P. Desnuelle

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.

Spreading of liposomes at the air/water interface.

Two types of film structure are formed when liposomes are spread at the air/water interface. At zero surface pressure, there is a slow transformation of the closed bilayered structure into a lipid monolayer. The internal content of the liposomes is released into the aqueous subphase. In contrast, when multilamellar liposomes are spread against a surface pressure, they retain their internal content at the air/water interface by forming multilayered structures. Among the liposomes which dipped through the interface an important fraction loses its internal content. During the spreading process at zero surface pressure, it seems that the outer layer of the liposome spreads with a better yield as compared with the inner layer. It is possible to use this spreading technique to determine the asymmetrical distribution of lipids across bilayers.

On porcine pancreatic colipase: Large scale purification and some properties

Abstract Two colipases (94–95 and 83–84 residues, respectively) preventing lipase inhibition by bile salts have been purified with a 30% yield from defatted porcine pancreas. In a tributyrin system at pH 6.0, NaTDC ∗ was found to inhibit lipase for concentrations (0.20–1.25 mM) below the critical micelle concentration. Ultracentrifugation assays on pure products were consistent with a bile salt-mediated conversion of colipase into another form possessing a higher MW and the ability to give with lipase a bile salt-resistant complex.

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.

The primary structure of porcine colipase II. II. The disulfide bridges

Abstract Three disulfide bridges out of a total of five were identified in porcine pancreatic colipase II by peptic or thermolysin digestion of the intact protein followed by separation of the cystine peptides by column chromatography and resolution of the cysteic acid fragments by diagonal electrophoresis. The position of the two other bridges could not be definitely ascertained because of the inability of the enzymes employed to cleave, to a detectable extent, the chain between the adjacent half-cystines 22 and 23. The two possible versions of the bidimensional structure of colipase suggest that the molecule is formed of two loosely bound “tails” and a heavily cross-linked central “core” probably containing the structure responsible for the colipase effect.

Amino acid sequence of horse colipase b

The complete sequence of the 96 residues composing horse colipase B has been determined by automated analysis of the intact protein, of two CNBr peptides and two tryptic peptides arising, respectively, from the citraconylated chain and from the unreduced protein. The single histidine of the protein is located at position 29 as in horse colipase A. His86, present in the C-terminal region of the pig cofactor and supposed to play a role in the folding molecule, is not conserved in horse B. Large pieces of the pig and horse B chains were found to be identical or very similar, especially the N-terminal sequence and the central segment Ala49-Cys65 including the three tyrosines of the molecule. The four lysines and the ten half cystines are also conserved.

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.

Spreading of biomembranes at the air/water interface

This paper presents the compression isotherms obtained by spreading membranes of intestinal brush border, human erythrocyte and Escherichia coli (cytoplasmic) at the air/water interface. Unilamellar membrane films were formed, with a good yield, at zero surface pressure, whereas multilamellar structures were formed at high surface pressure. Once formed, the films were particularly stable and could be manipulated without any detectable loss. With doubly-labelled E. coli cytoplasmic membrane, we could show that phospholipids and proteins spread, with the same yield, as a single unit. Moreover, we studied the influence of hydrolytic enzymes, chemical agents and cations on the compression isotherm of biomembranes. The resultant changes in architecture of membrane films can provide a very simple method of studying the influence of membrane packing on catalytic activity and protein conformation of membrane-bound proteins.

On the transient formation of an acetyl enzyme intermediate during the hydrolysis of P-nitrophenyl acetate by pancreatic lipase

Summary The hydrolysis of monomeric p-nitrophenyl acetate by lipase in the presence of 4% acetonitrile involves, like that catalyzed by ordinary esterases and some proteases, an acylation and a deacylation step (here rate-limiting). A transient acetyl lipase intermediate can be isolated. Various interfaces were found to exert a considerable accelerating effect in the lipase-p-nitrophenyl acetate-acetonitrile system.

The amino acid sequence of the hydrophobic anchor of rabbit intestinal brush border aminopeptidase N

The N-terminal sequence (14 residues) of the detergent form of rabbit intestinal aminopeptidase N was shown to be different from that of the protease form of the same enzyme and to be mostly hydrophobic. This finding is fully consistent with a previous assumption according to which this class of enzymes may be anchored to the brush border membrane by their N-terminus. This special mode of assembly may be facilitated by a positively charged lysine residue near the beginning of the sequence (Lys 4) just before an uninterrupted stretch of hydrophobic amino acids.