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.

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.

Characterization in rat pancreatic juice of a protein homologous to the human pancreatic stone protein.

1. The pancreatic stone protein (PSP, Mr 15,000) which has been discovered in human calculi derives from the native glycosylated forms of the protein (Mrs 17,500-22,000) which are present in human pancreatic juice through tryptic cleavage of the Arg 11-Ile 12 bond. 2. In the present study, a homologous native form of the protein (Mr 17,000) was purified from rat pancreatic juice. 3. Its N-terminal amino acid sequence was found to display a high degree of homology with that of the human native protein forms, apart from the fact that it was not glycosylated. 4. In rat as in human, tryptic cleavage of the Arg 11-Ile 12 bond transforms a soluble protein into one which is practically insoluble at neutral pH.

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.

Limited proteolyses in pancreatic chymotrypsinogens and trypsinogens.

In the 1950's, the specific cleavages of the peptide bonds occurring in bovine cationic chymotrypsinogen and trypsinogen were among the first examples of limited proteolyses. According to the split bond(s), the precursor is transformed into enzyme or different forms of zymogen or again into inert protein. The conversion of trypsinogen into trypsin triggers the activations of all the other enzyme precursors of pancreatic juice. In the pancreas, several factors oppose trypsinogen autoactivation, whereas, in the duodenum, all the conditions are favorable for trypsinogen activation by enteropeptidase.

Minireview on pancreatic lipase and colipase.

By hydrolyzing the dietary triacylglycerols, pancreatic lipase causes catalysis in heterogeneous medium. In vivo, lipase action cannot take place without colipase due to the presence of bile salts. The cofactor enables lipase anchoring to the water-lipid interface. The lipase-colipase system furnishes an excellent example of specific interactions (protein-protein and protein-lipid). The studies of lipase catalytic properties brought to light the importance of certain parameters related to the 'quality of the interface'. The structure-function relationship analyses revealed a certain number of functional amino acid residues in lipase and colipase involved either in the catalytic site of the enzyme or in the recognition sites (lipase-colipase and protein-interface). Comparisons of the sequences of lipases derived from different sources display interesting similarities in certain cases.

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.

Primary structure of the glycans of porcine pancreatic lipase.

The glycan primary structure of the main glycopeptide fraction obtained by pronase and carboxypeptidase A digestions of porcine pancreatic lipase has been investigated by 500-MHz 1H-NMR spectroscopy and methylation analysis. The results demonstrate that the glycopeptide fraction was a mixture containing the following structures: (formula; see text)

Complete amino acid sequence of an immunoreactive form of human pancreatic stone protein isolated from pancreatic juice.

The primary structure of a pancreatic stone protein form has been elucidated for the first time. The protein studied was the lowest-Mr form prepared from human pancreatic juice (PSP S1). The N-terminal sequence up to residue 65 had already been determined. The five peptides obtained after staphylococcal protease digestion of the carboxymethylated reduced and succinylated PSP S1 enabled the deduction of the entire sequence. The tryptic peptides arising from the digest of cyclohexanedione--treated PSP S1 and the amino acids released by carboxypeptidase P digestion of PSP S1 confirmed the data of the sequence. The peptides were purified by Sephadex filtration and, if required, by chromatography on DEAE-cellulose or thin-layer cellulose. The amino acid sequences of the peptides were determined with a sequencer. From the sequence data it was deduced that the PSP S1 polypeptide chain contains 133 amino acid residues and has a Mr of 15,000.

Cleavage of the Arg-Ile bond in the native polypeptide chain of human pancreatic stone protein.

The pancreatic stone protein (PSP) isolated from calculi (Mr 14,000) and the 5 protein forms (PSP S1-5) detected in pancreatic juice (Mr 14,000-19,000) derive from the same source differing seemingly in their carbohydrate contents or/and in their polypeptidic chain lengths. This kind of protein would inhibit in vivo CaCO3-crystal growth in pancreatic juice. PSP and PSP S1 N-terminal sequences are identical (NH2Ile-). This report demonstrates that: in PSP S2-5 the amino-terminal is blocked; the C-terminus is alike in every form; the single polypeptide chain of PSP S2-5 is converted into that of PSP S1 or PSP by the specific trypsin cleavage of the Arg-Ile bond.

Partial amino acid sequence of human pancreatic stone protein, a novel pancreatic secretory protein.

Pancreatic stone protein (PSP) is the major organic component of human pancreatic stones. With the use of monoclonal antibody immunoadsorbents, five immunoreactive forms (PSP-S) with close Mr values (14,000-19,000) were isolated from normal pancreatic juice. By CM-Trisacryl M chromatography the lowest-Mr form (PSP-S1) was separated from the others and some of its molecular characteristics were investigated. The Mr of the PSP-S1 polypeptide chain calculated from the amino acid composition was about 16,100. The N-terminal sequences (40 residues) of PSP and PSP-S1 are identical, which suggests that the peptide backbone is the same for both of these polypeptides. The PSP-S1 sequence was determined up to residue 65 and was found to be different from all other known protein sequences.

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.

Limited proteolysis of porcine pancreatic lipase. Lability of the Phe 335-Ala 336 bond towards chymotrypsin.

Mild chymotrypsin digestion of native lipase (449 amino acids) preferentially cleaved the Phe 335-Ala 336 bond. On SDS-gel electrophoresis, 3 major bands were observed: band 1 (52 kDa) representing native lipase, bands 2 and 3 (40 and 12 kDa) representing the two lipase fragments A and B. Fragment A does not retain lipase activity but maintains its ability to adsorb to interfaces. Fragment B was identified with the lipase C-terminal region (336-449). It does not exhibit any activity towards tributyrylglycerol emulsions and any ability to adsorb to interfaces.

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.

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.

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.

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.

Porcine pancreatic lipase. Sequence of the first 234 amino acids of the peptide chain.

The single polypeptide chain of about 460 amino acids of porcine pancreatic lipase (EC 3.1.1.3) has been fragmented into five peptides by cyanogen bromide cleavage [Rovery, M., Bianchetta, J. & Guidoni, A. (1973) Biochim. Biophys. Acta, 328, 391--395]. The sequence of the first three cyanogen bromide peptides (CNI, CNII, CNIII), including a total of 234 amino acids, was fully elucidated. Automatic or manual Edman degradation was performed on the different peptides. Fragmentations of the CN peptides were accomplished by digestions with trypsin (after citraconylation or 1,2-cyclohexanedione treatment), chymotrypsin and Staphylococcus aureus external protease. Hydrolysis of unreduced material by pepsin and thermolysin, performed in order to determine the S-S bridge positions, provided useful overlapping peptides. The glycan moiety of lipase is bound to Asn-166. The non-essential tyrosine specifically blocked by diisopropylphosphorofluoridate is Tyr-49 in a cluster of asparagine and glutamine residues. The existence of a highly hydrophobic sequence (206--217) at the C terminus of the CNII fragment is noteworthy.

An improved large scale procedure for the purification of porcine pancreatic lipase.

A modified procedure for purifying porcine pancreatic lipase (triacyglycerol acyl-hydrodrolase, EC 3.1.1.3) is described. In comparison to the previous procedure reported by Verger, R., de Hass, G.H., Sarda, L and Desnuelle, P. (1969) Biochim. Biophys. Acta 188, 272--282) it is more rapid, more reproducible and results in a purer enzyme preparation. No colipase could be detected in the mixture of isoenzymes and, naturally, in the different separated lipases. In this process, butanol treatment is omitted. After pancreas powder extraction, a batch procedure was used for adsorption of DEAE-cellulose. Sephadex filtration (pH 8.0) was made in a largeer size column. Finally the isoenzymes were separated on CM-cellulose as in the Verger procedure, but under slightly modified conditions. Lipase LB was fully homogeneous as judged by end group determination, gel electrophoresis (in the presence of absence of sodium dodecyl sulfate) and sedimentation equilibrium.