The fully active cholecystokinin analog (Thr,Nle)-CCK-9 was lipo-derivatized by N-terminal grafting of a dimyristoylglycerol moiety to induce tight interdigitation with cell membrane bilayers. While the parent CCK peptide was shown to interact only transiently with small unilamellar phospholipid vesicles, the lipo-CCK peptide, although self-aggregating into vesicles, inserts rapidly and quantitatively into phospholipid bilayers. Fluorescence and, even more so, NMR data are supportive for a chain reversal of the CCK moiety of the lipo derivative with embedment of the C-terminus into hydrophobic compartments of the bilayer. MD simulations allowed for a proposal of the folded form of CCK in bilayers with a helical array parallel to the interface and an amphipathic display of the side chains. In this model, the phenylalanine aromatic ring is heading the peptide molecule and may thus play a decisive role in the lateral penetration of the receptor at the water/lipid interface. In fact, despite the membrane-bound state, its binding affinity for rat pancreatic acini is comparable to that of the CCK peptide when tested after a 3-h equilibration period but 5-6-fold lower at 45 min, suggesting that the association rate is significantly lower than that of the unmodified CCK peptide. This can rationally be attributed to the tight interdigitation of the double-tailed lipo moiety with the membrane bilayer. Moreover, an escape of the lipopeptide into the extracellular aqueous phase is energetically highly unfavored; therefore, the receptor can only be reached by a membrane-bound two-dimensional migration. The observed difference in amplification between binding and amylase secretion may result from inadequate occupation of low-affinity CCK receptors, which leads then to poor couplings to G-proteins. Nevertheless the data confirm that lateral penetration of receptor structures is possible, and thus, preadsorption of peptide (neuro)hormones at the cell membrane bilayer may indeed represent the first step in the receptor recognition process.
The PACAP receptor (PACAP I receptor, selective for PACAP) and the PACAP II VIP1 receptor (recognizing PACAP and VIP with the same high affinity) were stably expressed in Chinese Hamster Ovary (CHO) cells. Cell lines expressing different receptor densities, as measured by binding saturation curves, were selected. Inositol phosphate production was stimulated dose dependently in all the cell lines by PACAP and VIP, and the order of potency of the agonists was identical to that of high affinity receptor occupancy. The stimulatory effect of a saturating peptide concentration was proportional to the total receptor density. At similar receptor densities, however, the PACAP receptor mediated stimulation was higher than the VIP receptor-mediated stimulation. Pretreatment of the cells with pertussis toxin for 8 h had no effect on receptor densities, did not alter the PACAP stimulated inositol phosphate synthesis by the cells expressing the PACAP I receptor but markedly inhibited the response of the cells expressing the PACAP II VIP1 receptor. Thus, the present results indicate that the two G(s)-coupled PACAP I and PACAP II VIP1 receptors may stimulate IP production. The maximal stimulation depended on the number of receptor expressed; the PACAP I and PACAP II VIP1 receptors probably activated the phospholipase C through G proteins of the G(q), and of the G(i)/G(o) families, respectively.
The effects of seven competitive atrial natriuretic peptide (ANP) receptor antagonists were compared on cultured human neuroblastoma NB-OK-1 cells expressing exclusively ANPA receptors, by evaluating their capacity to inhibit [125I]ANP binding and to suppress ANP-stimulated cyclic GMP elevation. In ANP analogues with a shortened Cys7-Cys18 bridge, Asp13 and a hydrophobic Tic residue at position 16 expressed antagonistic activity, while Ala16 provoked lower antagonistic potency and Phe16 induced receptor activation. The binding affinity of A71915 ([Arg6, Cha8]ANP-(6-15)-D-Tic-Arg-Cys-NH2), the most potent antagonist (with a pKi of 9.18 and a pA2 of 9.48) was only 22 times less lower than that of the agonist ANP-(1-28).
1. Five increasingly anionic variants (Pal -Pa,) of Caz+-dependent phospholipase A2 were purified to homogeneity from the venom of the lizard Heloderma suspecturn (Gila monster). The purification procedure was based on semi-preparative reverse-phase HPLC followed by anion-exchange HPLC and analytical reverse-phase HPLC.2. Their M , were 17000-18000, as deduced by SDS/PAGE. Specific activities tested by the capacity to hydrolyze phosphatidylcholines at pH 8.5 decreased as follows: Pa3 > Pa, > Pa, > Pal > Pa2. These activities showed the same optimum pH (9.0), were mainly of the phospholipase Az type and were lost upon p-bromophenacyl bromide treatment.3. All five phospholipases efficiently stimulated amylase release from dispersed rat pancreatic acini at pH 7.4, their potency decreasing as follows: Pa, > Pa, z Pa, > Pa3 z Pa,. No deleterious effect was apparent based on the lack of lactate dehydrogenase release. 4. The five variants, Pal -Pa5, differed significantly in amino acid composition and this, together with distinct antigenic properties of Paz and Pa5, establishes the subheterogeneity of this new type of phospholipase A2, despite the fact that the N-terminal amino acid sequence (31 residues) of Pal -Pa, was exactly the same.5. The full sequence of the major variant, Pa,, showed that this 142-amino-acid protein exhibited greater similarity to the bee venom enzyme than to any class I or class I1 secretory phospholipase A2 from snake venom and mammalian pancreas. While Pa, displayed the highly conserved region between Asp30 and Cys39 (the essential active site of all phospholipases A2), its salient original points included 10 half-cystine residues only, an incomplete N-terminal sequence, large changes in the putative calcium loop, several alterations after the active site and a C-terminal extension never seen in other phospholipases A2, with the only exception being bee venom. In 1984, Vandermeers et al. [l] purified, from the venom of the North American lizard Heloderrna suspecturn (Gila monster), a novel protein that was called pancreatic secretory factor because it induces high amylase release from dispersed rat pancreatic acini. In the same year this heat-stable lipophilic and relatively anionic protein was also characterized as a calcium-dependent phospholipase A2 [2]. In 1986, Sosa et al.[3] isolated a phospholipase A, from the venom of the Mexican beaded lizard Heloderma horridum horridum with an original N-terminal region that was probably related to the protein isolated from the venom of Heloderma suspecturn.Carrespandence to J. Christophe, Department of Biochemistry and Nutrition, Medical School, UniversitC Libre de Bruxelles, Boulevard de Waterloo I 1 5, B-1000 Brussels, BelgiumAhhreviutions. LDH, lactate dehydrogenase; Pal -Pa5, five phospholipase A2 variants from the lizard Helodermu suspecturn (Gila monster); T, C, CB and R peptides, trypsin, chymotrypsin, cyanogen bromide and arginine-preferring endopeptidase peptides, respectively. CmS-Pa5, S-carboxymethylated derivative of the Pa5 variant o...
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