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P Desnuelle

Publications and source records attributed to P Desnuelle.

At least 19 recordsLinked to original sources
PubMed1986

Amino acid sequence and disulfide bridges of subunit III, a defective endopeptidase present in the bovine pancreatic 6 S procarboxypeptidase A complex.

The sequence of the 240 amino acids and the position of the five S-S bridges of subunit III of the bovine pancreatic 6 S procarboxypeptidase A complex have been determined thus confirming its phylogenetic filiation with the pancreatic serine endopeptidase group. The subunit contains at equivalent positions all the elements of the catalytic site of these enzymes. The elements of a binding pocket very similar to that of porcine elastase I are also present in the protein thus accounting for its zymogen-like activity. The most obvious difference is the absence in the subunit of the two strongly hydrophobic amino acids (16 and 17 in the chymotrypsinogen numbering), which are known to participate in the stabilization of a fully functional binding pocket in active endopeptidases. Four of the five disulfide bridges of subunit III are homologous with those common to all pancreatic endopeptidases. In contrast the fifth bridge forms a very small loop of only four amino acids, which is not encountered in active endopeptidases. Other potentially lethal modifications in the structure of the subunit are not excluded.

Amino Acid Sequence↗
PubMed1982

Presence of free hydrophobic peptides in the brush border and basolateral membranes of pig enterocytes.

Short peptides containing approx. 60% of hydrophobic amino acids have been extracted by chloroform/methanol from purified brush border and basolateral membranes of pig enterocytes. These peptides can be separated from membrane lipids by thin-layer chromatography on Kieselgel plates using chloroform/methanol/water as developer. Their molecular weight is approx. 8000 as judged by SDS-gel electrophoresis. But, this value may be overestimated. They are devoid of cystine and methionine. They contain no N-terminal amino acid detectable by the dansyl and Edman degradation techniques. Extraction of papain-treated, right side out brush border vesicles led to mixtures containing the above peptides and the anchors which normally bind a variety of hydrolases to the external surface of the brush border. Peptides and anchors could not be separated by high performance thin-layer chromatography and SDS-gel electrophoresis. Their amino acid compositions were similar. However, several lines of evidence did not support the assumption that the peptides existing in non-treated brush border membranes can be identified to anchors left inside the bilayer after proteolytic cleavage of surface hydrolases. It is not yet known whether these peptides represent other hydrophobic fragments (leader or stop-transfer sequence, for instance) left in the membrane during the co-translational processing of certain proteins or constitute an independent population of molecules.

Amino Acids↗
PubMed1982

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.

Amino Acid Sequence↗
PubMed1981

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.

Amino Acid Sequence↗
PubMed1981

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.

Amino Acid Sequence↗
PubMed1981

Stabilization of the C-terminal part of pig and horse colipase by carboxypeptidase and trypsin inhibitors.

Pig and horse colipases have been purified by a common procedure using trypsin and carboxypeptidase inhibitors as stabilizers. Two forms of pig colipase were identified: a predominant A1 form with about 103-105 residues, and a minor slightly degraded A2 form in which the last two C-terminal residues, Asp and Ser, were lacking. This type of degradation is considerably slowed down by carboxypeptidase inhibitors. A total of four forms of the horse cofactor were characterized: two (A1 and B1) were probably isocolipases which differed by only a few substitutions. Both contained the same number of residues (about 96), an N-terminal valine and an Arg-Ser-Glu-(Glx)1,2-ArgC-terminal sequence. A2 and B2 were slightly degraded forms probably resulting from tryptic cleavage of the Arg-Ser bond in the above sequence. The presence of methionine in the horse cofactor allowed fragmentation by cyanogen bromide. The C-terminal fragment was composed of 16 or 17 residues and contained no histidine. The single histidine of horse B1 was found in the intermediary fragment between Met-18 and Met-(n-17). These data show that the C-terminal parts of both pig and horse colipases are still more exposed to proteolytic degradations than the N-terminal parts. Preliminary attempts to crystallize B1 were carried out.

Animals↗
PubMed1981

[Effect of the addition of hog pancreatic colipase on the permeability to glucose and the phase transition of phosphatidyl choline liposomes].

An interaction between porcine pancreatic coli-pase and lecithin liposomes is demonstrated by gel filtration assays. The extent of the colipase penetration into the phospholipid bilayer was assessed by permeability and calorimetry studies carried out on the liposome colipase complex. The addition of colipase to liposomes induces a three fold increase in the permeability to [6-H3] glucose. This result reflects a perturbation in the bilayer which may be the consequence of the colipase interaction. The phase transition temperature is not modified by the added colipase. This observation suggests that the perturbation brought by the protein does not affect the acyl chain packing of the bulk lipid. On the other hand the enthalpy of transition (delta H) is decreased from 8.9 to 7.8 kcal/mole by the addition of colipase to the lipid. This could be explained by the interaction of the colipase with neighbouring acyl chains which do not participate in the cooperative melting of the bulk lipid. In agreement with previous spectrophotometric observations, the present results are indicative of hydrophobic interactions between colipase and bilayer hydrocarbon chains.

Animals↗
PubMed1979

Lipolysis and lipid movement in a membrane model. Action of lipoprotein lipase.

The action of purified bovine milk lipoprotein lipase on tri[3H]oleoylglycerol and the effect of albumin on movement of lipolytic products at an argon-water interface were studied in a specially designed tricomparted trough. The amount of trioleoylglycerol applied was 14 times that needed to cover the surface of the aqueous subphase (0.1 M Tris . HCl, pH 7.4) with a monolayer. It is concluded that trioleoylglycerol was present in lenses on the surface of the aqueous subphase, that hydrolysis by lipoprotein lipase occurred in or near the lipid/argon-water interface, and that lipolytic products immediately located and spread throughout the interface, displacing substances with lower spreading pressures from the interface. Addition of albumin to the aqueous subphase accelerated markedly the desorption of oleic acid and monooleoylglycerol from the interface and thereby enhanced lipolysis. When albumin was not contiguous with the site of hydrolysis, oleic acid and monooleoylglycerol readily moved in the interface to the area of contact with albumin where they were desorbed from the interface. These findings support the hypothesis of transport of lipolytic products by lateral movement in cell membranes.

Animals↗
PubMed1978

Inhibition of pancreatic lipase by mixed micelles of diethyl p-nitrophenyl phosphate and bile salts.

Solubility and Sephadex filtration assays have shown that dissolved diethyl p-nitrophenyl phosphate can be included into bile salt micelles with a partition coefficient of 32 : 1. This inclusion is probably a prerequisite for the organophosphate to inhibit lipase. The essential role played by colipase confirms that the primary step in the inhibition is an interaction of lipase with bile salt containing micelles. Therefore, it appears that the requirements of lipase towards specific substrates and inhibitors are very similar. The inhibition rate strongly depends on the total bile salt concentration and on the micellar concentration of the organophosphate. This effect may be explained, at least qualitatively, by a competition between simple and mixed micelles for the binding of colipase and lipase.

Animals↗
PubMed1978

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.

Air↗
PubMed1978

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.

Calcium↗
PubMed1978

Adsorption and activation of pancreatic lipase at interfaces.

The first step of the lipase-catalyzed hydrolysis of insoluble long chain triglycerides is the adsorption of the enzyme to the interface. This adsorption, which is spontaneous when the interface is hydrophobic, is hindered by bile salts. Under these conditions, a small protein cofactor designated colipase adsorbs first and then anchors lipase at the interface. Interfacial adsorption enhances lipase activity, due, at least in part, to an acceleration of the rate-limiting deacylation step of the reaction. In this respect, lipase appears to be a most interesting model of an enzyme being activated by the presence of a lipid. The 3 steps of the heterogeneous catalysis induced by lipase, interfacial adsorption, interfacial activation and catalysis proper are under the control, respectively, of a serine hydroxyl group, a carboxyl and a histidine imidazole.

Adsorption↗
PubMed1977

Further studies of mode of action of lipolytic enzymes.

Pancreatic lipase and phospholipase A2 have been shown by the monomolecular film technique to be progressively inactivated when adsorbed at the interface of their respective substrates. This inactivation is faster for lipase than for phospholipase. It is also enhanced by low film pressures and film transfer. The use of radioactive phospholipase and lipase samples offered the possibility to measure the amount of enzyme adsorbed at a monomolecular film with a reasonable accuracy. This adsorption was found to be relatively slow under the conditions of the assays. The main conclusion drawn from these data is that the enzyme kinetics in presence of a substrate film, and probably also under bulk conditions, is controlled by an adsorption flux responsible for an initial lag period and an inactivation flux tending to decrease the reaction rate. The kinetics are linear only when both fluxes equilibrate.

Adsorption↗