M. Rovery

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.

The primary structure of porcine colipase II. I. The amino acid sequence

Abstract The sequence of the 84 residues of porcine colipase II was elucidated by a variety of techniques including sequencing of the first 21 residues with the aid of an automatic Sequenator, purification and analysis of 26 fragments resulting from the cleavage of carboxymethylated or oxidized colipase by trypsin (before and after citraconylation), chymotrypsin and pepsin. An important overlap was also found in a thermolysin digest. The chain shows two strongly hydrophobic regions, one in the N-terminal region and composed of three adjacent isoleucines (Ile2-Ile3-Ile4), the other in the central part and including the tyrosine residues of the molecule (Leu47-Tyr48-Gly49-Val50-Tyr51-Tyr52). The majority of the half-cystines (eight out of a total of ten) are also localized in the central part. Colipase I was definitely proved to differ from colipase II by the presence of a still unknown number of residues located on the C-terminal side of the chain.

Sur le trypsinogène et la trypsine de porc

Abstract A chromatographic procedure on carboxymethyl cellulose at pH 5.0 is described for the simultaneous purification of porcine trypsinogen and one of the porcine chymotrypsinogens. The yield calculated from the first extract of the gland in dilute sulfuric acid is 45% with a 2–3 fold purification. 1 kg of fresh pancreas gives at least 2.8 g of pure product. The amino acid compositions of bovine and porcine trypsinogens reveal some similarities. The molecular weight of both proteins is of the same order. But, a series of sizable differences of composition suggest that large regions of the molecules have in fact quite different structures. The diisopropylphosphoryl derivative of the trypsin formed by autoactivation of porcine trypsinogen can be purified by chromatography on carboxymethyl cellulose at pH 5.0. When the same technique is applied to active trypsin, a highly active, but slightly autolyzed, product is obtained. Autoactivation of porcine and bovine trypsinogens proceeds at about the same rate to give about the same maximal specific activity. Calcium ions have the same effect. In both cases, the activation process involves the specific splitting of a Lys-Ileu bond belonging to the N-terminal sequence of the chain and having on its left 4 aspartic acid residues. However, this bond is the 8th of the sequence in the case of porcine trypsinogen. The peptide set free is the octapeptide Phe-Pro-Thr-(Asp) 4 -Lys. Thus, the apparently essential region having the same structure in both trypsinogens is very narrowly restricted. Beyond it, structural modifications reflecting deep changes in the genetical information can be noted. Porcine trypsinogen and porcine trypsin have the same C-terminal sequence: Thr-(Ileu,Glu(NH 2 ))-Ala-Asp(NH 2 ). This identity is the first direct proof that trypsinogen activation does not involve any covalent modification in the C-terminal region of the chain.

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.

Contribution à l'étude du chymotrypsinogène b de boeuf

Abstract 1. 1. Our earlier preparations of bovine chymotrypsinogen B isolated by chromatography on O -(carboxymethyl)cellulose have been further purified by O -(carboxymethyl)Sephades chromatography. the product finally obtained (yield, 25%; 400 mg per kg fresh pancreas) is entirely free from deoxyribonuclease and behaves as an homogeneous protein when subjected to free-boundary electrophoresis, ultracetrifugation and equilibrium chromatography. Chromatography on O -(carboxymethyl)-Sephadex also appears to be a good technique for the purifition of bovine deoxyribonuclease. 2. 2. By gel filtration through Spehadex G-100 and ultracentriguation, a molecular weight of approx. 26000 was found for the pure chymotrypsinogen B. Thus, in contrast with earlier findings, the two chymotrypsinogens of bovine pancreas have nearly the same molecular weight. Comparison of the amino acid composition reveals other striking similarities between the two proteins. The number of residues per mole was found to be identical for 6 amino acids, very similar (if not identical) for 2 others, similar for 4 other and really different for only 6 amino acids. Chymotrypsinogen B contains a total of 243–250 residues, whereas chymotrypsinogen A contains 246 residues. 3. 3. The sequence of the first 17 residues of chymotrypsinogens A and B are identical except for the replacement of serine by alamine in position 14. 4. 4. During activation, trypsin splits the single chain of both precursors at the same arginyl-isoleucine bond at the same position in the chain (the 15th). However, this cleavage is considerably more rapid in the case of chymotrypsinogen B than for chymotrypsinogen A, indicating that this bond is still more “exposed” in chymotrypsinogen B. 5. 5. Furthermore, chymotrypsin B π , in contrast with chymotrypsin A π , does not autolyze its 13th bond, even in the absence of β-phenylpropionate. The stability of this bond in chymotrypsin B π is not due to the chemical nature of the neighboring residues, but probably to a different orientation of the short chain after activation. In any case, the formation of a chymotrypsin belonging to the δ-type is not a general feature of chymotrypsinogen “rapid” activation.

On the two anionic chymotrypsinogens of porcine pancreas.

A second anionic chymotrypsinogen (chymotrypsinogen B) was identified in porcine pancreas and purified from pancreatic juice by DEAE-cellulose chromatography and two Sephadex filtrations in buffers of different ionic strength. Its molecular weight (about 26 000) and amino acid composition were found similar to those of already known bovine chymotrypsinogen A, bovine chymotrypsinogen B and porcine chymotrypsinogen A. Like these three zymogens, its molecule is composed of a single peptide chain with an N-terminal half-cystine and a C-terminal asparagine. Activation by trypsin is associated with the cleavage of the 15th bond in the sequence Arg15-Ile16-Val17. The short chain of 15 residues arising from the cleavage has the same amino acid composition and probably the same sequence as porcine chymotrypsinogen A. The other anionic chymotrypsinogen of porcine origin (porcine chymotrypsinogen C) can also be purified from pancreatic juice by passage through DEAE-cellulose, Sephadex G-100 and CM-Sephadex. Sephadex filtration indicated for this protein a molecular weight of about 23 000. However, results obtained by ultracentrifugation and the amino acid composition were consistent with a markedly higher value (about 29 000). It was confirmed that porcine chymotrypsinogen C and Fraction II of bovine procarboxypeptidase A had a similar amino acid composition and that both were activated by the cleavage of an arginyl-valine bond. As for other chymotrypsinogens, this bond was the first basic bond in the N-terminal sequence of the molecule. But a preliminary investigation suggested that it was probably the 13th instead of the 15th, and that the amino acid composition of the short chain arising during activation was different. It appears, therefore, that two sub-groups can be discerned in the chymotrypsinogen group, one composed of bovine and porcine chymotrypsinogens A and B, and the other of bovine Fraction II and porcine chymotrypsinogen C.

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.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.

Cleavage of the ArgIle bond in the native polypeptide chain of human pancreatic stone protein

The pancreatic stone protein (PSP) isolated from calculi (M r 14000) and the 5 protein forms (PSP S1‐5) detected in pancreatic juice (M r 14000—19000) 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: (i) in PSP S2‐5 the amino‐terminal is blocked; (ii) the C‐terminus is alike in every form; (iii) 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.

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.

Le chymotrypsinogène a de porc purification et études de quelques propriétés

Abstract 1.5 g of a weakly cationic chymotrypsinogen (chymotrypsinogen A) and 5 g of trypsinogen can be obtained in essentially homogeneous form from 1 kg of fresh pig pancreas by a single chromatographic step on CM-cellulose. The molecular weight of the porcine chymotrypsinogen A is between 24 000 and 25 000 as determined by ultracentrifugation and by filtration through Sephadex G-100. The amino acid composition resembles those of bovine chymotrypsinogens A and B rather than that of porcine chymotrypsinogen C. Porcine chymotrypsinogen A and bovine chymotrypsinogens A and B contain the same number of histidines (2), tryptophans (8) and disulfide bridges (5), but porcine chymotrypsinogen A contains 5 tyrosines (instead of 4 in bovine A and 3 in bovine B), 2 methionines as in bovine A (compared with 4 in bovine B) and 16 prolines (compared with 9 in bovine A). The N-terminal sequence (first 17 residues) of porcine chymotrypsinogen A and bovine chymotrypsinogens A and B are the same with two exceptions: glutamine at position 7 of bovine chymotrypsinogens A and B is replaced by proline in the porcine chymotrypsinogen and serine at position 14 in porcine chymotrypsinogen A and bovine chymotrypsinogen A is replaced by alanine in bovine chymotrypsinogen B. The arrangement of the 16 residues in the vicinity of the “histidine loop” is identical with that already found by others in bovine chymotrypsinogens A and B. The three chymotrypsinogens studied so far are activated by the cleavage of the Arg15Ile16 bond. However, porcine chymotrypsin Aπ and bovine chymotrypsin Bπ, in contrast with bovine chymotrypsin Aπ, do not autolyze and, consequently, do not form a chymotrypsin δ.