Based on the known specific activity of the purified HPL, its activity was deduced from the ELISA measurements, and the PPL activity was obtained by subtracting the HPL activity from the total pancreatic lipase activity. This assay was established and validated using various samples containing pure PPL and recombinant HPL or PE, mixed or not with human duodenal juice. Samples collected in vivo from patients treated with PE were also tested. It was found that PPL did not affect the HPL ELISA, and the indirect PPL assay gave a measurement accuracy of 6.6% with the samples containing pure PPL and 10% with those containing PE. This assay was also used successfully to discriminate between PPL and the endogenous HPL present in the duodenal contents of patients with severe pancreatic insufficiency treated with PE. This method might provide a useful means of assessing the availability of PEs at their site of action, in the absence of a PPL-specific ELISA.

An enzymatically active truncated form (-55 N-terminal residues) of rabbit gastric lipase. Correlation between the enzymatic activity and disulfide bond oxydo-reduction state.

Rabbit gastric lipase (RGL) was subjected to proteolysis with trypsin and led to cleavage occurring at three defined sites (Lys-4, Arg-55 and Arg-229). The tryptic hydrolysate contained four fragments: Gly-230-Lys-379 (T1), Gly-56-Arg-229 (T2), Ser-5-Arg-55 (T3), as well as a 45 kDa molecular form consisting of peptides T1 and T2 linked by a disulfide bridge. The tryptic hydrolysate of RGL as well as the 55 N-terminal amino acid deleted forms conserved 30% of the initial enzymatic activity in a tributyrin assay. Two out of the three cysteine residues which are present in all the known gastric lipases were found to be involved in a disulfide bridge. Unlike HGL, RGL appears to have a heterogenous pattern of cysteine residues. The 30% enzymatic activity of RGL persisting after trypsin treatment may be attributable to the 45 kDa molecular form (with the Cys-227-Cys-236 or Cys-227-Cys-244 disulfide bridge). Trypsin-treated HGL, which was completely inactivated, showed that a single location of the disulfide bridge existed between cysteine residues 236 and 244. It can be concluded that the existence of one disulfide bridge is necessary to maintain the lipase activity of the 45 kDa form of RGL.

A conformational transition between an open and closed form of human pancreatic lipase revealed by a monoclonal antibody.

The interfacial activation of human pancreatic lipase (HPL) probably involves the motion of a lid covering the active site of the enzyme. Here we observed that the presence of either bile salts or a small proportion of water-miscible organic solvents (called activator compounds) considerably enhances the enzymatic activity of HPL on a monomeric solution of tripropionin. This finding suggests that the activator compounds may favor the opening of the lid. This hypothesis was checked by comparing the immunoreactivity of HPL and HPL with a mini-lid (HPL(-lid)) towards anti-HPL monoclonal antibodies (mAbs), in the presence and absence of the activator compounds. A single conformational mAb (248-31) fails to immunoprecipitate HPL in the presence of activator compounds and HPL covalently inhibited with diethyl p-nitrophenyl phosphate (DP.HPL). This loss of recognition of HPL by mAb 248-31 was probably due to the motion of the lid, since HPL(-lid) was always recognized in the presence or absence of activator compounds. Furthermore, two other mAbs (81-23 and 146-40) immunoprecipitated HPL similarly whether or not the activator compounds were present. MAb 248-31 therefore specifically recognizes HPL in the closed but not the open conformation.

Characterization of the serine reacting with diethyl p-nitrophenyl phosphate in porcine pancreatic lipase.

The position in porcine pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) of the serine reacting specifically with emulsified or micellar diethyl p-nitrophenyl phosphate has been investigated. This serine which appears to be involved in lipase adsorption to insoluble triglyceride interfaces, is at position 152 in the enzyme chain. The sequence around this amino acid is: His-Val-Ile-Gly-His-Ser-Leu-Gly.

Purification and molecular characterization of lamb pregastric lipase.

Lamb pregastric lipase (LPGL) was purified from pharyngeal tissues. The purification procedure was based on an aqueous extract containing 0.7% Tween 80 which was chromatographed on DEAE-cellulose anion-exchanger and adsorbed on HA-Ultrogel followed by gel filtration on Ultrogel AcA-54. The final enzymatic preparation, where the overall activity recovery was 3%, showed a single protein band on SDS-PAGE with a molecular mass of 50 kDa. LPGL is a glycoprotein containing approx. 14% (w/w) of carbohydrate. Extensive deglycosylation using peptide N-glycosidase F yielded a protein with an apparent molecular mass of 43 kDa. An uncontrolled proteolysis of LPGL during the purification lead to a 45 kDa form which was previously observed in human lysosomal acid lipase (HLAL) and rabbit gastric lipase (RGL). The labile bond X54-Leu55 was identified. Isoelectric focusing of LPGL reveals a major band corresponding to an isoelectric point of 4.8. The pure enzyme displayed specific activities of 950 U mg-1, 300 U mg-1 and 30 U mg-1 at pH 6.0, using tributyroylglycerol, trioctanoylglycerol and trioleoylglycerol as substrates, respectively. Using Western blot analysis, a cross-immunoreactivity of LPGL was observed with purified anti-human gastric lipase polyclonal antibodies. Determination of the amino-acid sequence of 62 residues revealed a high degree of homology with other known preduodenal lipases.

Porcine pancreatic lipase. Completion of the primary structure.

The complete primary structure of a lipase (triacylglycerol hydrolase; EC 3.1.1.3) is presented for the first time. The porcine pancreatic enzyme which was investigated is composed of a single chain of 449 amino acids. Upon fragmentation by CNBr, five peptides were obtained. The sequence of four of them (CN I-CN IV) has already been published. The present report deals with the arrangement of the 142 amino acids of the C-terminal peptide CN V, thus completing the analysis of the whole molecule. Special problems resulting from incomplete cleavage of some peptide bonds in CN V and aggregation of large peptides were overcome using Sephadex filtration of succinylated derivatives in 50% acetic acid, automated sequence analysis of peptide mixtures and subdigestion of material which could not be directly resolved. No obvious homology was found when the sequence of porcine lipase was compared with other protein, including pancreatic phospholipase A2 and colipase from the same species. However, a few similarities which might be significant were detected between the environment and relative position of certain half cystines in lipase and colipase, as well as between two tyrosine-rich regions existing in both proteins.

Nitration of the tyrosine residues of porcine pancreatic colipase with tetranitromethane, and properties of the nitrated derivatives.

The nitration of the long form (N-terminal valine) of porcine pancreatic colipase with tetranitromethane was investigated under a variety of conditions. Fractionation of the nitrated monomers on DE-cellulose led to well-defined derivatives containing one, two and three nitrotyrosines per mol. Automated Edman degradation of the nitrated peptides, especially that of the staphylococcal proteinase peptide (49-64) showed that Tyr-54 was nitrated very fast under all conditions. This residue was the only one to be nitrated in water. Partial nitration of Tyr-59 was induced by bile salt micelles, while both Tyr-59 and Tyr-58 reacted extensively in the presence of lysophosphatidylcholine micelles (in which tetranitromethane is concentrated 150-fold compared to water) or of a liquid tetranitromethane-water interface. The strong negative Cotton effect at 410 nm which has already been observed using unfractionated preparations of nitrated colipase (Behnke W.D. (1982) Biochim. Biophys. Acta 708, 118-123) is linked with the nitration of Tyr-59 and it is markedly reduced by taurodeoxycholate micelles, suggesting a conformational change induced by the micelles in the tyrosine region. Moreover, the pKa of the nitrotyrosine residues in nitrated colipase is the same as that of free nitrotyrosine (pKa = 6.8) and it is shifted to 7.6 in the presence of taurodeoxycholate micelles. Micelles protected colipase against polymerization during nitration. These data suggest that Tyr-58 and Tyr-59 are part of the interface recognition site of colipase. The participation of Tyr-55 in binding is not excluded. The upwards nitrotyrosine pKa shift in the colipase micelle complex may explain why nitrated colipase can reactivate lipase in a triacylglycerol-taurodeoxycholate system at pH 7.5.

Pancreatic lipase-related protein 1 (PLRP1) is present in the pancreatic juice of several species.

Pancreatic lipase-related protein 1 (PLRP1) was purified from human, canine, porcine and rat pancreatic juices. The four PLRP1s were identified using microsequencing methods after performing gel filtration on Ultrogel AcA-54 followed by chromatography on Heparin-Sepharose cation-exchanger. Polyclonal antibodies specific to human PLRP1 (HPLRP1) were raised in the rabbit using a synthetic decapeptide from HPLRP1. The results of Western blotting analysis showed that these antibodies recognized native HPLRP1 and recombinant HPLRP1 produced by insect cells, and cross-reacted only with rat PLRP1 (RPLRP1). No significant lipolytic activity was observed with native canine PLRP1 and recombinant HPLRP1 on various glycerides, phospholipid and vitamin esters, or on cholesterol esters. It was established for the first time that this protein is secreted in variable amounts by the adult exocrine pancreas of several species.

N-terminal sequence extension in the glycosylated forms of human pancreatic stone protein. The 5-oxoproline N-terminal chain is O-glycosylated on the 5th amino acid residue.

The pancreatic stone protein isolated from human calculi (PSP) derives from the immunoreactive protein forms detected in human pancreatic juice (PSP S2-5) through the tryptic cleavage of the Arg-11-Ile-12 bond. Among the eleven amino acids of the PSP S2-5 N-terminal extension Z-E-A-Q-T-E-L-P-Q-A-R, the first residue is an oxoproline and the fifth, a threonine, bears the single carbohydrate chain of the protein molecules. Variations in the glycan chain composition account for the differences in the Mr of PSP S2-5. The PSP S2-5 forms are very soluble in aqueous solutions between the pH values 5.0-9.0, whereas the proteolysated form is scarcely soluble.

Purification and some characteristics of chicken liver L-2-hydroxyacid oxidase A.

The isozyme A of L-2-hydroxyacid oxidase is a peroxisomal flavoenzyme that catalyzes the oxidation of short-chain aliphatic L-2-hydroxyacids in many tissues of higher organisms. A new purification procedure allowed us to obtain a 1400-fold purified enzyme from chicken liver. The N-terminal amino acid of the polypeptide chain was found to be blocked as that of spinach glycolate oxidase, contrastingly with that of rat kidney isozyme B. Its amino acid composition was comparable to that of other known L-2-hydroxyacid oxidases. Despite different substrate specificity, some immunological identity was observed between chicken liver L-2-hydroxyacid isozyme A and rat kidney isozyme B.

The disulfide bridges of the immunoreactive forms of human pancreatic stone protein isolated from pancreatic juice.

Following the complete sequence elucidation of human pancreatic stone protein (immunoreactive form PSP S1 isolated from pancreatic juice) [(1987) Eur. J. Biochem. 168, 201-207], the location of the three S-S bridges of the protein was investigated. The cystine-containing peptides, detected after the separation of the peptic or chymotryptic digests on SP-Sephadex or Sephadex G-50, were submitted to Edman degradation and/or to oxidation. The cysteic peptides after separation on SP-Sephadex or Sephadex G-50 were characterized by their amino acid compositions. The pairing of the half-cystines: Cys 3-Cys 14, Cys 31-Cys 129 and Cys 104-Cys 121 was determined. The same experiments carried out with PSP S2-5 (other immunoreactive forms) gave an identical characterization.

Acetylation of Lys-373 in porcine pancreatic lipase after reaction of the enzyme or its C-terminal fragment [corrected] with p-nitrophenyl acetate.

The reactions of lipase (449 amino acid residues) and lipase fragment (336-449) with p-nitrophenyl acetate have been studied from 2 different angles. In previous papers it has been shown that lipase and lipase fragment enzymatically hydrolyze p-nitrophenyl acetate. The amino acid residue of the catalytic site that is temporarily acetylated has not yet been characterized in lipase or lipase fragment. Besides this very fast enzymatic hydrolysis, acetylation reactions may take place on nucleophilic amino acid side-chain groups. In the present report, acetylated amino acid residues whose acetyl linkages were not cleaved after pH 7.5-8.5 incubations have been investigated. Several residues were acetylated in very low proportion, whereas lysine 373 was stoichiometrically acetylated in lipase and in lipase fragment. This specific acetylation may have been favored by the presence of a hydrophobic reversible binding site for p-nitrophenyl acetate near Lys-373. This acetylation did not greatly change the specific activity of lipase towards an emulsion of tributyrylglycerol in the presence of colipase, but under certain conditions it had an effect on the enzymatic hydrolysis of p-nitrophenyl acetate by the lipase fragment.

In vitro oxidative decarboxylation of L-2-hydroxy-4-methylthiobutanoic acid by L-2-hydroxy acid oxidase A from chicken liver.

The first step in the set of reactions responsible for the biological utilization of L-2-hydroxy-4-methylthiobutanoic acid, the methionine hydroxy analogue, in protein synthesis was investigated in vitro using pure L-2-hydroxy acid oxidase A from chicken liver. The reaction yielded no more than 20% of the corresponding alpha-keto acid, the well-known intermediate in methionine metabolism, and as much as 80% of the subsequent decarboxylation product, 3-methylthiopropionate, suggesting that L-2-hydroxy-4-methylthiobutanoic acid cannot be completely converted into methionine in vivo. It was therefore concluded that chicken liver L-2-hydroxy acid oxidase, a peroxisomal enzyme requiring flavin mononucleotide as a coenzyme, also has an oxidative decarboxylation activity in vitro, which was found to be NADH-dependent. The mechanism possibly underlying the successive conversion of the methionine hydroxy analogue into alpha-keto acid and 3-methylthiopropionate by this NADH:flavin oxidoreductase-decarboxylase activity is described.

Inhibitory effects of anions and active site amino acid sequence of chicken liver L-2-hydroxyacid oxidase A, a member of the FMN-dependent alpha-hydroxyacid oxidizing enzyme family.

Monocarboxylic acids with aliphatic chains were found to be mixed inhibitors of chicken liver L-2-hydroxyacid oxidase A when L-2-hydroxy-4-methylthiobutanoic acid was used as the substrate. The finding that the binding affinity of the enzyme for monocarboxylic acids was directly proportional to the number of carbon atoms in the chain strongly suggests that in addition to the electrostatic interaction due to the carboxyl moiety, hydrophobic forces may also be involved in the binding affinity of monocarboxylic acids to the enzyme's active site. Oxalate, a dicarboxylic acid, also resulted in a mixed-type inhibition of chicken liver L-2-hydroxyacid oxidase A, and, surprisingly, its binding affinity to the enzyme was found to be quite high as compared with monocarboxylic acids. This is probably due to the fact that the two carboxyl groups of oxalate give rise to electrostatic interactions with the positively charged side chains of two adjacent residues in the polypeptide chain. The inhibitory effects of other dicarboxylic acids was found to decrease as the number of carbon atoms in the chain increased. Oxamate was found however to be a novel type of potent inhibitor of the enzyme. All in all, these kinetic studies and the amino acid sequence determination in the active site region after limited proteolysis of the polypeptide chain definitely establish that chicken liver NADH/FMN containing L-2-hydroxyacid oxidase A is a member of the FMN-dependent alpha-hydroxyacid oxidizing enzyme family.

The histidines reacting with ethoxyformic anhydride in porcine pancreatic lipase: their relationships with enzyme activity.

The activities of porcine pancreatic lipase (449 amino acid residues) toward two different substrates, p-nitrophenylacetate and tributyrylglycerol, and their dependence on histidine ethoxyformylation were studied. In parallel, the ethoxyformylation of the lipase fragment constituting the C-terminal sequence of lipase (residues 336 to 449) was also investigated. This fragment was found to have retained the ability of lipase to catalyse p-nitrophenylacetate hydrolysis. The first histidine to react either in lipase or in the lipase fragment was His-354. The activities of the two compounds toward p-nitrophenyl-acetate were lost but that of the enzyme toward tributyrylglycerol was almost entirely retained. When a larger excess of ethoxyformic anhydride was used for the lipase reaction, 2.8 histidine residues were ethoxyformylated and characterised as His-354, His-156 and His-75, which resulted in an 85% inhibition of the tributyrylglycerol hydrolysis by the enzyme. Hydroxylamine treatment reactivated most of the lipase and lipase fragment. This is the first demonstration that the two lipase activities are not associated with the same active site. The loss of activity toward triacylglycerol hydrolysis suggests that His-156 and/or His-75 belong(s) to the active site or that a conformational change resulting from the ethoxyformylation renders the lipase inactive.

An inactive pancreatic lipase-related protein is activated into a triglyceride-lipase by mutagenesis based on the 3-D structure.

Both classical dog pancreatic lipase (DPL) and dog pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by the exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with DPLRP1 on any of the substrates tested: di- and tri-glycerides; phospholipids (PC); etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 A. Its structure is similar to that of the classical pancreatic lipase (PL) structures determined in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 was suspected of being responsible for the absence of enzymatic activity by inducing a steric clash with one of the acyl chain observed in the structures of chiral C11 alkyl phosphonate inhibitors, bound to the classical PL. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of the three known pancreatic lipase-related protein 1 (PLRP1), whereas Ala and Pro residues are always present at the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic-related protein 1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on tributyrin (1800 U/mg) as well as trioctanoin (2250 U/mg) and its activity is low in the presence of taurodeoxycholate and stimulated in the presence of colipase. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the PLRP1 lipases.

Comparative oxidative rates of methionine hydroxy analogue in rat and chicken kidney.

1. L-2-HAOX A and L-2-HAOX B were purified from chicken and rat kidney with purification factors of 550 and 45, respectively. 2. It was established that both enzymes were tetrameric (M(r) = 169,000) and consisted of 40 kDa monomers. 3. While chicken kidney L-2-HAOX A is N-terminally blocked like spinach glycolate oxidase and chicken liver L-2-HAOX A, L-2-HOAX B begins with a Pro residue. 4. The kinetic parameters of L-HMB oxidation by L-2-HAOX A and B were determined. 5. The results are particularly interesting as regards L-HMB oxidation by L-2-HAOX B.

Hydrolysis of p-nitrophenyl acetate by the peptide chain fragment (336-449) of porcine pancreatic lipase.

The incubation of porcine pancreatic lipase (449 amino acids) with chymotrypsin led to the preferential cleavage of the Phe-335-Ala-336 bond [Bousset-Risso et al. (1985) FEBS Lett. 182, 323-326]. Up to now it has not been possible to isolate the fragment (1-335) whereas fragment (336-449) was purified. This fragment does not display any activity towards the specific substrates of lipase, triacylglycerols, either in the aggregate form or monomeric solution, but like lipase it hydrolyzes p-nitrophenyl acetate. The biphasic kinetics of the release of p-nitrophenol by the fragment with different concentrations of p-nitrophenyl acetate ([S] greater than [E]) are very similar to those of lipase and other esterases. The initial burst is equal to 1 mol p-nitrophenol/mol fragment (when [S] = infinity). Ethoxyformic anhydride only reacts with 1 mol histidine out of the 2 mol that the fragment contained. The activity of the fragment towards p-nitrophenyl acetate hydrolysis is inhibited after ethoxyformic anhydride reaction as in the case of lipase. The results presented led to the hypothesis that in the area (336-449) a part of the active-site structure of the lipase molecule is included. It would seem that fragment (336-449) is a functional domain of lipase.

Human pancreatic lipase: an exposed hydrophobic loop from the C-terminal domain may contribute to interfacial binding.

Epitope mapping was performed using four anti-HPL monoclonal antibodies (mAb's 81-23, 146-40, 315-25, and 320-24) directed against human pancreatic lipase (HPL). Three HPL mutants produced in insect cells were tested for this purpose: (i) N-HPL, which consists of only the N-terminal domain of HPL, (ii) HPL(-lid), in which a short loop consisting of 5 amino acid residues replaces the full-length 23-residue lid domain present in HPL, and (iii) N-GPLRP2/C-HPL chimera, a chimeric mutant consisting of the N-terminal domain of the guinea pig pancreatic lipase related protein 2 (GPLRP2) fused to the C-terminal domain of HPL. The C-terminal domain of HPL (C-HPL) was prepared in a pure form after performing chymotryptic digestion of HPL. The mAb 146-40 recognizes HPL, HPL(-lid), and N-HPL but not GPLRP2, N-GPLRP2/C-HPL chimera, or the C-HPL. The antibody mAb 146-40 therefore specifically recognizes the N-terminal domain of HPL, and the epitope recognized does not include the amphiphilic lid. On the other hand, mAb's 81-23, 315-25, and 320-24 react specifically to the C-terminal domain of HPL, since they recognize HPL, HPL(-lid), the N-GPLRP2/C-HPL chimera, and the C-HPL but not N-HPL or GPLRP2. It was further established that these three mAb's recognize the same conformational epitope, the structure of which is stabilized by the N-terminal domain in the presence of SDS at concentrations greater than its critical micellar concentration. This conformational epitope was found to be located in the vicinity of Met 397 and Arg 414. These two residues delineate a highly exposed peptide stretch extending from the HPL C-terminal domain, which includes a hydrophobic surface loop (beta5'). Kinetic studies on the HPL/mAb's complexes showed that the lipase activity was much lower in these complexes than in HPL. The results of the present study suggest for the first time that the beta5' loop from the C-terminal domain may be involved in the interaction of HPL with a lipid/water interface.

Porcine pancreatic lipase. The disulfide bridges and the sulfhydryl groups.

Following complete sequence analysis of the 449 amino acids in porcine pancreatic lipase [J. De Caro et al. (1981) Biochim. Biophys. Acta, 671, 129-138], the position of the six disulfide bridges and of the two free thiols of the protein was investigated using a variety of techniques. Three bridges (Cys-4--Cys-10, Cys-237--Cys-261 and Cys-433--Cys-449) were easily identified in the peptic digest of lipase at pH 2.0. In the latter digest, two other bridges (Cys-285--Cys-296 and Cys-299--Cys-304) were also identified by means of the cystine peptide constituted by two peptide segments: Ala281-Gly-Phe-Pro-Cys-Asp-Ser287 and Thr292-Ala-Asn-Lys-Cys-Phe-Pro-Cys-Pro-Ser-Glu-Gly-Cys-Pro-Gln-Met307. A disulfide bridge, formed by Cys-285 and one of the three half-cystines of the other segment, connected the two peptide moieties. A second disulfide bridge linked the two remaining half-cystines. It was not possible to split any peptide bond between Cys-296 and Cys-304 with proteolytic enzymes. The determination of the pairing of the four half-cystines was resolved as follows. Bond Lys-295--Cys-296 was cleaved with trypsin. A single cycle of Edman degradation was then performed on the peptide compound, thus freeing, in particular, Cys-296 of the peptide bond (296-297). Cys-296 only retained its S-S connection with the half-cystine partner. At the completion of the above operations, the two peptide segments (282-287) and (297-307) could be separated. Consequently it was concluded that Cys-285 is linked to Cys-296. Therefore Cys-299 and Cys-304 are paired. The most reactive SHI group of the enzyme was localized on Cys-181 by condensation of the native protein with radioactive N-ethylmaleimide. The alkylation of the SHII group required previous denaturation of the molecule at alkaline pH. The results obtained for the characterization of the SHII group suggested the existence of two isomeric forms, the SHII being either on Cys-101 or Cys-103 and the bridge alternately between Cys-90--Cys-103 or Cys-90--Cys-101. It is not yet known whether these two forms pre-exist in native lipase or result from an exchange reaction. The bridge Cys-90--Cys-101 was characterized in a thermolytic digest of a cyanogen bromide fragment (CN II) of the protein. However, another bridge involving Cys-181 was also found in the digest. This bridge is considered as being an artefact. It is possible that considering the treatments undergone by the large peptide (CN II), the SH groups of lipase were oxidized and transformed in an S-S bridge. The disulfide bridges of lipase form relatively small loops along the main chain. This arrangement is consistent with a high flexibility of the molecule. As reported earlier [R. Verger et. al. (1971) Biochim. Biophys. Acta. 242, 580-592], the SHI group is not essential for lipase activity. The role of the SHII group should be more precisely investigated.