C-terminal Position Research Articles

Differential Effects of Integrin α Chain Mutations on Invasin and Natural Ligand Interaction

To determine if recognition of the Yersinia pseudotuberculosis invasin protein and natural substrates requires identical integrin residues, a region of the human alpha3 integrin chain predicted to be involved in substrate adhesion was targeted for mutation. One point mutation located in a region of the third N-terminal repeat of the alpha3 chain, alpha3-W220A, failed to promote adhesion to the natural alpha3 beta1 substrate epiligrin but maintained near wild type levels of adhesion to invasin. A second nearby mutation, alpha3-Y218A, which showed no detectable adhesion to epiligrin, was only partially attenuated for invasin binding as well as invasin-mediated bacterial uptake. A third substitution, alpha3-D154A, predicted to be in the second N-terminal repeat not known to be implicated in cell adhesion, was competent for invasin-promoted adhesion events and appeared to encode a receptor of increased activity, as it had a higher efficiency than wild type receptor for adhesion to epiligrin. Cell lines expressing this derivative were not recognized by a function blocking anti-alpha3 antibody, indicating that the second and third repeats of the alpha3 chain are either closely linked in space or the second repeat can modulate activity of the third. Differential effects on substrate adhesion do not appear to be associated with all integrin alpha chain mutations, as alpha4 chain mutations affecting the divalent cation binding domains depressed adhesion to invasin to a significant extent.

Sequential DNA damage-independent and -dependent activation of NF-kappaB by UV.

NF-kappaB activation in response to UV irradiation of HeLa cells or of primary human skin fibroblasts occurs with two overlapping kinetics but totally different mechanisms. Although both mechanisms involve induced dissociation of NF-kappaB from IkappaBalpha and degradation of IkappaBalpha, targeting for degradation and signaling are different. Early IkappaBalpha degradation at 30 min to approximately 6 h is not initiated by UV-induced DNA damage. It does not require IkappaB kinase (IKK), as shown by introduction of a dominant-negative kinase subunit, and does not depend on the presence of the phosphorylatable substrate, IkappaBalpha, carrying serines at positions 32 and 36. Induced IkappaBalpha degradation requires, however, intact N- (positions 1-36) and C-terminal (positions 277-287) sequences. IkappaB degradation and NF-kappaB activation at late time points, 15-20 h after UV irradiation, is mediated through DNA damage-induced cleavage of IL-1alpha precursor, release of IL-1alpha and autocrine/paracrine action of IL-1alpha. Late-induced IkappaBalpha requires the presence of Ser32 and Ser36. The late mechanism indicates the existence of signal transfer from photoproducts in the nucleus to the cytoplasm. The release of the 'alarmone' IL-1alpha may account for some of the systemic effects of sunlight exposure.

Substrate and binding specificity of aspartic proteases with milk clotting properties.

The hydrolysis of whole casein and isolated casein components were investigated with the purpose of obtaining information concerning the kinetic and specifty of aspartic proteases in rennin, pepsin and 4 microbial rennet substitutes. The velocity of hydrolysis decreased rapidly within the first hour. However, the hydrolysis was not completed after 2 days. A mathematical description of the slope of hydrolysis is possible by use of exponential equations. More than 40 peptides were detected by capillary electrophoresis or PAGE. The characterization of the C- and N-terminal amino acids of peptides shows that the hydrolysis of any peptide bond depends mainly on the structure of the C-terminal side chains of the amino acids. The detection of the basic amino acids lysin and arginin in the C-terminal position of peptides is a new result, furthering the knowledge about the specificity of aspartic proteases. Differences in the reaction velocity or in the extent of hydrolysis are one of the possible explanations for the described differences in the rennet curd yield. It was concluded that the rennet enzymes are active also in the later phases of cheese ripening and are able to support the action of cheese ripening flora.

A comparison of histidine protecting groups in the synthesis of peptide-oligonucleotide conjugates

Abstract Protection of the imidazole ring of the histidine residue is not required for the elongation of an oligonucleotide chain at the side chain hydroxyl group of an amino acid residue by the phosphite triester approach. For the assembly of the peptide chain, the 2,4-dinitrophenyl group is the best alternative when histidine is placed at the C-terminal position.

The b and δ Subunits of the Escherichia coli ATP Synthase Interact via Residues in their C-terminal Regions

An affinity resin for the F1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b24-156, a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F1. Truncated forms of b24-156, in which one or four residues from the C terminus were removed, competed poorly for F1 binding, suggesting that these residues play an important role in b-F1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b24-156 resulted in a disruption of its association with the purified delta subunit of the enzyme. To determine whether these residues interact directly with delta, cysteine residues were introduced at various C-terminal positions of b and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and delta were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b24-156-F1 complex and the membrane-bound F1F0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue delta subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that the b and delta subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F1 sector to the b subunit of F0.

N- and C-terminal structure-activity study of angiotensin II on the angiotensin AT2 receptor.

The predominant angiotensin II receptor expressed in the human myometrium is the angiotensin AT2 receptor. This preparation was used for a structure-activity relationship study on angiotensin II analogues modified in positions 1 and 8. The angiotensin AT2 receptor present on human myometrium membranes displayed a high affinity (pKd = 9.18) and was relatively abundant (53-253 fmol/mg of protein). The pharmacological profile was typical of an angiotensin AT2 receptor with the following order of affinities: (angiotensin III > or = angiotensin II > angiotensin I > PD123319 > angiotensin-(1-7) > angiotensin-(1-6) approximately angiotensin IV >> Losartan). Modifications of the N-terminal side chain and of the primary amine of angiotensin II were evaluated. Neutralisation of the methylcarboxylate (Asp) to a methylcarboxamide (Asn) or to a hydroxymethyl (Ser) or substitution for a methylsulfonate group (cysteic acid) improved the affinity. Extension from methylcarboxylate (Asp) to ethylcarboxylate (Glu) did not affect the affinity. Introduction of larger side chains such as the bulky p-benzoylphenylalanine (p-Bpa) or the positively charged Lys did not substantially affect the affinity. Complete removal of the side chain (angiotensin III), however, resulted in a significant affinity increase. Removal or acetylation of the primary amine of angiotensin II did not noticeably influence the affinity. Progressive alkylation of the primary amine significantly increased the affinity, betain structures being the most potent. It appears that quite important differences exist between the angiotensin AT1 and AT2 receptors concerning their pharmacological profile towards analogues of angiotensin II modified in position 1. On position 8 of angiotensin II, a structure-activity relationship on the angiotensin AT2 receptor was quite similar to that observed with angiotensin AT1 receptor. Bulky, hydrophobic aromatic residues displayed affinities similar to or even better than [Sarcosine1]angiotensin II. Aliphatic residues, especially those of reduced size, caused a significant decrease in affinity especially [Sarcosine1, Gly8]angiotensin II who showed a 30-fold decrease. Introduction of a positive charge (Lys) at position 8 reduced the affinity even further. Stereoisomers in position 8 (L-->D configuration) also induced lower affinities. The angiotensin AT2 receptor display a structure-activity relationship similar to that observed on the AT1 receptor for the C-terminal position of the peptide hormone. Position 1 structure-activity relationships are however fundamentally different between the angiotensin AT1 and AT2 receptor.

Linear oligopeptides. Part 406.1 Helical screw sense of peptide molecules: the pentapeptide system (Aib)4/L-Val[L-(αMe)Val] in solution

A variety of N α-blocked pentapeptide esters, each containing four helicogenic, achiral α-aminoisobutyric acid residues and one chiral L-valine or C α-methyl-L-valine residue in the N-terminal, internal or C-terminal position of the sequence, have been synthesized by solution methods and fully characterized. The results of a solution conformational analysis, performed by using FTIR absorption and 1H NMR techniques, favour the conclusion that all of the pentapeptides examined fold into a 310-helical structure. In addition, a CD study of the N α-para-bromobenzoylated peptides strongly supports the view that the prevailing screw sense of the 310-helical structure that is formed is strongly dependent upon the position in the sequence of the single chiral C α-tri- or C α-tetrasubstituted α-amino acid.

Bacterial resistance to vancomycin: overproduction, purification, and characterization of VanC2 from Enterococcus casseliflavus as a D-Ala-D-Ser ligase.

The VanC phenotype for clinical resistance of enterococci to vancomycin is exhibited by Enterococcus gallinarum and Enterococcus casseliflavus. Based on the detection of the cell precursor UDP-N-acetylmuramic acid pentapeptide intermediate terminating in D-Ala-D-Ser instead of D-Ala-D-Ala, it has been predicted that the VanC ligase would be a D-Ala-D-Ser rather than a D-Ala-D-Ala ligase. Overproduction of the E. casseliflavus ATCC 25788 vanC2 gene in Escherichia coli and its purification to homogeneity allowed demonstration of ATP-dependent D-Ala-D-Ser ligase activity. The kcat/Km2 (Km2 = Km for D-Ser or C-terminal D-Ala) ratio for D-Ala-D-Ser/D-Ala-D-Ala dipeptide formation is 270/0.69 for a 400-fold selection against D-Ala in the C-terminal position. VanC2 also has substantial D-Ala-D-Asn ligase activity (kcat/Km2 = 74 mM-1min-1).

Purification and characterization of aminopeptidase P from Lactococcus lactis subsp. cremoris.

Aminopeptidase P was purified 65.3-fold from the cytoplasm of Lactococcus lactis subsp. cremoris AM2 with a 5.8% yield. The purified enzyme was found to consist of one polypeptide chain with a relative molecular mass of 41,600. Metal chelating agents were found to be inhibitory and Mn2+ and Co2+ stimulated activity 7-fold and 6-fold respectively. The purified enzyme removed the N-terminal amino acid from peptides only where proline (and in one case alanine) was present in the penultimate position. No hydrolysis was observed either with dipeptides even when proline was present in the C-terminal position or when either N-terminal proline or pyroglutamate was present preceding a proline residue in the penultimate position of longer peptides. On the basis of this substrate specificity either aminopeptidase P or post-proline dipeptidyl aminopeptidase are necessary along with a broad specificity aminopeptidase to effect complete hydrolysis of casein-derived peptides containing a single internally placed proline residue. However, both aminopeptidase P and post-proline dipeptidyl aminopeptidase would be required together with a broad specificity aminopeptidase in order to completely hydrolyse casein-derived peptides that contain two internally placed consecutive proline residues. As bitter casein-derived peptides are likely to contain either single prolines or pairs of prolines, aminopeptidase P appears to be an important enzyme for debittering.

Increased Incorporation of Chimeric Human Immunodeficiency Virus Type 1 gp120 Proteins into Pr55gagVirus-like Particles by an Epstein–Barr Virus gp220/350-Derived Transmembrane Domain

Noninfectious Pr55gagvirus-like particles containing high quantities of oligomeric human immunodeficiency virus type 1 (HIV-1) envelope (Env) proteins represent potential candidate immunogens for a vaccine against HIV-1 infection. Thus, chimericenvgenes were constructed encoding the HIV-1 exterior glycoprotein gp120 which was covalently linked at different C-terminal positions to a transmembrane domain (TM) from the Epstein–Barr virus (EBV) major Env glycoprotein gp220/350. All chimeric Env-TM polypeptides as well as the wild-type HIV Env proteins were equally produced and incorporated at the outer surface of insect cells using the baculovirus expression system. In the presence of coexpressed HIV Pr55gagpolyproteins significantly decreased amounts of wild-type Env proteins were presented at the cell surface, whereas the membrane incorporation of the Env-TM chimeras was not affected. Biochemical and immunoelectron microscopical analysis of particles that were efficiently released from these cells displayed the incorporation of both wild-type Env and chimeric Env-TM proteins on the surface of VLPs. However, the quantities of particle-associated chimeric Env-TM proteins exceeded those of incorporated wild-type Env proteins by a factor of 5–10. Chemical cross-linking and subsequent polyacrylamide gel electrophoresis of VLP-entrapped Env proteins revealed that the chimeric Env-TM proteins form homodimers and a higher-order oligomer, similar to that observed for wild-type Env proteins. Thus, the results of this study clearly demonstrate that the replacement of the gp41 transmembrane protein of gp160 by a heterologous, EBV gp220/350-derived membrane anchor provides an effective strategy to incorporate high quantities of oligomeric HIV gp120 proteins on the surface of Pr55gagvirus-like particles.

Expression of a mutant alpha-zein creates the floury2 phenotype in transgenic maize.

The maize floury2 mutation results in the formation of a soft, starchy endosperm with a reduced amount of prolamin (zein) proteins and twice the lysine content of the wild type. The mutation is semidominant and is associated with small, irregularly shaped protein bodies, elevated levels of a 70-kDa chaperone in the endoplasmic reticulum, and a novel 24-kDa polypeptide in the zein fraction. The 24-kDa polypeptide is a precursor of a 22-kDa alpha-zein protein that is not properly processed. The defect is due to an alanine-to-valine substitution at the C-terminal position of the signal peptide, which causes the protein to be anchored to the endoplasmic reticulum. We postulated that the phenotype associated with the floury2 mutation is caused by the accumulation of the 24-kDa alpha-zein protein. To test this hypothesis, we created transgenic maize plants that produce the mutant protein. We found that endosperm in seeds of these plants manifests the floury2 phenotype, thereby confirming that the mutant alpha-zein is the molecular basis of this mutation.

Isolation and characterization of new C-terminal substitution mutations affecting secretion of polygalacturonase in Erwinia carotovora ssp. carotovora

An intact C-terminus was previously shown to be required for stability and secretion of the polygalacturonase (PehA) in Erwinia carotovora ssp. carotovora. Here we have analyzed the effects of amino acid (aa) substitutions generated to five C-terminal positions of PehA. Conservation of two hydrophobic and one non-hydrophobic residue (V372, V374 and N371, respectively) was found to be essential for maintenance of the protein stability. As an exception, one of the mutants (V372G) did not show major effects on protein stability, as determined by immunoblots and enzyme activity assay, yet it prevented the secretion completely. We conclude that the C-terminus of PehA is directly involved in the formation or stabilization of a conformation-sensitive structure needed for recognition of the protein as secreted.

Zur Festphasenpeptidsynthese des Makrophagen-migrationsinhibierenden Faktors der Maus (mMIF): Darstellung von Peptidfragmenten zur konvergenten Totalsynthese

Solid Phase Peptide Synthetic Approaches to Mouse Macrophage Migration Inhibitory Factor (mMIF): Synthesis of Peptide Fragments for a Convergent Solid Phase Peptide Synthetic Strategy For a convergent solid phase synthesis of mMIF, the sequence was separated into 10 fragments leading C-terminal Pro and Gly positions. Racemization of Cys(Trt) was observed during the in situ activation process using TBTU/DIEA in stepwise SPPS and also during a fragment condensation of the protected fragment mMIF(53–69) with the MIF(62–69) peptide resin. To overcome “difficult sequence” problems arising during several stepwise syntheses different coupling methods were performed.

Efficient Insertion of Odd-numbered Transmembrane Segments of the Tetracycline Resistance Protein Requires Even-numbered Segments

Functional membrane insertion elements in the pBR322 tetracycline resistance protein were identified by comparing the ability of odd-numbered transmembrane segments and their attached periplasmic loops to insert into the membrane individually or when combined with the next even-numbered segment in the tetracycline resistance protein sequence. The efficiency with which individual odd-numbered segments and periplasmic loops inserted was probed by treating proteins truncated at the distal ends of periplasmic loops P2-P6 with carboxypeptidases and endoproteases in inside-out membrane vesicles. Insertion of odd-numbered segments and attached loops is inefficient when they occupy a C-terminal position in the protein. The C-terminal odd-numbered segment and loop sequences of 34-54% of the molecules of periplasmic loop truncation mutants could be removed by carboxypeptidase Y. In contrast, odd-numbered segments and loops insert efficiently if the next even-numbered segment in the sequence is present. In such cytoplasmic loop truncation mutants, only the cytoplasmic tail sequences of the proteins could be removed by carboxypeptidases. Remarkably, insertion of individual odd-numbered segments and loops is inefficient even though free energies for insertion of these sequences are highly favorable. The results indicate that pairs of adjacent segments, possibly "helical hairpins," are necessary for efficient membrane insertion of the tetracycline resistance protein.

Specificity and degeneracy in peptide binding to HLA-B7-like class I molecules.

The HLA-B7-like binding supertype includes several different HLA-B molecules. Herein, the primary and secondary anchor specificities of the five most common HLA-B7-like molecules (B*0702, B*3501, B51, B*5301, and B*5401) were defined by the use of molecular binding assays, analogue peptides, and large sets of peptides corresponding to naturally occurring sequences. All five B7-like molecules analyzed preferentially bound 9-mers, with a stringent requirement for proline in position 2, while a variety of hydrophobic or aromatic residues were well tolerated at the C-terminal anchor position. Although most peptides bound in an allele-specific fashion, approximately 20% of the binders identified were degenerate and bound at least three of the five B7-like molecules analyzed with affinities of 500 nM or less. It was also noted that, in general, peptides that bind with high affinity to any given one B7-like molecule were also most frequently capable of degenerate binding. Prominent roles for secondary anchors in positions 1 and 3 were observed for most B7-like molecules, and secondary anchor motifs were utilized to derive an HLA-B7-like supermotif. The validity of this B7-like supermotif was tested by a blind prediction set. Finally, the B7-like supermotif was utilized to derive a general strategy for rationally engineering peptide analogues of naturally occurring sequences with greatly increased binding affinity and degeneracy. Such engineered supermotif binding peptides may be of significant utility in the development of peptide-based vaccines against chronic viral diseases and cancer.

Two neurotoxins (BmK I and BmK II) from the venom of the scorpion Buthus martensi Karsch: purification, amino acid sequences and assessment of specific activity

Two neurotoxins, BmK I and BmK II, were purified from the venom of the Chinese scorpion Buthus martensi Karsch. The complete amino acid sequences of both toxins, each containing 64 amino acid residues, were determined by the automatic sequencing of reduced and S-carboxymethylated toxins and their peptides, obtained after cleavage with TPCK-treated trypsin and Staphylococcus aureus V 8 protease, respectively. Toxicity as minimum lethal dose tested by i.c.v. injection in mice showed that BmK I was six times more potent than BmK II. Only two amino acid replacements were found: at position 59 Val in BmK I was replaced by Ile in BmK II, and at position 62 a basic Lys residue in BmK I was substituted by a neutral Asn residue in BmK II. These features suggest that the positively charged residue (Lys or Arg) in the C-terminal position 62 (or 61 or 63) may also play an important role in facilitating the interaction between scorpion neurotoxins and the receptor on sodium channels. The effects of BmK I on nerve excitability were examined with the crayfish axon using intracellular recording and voltage-clamp conditions. The results indicate that BmK I preferentially blocks the sodium channel inactivation process. Thus,

Synthesis and activities of cyclic thrombin-receptor-derived peptide analogues of the Ser42-Phe-Leu-Leu-Arg46 motif sequence containing d-Phe and/or d-Arg

Cyclic analogues of the active thrombin receptor peptide SFLLR (TRP42–46) containingd-Phe and/ord-Arg have been prepared by the solid-phase method, purified by reversed-phase HPLC and bioassayed in a rat smooth muscle contractile assay. Cyclization was achieved by forming an amide linkage between the-NH2 and-COOH groups of the two leucine residues located at the N- and C-terminal positions of the linear protected precursor H2N-Leu-Arg(Pmc)-Y-Phe-Leu-OH (Y=Gly,Acp) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoroborate borate (HBTU) or 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) as coupling reagents andN,N′-diisopropylethylamine (DIPEA) in high dilution. Their structure was confirmed by fast by fast atom bombardment mass spectrometry and NMR methods. The cyclic peptides c-fLLrG, c-fLLRG, c-FLLrG and c-fLLrAcp, c-FLLrAcp so synthesized were assessed for their contractile activity in a rat gastric longitudinal muscle bioassay system which has been used previously to evaluate the biological activities of linear thrombin-receptor-derived polypeptides such as SFLLR (P5) and SFLLR-NH2 (P5-NH2).

Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA.

Two residues of Escherichia coli elongation factor Tu involved in binding of aminoacyl-tRNA were identified and subjected to mutational analysis. Lys-89 and Asn-90 were each replaced by either Ala or Glu. The four single mutants were denoted K89A, K89E, N90A, and N90E, respectively. The mutants were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay. Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of elongation factor Tu in complex with aminoacyl-tRNA, which suggested an important role of Lys-89 and Asn-90 in tRNA binding. Furthermore, our results indicate helix B to be an important target site for nucleotide exchange factor EF-Ts. Also the mutants His-66 to Ala and His-118 to either Ala or Glu were characterized in an in vitro translation assay. Their functional roles are discussed in relation to the structure of elongation factor Tu in complex with aminoacyl-tRNA.

Comparative efficiency of simple lipopeptide constructs for in vivo induction of virus-specific CTL

We have previously shown that virus-specific CTL responses can be elicited in vivo by injecting, without adjuvant, 12–40 amino acid-long peptides, modified in C-terminal position by a simple lipidic amino acid. In this paper, we have studied the chemical accessibility, and the ability to induce in mice a CTL response, of a series of lipopeptides derived from the HIV-1 env (312–327) or (302–335) sequences. We showed that a single modification of these peptides by a lipidic amino acid, preferably in C-terminal position, results in the ability to reproducibly induce, without adjuvant, a relevant CTL response. No clear discrimination appeared concerning the nature of the lipidic modification. Our findings indicate that modification of a relatively long peptide by a N ε-palmitoyl-L-Lysylamide can be achieved by conventional methods of synthesis and characterization, offering the possibility to develop low-cost synthetic vaccines in models in which the CTL component is of importance.

Ligand binding and functional effects of systematic double d-amino acid residue substituted neuropeptide Y analogs on Y1 and Y2 receptor types

In order to identify the signal epitopes of the neuropeptide Y (NPY) molecule, the conformation of the NPY molecule was pertubated by a systematic double d-amino acid replacement of neighbouring residues. These NPY-analogs were examined for receptor affinity and on biological activity. The rat cerebral cortex and hippocampus were used for binding characteristics on Y1 and Y2 binding sites, respectively, while the isolated guinea pig caval vein and rat vas deferens were used in functional characterization of Y1 and Y2 receptors, respectively. The NPY analogs were examined as ligands at [ 3H]NPY binding sites in homogenates of the rat brain. Pairwise d-substitutions of either of the first 6 amino acid residues in the N-terminal part of the molecule resulted in a 20–100-fold loss of affinity for Y1 binding sites compared with the native peptide. In comparison, the same analogs displayed affinities, which were about 8–40 times lower than NPY itself at Y2 binding sites. Especially [ d-Ser 3, d-Lys 4]NPY had a low affinity to Y1 and Y2 binding sites. For many of the pairwise d-amino acid substituted NPY analogs, there were similar affinities for Y1 and Y2 binding sites in the cerebral cortex and hippocampus, respectively. d-Amino acid residue substitutions in positions 7 and 8 did essentially not affect the affinity to either type of binding site, while such replacements in positions 19 and 20 resulted in a drastic loss of affinity to both types of NPY binding site. In contrast, [ d-Tyr 21, d-Ser 22]NPY was only slightly less potent than NPY itself on either type of binding site. Pairwise d-amino acid substitutions in the C-terminal (positions 27 to 36) decreased the affinity to Y1 and Y2 binding sites by 2 to 3 orders of magnitude. In the guinea pig vena cava the d-amino acid substituted NPY analogs evoked a concentration-dependent contraction with an rank order of potency similar to that of the respective analog at Y1 binding sites in the cerebral cortex. Similarly, in the rat vas deferens the d-amino acid substituted NPY analogs evoked a concentration-dependent inhibition of the electrically-stimulated twitches with a rank order of potency similar to that of the respective analog at Y2 binding sites in the hippocampus. However, d-amino acid replacements in positions 25 and 26 resulted in an analog which was virtually inactive in the vas deferens, but almost equipotent with NPY in the vena cava. In conclusion, the present study has shown that N-terminal double d-amino acid substitutions in the NPY molecule reduced the binding affinity to and activation more of the Y1 receptor, than of the Y2 receptor, while both receptors were quite sensitive to double d-amino acid changes in positions 19 and 20 and in the C-terminal end of the NPY molecule.