An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae.
ABSTRACT: Competence for genetic transformation in Streptococcus pneumoniae has been known for three decades to arise in growing cultures at a critical cell density, in response to a secreted protease-sensitive signal. We show that strain CP1200 produces a 17-residue peptide that induces cells of the species to develop competence. The sequence of the peptide was found to be H-Glu-Met-Arg-Leu-Ser-Lys-Phe-Phe-Arg-Asp-Phe-Ile-Leu-Gln-Arg- Lys-Lys-OH. A synthetic peptide of the same sequence was shown to be biologically active in small quantities and to extend the range of conditions suitable for development of competence. Cognate codons in the pneumococcal chromosome indicate that the peptide is made ribosomally. As the gene encodes a prepeptide containing the Gly-Gly consensus processing site found in peptide bacteriocins, the peptide is likely to be exported by a specialized ATP-binding cassette transport protein as is characteristic of these bacteriocins. The hypothesis is presented that this transport protein is encoded by comA, previously shown to be required for elaboration of the pneumococcal competence activator.
Project description:The known amino acid sequences at the two sites on phosphorylase kinase that are phosphorylated by cyclic AMP-dependent protein kinase were extended. The sequences of 42 amino acids around the phosphorylation site on the alpha-subunit and of 14 amino acids around the phosphorylation site on the beta-subunit were shown to be: alpha-subunit Phe-Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly-Gly-His-Ser-Leu-Gly-Ala-Asp-Leu-Met-Ser-Pro-Ser-Phe-Leu-Ser-Pro-Gly-Thr-Ser-Val-Phe(Ser,Pro,Gly)His-Thr-Ser-Lys; beta-subunit, Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-VALIle-Tyr-Glu-Pro-Leu-Lys. The sites on histone H2B which are phosphorylated by cyclic AMP-dependent protein kinase in vitro were identified as serine-36 and serine-32. The amino acid sequence in this region is: Lys-Lys-Arg-Lys-Arg-Ser32(P)-Arg-Lys-Glu-Ser36(P)-Tyr-Ser-Val-Tyr-Val- [Iwai, K., Ishikawa, K. & Hayashi, H. (1970) Nature (London) 226, 1056-1058]. Serine-36 was phosphorylated at 50% of the rate at which the beta-subunit of phosphorylase kinase was phosphorylated, and it was phosphorylated 6-7-fold more rapidly than was serine-32. The amino acid sequences when compared with those at the phosphorylation sites of other physiological substrates suggest that the presence of two adjacent basic amino acids on the N-terminal side of the susceptible serine residue may be critical for specific substrate recognition in vivo.
Project description:The N-terminal 72 residues of an integral membrane fragment, P5, of the human erythrocyte anion-transport protein, which is known to be directly involved in the anion-exchange process, was shown to have the following amino acid sequence: Met-Val-Pro-Lys-Pro-Gln-Gly-Pro-Leu-Pro-Asn-Thr-Ala-Leu-Leu-Ser-Leu-Val-Leu-Met -Ala-Gly-Thr-Phe-Phe-Phe-Ala-Met-Met-Leu-Arg-Lys-Phe-Lys-Asn-Ser-Ser-Tyr-Phe-Pro-Gly-Lys-Leu-Arg-Arg-Val-Ile-Gly-Asp-Phe-Gly-Val-Pro-Ile-Ser-Ile-Leu-Ile-Met-Val-Leu-Val-Asp-Phe-Phe-Ile-Gln-Asp-Thr-Tyr-Thr-Gln- The structure of this fragment was analysed, with account being taken of the constraints that apply to the folding of integral membrane proteins and the topographical locations of various sites in the sequence. It was concluded that this sequence forms two transmembrane alpha-helices. These are probably part of a cluster of amphipathic transmembrane alpha-helices, which could comprise that part of the protein responsible for transport activity. The presently available evidence relating to the anion-exchange process was considered with the structural features noted in this study and a possible molecular mechanism is proposed. In this model the rearrangement of a network of intramembranous charged pairs mediates the translocation of an anion between anion-binding regions at each surface of the membrane, which are composed of clusters of positively charged amino acids. This model imposes a sequential exchange mechanism on the system. Supplementary material, including Tables and Figures describing the compositions of peptides determined by amino acid analysis and sequence studies, quantitative and qualitative data that provide a residue-by-residue justification for the sequence assignment and a description of modifications to and use of the solid-phase sequencer has been deposited as Supplementary Publication SUP 50123 (12 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained as indicated in Biochem. J. (1983) 209, 5.
Project description:Insulin was isolated from an extract of the pancreas of a urodele, the three-toed amphiuma (Amphiuma tridactylum), and its primary structure established as Ala-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-His10-Asn-Thr-Cys- Ser-Leu-Asn-Gln-Leu-Glu-Asn20-Tyr-Cys-Asn for the A-chain and Ile-Thr-Asn-Gln-Tyr-Leu-Cys-Gly-Ser-His10-Leu-Val-Glu-Ala- Leu-Tyr-Leu-Val-Cys-Gly20-Asp-Arg-Gly-Phe-Phe-Tyr-Ser-Pro-Lys for the B-chain. The N-terminus of the A-chain is extended by two amino acids (Ala-Arg) relative to all other known insulins suggesting an anomalous pathway of post-translational processing in the region of the C-peptide/A-chain junction of proinsulin. In common with chicken and Xenopus insulins, which contain a HisA8, amphiuma insulin was more potent (approx. 5-fold) than porcine insulin in inhibiting the binding of [125I-TyrA14]insulin to the soluble human insulin receptor from transfected 293EBNA cells (an adenovirus-transformed human kidney cell line). This result is consistent with previous data showing that insulin analogues extended at GlyA1 by uncharged groups have reduced binding affinity whereas high affinity is preserved in analogues extended by basic amino acid residues.
Project description:As a folk medicine of the Jingpo minority in Yunnan province, the venom of <i>Vespa magnifica</i> has been commonly used for the treatment of rheumatoid arthritis. Quality standardization of the wasp venom is a necessary step for its pharmaceutical research and development. To control the quality of the wasp venom, a method based on high-performance liquid chromatography (HPLC) was developed for chemical fingerprint analysis. In the chromatographic fingerprinting, chemometrics procedures, including similarity analysis (SA), hierarchical clustering analysis (HCA), and principal component analysis (PCA), were applied to classify 134 batches (S1-S134) of wasp venom from different origins. The HPLC fingerprint method displayed good precision (Relative standard deviation, RSD < 0.27%), stability (in 16 h, RSD < 0.34%), and repeatability (RSD < 1.00%). Simultaneously, four compounds (VMS1, VMS2, VMS3, and VMS4) in the wasp venom were purified and identified. VMS1 was 5-hydroxytryptamine, and the other compounds were three peptides that were sequenced as follows: Gly-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg-Ile-Asp-NH<sub>2</sub> (VMS2), Ile-Asn-Leu-Lys-Ala-Ile-Ala-Ala-Leu-Ala-Lys-Lys-Leu-Leu-NH<sub>2</sub> (VMS3), and Phe-Leu-Pro-Ile-Ile-Gly-Lys-Leu-Leu-Ser-Gly-Leu-Leu-NH<sub>2</sub> (VMS4). The quantifications for these components were 110.2 mg/g, 26.9 mg/g, 216.3 mg/g, and 58.0 mg/g, respectively. The results of this work indicated that the combination of the chemical fingerprint and quantitative analysis offers a reasonable way to evaluate the quality of wasp venom.
Project description:Fermented milks with strong angiotensin I-converting enzyme (ACE)-inhibitory activity were obtained through a culture with Lactobacillus helveticus KLDS.31 and Lactobacillus casei KLDS.105 with a fermentation and storage temperature of 37 °C. Ultrafiltration fractions with a molecular weight less than 3 kDa in fermented milk whey exhibited the strongest inhibitory activity. Correspondingly, a gastrointestinal digestion experiment showed retention of the bioactivity of these fractions with pepsin and trypsin treatment. Four ACE-inhibitory peptides from fermented milk were isolated, purified by two-step reverse chromatography, and sequenced. Furthermore, the interaction mechanisms between ACE and four isolated peptides were investigated by a molecular docking method and the Independent Gradient Model. Experimental determination of IC50 was done to verify theoretical results. The inhibitory peptide interacted with ACE as follows: Lys-Pro-Ala-Gly-Asp-Phe > Lys-Ala-Ala-Leu-Ser-Gly-Met > Lys-Lys-Ala-Ala-Met-Ala-Met > Leu-Asp-His-Val-Pro-Gly-Gly-Ala-Arg.
Project description:Insect peptidyl-dipeptidase A [angiotensin I-converting enzyme (ACE)] is a soluble single-domain peptidyl-dipeptidase that has many properties in common with the C-domain of mammalian somatic ACE and with the single-domain mammalian germinal ACE. Mammalian somatic ACE is important in blood homoeostasis, but the role of ACE in insects is not known. Immunocytochemistry has been used to localize ACE in the neuroendocrine system of the locust, Locusta migratoria. Staining was observed in five groups of neurosecretory cells in the brain and suboesophageal ganglion, in the nervi corpori cardiaci, the storage part of the corpora cardiaca and in the nervi corpori allati. In three groups of neurosecretory cells, ACE co-localized with locustamyotropins, suggesting a possible role for the enzyme in the metabolism of these neuropeptides. We demonstrate in vitro a novel activity of ACE that removes pairs of basic amino acid residues from a locustamyotropin peptide extended at the C-terminus with either Gly-Lys-Arg or Gly-Arg-Arg, corresponding to a consensus recognition sequence for endoproteolysis of prohormone proteins by prohormone convertases. The low Km and high kcat values (Km 7.3 and 5.0 microM, kcat 226 and 207 s-1 for the hydrolysis of Phe-Ser-Pro-Arg-Leu-Gly-Lys-Arg and Phe-Ser-Pro-Arg-Leu-Gly-Arg-Arg, respectively) obtained for the hydrolysis of these two peptides by insect ACE means that these peptides, along with mammalian bradykinin, are the most favoured in vitro ACE substrates so far identified. The discovery of this in vitro prohormone-processing activity of insect ACE provides a possible explanation for the intracellular co-localization of the enzyme with locustamyotropin peptides, and provides evidence for a new role for ACE in the biosynthesis of peptide hormones and transmitters.
Project description:The reaction of holo-(D-glyceraldehyde 3-phosphate dehydrogenase) (EC 18.104.22.168) from rat skeletal muscle with [14C]butanedione in 0.05 M-NH4HCO3, pH 8.0, resulted in modification (*) of two arginine residues per subunit with a concomitant loss of catalytic activity. From a tryptic digest of the modified protein two radiolabelled peptides were isolated, with the following sequences: (1)Val-Ile-Ile-Asn-Ala-Pro-Thr-Ala-Asp-Ala(Glx,Met,Leu,Phe,Met)Gly-Val-Asx-Arg- Glx(His,Tyr)Ser-Lys and (2) Asp-Ala-Gly-Ala-Thr-Ile-Ala-Leu(Asx,Glx,Arg,Phe,Val)Lys. By comparison of the data with the known sequences of homologous enzymes, the localization of the modified residues was established. The first peptide was identified as corresponding to residues 116--139, the second to residues 293--306. Experimental evidence from this and previous studies suggests that arginine-134 is important for the catalytic activity of the rat muscle enzyme, being involved in structural rearrangements accompanying the organization of the active centre on the binding of coenzyme and substrate.
Project description:An adrenocorticotrophic hormone (ACTH) was isolated from extracts of the pars distalis of the pituitary of the dogfish Squalus acanthias by gel filtration and ion-exchange chromatography. It had 15% of the potency of human ACTH in promoting cortico-steroidogenesis in isolated rat adrenal cells. Sequence analysis revealed it to be a nonatria-contapeptide with the following primary structure: Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Met-Gly-Arg-Lys-Arg-Arg-Pro-Ile-Lys-Val-Tyr-Pro-Asn-Ser-Phe-Glu-Asp-Glu-Ser-Val-Glu-Asn-Met-Gly-Pro-Glu-Leu. The N-terminal tridecapeptide sequence was identical with the proposed structure of dogfish alpha-melanocyte-stimulating hormone (alpha-MSH). On comparison with human ACTH eleven amino acid differences were seen, nine of which are in the 20-39 region of the molecule which is not essential for the steroidogenic activity of ACTH. A peptide identical with the 18-39 portion of this new ACTH was similarly isolated from the neurointermediate lobe of the pituitary where considerable amounts of dogfish alpha-MSH were found. This supported our view that ACTH as well as having a distinct biological role of its own is also the precursor of alpha-MSH.
Project description:We have demonstrated that the S'(1) and S'(2) subsites of human tissue kallikrein (hK1) play determinant roles in the recognition and hydrolysis of substrates. The presence of serine at position P'(1) and arginine at P'(2) resulted in the best substrate, Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, which was derived from the kallistatin reactive-centre loop sequence and quencher groups o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp). Serine and arginine are also the residues at positions P'(1) and P'(2) in human kininogen, from which hK1 releases Lys-bradykinin. Several peptide analogues of Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, in which the Ser and Arg residues were substituted with various other amino acids, were synthesized and tested as substrates. Most of them were hydrolysed slowly, although they showed significant binding to hK1, as demonstrated by their competitive inhibition constants (K(i)). Using this information, six peptides were designed, synthesized and assayed as inhibitors of hK1. Abz-Lys-Phe-Phe-Pro-Arg-Gln-EDDnp, Abz-Lys-Phe-Arg-Pro-Arg-Gln-EDDnp and acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH(2) inhibited hK1 in the range 20-30 nM (letters in italics denote the D-form of the amino acid). The peptide acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH(2) was a weak inhibitor for other serine proteases, as indicated by the higher K (i) values compared with hK1, but this peptide was a potent inhibitor of human plasma kallikrein, which has a K (i) value of 8 nM. This result was surprising, since this enzyme is known to be a restricted arginyl-hydrolase. In conclusion, acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH(2) can be used as a leader compound to design specific inhibitors for hK1, plasma kallikrein, or for both at same time, if the inhibition of kinin release is the main goal.
Project description:Upon exposure to the bacterial chemotactic peptide fMet-Leu-Phe, human neutrophils release lysozyme and generate superoxide anions (O2.-). The synthetic lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3Cys), which is derived from the N-terminus of bacterial lipoprotein, when attached to Ser-(Lys)4 [giving Pam3Cys-Ser-(Lys)4], activated O2.- formation and lysozyme release in human neutrophils with an effectiveness amounting to about 15% of that of fMet-Leu-Phe. Palmitic acid, muramyl dipeptide, lipopolysaccharide and the lipopeptides Pam3Cys-Ala-Gly, Pam3Cys-Ser-Gly, Pam3Cys-Ser, Pam3Cys-OMe and Pam3Cys-OH did not activate O2.- formation. Pertussis toxin, which ADP-ribosylates guanine-nucleotide-binding proteins (G-proteins) and functionally uncouples formyl peptide receptors from G-proteins, prevented activation of O2.- formation by fMet-Leu-Phe and inhibited Pam3Cys-Ser-(Lys)4-induced O2.- formation by 85%. Lipopeptide-induced exocytosis was pertussis-toxin-insensitive. O2.- formation induced by Pam3Cys-Ser-(Lys)4 and fMet-Leu-Phe was enhanced by cytochalasin B, by a phorbol ester and by a diacylglycerol kinase inhibitor. Addition of activators of adenylate cyclase and removal of extracellular Ca2+ inhibited O2.- formation by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 to different extents. Pam3Cys-Ser-(Lys)4 synergistically enhanced fMet-Leu-Phe-induced O2.- formation and primed neutrophils to respond to the chemotactic peptide at non-stimulatory concentrations. Our data suggest the following. (1) Pam3Cys-Ser-(Lys)4 activates neutrophils through G-proteins, involving pertussis-toxin-sensitive and -insensitive processes. (2) The signal transduction pathways activated by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 are similar but not identical. (3) In inflammatory processes, bacterial lipoproteins and chemotactic peptides may interact synergistically to activate O2.- formation, leading to enhanced bactericidal activity.