Peptide VI Research Articles

The relationship between micelle formation and biological activity of peptide 562–572 of luteinizing hormone receptor modified with decanoyl radicals

Lipophilic derivatives of peptides corresponding to cytoplasmic regions of G protein-coupled receptors (GPCR) can act as intracellular agonists. Our previous work showed that peptides corresponding to residues 562–572 of luteinizing hormone receptor and modified with palmitate or decanoate at the C-terminus activate adenylate cyclase in rat testes. The stimulating effect of peptide 562–572 modified with decanoates at both the N- and the C-termini (peptide IV) reached its maximum at the peptide concentration of 10–5 M and diminished with further increase in its concentration. It was supposed that this effect was due to peptide IV ability to form micelles. To verify this hypothesis, the relationship between biological activity, hydrophobicity, and ability to form micelles was investigated for peptide IV and other acylated derivatives of peptide 562–572, including those carrying C-terminal decanoate (peptide III) and palmitate (peptide VI) moieties. It was found that the stimulating effect of peptide IV taken in the concentration of 10–5 M on adenylate cyclase activity in plasma membranes of rat testes and ovaries was only slightly lower than that of peptide VI and higher than the effect of peptide III. At the concentration of 10–3 M, the effect of peptide IV was 20–27% lower and amounted to only 50–51 and 87–88% of the effects of peptides VI and III, respectively. In spite of its high hydrophobicity, peptide IV was characterized with an abnormally low retention time when eluted from a Nucleosil C8 column during reverse-phase HPLC: it was even lower than the retention time of nonmodified peptide 562–572. However, the retention time of peptide IV, but not of other peptides, increased significantly when the eluent contained a higher proportion of trifluoroacetic acid, which disrupts micellelike structures (0.5 instead of 0.1%). The surface tension of peptide IV solution in water slightly decreased with increasing peptide concentration, but rapidly dropped and reached a plateau at the concentration of 7 × 10–6 M, which indicates the beginning of micelle formation. Thus, peptide IV in the concentrations above 10–5 M forms micelles, which prevents it from interacting with the receptor. The ability of GPCR peptides to aggregate and form micelles should be taken into account in the development of their new membrane-active analogs.

Identification of neuropeptides from brains of larval Manduca sexta and Lacanobia oleracea using MALDI-TOF mass spectrometry and post-source decay

The occurrence of neuropeptides in the brain of larvae of the tobacco hawkmoth, Manduca sexta, and tomato moth, Lacanobia oleracea, was investigated using matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and post-source decay (PSD). Methanolic extracts of 100 brains separated by reversed-phase high performance liquid chromatography yielded numerous ion peaks, some of which were common to both species. In M. sexta six [M+H] + ions were in agreement with peptides previously structurally characterised from M. sexta (FLRF-amides I, II and III, M. sexta allatostatin, CAP 2b and myoinhibitory peptide VI), whereas a further five corresponded to other known lepidopteran peptides (cydiastatins 3 and 4, helicostatins 1 and 6 and helicokinin II). Of these the identities of FLRF-amide I, cydiastatins 3 and 4 and CAP 2b were confirmed by PSD analysis. Fourteen [M+H] + ions corresponding to known lepidopteran peptides (FLRF-amide I, cydiastatins 2, 3 and 4, helicostatins 1, 5, 6, 7 and 9, CCAP, CAP 2b, M. sexta allatostatin and myoinhibitory peptide VI) were measured in L. oleracea brain extracts. From this insect, cydiastatins 3 and 4, helicostatin 5 and FLRF-amide I were identified by PSD. These peptides had not previously been structurally characterised from L. oleracea.

Synthesis and opioid activity of novel tetrapeptides analogous to sequence (1–4) of dermorphin

Seven new tetrapeptides analogous to (1–4) sequence of dermorphin were synthesized and evaluated for their opioid activity. The peptides were synthesized by the solution phase method. Their opioid activity revealed that peptides II and V were the most potent in the analgesia test as well as in the peripheral assays. Peptide II was most active in the guinea pig ileum assay, whereas peptide VI was 2763 times more selective for μ-receptors.

Macrocyclic Peptides VI. Complex Formations and Conformations of an Ionophorous Cyclic Octapeptide Containing N,N′-Ethylene-Bridged (S)-Leucyl-(S)-leucine and Glycine in Acetonitrile

Macrocyclic Peptides VI. Complex Formations and Conformations of an Ionophorous Cyclic Octapeptide Containing N , N ′-Ethylene-Bridged ( S )-Leucyl-( S )-leucine and Glycine in Acetonitrile

Fatty peptides VI. Penicillin and cephalosporin esters with increased lipophilic character

Several series of methoxycarhonyl alkyl esters, 2b-d, 3b and 4b-d, with increased lipophilicity, were prepared by conjugating penicillin G, ampicillin and cefuroxime, respectively. The suitability of this type of ester linkage as a pro-drug linkage for the carboxylic acid group of β-lactam antibiotics was determined both in vitro and in vivo. Only one compound, the penicillin G conjugate 2b showed a weak activity in vitro against the sensitive Staphylococcus aureus strain (663E), Pseudomonas and clostridia. The remaining conjugates 2c, d, 3b and 4b-d were inactive. Conjugates 2b-c and 4b exhibited antibiotic activity against S. aureus 663E following subcutaneous administration in the mouse. The most active conjugates 2b and 4b were methyl octanoyl derivatives of penicillin G and cefuroxime. It can be assumed that the secondary alcohol ester linkage was cleaved in vivo to afford active, presumably the parent, antibiotic. The penicillin G conjugate 2b-d and the ampicillin conjugate 3b were orally active, conjugate 3b was more active than the parent ampicillin, and cefuroxime conjugates 4c and d were orally inactive. Conjugation of a lipidic moiety via a secondary alcohol ester linkage may improve the absorption of β-lactam antibiotics. There appeared to be a preference for short alkyl chains for oral and subcutaneous activity in this series of conjugates, therefore, it can be assumed that the longer alkyl chains in compounds 2d, 4c and d protect the ester bond from esterases.

Improved coupling of protected peptides on the kaiser oxime resin using bop activation

Protected peptides and amino acids can be coupled to resin-bound amine in high yield with low levels of racemization using BOP activation. The synthesis of the 16 amino acid protected peptide VI derived from the ice nucleation protein is described and experimental procedures for these couplings are provided.

Phosphorylation of rabbit skeletal muscle glycogen synthase by casein kinase 1: Evidence of phosphorylation sites specific for casein kinase 1

Casein kinase 1 phosphorylated rabbit skeletal muscle glycogen synthase at both seryl and threonyl residues. With glycogen synthase phosphorylated up to 7.5 mol phosphate/mol subunit, about 26% of the phosphate was present in the N-terminal cyanogen bromide fragment (CB1) and 74% in the C-terminal fragment (CB2). Both fragments contained phosphothreonine (11 to 14%) in addition to phosphoserine. When 32P-labeled glycogen synthase was totally digested with trypsin and chromatographed on reversephase high-performance liquid chromatography, seven phosphopeptides were observed. Peptide I eluted in the vicinity of the peptide containing site 1a, peptide II coincided with sites 4 + 5, peptides III and IV eluted in the region corresponding to sites 3a + 3b + 3c, peptide V appeared slightly after the peptide containing site 1b and peptide VII behaved as the peptide containing site 2, whereas peptide VI did not coincide with any of the known phosphopeptides. Limited trypsinization prior to analysis by HPLC led to the disappearance of peaks V and VI without altering peaks I to IV and VII. Only peaks I and VII remained when limited chymotrypsinization was performed prior to HPLC analysis. Chromatography on HPLC of the fragments derived from complete trypsinization of CB2 showed the presence of peaks II to VI. Phosphoamino acid analysis of the different peptides demonstrated the presence of quantitative amounts of phosphothreonine in peptides V, VI, and VII. These results indicate that multiple phosphorylation sites for casein kinase 1 must exist in both the N-terminal and C-terminal regions of glycogen synthase, some of which would only be labeled by casein kinase 1.

Lactic Acidosis and Mitochondrial Myopathy Associated with Deficiency of Several Components of Complex III of the Respiratory Chain

Summary: We have studied a 17-year-old girl with lactic acidosis (3-18 mEq/liter) and progressive muscle weakness since 9 years of age. Morphological findings in muscle were of a typical ragged red myopathy with multiple collections of bizarre mitochondria, some containing paracrystalline inclusions. The carnitine content of serum and muscle was normal, as were the activities of carnitine palmitoyltransferase, carnitine octanoyltransferase, and carnitine acetyltransferase in the patient's muscle. Measurement of the enzymes of oxidative phosphorylation in both crude muscle homogenates and mitochondrial fractions showed close to normal activities of cytochrome c oxidase, succinate dehydrogenase, and ATPase. In contrast, succinate cytochrome c reductase activity was greatly reduced in the patient, being 0.035 μmol/min/g tissue in whole muscle (controls 1.16 ± 0.47 μmol/min/g tissue) and 8 nmol/min/mg protein in the mitochondria (control, 340 nmol/min/mg protein). Rotenonesensitive NADH-cytochrome c reductase was also undetectable in the patient's mitochondria. Spectral analysis of cytochromes showed decrease of reducible cytochrome b to 16% of the control. These results indicate a defect of ubiquinol-cytochrome c reductase or the cytochrome bc1 segment (complex III) of the electron transport chain. Antibody-binding studies of the individual components of complex III showed additional deficiencies of core proteins I and II and peptide VI, indicating a more widespread defect of complex III than was evident from spectral analysis and enzyme activity measurements alone. Urine organic acid analysis after fasting and following a medium chain triglyceride load showed unusually high levels of lactate and 3-hydroxybutyrate, lower than expected levels of acetoacetate and dicarboxylic acids, and the presence of several other metabolites suggesting a disturbed citric acid cycle and redox state. Thus, the defect in this patient may be more widespread than is apparent from the clinical presentation.

N‐Acylamino acids and peptides VI. A Simple synthesis of N‐acylglycines of the benzoyl‐ and cinnamyl type

AbstractA very simple and fast method for the synthesis of N‐acylamino acids and peptides from the benzoyl‐ and cinnamyl type is described. The method is based on the use of N‐hydroxysuccinimide as reagent (active ester method).The characteristics and chromatographic parameters of some new benzoylglycines are given.

Mass spectrometric studies of peptides—VI:. Applications of metastable ion and collisional activation spectra

AbstractAn unambiguous differentiation between leucine and isoleucine residues in peptides is possible with unimolecular metastable ion (MI) and collisional activation (CA) spectra. The immonium ion \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm [Y}\!\mathop {\rm N}\limits^{\rm + } {\rm H = CHC}_{\rm 4} {\rm H}_{\rm 9} {\rm ]} $\end{document}, which is present in the normal mass spectra of all leucine or isoleucine containing peptides, yields MI spectra with which this differentiation can be made with high sensitivity. The MI and CA spectra of other peptide fragments are also applicable in particular cases. MI and CA spectra can also be used to obtain information on the amino acide sequences of individual ions. In general more extensive sequece information is available from CA than from MI spectra. This information appears to be particularly promising for the sequencing of oligopeptide mixtures or of ions produced by field or chemical ionization.

Steroid Binding Properties of Some Peptide Fragments of Bovine Serum Albumin Obtained on Peptic Digestion

Abstract To elucidate the structural characteristics of bovine serum albumin (BSA) which allow for steroid binding, BSA was digested with pepsin under conditions which afford maximal retention of steroid binding activity consistent with a maximal degree of peptide fragmentation. Digestion with pepsinBSA (w/w), 1:3000, pH 3.0, for 2 hours at 25° afforded a complex mixture of peptide fragments with molecular weights of about 14,000 (a major class of components), and 27,000 and 34,000 (minor classes) estimated on sodium dodecyl sulfate gel electrophoresis of the digest. As much as 40 to 50% of the initial steroid binding activity was retained in the BSA digest; the steroids studied were progesterone, testosterone, and 17β-estradiol. Steroid binding activity was empirically defined as the product of the ratio of bound to unbound steroid times the reciprocal of the total protein or peptide concentration (gram per liter) in an assay system containing Sephadex G-25 or G-10 under conditions of equilibrium. The crude BSA digest was treated successively with 2% and 10% trichloroacetic acid; the respective trichloroacetic acid-precipitable peptides were chromatographed on Sephadex G-75. The 2% trichloroacetic acid chromatographic fractions exhibited a relatively high progesterone binding activity, and also appreciable testosterone and estradiol binding, whereas successive chromatographic fractions of the 10% trichloroacetic acid fraction exhibited diminishing steroid binding activity. Binding activity correlated with the absorbance ratio, 280:258 nm, of the respective chromatographic fractions, suggesting that peptide fragments which are richer in tyrosine tend to retain steroid binding activity to a greater degree. Although the bulk of the peptide material remains to be resolved, two peptide fragments, KL and VI, were isolated from the 2% trichloroacetic acid and 10% trichloroacetic acid fractions, respectively. Peptide KL (mol wt 10,050) is rich in tyrosine. The equilibrium constant, nk, for the formation of a complex of peptide KL with progesterone, testosterone, or 17β-estradiol at 25° was about 0.44, 0.18, or 0.33 x 104 m-1, respectively. Peptide VI (mol wt 2766) appears to be identical with the NH2-terminal or Asp fragment of BSA previously isolated by Peters and Hawn ((1967) J. Biol. Chem. 242, 1566). Peptide VI contains no tyrosine and exhibits very little steroidbinding affinity.