The delta-selective opioid peptide dermenkephalin and the mu-selective hybrid peptide dermenkephalin-[1-4]-dermophin-[5-7] display strikingly different conformations despite identical tetrapeptide N-termini. A quantitative 2-D NMR and molecular modeling analysis.

The selective recognition of the aminoterminal binding pharmacophore Tyr-D-Xaa-Phe of the opioid heptapeptide dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (DRM)1, and of dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (DREK), by the mu-opioid receptor and delta-opioid receptor, respectively, depends upon the constitution / conformation of the C-terminal tripeptide. The hybrid peptide DREK-[1-4]-DRM-[5-7] is very potent at, and exquisitely selective for the mu-opioid receptor, and differs only from dermenkephalin by its C-terminal tripeptide. Comparison of the structural features of DREK-[1-4]-DRM-[5-7] and dermenkephalin by nmr analysis and molecular modeling revealed striking differences, as well in the trans (Tyr5 - Pro6) isomer (population 75%) than in the cis isomer.. Whereas the folded C-terminal tail of dermenkephalin influenced the tertiary structure of the N-terminal tetrapeptide and placed the Tyr1 and Phe3 aromatic rings in definite orientations that are best suited for the delta-receptor, there were only weak contacts, as shown by NOE data, between the aminoterminal and carboxyterminal parts of the hybrid peptide. This promoted increased flexibility of the whole backbone and relaxed orientations for the side-chains of Tyr1 and Phe3 that are compatible with the mu-receptor but unsuitable for the delta-receptor. The steric hindrance introduced by Pro6 in DREK-[1-4]-DRM-[5-7], plus the absence of large hydrophobic side-chains in positions 5 and 6 may prevent close contacts between the N-terminal and C-terminal domains and reorientation of the main pharmacophoric elements Tyr1 and Phe3.

Solution conformations of deltorphin-I obtained from combined use of quantitative 2D-NMR and energy calculations: a comparison with dermenkephalin.

Deltorphin-I, Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 and dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, two highly related opioid peptides from frog skin, display very similar N-termini but strikingly different C-terminal tails. Nevertheless, both peptides are highly potent at, and exquisitely selective for the delta-opioid receptor. To identify common determinants concuring to the remarkably efficient targeting of deltorphin-I and dermenkephalin, combined use of quantitative two-dimensional nuclear magnetic resonance (53 dipolar interactions studied at four temperatures) and energy calculations using simulated annealing generated five groups of deltorphin-I conformers. These groups were pooled into two families whose overall conformation could be described either by a left-handed helix (Family I) or by a big loop (Family II), both stabilized by H-bonds. Proximity of D-Ala2-Phe3-Asp4 and Val5-Val6-Gly7 triads is an obvious structural similarity between almost all groups in both families of structures. Whereas differences between the two families originated mostly from a transition at psi Asp4 backbone dihedral angle, the backbone structures at segment 1-4 are similar and spatial arrangements of Tyr1 (t) and Phe3 (g-) are identical in one group of each family. Moreover, these two groups have a N-terminal tetrapeptide whose conformation most closely resembles that of a well-defined group of structures for dermenkephalin. Altogether, these results suggest that conformational attributes that are common to dermenkephalin and deltorphin-I, i.e., the backbone conformation of the N-terminal tetrapeptide and preferential orientations in the side-chain of Tyr1 (t) and Phe3 (g-) underlie their ability to bind with high selectivity to the delta-opioid receptor.

Bile salt-dependent lipase biosynthesis in rat pancreatic AR 4-2 J cells. Essential requirement of N-linked oligosaccharide for secretion and expression of a fully active enzyme.

This study used the rat pancreatic AR 4-2 J cell line as a model system to investigate the role of glycosylation in bile salt-dependent lipase (BSDL) secretion and esterolytic activity. Results indicated that AR 4-2 J cells synthesized BSDL, the esterolytic activity of which was the greater part of the cell activity toward 4-nitrophenylcaproate. The protein thus expressed was glycosylated and had a molecular mass approximately 74 kDa. Exposure of these cells to tunicamycin significantly decreased [3H]mannose incorporation, while [35S]methionine incorporation in trichloroacetic acid-precipitable material was not modified. Tunicamycin treatment of AR 4-2 J cells lead to a lower molecular mass form (approximately 70 kDa) of BSDL which did not incorporate [3H]mannose. The nonglycosylated low M(r) form of the enzyme was not secreted as shown by the decreasing activity in cell-free medium which paralleled the time-dependent secretion of the enzyme. Tunicamycin had no effect on BSDL synthesis. Nevertheless, the nonglycosylated BSDL was apparently not degraded in any cell compartment as shown in part by the enzyme activity accumulation within cells upon brefeldin A treatment. The BSDL expressed by AR 4-2 J cells was characterized by a Km of 68 +/- 30 microM and a kcat of 106 +/- 19 min-1. The sequestrated BSDL due to tunicamycin treatment of cells presents a significant increase of Km of over 10 times to 757 +/- 303 microM. kcat was affected by a factor of approximately 4-445 +/- 22 min-1. These data correlated with an approximately 2.5-fold decrease of the esterolytic activity following inhibition of the N-glycosylation of the protein. The nonglycosylated BSDL was less stable to temperature than the native form. Processing inhibitors (castanospermine, 1-deoxymannojirimycin, and swainsonine) had no effect either on the enzyme activity or on its secretion. Results suggested first that the transfer of the oligosaccharide precursor to the nascent BSDL is essential for the folding of the fully active BSDL. Second that the glycan structure is required for the enzyme secretion.

Aminopeptidases and proteolipids of intestinal brush border.

The presence of human blood group A determinants has been shown on the A+ rabbit intestinal brush border glycoproteins, particularly hydrolases. Sugar compositions of aminopeptidases N from A+ and A- rabbits were compatible with the presence in these molecules of eight N-linked glycans and of two O-linked glycans bearing the A determinants in A+ animals. The exact relative molecular masses of hydrophobic domain(s) of aminopeptidases N and A from pig and rabbit intestinal brush border have been determined by an isotopic dilution technique. The values obtained were compatible with the anchorage in the membrane of the monomeric rabbit enzymes, or of each subunit of the dimeric pig enzymes, by their N-terminal sequences, composed of 20-25 hydrophobic amino acids. This N-terminal hydrophobic sequence (14 residues) has been determined for rabbit aminopeptidase N. Short peptides containing approximately 60% hydrophobic amino acids have been extracted by chloroform-methanol from purified brush border and basolateral membranes of pig enterocytes. Their molecular properties were very similar to those of the aminopeptidase anchors released by trypsin treatment of detergent-extracted enzymes. However, several lines of evidence failed to support the assumption that these free hydrophobic peptides can be identified with anchors left inside the bilayer after proteolytic cleavage of surface hydrolases.

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.

Subunit structured of pig small-intestinal brush-border aminopeptidase N.

Aminopeptidase N (EC 3.4.11.2), when isolated from pig intestine in either the proteinase- or detergent-released form, frequently appears to contain three polypeptide chains, here termed alpha, beta and gamma. We have established by an immunological technique that the beta- and gamma-polypeptides are derived from the alpha-chain and that the intact enzyme is a dimer, alpha 2. Each alpha-chain of the detergent form was shown to contain a hydrophobic anchor peptide about 35 amino acid residues in length, which included the N-terminal sequences. A peptide bond in the alpha-chain was very sensitive to proteolysis. Its cleavage generated the commonly observed forms: alpha beta gamma and beta 2 gamma 2. The gamma-fragment, which lacked the anchor peptide, was derived from the C-terminal part of the alpha-chain.

Enzymatic and immunological properties of the protease form of aminopeptidase N and A from pig and rabbit intestinal brush border.

Immunological homology was shown between the active site regions of pig and rabbit aminopeptidases N and between those of the corresponding aminopeptidases A. However, no homology was detectable between the aminopeptidases N and A (EC 3.4.11.-) in a given species. The dimeric structure of pig aminopeptidases did not significantly modify their catalytic properties in aqueous solution compared to those of the monomeric rabbit enzymes. Only a slight difference in binding conditions was noted in the case of aminopeptidases N. Aminopeptidase A activity towards acidic substrates was enhanced by physiological concentrations of Ca2+ while that towards neutral substrates was considerably reduced. Therefore, acidic amino acid residues in proteins and peptides may be assumed to be mostly split off in vivo by aminopeptidase A, neutral residues by aminopeptidases N and basic residues by both enzymes. The respective specificity of aminopeptidase A and N for acidic and neutral amino acid residues was found to be mainly due to a more productive binding mode of the substrate rather than to a better affinity.

Purification and characterization of the rabbit intestinal brush-border aminopeptidase A.

The papain form of rabbit intestinal brush-border aminopeptidase A has been purified to homogeneity. It is a monomeric enzyme of molecular weight 170 000. It represents 3.5% of the total proteins of the membrane. Its specificity slightly overlaps with that of aminopeptidase N.

Purification and characterization of an aminopeptidase A from hog intestinal brush-border membrane.

The aminopeptidase A of the porcine intestinal brush-border membrane has been purified following solubilization by trypsin (p-form) or Emulphogen (d-form). Full purification of d-amino-peptidase A required the use of anti-impurities immunoabsorbant chromatography. The d-amino-peptidase A constitutes about 4% of the total proteins of the membrane, compared to 8-12% for another, already characterized, brush-border aminopeptidase N. Both d-form and p-form of aminopeptidase A have been clearly shown to be dimeric. Experimental evidence is presented favoring the view that they are symmetrical dimers, with the consequence that each of the two subunits of the d-form possesses an hydrophobic anchor holding them at the membrane surface. As already demonstrated for several other brush border hydrolases, the hydrophobic anchor is N-terminal in porcine intestinal aminopeptidase A. The molecular weight of the peptide including the anchor liberated by trypsin during the conversion of the d-form into the p-form has been estimated by an isotopic dilution method to be about 4500 (42 residues). This value which compares well with those recently obtained in the case of rabbit aminopeptidase N (3700-3800; 36-38 residues), indicates that the anchor is much shorter than believed earlier. A preliminary survey of the specificity of both aminopeptidases A and N towards four synthetic amino acid p-nitroanilides confirms that aminopeptidase A mostly cleaves acidic residues. Its activity towards neutral residues is much lower, but probably significant in certain cases.

Ovine pancreatic lipase : purification and some properties.

Lipase has been isolated from sheep pancreas. The lipoprotein complex formed in pancreas homogenates by the enzyme and endogenous lipids is split by treatment with acetone. Lipase is further purified by ion-exchange chromatography and gel filtration. The molecular weight and the amino-acid composition of ovine lipase are very similar to that of the porcine and bovine enzymes. As previously found in bovine lipase, no carbohydrate is covalently bound to the polypeptide chain which has a N-terminal residue of lysine. The study of the catalytic properties of ovine pancreatic lipase indicates that the enzyme is fully activated by colipase from various species in the presence of conjugated bile salt micellar solutions.