A. Guidoni

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.

Chemical structure of porcine pancreatic lipase end group determination and cyanogen bromide peptides

Abstract Results on primary structure of porcine lipase are reported. The two isoenzyme lipases A and B have identical terminal residues NH2 serine and half cystine COOH. Cleavage of the molecule by CNBr was investigated on the major form, lipase B.

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.

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.