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Several bioactive peptides were isolated from some annelids. Each of the annelidan peptides seemed to be a member of a previously found molluscan peptide family.
An endo-acting proline-specific oligopeptidase (prolyl oligopeptidase [POPase], EC 3.4.21.26) was purified to homogeneity from the Triton X-100 extracts of cells of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised five successive fast protein liquid chromatography steps. The POPase is a cell-associated 75- to 77-kDa protein with an isoelectric point of ca. 6.5. The enzyme hydrolyzed (optimum pH 6.5) the Pro-pNA bond in carbobenzoxy-Gly-Pro-p-nitroanilide (Z-Gly-Pro-pNA) and bonds at the carboxyl side of proline in several human bioactive peptides, such as bradykinin, substance P, neurotensin, angiotensins, oxytocin, vasopressin, and human endothelin fragment 22-38. The minimum hydrolyzable peptide size was tetrapeptide P3P2P1P'1, while the maximum substrate size was ca. 3 kDa. An imino acid residue in position P1 was absolutely necessary. The hydrolysis of Z-Gly-Pro-pNA was potently inhibited by the following, with the Ki(app) (in micromolar) in parentheses: insulin B-chain (0.7), human endothelin-1 (0.5), neuropeptide Y (1.7), substance P (32.0), T-kinin (4.0), neurotensin (5.0), and bradykinin (16.0). Chemical modification and inhibition studies suggest that the POPase is a serine endopeptidase whose activity depends on the catalytic triad of COOH ... Ser ... His but not on a metal. The amino acid sequence around the putative active-site serine is Gly-Gly-Ser-Asn-Pro-Gly. The enzyme is suggested to contain a reactive cysteinyl residue near the active site. Amino acid residues 4 to 24 of the first 24 N-terminal residues showed a homology of 71% with the POPase precursor from Flavobacterium meningosepticum and considerable homology with the Aeromonas hydrophila POPase. The ready hydrolysis of human bioactive peptides at bonds involving an imino acid residue suggests that enzymes like POPase may contribute to the chronicity of periodontal infections by participating in the peptidolytic processing of those peptides.
The effects of discodermin A (DC-A), a novel marine bioactive peptide extracted from sea sponge Discodermia kiiensis, on the vascular smooth muscle cells and tissues were examined. Analysis with a confocal laser microscope showed that DC-A (0.1-30 microM) permeabilized the plasma membrane of A10 cells to the non-permeable fluorescent agents, ethidium homodimer-1 (MW = 857) and calcein (MW = 623), in a concentration-dependent manner. In the vascular tissue treated with 30 microM DC-A, addition of a micromolar concentration of Ca(2+) evoked a sustained contraction in the presence of ATP, suggesting that DC-A increased the permeability of the membrane to Ca(2+) and ATP. DC-A at higher concentrations (30 microM) significantly increased the leakage of lactate dehydrogenase (140 kD) from the vascular tissue. These results suggest that DC-A has a permeabilizing effect on the plasma membrane possibly by interacting with plasma membrane phospholipids with its six successive hydrophobic amino acid residues at N-terminal.
Many kinds of bioactive peptides which might prevent lifestyle-related diseases are released from food proteins after enzymatic digestion. Inhibitory peptides for angiotensin I-converting enzyme (ACE) having anti-hypertensive effect have been isolated from enzymatic digests of various food proteins. LKPNM, which was isolated from the thermolysin digest of dried bonito was activated 8-fold by ACE itself and showed a prolonged effect after oral administration. Two vasorelaxing peptides, ovokinin and ovokinin(2-7), showing antihypertensive effect after oral administration were obtained from ovalbumin digests. We found that low molecular weight peptides derived from food proteins lowered serum cholesterol without increasing excretion of cholesterol and bile acids. An immunostimulating peptide isolated from an enzymatic digest of soybean protein prevented alopecia induced by cancer chemotherapy.
Hemopressin (PVNFKFLSH), a novel bioactive peptide derived from the alpha1-chain of hemoglobin, was originally isolated from rat brain homogenates. Hemopressin causes hypotension in anesthetized rats and is metabolized in vivo and in vitro by endopeptidase 24.15 (EP24.15), neurolysin (EP24.16), and angiotensin-converting enzyme (ACE). Hemopressin also exerts an antinociceptive action in experimental inflammatory hyperalgesia induced by carrageenin or bradykinin via a mechanism that is independent of opioids. These findings suggest that this peptide may have important regulatory physiological actions in vivo.
A bioactive peptide that extensively prolongs action potentials (APs) in frog nerve has been isolated and purified from the venom of the scorpion Buthus martensii Karsch (BMK). The peptide, designated as BMK 18(2), was purified using gel filtration, ion exchange, FPLC, and HPLC chromatography. APs recorded in the presence of nanomolar concentrations of the peptide were extensively prolonged with some attenuation in their heights. The N-terminal sequence of BMK 18(2) was found to be: VRDAYIAEDYD-VYH-ARDA. Sequence similarity comparisons to other alpha-scorpion toxins suggest that the two blanks in the sequences are cysteines. The molecular weight (M.W.) of BMK 18(2) was determined by LC/MS/MS to be 7185 Da. Since the peptide prolongs APs when both K+ and Ca++ channels were blocked and shows sequence similarity to other alpha-neurotoxins, it appears likely that BMK 18(2) acts to alter Na channel inactivation to produce its effect.
AIM: To screen bioactive peptides that mimic the epitope of gastric cancer associated antigen. METHODS: Anti-gastric cancer monoclonal antibody (mAb) MG7 was purified by ion chromatography, and then coated on ELISA plate. By using the mAb as selective molecular, a 12-meres phage displaying peptide library was biopanned, and the positive clones were selected. RESULTS: 12 positive phage clones were selected. CONCLUSION: A candidate mimic epitope of gastric cancer associated antigen was identified, which provided the foundation for developing tumor peptide vaccine.
In recent years, there has been increasing interest in de novo design and construction of novel synthetic peptides that mimic protein secondary structures, i.e., turns, helices and sheets. The unique structural influences exerted by unsubstituted, non-coded, non-chiral beta-amino acid, i.e., beta-alanine (beta-Ala; 3- or beta- aminopropionic acid) on peptide backbone, when inserted into peptide chain comprised alpha-amino acids, offer an excellent opportunity to design and construct diverse well-defined three-dimensional structures. Our current understanding of folding-unfolding behavior of the beta-Ala residues relies primarily from an examination of conformational preferences of a large number of short cyclic- as well as acyclic beta-Ala containing peptides investigated using single crystal X-ray diffraction analysis. In addition, theoretical conformational energy calculations and different spectroscopic techniques: 1H NMR, FT-IR and CD, have also been employed although, to a lesser extent. The obtainable results tend to reveal overwhelming preferences of the beta-Ala moiety for the folded gauche (mu approximately +/-65+/-10 degrees conformation in cyclic- and for an extended trans (mu approximately +/-165+/-10 degrees) as well as gauche (mu approximately +/-65+/-10 degrees) orientations in acyclic beta-Ala containing peptides. The results also indicate that in short linear beta-Ala containing peptides, the specific influence of selective neighboring side-chain substituents e.g. linear- or cyclic symmetrically C(alpha,alpha)-disubstituted glycines and other conformational constraints, may be significant in controlling the overall folded-unfolded topographical features across the two methylene units (-CbetaH2-CalphaH2-) of the beta-Ala residue. Taking into consideration the wide occurrence of beta-Ala moiety in animal and plant kingdoms and the remarkable structural versatility of the peptides incorporating beta-Ala residue(s), together with appreciable resistance towards enzymatic degradation, hold strong promise for biophysicists and biochemists not only to design molecules that fold to mimic protein secondary structures but also to develop potent peptide analogs and peptidomimetics displaying unique pharmaceutical properties.
The systematic preparation of synthetic peptide combinatorial libraries (SPCLs), each composed of tens of millions of peptides that can be screened in existing diagnostically or pharmacologically relevant in vitro assay systems, is reviewed. The identification of optimal peptide sequences has been achieved through the screening in solution of SPCLs, each element of which is composed of more than 100,000 nonsupport-bound peptides in equimolar representation, along with an iterative synthesis and screening process. Examples are presented in which an SPCL, composed in total of 52,128,400 acetylated hexa-peptides, is used along with an iterative selection process to precisely identify the antigenic determinant of a peptide recognized by a monoclonal antibody using competitive enzyme-linked immunosorbent assay. This same library was also used to develop highly potent antimicrobial peptides in bacterial growth inhibition assays. A separate non-acetylated SPCL was used to screen and identify high affinity peptide ligands using an opiate radio-receptor binding assay.
The order of secretion of newly synthesized and older bioactive peptides was investigated using primary rat intermediate pituitary melanotropes, which synthesize, store, and secrete peptides derived from pro-ACTH/endorphin (PAE; also POMC). PAE-derived peptides produced by the cells were biosynthetically labeled by incubating the cells with radioactive amino acids at various times preceding the period during which secretion was examined; secreted and cellular peptides were characterized and quantitated by immunoprecipitation, using affinity-purified antibodies to selected regions of PAE, followed by polyacrylamide gel electrophoretic analysis. Release in the absence of secretagogues (basal or constitutive release) was compared to release in the presence of maximally effective levels of 8-bromo-cAMP and BaCl2 (stimulated or regulated release). Both cell types showed short-lived preferential basal release of newly synthesized and not fully mature peptides (less than 2-3 h old). Conversely, the cells showed preferential stimulated secretion of older peptides. A process of maturation occurred, taking 2-4 h, after which the secretion of newly synthesized and older peptides in response to secretagogues was nearly indistinguishable for the smallest product peptides. The data support a model of gradual processing of peptides from precursors into smaller products and maturation from molecules only available for basal release into peptides available for stimulated secretion as well as for basal release. Basal secretion was found to include mature peptides as well as intermediates and precursor molecules. The data do not support the existence of any preferential regulated secretion of newly synthesized peptides.
The degradation of several bioactive peptides and proteins by purified human dipeptidyl peptidase IV is reported. It was hitherto unknown that human gastrin-releasing peptide, human chorionic gonadotropin, human pancreatic polypeptide, sheep prolactin, aprotinin, corticotropin-like intermediate lobe peptide and (Tyr-)melanostatin are substrates of this peptidase. Kinetic constants were determined for the degradation of a number of other natural peptides, including substance P, the degradation of which has been described earlier in a qualitative manner. Generally, small peptides are degraded much more rapidly than proteins. However, the Km-values seem to be independent of the peptide chain length. The influence of the action of dipeptidyl peptidase IV on the biological function of peptides and proteins is discussed.
A novel bioactive peptide was isolated from the tryptic digest of rice soluble protein based on ileum-contracting and anti-opioid activities in the isolated guinea pig ileum. The structure of the peptide was Gly-Tyr-Pro-Met-Tyr-Pro-Leu-Pro-Arg, and it was named oryzatensin. Oryzatensin showed a biphasic ileum-contraction, which was characterized by a rapid contraction followed by a slower one. The latter was mediated by the cholinergic nervous system because it was inhibited by tetrodotoxin and atropine. Although oryzatensin showed weak affinity for mu-opioid receptors, the apparent anti-opioid activity seemed to be associated with the slower contraction. On the other hand, oryzatensin showed phagocytosis-promoting activity for human polymorphonuclear leukocytes and augmented the production of superoxide anion by human peripheral leukocytes.
A modern method is reported for the assignment of absolute configuration for peptidomimetics in bioactive peptides by use of 1H-NMR parameters in solution. Four peptide systems incorporating either retro-inverso modifications or 2-aminocyclopentanecarboxylic acid (2-Ac5c) as a peptidomimetic for proline are discussed. (1) Two 14-membered cyclic dermorphin analogs Tyr-c[D-A2bu-Phe-gPhe-(S and R)-mLeu] with a reverse amide bond between gPhe and mLeu residues where gPhe denotes a gem-diamino analog of Phe and mLeu refers to a malonyl analog of Leu. (2) Two cyclic hexapeptides related to somatostatin, c[gSar6-(S and R)-mPhe7-D-Trp8-Lys9-Thr10-Phe11], with a reverse amide bond between the gSar and mPhe residues where the gSar and mPhe denote the gemdiamino and malonyl analogs of the Sar and Phe residues, respectively. The superscript numbers refer to positions in native somatostatin. (3) Cyclic hexapeptide somatostatin analogs containing 2-Ac5c [trans-(1S,2S)-2-Ac5c, trans-(1R,2R)-2-Ac5c,cis-(1R,2S)-2-Ac5c, and cis-(1S,2R)-2-Ac5c] in place of proline c[(2-Ac5c)6-Phe7-D-Trp8-Lys9-Thr10-Phe11]. (4) Morphiceptin related analogs incorporating a cis-2-Ac5c residue as shown in Tyr-cis-2-Ac5c-Phe-Val-NH2. The methodology described in this investigation could be applied to a wide variety of peptide systems.
The role of various bioactive peptides in the control of secretion of hypothalamic somatostatin into the hypophysial portal blood was examined in anesthetized rats. Hypophysial portal blood was withdrawn at a rate of 5.0 microliter/min into a chilled tube through a cannula placed over the stump of the pituitary stalk and segmented every 20 min by air bubbles. Immunoreactive somatostatin (IRS) in the plasma was extracted with acetic acid and acetone and quantified by RIA. Basal levels (mean +/- SE) of plasma IRS in the hypophysial portal blood were 646 +/- 36 and 317 +/- 44 pg/ml in urethane- and pentobarbital-anesthetized rats, respectively. Under urethane anesthesia, injection of synthetic neurotensin into the lateral ventricle at various doses in the range of 0.016--2 microgram/rat caused a significant and dose-related increase of plasma IRS levels in the hypophysial portal blood, and this effect of neurotensin was significantly (P less than 0.05) suppressed by pretreatment with diphenhydramine (1 mg/100 g BW, iv), a histamine receptor blocker. Enhancement of IRS release by neurotensin was also observed in pentobarbital-anesthetized rats. Intraventricular injection of substance P (10 microgram/rat), beta-endorphin (1 and 5 microgram/rat), or [Met5]enkephalin had no effect on the level of somatostatin in the hypophysial portal blood of urethane-anesthetized rats. These results suggest a release of hypothalamic somatostatin into the hypophysial portal blood in response to intraventricular administration of neurotensin, probably by a histaminergic mechanism.
A novel bioactive peptide with the C-terminal RFamide was isolated from the brain of the Japanese crucian carp, Carassius auratus langsdorfii, by using the intestine of the fish as the bioassay system. The primary structure of the peptide was determined to be SPEIDPFWYVGRGVRPIGRFamide and it was designated Carassius RFamide (C-RFa). The sequence of C-RFa was found to be significantly homologous to the molluscan neuropeptide termed Achatina cardioexcitatory peptide 1 (ACEP-1). Both peptides have RPXGRFamide structure in their C-terminal moieties. C-RFa was found to have an excitatory effect on visceral muscle tissues of fish, newt, quail, and rat. In the stomach of the fish, Zacco temminckii, IGRFamide (C-RFa 17-20) was shown to be the minimal structure required for the excitatory effect and PIGRFamide (C-RFa 16-20) to be almost equipotent with the parent peptide C-RFa.
OBJECTIVE: Food contains a number of peptides with potential bioactivity. We previously found ng/mliter to mcg/mliter quantities of cyclo(His-Pro)-like immunoreactivity in a number of foods and nutritional supplements. A number of activities have been attributed to cyclo(His-Pro) (CHP), including appetite inhibition and inhibition of insulin secretion in vitro. We wondered whether the cyclo(His-Pro)-like immunoreactivity present in nutritional supplements might be absorbed and, if so, whether parameters of insulin secretion would be altered. METHODS: After performing a pilot study which suggested some common nutritional supplements contain CHP, a follow-up study was done to confirm and expand the findings of the pilot study. Eight fasting volunteers ingested approximately 250 mL of a CHP-containing supplement one day, and then an equienergetic CHP-free supplement the next. RESULTS: Blood drawn for CHP, insulin, glucose, and C-peptide a number of times on both days revealed that when volunteers ingested CHP-containing supplements, CHP levels at 120 minutes were significantly higher than baseline (7.69 +/- 0.50 pmol/mL vs. 9.18 +/- 0.48 pmol/mL; p = 0.011 in the CHP group and 7.90 +/- 0.85 pmol/mL vs. 7.22 +/- 0.73 pmol/mL, p > 0.3 in the CHP-free group) and significantly higher than levels achieved when they drank CHP-free supplements. Levels of glucose, insulin, and C-peptide were not different in the two groups. CONCLUSION: CHP in nutritional supplements may be absorbed when ingested orally and does not grossly affect glucose or parameters of insulin secretion.
Cyanobacteria synthesize several types of bioactive secondary metabolites. Anabaena strain 90 produces three types of bioactive peptides, microcystins (inhibitors of protein phosphatases 1 and 2A), anabaenopeptilides, and anabaenopeptins (serine protease inhibitors). To investigate the role of the anabaenopeptilides in Anabaena, wild-type strain 90 (WT) and its anabaenopeptilide deficient mutant (MU) were cultured with various light and phosphate levels to evaluate the effects and coeffects of these growth factors on the concentrations of the three classes of peptides and the growth characteristics. WT and MU grew in comparable ways under the different growth conditions. The total peptide concentration in WT was significantly higher than that in MU (2.5 and 1.4 microg/mg [dry weight], respectively). Interestingly, the average concentration of anabaenopeptins was significantly higher in MU than in WT (0.59 and 0.24 microg/mg [dry weight], respectively). The concentration of microcystins was slightly but not statistically significantly higher in MU than in WT (1.0 and 0.86 microg/mg [dry weight], respectively). In WT, the highest peptide concentrations were usually found after 13 days in cultures grown at medium light intensities (23 micromol m(-2) s(-1)) and with the highest phosphate concentrations (2,600 microg liter(-1)). In MU, the highest peptide concentrations were found in 13-day-old cultures grown at medium light intensities (23 micromol m(-2) s(-1)) and with phosphate concentrations greater than 100 microg liter(-1). The higher concentrations of anabaenopeptins in MU may compensate for the absence of anabaenopeptilides. These findings clearly indicate that these compounds may have some linked function in the producer organism, the nature of which remains to be discovered.
The anterior pituitary (AP) has been shown to contain a wide variety of bioactive peptides: brain-gut peptides, growth factors, hypothalamic releasing factors, posterior lobe peptides, opioids, and various other peptides. The localization of most of these peptides was first established by immunocytochemical methods and some of the peptides were localized in identified cell types. Although intracellular localization of a peptide may be the consequence of internalization from the plasma compartment, there is evidence for local synthesis of most of these peptides in the AP based on the identification of their messenger-RNA (mRNA). In several cases the release of the peptide from the AP cell has been shown and regulation of synthesis, storage and release have also been described. Because the amount of most of the AP peptides is very low (except for POMC peptides and galanin), endocrine functions are not expected. There is more evidence for paracrine, autocrine, or intracrine roles in growth, differentiation, and regeneration, or in the control of hormone release. To demonstrate such functions, in vitro AP experiments have been designed to avoid the interference of hypothalamic or peripheral hormones. The strategy is first to show a direct effect of the peptide after adding it to the in vitro system and, secondly, to explore if the endogenous AP peptide has a similar action by using blockers of peptide receptors or antisera immunoneutralizing the peptide.