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PubMed1999

Incorporation of heparin-binding peptides into fibrin gels enhances neurite extension: an example of designer matrices in tissue engineering.

The goal of this work was to improve the potential of fibrin to promote nerve regeneration by enzymatically incorporating exogenous neurite-promoting heparin-binding peptides. The effects on neurite extension of four different heparin-binding peptides, derived from the heparin-binding domains of antithrombin III, neural cell adhesion molecule and platelet factor 4, were determined. These exogenous peptides were synthesized as bi-domain peptide chimeras, with the second domain being a substrate for factor XIIIa. This coagulation transglutaminase covalently bound the peptides within the fibrin gel during coagulation. The heparin-binding peptides enhanced the degree of neurite extension from embryonic chick dorsal root ganglia through 3-dimensional fibrin gels, and the extent of enhancement was found to correlate positively with the heparin-binding affinity of the individual domains. The enhancement could be inhibited by competition with soluble heparin, by degradation of cell-surface proteoglycans, and by inhibition of the covalent immobilization of the peptide. These results demonstrate an important potential role for proteoglycan-binding components of the extracellular matrix in neurite extension and suggest that fibrin gels modified with covalently bound heparin-binding peptides could serve as a therapeutic agent to enhance peripheral nerve regeneration through nerve guide tubes. More generally, the results demonstrate that the biological responses to fibrin, the body's natural wound healing matrix, can be dramatically improved by the addition of exogenous bioactive peptides in a manner such that they become immobilized during coagulation.-Sakiyama, S. E., Schense, J. C., Hubbell, J. A. Incorporation of heparin-binding peptides into fibrin gels enhances neurite extension: an example of designer matrices in tissue engineering.

Amino Acid Sequence↗

PubMed2002

An integrated vector system for cellular studies of phage display-derived peptides.

Peptide phage display is a method by which large numbers of diverse peptides can be screened for binding to a target of interest. Even when successful, the rate-limiting step is usually validation of peptide bioactivity using living cells. In this paper, we describe an integrated system of vectors that expedites both the screening and the characterization processes. Library construction and screening is performed using an optimized type 3 phage display vector, mJ(1), which is shown to accept peptide libraries of at least 23 amino acids in length. Peptide coding sequences are shuttled from mJ(1) into one of three families of mammalian expression vectors for cell physiological studies. The vector pAL(1) expresses phage display-derived peptides as Gal4 DNA binding domain fusion proteins for transcriptional activation studies. The vectors pG(1), pG(1)N, and pG(1)C express phage display-derived peptides as green fluorescent protein fusions targeted to the entire cell, nucleus, or cytoplasm, respectively. The vector pAP(1) expresses phage display-derived peptides as fusions to secreted placental alkaline phosphatase. Such enzyme fusions can be used as highly sensitive affinity reagents for high-throughput assays and for cloning of peptide-binding cell surface receptors. Taken together, this system of vectors should facilitate the development of phage display-derived peptides into useful biomolecules.

Alkaline Phosphatase↗

PubMed1998

High expression of neutral endopeptidase in idiopathic diffuse hyperplasia of pulmonary neuroendocrine cells.

Idiopathic diffuse hyperplasia of pulmonary neuroendocrine cells (IDHPNC) is a clinicopathological entity characterized by a diffuse hyperplasia of neuroendocrine cells involving distal bronchi and bronchioles. The pathogenesis of this syndrome remains unknown. The hyperplastic neuroendocrine (NE) cells contain multiple neuropeptides, including the bombesinlike peptides (BLP), which are likely important in the pathogenesis of the disorder by stimulating proliferation of fibroblasts in a paracrine fashion and the NE cells themselves in an autocrine manner. Neutral endopeptidase (NEP) is a cell-surface enzyme that hydrolyzes BLP and other bioactive peptides. Low or undetectable NEP is present in many primary lung cancers and cell lines. Low NEP expression could increase neuropeptide-induced autocrine effects by increasing local levels of neuropeptides. We hypothesized that IDHPNC was associated with low or absent NEP expression. NEP expression was assayed in patients with IDHPNC (n = 3) and was compared with expression in patients with idiopathic pulmonary fibrosis (n = 5), hypersensitivity pneumonitis (n = 5), and normal lung (n = 4) using immunohistochemistry, ELISA, activity assay, and Western blot analysis. By these assays, NEP expression was highest in lungs affected by IDHPNC. NEP mRNA, as assessed in IDHPNC lung tissue by RT-PCR, was the expected size and free of mutation between bp 238-2437. Therefore, IDHPNC is unlikely to be the result of a defect in NEP expression. The apparent increase in NEP expression in lung tissue from patients with IDHPNC may reflect a compensatory increase that partly counteracts abundant neuropeptides, including BLP, present in this disorder.

Aged↗

PubMed2004

In vitro and in vivo evaluation of 111In-DTPAGlu-G-CCK8 for cholecystokinin-B receptor imaging.

UNLABELLED: Regulatory peptides and their analogs are being extensively investigated as radiopharmaceuticals for cancer imaging and therapy. Receptors of the cholecystokinin family have been shown to be overexpressed in different types of neuroendocrine tumors. The purposes of this study were to evaluate the cholecystokinin octapeptide amide (CCK8) peptide tagged with a diethylenetriaminepentaacetic acid derivative (DTPAGlu) and to test whether a (111)In-labeled conjugate ((111)In-DTPAGlu-G-CCK8, a derivative containing the chelating agent DTPAGlu bound through a glycine linker at the N-terminal end of the bioactive peptide CCK8) is suitable for cholecystokinin-B receptor (CCKBR) imaging. METHODS: CCK8 was synthesized by solid-phase techniques and covalently coupled to DTPAGlu through a glycine linker at its amino terminus. The compound was labeled with (111)In. The radiochemical purity and stability of the compound were assessed by chromatographic methods. NIH-3T3 and A431 cells overexpressing CCKBR were used to characterize the in vitro properties of the compound. Nude mice bearing control and CCKBR-overexpressing A431 xenografts were used as an in vivo model. RESULTS: DTPAGlu-G-CCK8 showed rapid and efficient labeling with (111)In. The radiolabeled conjugate showed specific binding to both cell lines overexpressing CCKBR. Binding was saturable, with a dissociation constant of approximately 20 nmol/L in both cell systems. Both cell lines showed internalization of the ligand after interaction with the receptor. Biodistribution studies showed rapid localization of (111)In-DTPAGlu-G-CCK8 on CCKBR-overexpressing A431 xenografts that was severalfold higher than that on control tumors at all time points tested. Unbound activity showed rapid clearance of over 80% through the kidneys by 30 min after injection. The labeled peptide conjugate was very stable in serum but showed a rapid breakdown after injection. Incubation with kidney homogenates suggested that most breakdown occurred in the kidneys, favoring the clearance of unbound activity. CONCLUSION: Our findings indicate that the in vitro and in vivo characteristics of (111)In-DTPAGlu-G-CCK8 are favorable for CCKBR imaging, as the peptide shows high-affinity binding to the receptor, is internalized in CCKBR-expressing cells, and shows avid uptake in CCKBR-overexpressing xenografts, with rapid clearance of unbound radioactivity through the kidneys. Furthermore, the ease of synthesis, high labeling efficiency, and chemical stability of DTPAGlu make this chelating moiety an ideal candidate for widespread use in peptide radiolabeling for nuclear medicine applications.

3T3 Cells↗

PubMedDate not supplied

Reduced expression of a novel peptide, prostacyclin-stimulating factor, in the kidneys of streptozotocin-induced diabetic rats.

Prostacyclin (PGI2) produced by vascular endothelial cells (ECs) is a potent vasoactive prostanoid involved in maintenance of vessel wall homeostasis. Reduced PGI2 synthesis by vascular ECs could be a mechanism of pathogenesis in the development of vascular lesions such as diabetic angiopathy. Recently, we purified and cloned a novel bioactive peptide, PGI2-stimulating factor (PSF), which stimulates PGI2 production by vascular ECs. PSF may act on vascular ECs in a paracrine and/or autocrine fashion to regulate PGI2 synthesis. Decreased PSF production in the vessel wall may result in an imbalance of prostanoid synthesis, leading to the development of vascular lesions such as diabetic angiopathy. Our immunohistochemical study demonstrated that PSF is located in vascular resident cells such as vascular smooth muscle cells (SMCs) and ECs, as well as in bronchial SMCs. Moreover, PSF mRNA was found to be expressed in various tissues in Wistar rats, particularly in the kidneys and lungs. The present study demonstrated that streptozotocin (STZ)-induced diabetic rats showed less PSF mRNA expression in the kidneys (PSF mRNA/28S rRNA ratio; STZ versus control; 1.7+/-0.2 versus 2.5+/-0.2, p < 0.05) and reduced immunohistochemical staining for PSF in arteries in the kidney. However, in the lungs, there were no changes in tissue PSF mRNA expression (STZ versus control; 10.9+/-0.9 versus 11.5+/-1.0, NS) or in the extent of PSF staining in bronchial SMCs of STZ-induced diabetic rats. These findings suggest that decreased expression of PSF in renal vessels of STZ-induced diabetic rats may cause an imbalance of prostanoid synthesis, leading to the development and progression of vascular damage in the kidney.

Animals↗

PubMed1999

Bradykinin-potentiating peptides and C-type natriuretic peptides from snake venom.

Cloning of cDNAs encoding bradykinin-potentiating peptides (BPPs)-C-type natriuretic peptide (CNP) precursor or its homologue was performed for cDNA libraries of Bothrops jararaca (South American snake), Trimeresurus flavoviridis, Trimeresurus gramineus and Agkistrodon halys blomhoffi (Asian snakes), all belonging to Crotalinae subfamily. Each cDNA library was constructed from the venom glands of a single snake to preclude ambiguity by intraspecies variation in venom components. Thirteen positive clones derived from B. jararaca were divided into two types depending on restriction sites. Differences in the nucleotide sequence arise at three locations and two of them accompanied amino acid conversions. Despite the differences, both types of cDNA clones encode the BPP-CNP precursor of 256 amino acid residues. Sequence analysis demonstrated that cDNA clones from three Asian snakes encode homologues of the BPP-CNP precursor from B. jararaca. In a precursor polypeptide, a signal sequence (approximately 25 aa) at the N-terminus is followed by sequences of BPP or the analogue (5-13 aa) with flanking spacer sequences (indefinite number of aa), an intervening linker sequence (approximately 144 aa) with unidentified function, and a CNP sequence (22 aa) with a preceding processing signal sequence (10 aa). cDNA clones from A. halys blomhoffi encode two distinct peptides in place of BPP, and T. flavoviridis and T. gramineus were shown to have considerably different sequences in the BPP domain from those known as BPP sequences. The present results provide evidence for a wide distribution of the orthologous gene expressing a series of bioactive peptides among Crotalinae subfamily.

3' Untranslated Regions↗

PubMed2006

Optimization of field-amplified sample injection for analysis of peptides by capillary electrophoresis-mass spectrometry.

A versatile experimental approach is described to achieve very high sensitivity analysis of peptides by capillary electrophoresis-mass spectrometry with sheath flow configuration based on optimization of field-amplified sample injection. Compared to traditional hydrodynamic injection methods, signal enhancement in terms of detection sensitivity of the bioanalytes by more than 3000-fold can be achieved. The effects of injection conditions, composition of the acid and organic solvent in the sample solution, length of the water plug, sample injection time, and voltage on the efficiency of the sample stacking have been systematically investigated, with peptides in the low-nanomolar (10(-9) M) range readily detected under the optimized conditions. Linearity of the established stacking method was found to be excellent over 2 orders of magnitude of concentration. The method was further evaluated for the analysis of low concentration bioactive peptide mixtures and tryptic digests of proteins. A distinguishing feature of the described approach is that it can be employed directly for the analysis of low-abundance protein fragments generated by enzymatic digestion and a reversed-phase-based sample-desalting procedure. Thus, rapid identification of protein fragments as low-abundance analytes can be achieved with this new approach by comparison of the actual tandem mass spectra of selected peptides with the predicted fragmentation patterns using online database searching algorithms.

Amino Acid Sequence↗

PubMed2002

ProSAAS and prohormone convertase 1 are broadly expressed during mouse development.

ProSAAS (encoded by mouse gene Pcsk1n) is a recently described neuroendocrine secretory pathway protein that is cleaved into smaller peptides that may function in cell-cell signalling. ProSAAS and its processing intermediates are also potent inhibitors of prohormone convertase 1 (PC1), which is encoded by mouse gene Pcsk1. In order to gain insight into the function of proSAAS, we have examined the distribution of several proSAAS-derived peptides and PC1 by immunohistochemistry throughout mouse development. The distribution patterns of both SAAS and PC1 are broad from E9 to E11, with some enrichment in neural tube-derived tissues. By E15, the expression of SAAS is largely restricted to neuroendocrine tissues known to produce bioactive peptides. In general, the expression pattern of PC1 overlaps with that of SAAS and other proSAAS-derived peptides, consistent with the hypothesis that proSAAS functions as an endogenous PC1 inhibitor.

Animals↗

PubMed2003

Human hemoglobin-derived peptides exhibit antimicrobial activity: a class of host defense peptides.

Hemoglobin is a known source of biologically active peptides with various functions. In the present study, we report for the first time the existence of natural processed hemoglobin fragments exhibiting antimicrobial activity in humans. Two antimicrobial hemoglobin-derived peptides were purified from a human placental peptide library by consecutive chromatographic steps tracking the maximum growth inhibitory activity against Escherichia coli BL21. These peptides, consisting of 17 and 36 amino acid residues, were identified as being C-terminal fragments of gamma-hemoglobin and beta-hemoglobin, respectively. The antimicrobial beta-hemoglobin fragment was also purified from lysed erythrocytes, demonstrating that proteolytic degradation of hemoglobin into small bioactive peptides already starts inside erythrocytes. The identified peptides inhibit the growth of Gram-positive and Gram-negative bacteria and yeasts in micromolar concentrations. Moreover, by LPS-binding, the beta-hemoglobin fragment reduces biological activity of endotoxins. In contrast, even at high concentrations, the identified antimicrobial hemoglobin peptides do not exhibit toxic activity on human primary blood cells. We conclude that antimicrobial hemoglobin-derived peptides could be important effectors of the innate immune response killing microbial invaders.

Anti-Bacterial Agents↗

PubMed2000

Regulation of aminopeptidase A in human brain tumor vasculature: evidence for a role of transforming growth factor-beta.

Angiotensin peptides are potent vasoconstrictors, cell growth factors, and neuromodulators in normal and pathological situations. To assess the potential role of the angiotensins in brain tumor-associated vessels, the expression of the enzymes of the angiotensin cascade were evaluated in these tumors. The production of these bioactive peptides is dependent on the activities of exopeptidases, including several aminopeptidases and carboxypeptidases, producing angiotensin (Ang) I, II, III, IV and Ang 1-7. Human cerebral parenchymal and glioblastoma cells expressed renin, and tumor vasculature, but not glioblastoma cells, expressed angiotensin-converting enzyme. High aminopeptidase A (APA) activity, but no aminopeptidase N/B activity, was observed in human brain tumor vasculature, suggesting a predominant production of Ang III. Grafting of rat glioma cells in rat brains yielded tumors with high APA and low aminopeptidase N/B activities in tumor vessels, confirming human results. Tumor growth and APA activity in tumor vessels were not affected by chronic angiotensin-converting enzyme inhibition. The brain-derived EC219 endothelial cells expressed high APA activity, which was not involved in endothelial cell proliferation, but was down-regulated by exposure of cells to transforming growth factor-beta (TGF beta) or to TGF beta-secreting tumor cells, suggesting a role for this peptide in the control of APA activity in cerebral vasculature. Thus, APA is a potential marker of chronic dysfunction, involving loss of TGF beta function, of the metabolic blood-brain barrier, but not of neovascularization.

Aminopeptidases↗

PubMed1990

Co-localization of EGF transcripts and peptides by combined immunohistochemistry and in situ hybridization.

There is increasing evidence that autocrine- and paracrine-acting growth factors participate in cell and tissue development, maintenance, and renewal. Recent advances in histochemical techniques have facilitated the localization of growth factor messenger RNAs or polypeptides in tissue sections. However, the spatial relationships between the sites of growth factor transcription, translation, and post-translational processing to functional bioactive peptides have been difficult to correlate because each method of detection requires separate tissue sections. We undertook the simultaneous detection of epidermal growth factor (EGF) transcripts and EGF epitopes by combining immunohistochemistry methods with in situ hybridization. Adult mouse submandibular gland was chosen as a representative model because it contains sites of EGF biosynthesis which may participate in mediating the development, maintenance, and renewal of the organ through autocrine or paracrine mechanism(s). Granular duct (GD) cells demonstrated the presence of both EGF transcripts and EGF peptides. In contrast, the interstitial cells lying adjacent to glandular epithelium also contained relatively high levels of EGF transcripts, although no mature EGF peptides were detected. The experimental approach of co-localization and the resulting data indicate previously unreported sites of EGF transcription in glandular interstitial cells, which may provide molecular information required for the morphogenesis and differentiation of adjacent glandular epithelium.

Animals↗

PubMed2005

Molecular mechanisms of action of the soy isoflavones includes activation of promiscuous nuclear receptors. A review.

Consumption of soy has been demonstrated to reduce circulating cholesterol levels, most notably reducing low-density lipoprotein (LDL) cholesterol levels in hypercholesterolemic individuals. The component or components that might be responsible for this effect is still a matter of debate or controversy among many researchers. Candidate agents include an activity of soy protein itself, bioactive peptides produced during the digestive process, or the soy isoflavones. Although soy intake may provide other health benefits including preventative or remediative effects on cancer, osteoporosis and symptoms of menopause, this review will focus on isoflavones as agents affecting lipid metabolism. Isoflavones were first discovered as a bioactive agent disrupting estrogen action in female sheep, thereby earning the often-used term 'phytoestrogens'. Subsequent work confirmed the ability of isoflavones to bind to estrogen receptors. Along with the cholesterol-lowering effect of soy intake, research that is more recent has pointed to a beneficial antidiabetic effect of soy intake, perhaps mediated by soy isoflavones. The two common categories of antidiabetic drugs acting on nuclear receptors known as peroxisome proliferator activated receptors (PPARs) are the fibrates and glitazones. We and others have recently asked the research question 'do the soy isoflavones have activities as either "phytofibrates" or "phytoglitazones"?' Such an activity should be able to be confirmed both in vivo and in vitro. In both the in vivo and in vitro cases, this action has indeed been confirmed. Further work suggests a possible action of isoflavones similar to the nonestrogenic ligands that bind the estrogen-related receptors (ERRs). Recently, these receptors have been demonstrated to contribute to lipolytic processes. Finally, evaluation of receptor activation studies suggests that thyroid receptor activation may provide additional clues explaining the metabolic action of isoflavones. The recent advances in the discovery and evaluation of the promiscuous nuclear receptors that bind many different chemical ligands should prove to help explain some of the biological effects of soy isoflavones and other phytochemicals.

Animals↗

PubMed2000

Identification of ostrich and chicken cholecystokinin cDNA and intestinal peptides.

The cDNAs encoding the preprohormones of the regulatory peptides cholecystokinin (CCK) and the related gastrin have been identified in a number of vertebrate species. However, from birds only chicken preprogastrin is known. In the present study preproCCK cDNA was identified in two species of birds, ostrich and chicken. In addition, the molecular forms of the bioactive peptides expressed in the small intestine were characterized. Both preproCCKs contain mono basic processing sites for the production of CCK-70 and -8 as seen in turtle and bullfrog. However, compared to these species an unusually large proportion was processed to the small forms CCK-7 and -8 and only minute amounts to larger forms. The encoded preprohormones are very similar to each other and to turtle CCK. Furthermore, they also show a high degree of similarity to the CCKs identified in more distant vertebrates. This confirms that CCK is highly conserved among vertebrates while the structure of gastrin, the other member of the CCK/gastrin family, is considerably more variable.

Amino Acid Sequence↗

PubMed2003

Liver fluke (Fasciola hepatica)-derived peptides activate rat peritoneal mast cells.

Short peptides with sequences derived from those found in the tegumental antigen of Fasciola hepatica have been synthesised. Incubation of some of these peptides with rat peritoneal mast cells resulted in the degranulation of the cells as measured by a histamine release assay. This activity was shown to be associated with the proline-lysine-proline motif, which is responsible for the induction of mast cell degranulation by the mammalian bioactive peptide substance P. Studies on the mode of action of the fluke-derived peptide indicated that it was operating through the same biochemical pathways as substance P. The implications of these findings for the development of immune responses during parasite infections are discussed.

Animals↗

PubMed2006

Yak milk casein as a functional ingredient: preparation and identification of angiotensin-I-converting enzyme inhibitory peptides.

Yak milk casein derived from Qula, a traditional Tibetan acid curd cheese, was hydrolyzed by six commercially available proteases (Trypsin, Pepsin, Alcalase, Flavourzyme, Papain and Neutrase). These hydrolysates were assayed for their inhibitory activity of Angiotensin-I-converting enzyme (ACE). The hydrolysates obtained by Neutrase from Bacillus amyloliquefaciens showed the highest ACE inhibitory activity. The IC50 value of Neutrase-hydrolysate was 0.38 mg/ml. The hydrolysate obtained by Neutrase was further separated by consecutive ultra-filtration with 10 kDa and then with 6 kDa molecular weight cut-offs into different permeated parts and fractionated by gel filtration chromatography with a Sephadex G-25 column. The active fraction was subjected to RP-HPLC, in which five peaks were purified and identified. Amino acid sequence analysis confirmed that the peptides and origins were as follows: YQKFPQY (alphas2-CN; f89-95), LPQNIPPL (beta-CN; f70-77), SKVLPVPQK (beta-CN; f168-176), LPYPYY (kappa-CN; f56-61) and FLPYPYY (kappa-CN; f55-61). Their amino acid sequences matched well with those of known bioactive peptides from bovine casein. The results indicated that yak milk casein could be a resource to generate antihypertensive peptides and be used as multifunctional active ingredients for many value-added functional foods as well as a traditional food protein.

Amino Acid Sequence↗

PubMed1982

Lipid-induced ordered conformation of some peptide hormones and bioactive oligopeptides: predominance of helix over beta form.

The conformation of several naturally occurring peptide hormones and bioactive oligopeptides in phospholipid solutions was studied by circular dichroism. Phosphatidylcholine induced a partial helix in human gastrin I at neutral pH, but phosphatidylserine did not unless the five consecutive glutamic acid residues in gastrin were protonated. Reduced somatostatin with two lysines and substance P with one arginine and one lysine were partially helical in phosphatidylserine, but not phosphatidylcholine, solution. Both lipids induced a helical conformation in glucagon and its COOH-terminal fragment (19-29) probably because the helical segment is primarily located at the uncharged COOH terminus. Thus, polypeptides with a helix-forming potential can have the helical conformation only when the peptides carry no charge or charges opposite to those on the polar head of the lipid. Renin substrate, which has potentials for the beta form and beta turn, seemed to form a mixture of the two conformations in phosphatidylserine solution. Angiotensin I with a strong probability for the beta form adopted the beta form in phosphatidylserine solution and sleep peptide with no structure-forming potential remained unordered in lipid solutions. The helix usually predominated over the beta form in lipid solutions if the peptide has potentials for both conformations. This could account for the preponderance of helices in bacteriorhodopsin of the purple membrane, which according to its amino acid sequence would have favored the beta form.

Amino Acid Sequence↗

PubMed1991

The effect of a membrane potential on the interaction of mastoparan X, a mitochondrial presequence, and several regulatory peptides with phospholipid vesicles.

Recently the pH gradient evoked by a K+ diffusion potential was shown to translocate a synthetic monobasic amphipathic hexapeptide across the bilayer of lipid vesicles (De Kroon, A.I.P.M., Vogt, B., Van 't Hof, R., De Kruijff, B. and De Gier, J. (1991) Biophys. J. 60, in press). Here this observation is extended by studying the effect of a membrane potential on a set of bioactive peptides. The panel of peptides comprises the toxin mastoparan X, a tryptophan-containing analogue of the presequence of the mitochondrial protein cytochrome oxidase subunit IV (preCoxIV(1-25)W18), and the regulatory peptides ACTH(1-24), alpha-MSH, ACTH(1-10), dynorphin A, bombesin, and LHRH. The interaction of these peptides with phospholipid vesicles has been measured using the intrinsic tryptophan residue as fluorescent probe. In the absence of a K+ diffusion potential only mastoparan X and the presequence show considerable binding to vesicles consisting of phosphatidylcholine (PC). In contrast, under these conditions all peptides display affinity for vesicles consisting of the acidic phospholipid cardiolipin (CL), the extent of which depends on the net positive charge of the peptide. Application of a K+ diffusion potential to large unilamellar vesicles (LUV) consisting of PC results in a time dependent tryptophan fluorescence increase for mastoparan X, which is accelerated upon incorporating increasing amounts of CL into the LUV. A similar fluorescence increase in response to a K+ diffusion potential was observed for the above model peptide. Yet the mechanism resulting in the fluorescence increase of mastoparan X is completely different from that of the hexapeptide. Binding experiments indicate that a membrane potential-induced enhanced binding of the peptide to the outer surface of the vesicles contributes to the fluorescence increase. PreCoxIV(1-25)W18, dynorphin A, and ACTH(1-24) show fluorescence responses upon applying a membrane potential that are consistent with that of mastoparan X, whereas the other peptides tested do not respond up to a LUV CL content of 50%. The results tentatively suggest that the membrane potential only affects a peptide when it has the ability to adopt a stable membrane bound conformation.

Amino Acid Sequence↗

PubMed1996

Cyclodextrins as templates for the presentation of protease inhibitors.

Mono(6-succinylamido-6-deoxy)-beta-cyclodextrin was synthesized by classical carbohydrate chemistry and used as a template mono-functionalized with the linear, fully flexible 4C-spacer carboxylate for covalent linkage of the calpain inhibitor leucyl-leucyl-norleucinal. Spectroscopic analyses of the conjugate do not support a self-inclusion of part of the hydrophobic peptide tail, but confirm its intra- or intermolecular interaction with the template moiety that leads to full water solubility. The inhibitory potency of the beta-cyclodextrin/peptide aldehyde construct was compared with that of the parent Ac-Leu-Leu-Nle-H against cathepsin B and calpain. Despite the large size of the template the inhibition of cathepsin B was only slightly reduced in full agreement with the X-ray structure of this enzyme which shows full accessibility of the S-subsites. For this enzyme the 4C-spacer is apparently sufficient to guarantee optimal interaction of the peptide tail with the binding cleft. Conversely, for mu-calpain a significantly decreased inhibitory potency was obtained with the conjugate suggesting steric interference of the template in the binding process. These results show that the beneficial properties of the cyclodextrin template can be retained in conjugates with bioactive peptides if attention is paid to optimize in each case the size and nature of the spacer for optimal recognition of the grafted biomolecule.

Calpain↗