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At least 793 records · Page 44Linked to original sources

PubMed1993

Cetyltrimethylammonium chloride as a surfactant buffer additive for reversed-polarity capillary electrophoresis-electrospray mass spectrometry.

The rapid analysis of picomole quantities of various cationic molecules (laser dyes, tripeptides and larger bioactive peptides) has been achieved by on-line capillary electrophoresis-electrospray mass spectrometry (CE-ES-MS). Use of the cationic surfactant, cetyltrimethylammonium chloride (CTAC) in the CE buffer greatly facilitated the analyses. Under reversed-polarity conditions (negative potential at the source vial), CTAC induces electroosmotic flow towards the mass spectrometer, presumably due to the creation of a cationic layer on the inner surface of the fused-silica capillaries. CTAC diminishes analyte-capillary wall interactions, allowing efficient separations and symmetrical peak shapes. It may be used over a wide range of pH values without loss of electroosmotic flow. Added selectivity, provided by the surfactant properties of CTAC, played a critical role in resolving closely related tripeptides as well as larger (five to thirteen amino acid units) peptides. Above the critical micelle concentration of CTAC, interactions with the pseudostationary micellar phase increased selectivity even for ionic analytes.

Amino Acid Sequence↗

PubMed1996

Complete capillary electrophoretic separation of substance P and its metabolites at neutral pH using ionic run buffer additives.

Substance P (SP), a bioactive peptide that is present in the extracellular fluid of the brain at picomolar concentrations, has been proposed to be a neurotransmitter and a neuromodulator in the central and peripheral nervous systems. This paper describes the development of analytical methodology for the separation of SP and its metabolites by capillary electrophoresis at neutral pH. Since SP is a cationic peptide, it tends to adsorb to silica surfaces. In this work, phytic acid was employed as a run buffer additive to eliminate the interaction of SP and its cationic N-terminus metabolites with ionized silanol groups. The separation of the C-terminus metabolites was then accomplished through the use of sulfobutyl ether beta-cyclodextrin, which imparted a negative charge to these otherwise neutral metabolites through complexation of the phenylalanine residue. The final separation buffer consisted of 150 mM boric acid, 15 mM phytic acid and 5 mM SBE(IV)beta-cyclodextrin at pH 7. Under these conditions, it was possible to separate SP from all eight of its reported metabolites.

Amino Acid Sequence↗

PubMed1985

Somatostatin: a metabolic regulator.

Somatostatin, the hypothalamic release-inhibiting factor, has been found to stimulate gluconeogenesis in rat kidney cortical slices. Stimulation by somatostatin was linear and dose-dependent. Other bioactive peptides such as cholecystokinin, gastrointestinal peptide, secretin, neurotensin, vasoactive intestinal peptide, pancreatic polypeptide, beta endorphin and substance P did not affect the renal gluconeogenic activity. Somatostatin-induced gluconeogenesis was blocked by phentolamine (alpha adrenergic antagonist) and prazosin (alpha1 adrenergic antagonist) but not by propranolol (beta adrenergic antagonist) and yohimbine (alpha2 adrenergic antagonist) suggesting that the effect is via alpha1 adrenergic stimuli. Studies on the involvement of Ca2+ revealed that tissue depletion and omission of Ca2+ from the reaction mixture would abolish the stimulatory effect of somatostatin. Furthermore, somatostatin enhanced the uptake of 45calcium in renal cortical slices which could be blocked by lanthanum, an inhibitor of Ca2+ influx. It is proposed that the stimulatory effect of somatostatin on renal gluconeogenesis is mediated by alpha1 adrenergic receptors, or those which functionally resemble alpha1 receptors and that the increased influx of Ca2+ may be the causative factor for carrying out the stimulus.

Adrenocorticotropic Hormone↗

PubMed1987

Immunochemical analysis of neuropeptides.

Measurement of neuropeptides requires assays that take into account the basic characteristics of bioactive peptides, i.e. the structural homology ("families"); the molecular heterogeneity of a given neuropeptide system; the widespread synthesis in different neurons and cells; and cellspecific processing of the primary translationproduct. Development of libraries of sensitive radioimmunoassays (RIAs), each of which is monospecific for essential sequences of propeptides, comply with some of the needs. Processing-site specific RIAs have proven particularly useful in combination with chromatography and enzymography.

Antibodies↗

PubMed1992

A novel radioligand [125I]BQ-3020 selective for endothelin (ETB) receptors.

A linear endothelin (ET) analog, N-acetyl-LeuMetAspLysGluAlaValTyrPheAlaHisLeu-AspIleIleTrp (BQ-3020), is highly selective for ETB receptors. BQ-3020 displaces [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in porcine cerebellar membranes (IC50: 0.2nM) at a concentration 4,700 times lower than that to ETA receptors (selective to ET-1) on aortic vascular smooth muscle cells (VSMC) (IC50: 940nM). BQ-3020 as well as ET-1 and ET-3 elicits vasoconstriction in the rabbit pulmonary artery. The ETA antagonist BQ-123 failed to inhibit this BQ-3020-induced vasoconstriction. Furthermore, BQ-3020 elicits endothelium-dependent vasodilation. These data indicate that BQ-3020 has ETB agonistic activity. The radioligand [125I]BQ-3020 binds to cerebellar membranes at single high affinity sites (Kd = 34.4pM), whereas it scarcely binds to VSMC. [125I]BQ-3020 binding to the cerebellum was displaced by BQ-3020, ET-1 and ET-3 in a nonselective manner (IC50: 0.07-0.17nM). However, the binding of [125I]BQ-3020 was insensitive to the ETA antagonist BQ-123 and other bioactive peptides. Both [125I]ET-1 and [125I]BQ-3020 show slow onset and offset binding kinetics to ETB receptors. These data indicate that the radioligand [125I]BQ-3020 selectively labels ETB receptors and that the slow binding kinetics of ET-1 are dependent on the peptide sequence from Leu6 to Trp21, but not on the structure formed by its two disulfide bridges.

Amino Acid Sequence↗

PubMed1996

Synthesis and biological properties of gamma-glutamyl-dermorphin, a prodrug.

The possibility of using the gamma-glutamyl-transpeptidase system for transformation of inactive propeptide, gamma-glutamyl-neuropeptides into active neuropeptides has been tested on dermorphin and its gamma-glutamyl analogue. Gamma-glutamyl-dermorphin 2 showed little affinity for opioid receptors. Nonetheless, systemic (intraperitoneal (i.p.), or intravenous (i.v.)) application of this compound induced significant antinociceptive effects, although ten to twenty-fold higher doses were required compared to the parent dermorphin 1. On the other hand, the analogue 2 showed high, antinociceptive activity when injected intrathecally (i.t.). When compared to dermorphin, 2 was one third as potent, but did show a significant prolonged duration of the effect. These results suggest that in the periphery, the peptidase metabolism which results in degradation of bioactivity, is offset by gamma-glutamyl-transpeptidase (GGTP) activity that liberates bioactive peptide 2. On the other hand, in the central nervous system, the activity of gamma-glutamyl-transpeptidase system seems to be more effective than other peptidase systems, resulting in formation of active peptide 2 in a significant amount. These data suggests that gamma-glutamyl analogues of neuropeptides can be considered as potential prodrugs, especially for synthetic analogues which themselves are resistant to peptidase action.

Analgesics, Opioid↗

PubMed1992

Effect of calcium and phosphate ions on hemolysis induced by Pyrularia thionin and Naja naja kaouthia cardiotoxin.

Pyrularia thionin is a strongly basic bioactive peptide of 47 amino acids isolated from nuts of Pyrularia pubera. It is hemolytic, cytotoxic and activates an endogenous phospholipase A2 in 3T3 cells. Earlier studies have shown that the cardiotoxin from Naja naja kaouthia has similar activities and binds to the same site as Pyrularia thionin. Since the peptides appear to bind to the phospholipids of cell membranes to elicit their cellular responses, the effect of modifying the electrostatic environment was studied by separately adding phosphate ion and Ca2+, and by removing Ca2+ from the membrane by treatment with EGTA. Analysis of erythrocyte hemolysis for both Pyrularia thionin and cardiotoxin shows that the reactions follow Michaelis-Menten kinetics, with the peptides serving as the substrate. The basal rate of hemolysis in physiological saline is markedly increased by the addition of phosphate in the 5-10 mM range and also by removing membrane-bound Ca2+ by incubation of the cells with 10 mM EGTA. These treatments do not change the apparent K(m) values, but increase the V(max), indicating that more binding sites are made available by these treatments. On the other hand, added Ca2+ in the 5-10 mM range competitively inhibits the reaction by inhibiting the binding of the peptide to the membrane.

Antimicrobial Cationic Peptides↗

PubMed1984

Transforming growth factor-beta controls receptor levels for epidermal growth factor in NRK fibroblasts.

NRK fibroblasts exposed to transforming growth factor-beta (TGF-beta) show increased binding of radiolabeled epidermal growth factor (EGF) relative to untreated cells. The binding of another growth factor, rat insulin-like growth factor-II, is unaffected. The increase in EGF binding induced by TGF-beta is not due to inhibition of EGF processing nor to an alteration in the affinity of plasma membrane EGF receptors. However, treatment of the cells with TGF-beta does cause a rapid increase in the number of plasma membrane receptors for EGF. TGF-beta has little effect on the rate of overall protein synthesis, but the increase it induces in EGF binding can be completely inhibited by cycloheximide and tunicamycin. Thus a selective synthetic mechanism underlies TGF-beta action. Cells incubated with TGF-beta also show altered down regulation of their EGF receptors in response to the ligand; concentrations of EGF that can induce strong biological responses no longer decrease the plasma membrane receptor level below the basal state. These results agree well with the known specificity and synergism of the interaction between TGF-beta and EGF. Moreover, they describe a mechanism of growth control in which bioactive peptides act coordinately through a regulatory effect on the number of cell-surface receptors.

Animals↗

PubMed1989

Amino-terminal sequences of prosomatostatin direct intracellular targeting but not processing specificity.

Rat preprosomatostatin (rPPSS) is processed to two bioactive peptides, somatostatin-14 and somatostatin-28. In anglerfish islets, the two peptides are synthesized by distinct cell types and are derived from different precursors, anglerfish preprosomatostatin-1 (a(I)PPSS) and anglerfish preprosomatostatin-2 (a(II)PPSS). To determine the basis of the differential processing, we introduced a(I)PPSS or a(II)PPSS expression vectors into mammalian endocrine cell lines that can accomplish both patterns of processing. Both precursors were processed identically, indicating that cellular factors must determine the processing pattern. Although similar processing sites are present in both precursors, high levels of unprocessed anglerfish prosomatostatin-2 were secreted constitutively from the transfected cells. A hybrid protein containing the leader sequence and a portion of the pro-region of rPPSS fused to the carboxy-terminal third of a(II)PPSS was processed and secreted via a regulated pathway. We conclude that the amino-terminal 78 residues of rPPSS contain sufficient information to correct the targeting deficiency of a(II)PPSS in mammalian endocrine cell lines.

Adrenal Cortex↗

PubMed1996

Conditions which promote mineralization at the bone-implant interface: a model in vitro study.

This in vitro study was an investigation of osteoblast functions on glass substrates modified with the bioactive peptide Arg-Gly-Asp-Ser (RGDS) in the absence and presence of recombinant human Osteogenic Protein-1 (OP-1); control substrates were plain glass, glass modified with amine groups, and glass modified with the non-adhesive peptide Arg-Asp-Gly-Ser. In serum-free cell culture medium, osteoblasts adhered in greater numbers (P < 0.1) to glass modified with RGDS, compared to adhesion on all other substrate types tested in the present study. In the presence of serum proteins, osteoblasts adhered similarly to all substrate types examined, in the absence or presence of 100 ng ml-1 OP-1. The presence of 100 ng ml-1 OP-1 inhibited (P < 0.1) 72 h proliferation of sparsely seeded (2500 cells cm-2) cultures on all substrates examined in the present study. OP-1 (100 ng ml-1) promoted 21 day mineralization on all substrates examined; in addition, mineralization was further enhanced in osteoblast cultures grown on glass modified with the adhesive peptide RGDS. The present study establishes conditions which can be utilized in the design of dental/orthopaedic biomaterials which elicit timely, specific responses from surrounding bone tissue.

Amino Acid Sequence↗

PubMed1994

Functional effects of a family of galanin antagonists on the cardiovascular system in anaesthetised cats.

Previous studies have shown that injection of galanin (GAL: 6.2 nmol/kg) causes prolonged inhibition of cardiac vagal action in anaesthetised cats. Stimulation of the cardiac sympathetic nerve (16 Hz for 5 min) also produces inhibition of cardiac vagal action, an effect which has been proposed to be due to the release of endogenous GAL from sympathetic nerves. In a previous study we tested galantide (M15) and in this study we compared galantide with two other GAL antagonists for their GAL antagonist activity in our experimental model. Each of these incorporate the N-terminal fragment GAL 1-13 and a C-terminal portion of another bioactive peptide and all are C-terminally amidated. GAL 1-13 Substance P 5-11 amide (galantide: M15: 62 nmol/kg and 156 nmol/kg), GAL 1-13 Spantide amide (C7: 156 nmol/kg) and GAL 1-13 NPY 24-36 amide (M32a: 62 nmol/kg) all significantly reduced the cardiac vagal inhibitory effect of exogenous GAL and also reduced the effect of sympathetic stimulation on subsequent cardiac vagal slowing, giving strong support to our hypothesis that GAL is involved in this phenomenon. No antagonist reduced the depressor effect of GAL. This study demonstrates the GAL antagonist properties of these agents on autonomic neuroeffector functions making them useful tools in elucidating further functions of endogenous GAL.

Analysis of Variance↗

PubMed1994

BON cells display the intestinal pattern of neurotensin/neuromedin N precursor processing.

Antisera towards the bioactive peptides, neurotensin (NT, 13 residues) and neuromedin N (NMN, 6 residues), as well as towards three regions of their 147-residue canine precursor were used to identify and to quantitate precursor-derived peptides in extracts of human BON cells. This cell-line, which was obtained from a human pancreatic carcinoid tumor, constitutively expresses NT/NMN mRNA and secretes NT. Quantitation of seven precursor-derived peptides led us to conclude that BON cells display the intestinal pattern of NT/NMN precursor processing, which is primarily characterized by the production of a large molecular (125 amino acid) form of NMN. Four large molecular components, identified by immunochemical analyses and Western blotting, displayed physico-chemical properties which, for the most part, were consistent with the structures predicted from the partially-known human mRNA sequence. However, as shown previously for these peptides in canine gut, the empirically determined M(r) and pI values were slightly higher than those predicted solely from the amino acid content, perhaps due to the presence of additional substituents. These results suggest that BON cells may provide a good in vitro model in which to study the regulation of intestinal NT/NMN precursor processing and the nature of the enzyme(s) involved.

Amino Acid Sequence↗

PubMed1996

Octreotide differentially modulates human Caco-2 intestinal epithelial cell proliferation and differentiation by decreasing intracellular cAMP.

Somatostatin modulates gastrointestinal mucosal growth and differentiation indirectly via inhibition of bioactive peptides and directly by less well understood mechanisms. We studied the direct effects of the somatostatin analog octreotide on proliferation, brush-border enzyme activity, cell-matrix interactions and intracellular cAMP in Caco-2 human intestinal epithelial cells. Proliferation was assessed by cell counting and [3H]thymidine uptake. The brush-border enzymes alkaline phosphatase (AP) and dipeptidyl dipeptidase (DP) were quantitated by synthetic substrate digestion. Adhesion and migration on purified matrix proteins were also measured. Octreotide (10(-9)-10(-5)M) shortened doubling time (46.5 +/- 6.2% at 10(-5) M, n = 20, P < 0.0001) and stimulated [3H]thymidine uptake. Octreotide decreased intracellular cAMP by 19.4 +/- 5.0% (n = 7, P < 0.0001) while dibutyryl-cAMP (10(-6) M) prolonged doubling time by 10.1 +/- 1.5% (n = 8, P < 0.0001), and blocked the octreotide effect. Octreotide decreased AP and DP with maximal effect at 10(-6) M (36.8 +/- 8.3% and 20.5 +/- 9.1%, n > 7, P < 0.0005 respectively). However, mitomycin proliferative blockade prevented octreotide inhibition of AP and DP, suggesting that the mitogenic effects of octreotide had simply decreased average maturity of the cells. Octreotide did not alter Caco-2 adhesion, EGF-or matrix-modulated motility, or integrin surface expression. Octreotide appears to directly stimulate Caco-2 proliferation by decreasing cAMP. These proliferative effects modulate Caco-2 differentiation but do not affect cell-matrix interactions.

Alkaline Phosphatase↗

PubMedDate not supplied

Identification of His5,Trp7,Tyr8-GnRH (chicken GnRH II) in amphibian brain.

GnRH immunoreactive and bioactive peptides in Xenopus laevis brain extract were investigated by high performance liquid chromatography (HPLC), radioimmunoassay with region-specific antisera raised against GnRH (mammalian), His5,Trp7,Tyr8-GnRH (chicken II) and Tyr3,Leu5,Glu6,Trp7,Lys8-GnRH (lamprey), and by assessment of biological activity. Two immunoreactive peptides eluted in the same positions as GnRH and His5,Trp7,Tyr8-GnRH respectively in HPLC systems which were specifically designed to separate four known natural vertebrate GnRHs (mammalian, chicken I and II, salmon). The immunological properties of these two immunoreactive peaks, determined by relative interaction with three region-specific antisera raised against mammalian GnRH and two specific His5,Trp7,Tyr8-GnRH antisera, were identical to those of GnRH and His5,Trp7,Tyr8-GnRH. The immunoreactive peak co-eluting with His5,Trp7,Tyr8-GnRH represented approximately one-third of the total brain GnRH. Both immunoreactive peaks stimulated luteinizing hormone (LH) release in a chicken dispersed pituitary cell bioassay, and the amounts of LH release stimulated by the two peaks were appropriate for these peaks being GnRH and His5,Trp7,Tyr8-GnRH. A small hydrophobic peak with GnRH immunoreactivity eluted in the same position as Trp7,Leu8-GnRH (salmon), while Gln8-GnRH (chicken I) and lamprey GnRH were not detected. Two additional rather hydrophilic peptides cross-reacted with a COOH-terminus-directed antiserum and had LH-releasing activity. LH-releasing activity was also detected in hydrophobic HPLC fractions. In summary, these data provide evidence for the presence of both GnRH and a second peptide with properties identical to His5,Trp7,Tyr8-GnRH in X. laevis brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Evidence that enzymatic conversion of N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate, a specific inhibitor of endopeptidase 24.15, to N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala is necessary for inhibition of angiotensin converting enzyme.

N-[1(R,S)-Carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB) is a potent, substrate-related, specific inhibitor of endopeptidase 24.15, an enzyme involved in the metabolism of bioactive peptides including bradykinin, neurotensin, and proenkephalin, and prodynorphin-derived enkephalin precursors. The observation that this inhibitor causes a pronounced decrease in blood pressure after intravenous infusion into normotensive rats posed the question of the mechanism of this hypotensive response. It was suggested previously that cFP-AAF-pAB is an inhibitor of angiotensin converting enzyme (ACE) and that this function can account for the hypotensive response to the inhibitor. We present here evidence that cFP-AAF-pAB has no intrinsic ACE-inhibitory activity. The previously observed inhibition is shown to be dependent on cleavage of the Ala-Phe bond in the inhibitor by endopeptidase 24.11 (enkephalinase, EC 3.4.24.11), a contaminant of some ACE preparations.

Amino Acid Sequence↗

PubMed1995

Degradation of lysylbradykinin by endopeptidase 24.11 and endopeptidase 24.15.

Lysylbradykinin (LBK), a potent bioactive peptide with pleiotropic actions, is the major kinin generated in the extravascular space. To explore possible mechanisms of inactivation of this peptide in tissues, we evaluated its degradation by endopeptidase 24.11 (EP 24.11) and endopeptidase 24.15 (EP 24.15), two zinc metalloenzymes widely distributed in tissues. EP 24.11 cleaved LBK at the Gly5-Phe6 and Pro8-Phe9 bonds, whereas EP 24.15 cleaved the Phe6-Ser7 bond. Determination of kinetic constants for degradation of LBK by the two enzymes yielded kcat/Km ratios of 5.2 x 10(5) and 8.4 x 10(5) for EP 24.15 and EP 24.11, respectively, indicating that LBK is a good substrate for both enzymes. The findings demonstrate that both EP 24.11 and EP 24.15 efficiently degrade LBK and thus may contribute to the inactivation of this peptide in tissues.

Amino Acid Sequence↗

PubMed1991

Dual presence of chromogranin A-like immunoreactivity in a population of endocrine-like cells and in nerve fibers in the human anal canal.

Light microscopic immunohistochemistry was used to investigate the presence and distribution of chromogranin A-like immunoreactivity in the human anal canal. In the anal transitional zone (ATZ), anal duct and anal gland epithelium, a varied number of mostly elongated cells strongly stained for CGA, using an antibody directed to a highly species-conserved region of the CGA molecule or the monoclonal antibody LK2H10. The density of CGA-immunoreactive (ir) cells strikingly increased from the ATZ epithelium towards the anal gland epithelium. CGA-ir cells possessed single processes running perpendicularly to reach the epithelial surface and exhibited basal ramifications that extended parallel to the basal lamina. The number of CGA-ir cells in anal glands exceeded CGA-ir cells in the crypt-bearing colorectal-type mucosa. The abundant population of CGA-ir cells in the anal canal most likely represents a population of specialized endocrine or paracrine cells. CGA-like immunoreactivity was also present in anocutaneous Merkel cells. A sparse number of vascular and non-vascular CGA-ir varicose nerve fibers was present throughout the layers and rostrocaudal divisions of the anal canal and in the perianal skin. Proposed functions of CGA in neuroendocrine cells and nerves of the anal canal include calcium binding and regulation, secretory granule matrix formation, and generation of bioactive peptides.

Amino Acid Sequence↗

PubMed1993

Basal and stimulated cyclic adenosine 3',5'-monophosphate production in rat adenopituitary homogenates: direct determination by high-performance liquid chromatography.

A sensitive and reliable procedure to quantify cAMP in biological samples, without involving radioisotopes, is described. After incubation, the assay mixture was boiled for 4 min, centrifuged, filtered and directly injected onto a mu Bondapak C18 column (10 microns particle size; 150 mm x 3.9 mm I.D.). cAMP was resolved within 40 min in isocratic mode using a mobile phase composed of 96% of 0.01 M ammonium acetate, pH 6.6 and 4% of acetonitrile-water &70:30, v/v) at a flow-rate of 0.5 ml/min. Detection was monitored at 254 nm and quantification was achieved by peak surface integration. The lowest detection limit was 1.6 pmol (signal-to-noise ratio = 2.5). The potential of this assay to perform structure-activity of bioactive peptides is illustrated with growth hormone-releasing factor in rat adenopituitary homogenates.

Adenylyl Cyclases↗