ABSTRACT: Tryptic glycopeptides were purified from the sialic acid-free variant of ovomucoid, O1, and its CNBr fragments. The amino acid sequences adjacent to the four major sites of carbohydrate (Carb.) attachment were: (1), Phe-Pro-Asn(Carb.)-Ala-Thr-Asp-Lys-Glu-Gly-Lys; (2), Ala-Try-Ser-Ile-Glu-Phe-Gly-Thr-Asn (Carb.)-Ile-Ser-Lys; (3), Glu, Thr-Val-Pro-Met-Asn(Carb.)-cys-Ser; (4), Ser-Ser-Tyr-Ala-Asn (Carb.)-Thr-Thr-Ser-Glu-Asp-Gly-Lys, Glycosylated Asn residues were located at position 10, between residues 49 and 60, and at positions 69 and 75, in the primary sequence. All of these carbohydrate groups contained GlcNAc, Man and Gal in the approximate molar proprotions 5:3:0.5. A further glycopeptide containing His was isolated in low yield, suggesting that some carbohydrate is attached at a fifth site. Two of the carbohydrate-attachment sites (Asn-10 and Asn-75) occur in sequences that show internal homologies. These are presumed to have evolved as a consequence of partial gene duplication. Three of the carbohydrate-attachment sites occur in similar positions to the carbohydrate groups in quail ovomucoid [Laskowski (1976) Protides Biol. Fluids Proc. Colloq. 23, in the press]. Prediction of peptide conformation from the sequence data by the method of Chou & Fasman [(1974) Biochemistry 13, 222-225] indicated that four glycosylated Asn residues in hen ovomucoid are very close to groups of amino acids that occur with high frequency in beta-turns. The possible significance of peptide-chain conformation in the attachment of carbohydrate to glycoproteins is briefly discussed.
Project description:The holostean fishes are the extant representatives of the primitive ray-finned fishes from which the present-day teleosts may have evolved. The primary structure of insulin from a holostean fish, the bowfin (Amia calva), was established as: A-chain: Gly-Ile-Val-Glu-Gln-Cys-Cys-Leu-Lys-Pro-Cys-Thr-Ile-Tyr-Glu-Met-Glu- Lys-Tyr-Cys-Asn B-chain: Ala-Ala-Ser-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Phe-Leu- Val-Cys-Gly-Glu-Ser-Gly-Phe-Phe-Tyr-Asn-Pro-Asn-Lys-Ser This amino acid sequence contains several substitutions (methionine at A16, phenylalanine at B16 and serine at B22) at sites that have been strongly conserved in other vertebrate species and that may be expected to influence biological activity. Consistent with this prediction, bowfin insulin was approx. 14-fold less potent than pig insulin in inhibiting the binding of [125I-Tyr-A14](human insulin) to transfected mouse NIH 3T3 cells expressing the human insulin receptor.
Project description:An anti-epilepsy peptide (AEP) was isolated and purified from venom of the scorpion Buthus martensii Karsch. The purification procedure included CM-Sephadex C-50 chromatography, gel filtration on Sephadex G-50 and DEAE-Sephadex A-50 chromatography. Its homogeneity was demonstrated by pH 4.3 polyacrylamide-disc-gel electrophoresis, focusing electrophoresis and SDS/polyacrylamide-disc-gel electrophoresis. The Mr of this peptide, calculated from measurements in SDS/15%-polyacrylamide-disc-gel and SDS/20%-polyacrylamide-disc-gel electrophoresis, is 8300. The isoelectric point is 8.52 by pH 8-9.5-range isoelectric focusing. No haemorrhagic or toxic activities were found. No toxicity was found even after the dose reached 28 mg/kg. The pharmacological tests showed that the AEP had no effect on heart rate, blood pressure or electrocardiogram, but strongly inhibited epilepsy induced by coriaria lactone and cephaloridine. The fluorescence spectrum showed that the peptide has a strong emission peak at 337 nm. Amino acid analysis suggested that the AEP is composed of 66 residues from 18 amino acids and has an Mr of 8290. The sequence of the first 50 N-terminal residues is as follows: Asp-Gly-Tyr-Ile-Arg-Gly-Ser-Asp-Asn-Cys-Lys-Val-Ser-Cys-Leu-Leu-Gly-Asn- Glu-Gly - Cys-Asn-Lys-Glu-Cys-Arg-Ala-Tyr-Gly-Ala-Ser-Tyr-Gly-Tyr-Cys-Trp-Thr-Val- Lys-Leu - Ala-Gln-Asp-Cys-Glu-Gly-Leu-Pro-Asp-Thr-.
Project description:Two glycoforms of a secretable human thrombomodulin mutant [TMD1-105 and TMD1-75; Parkinson, Grinnell, Moore, Hoskins, Vlahos & Bang (1990) J. Biol. Chem. 265, 12602-12610] were expressed in human 293 cells and used to study the role of glycosylation in the functions of this endothelial-cell thrombin receptor. Carbohydrate content analysis and intrinsic labelling with [3H]glucosamine and [35S]sulphate showed that TMD1-105 contained a chondroitin sulphate whereas TMD1-75 did not. Other than chondroitin sulphate, the carbohydrate contents of the two glycoforms were identical, indicating similar glycosylation patterns at other O-linked and N-linked sites in the two glycoforms. The properties of TMD1-105 were converted into those of TMD1-75 by chondroitin ABC lyase digestion. Trypsin digestion of labelled TMD1-105 permitted isolation of two overlapping peptides that contained chondroitin sulphate, spanned the entire O-glycosylation domain and had O-glycosylation sites at Ser-492, Ser-498, Thr-500, Thr-504 and Thr-506. The chondroitin sulphate-attachment site was assigned to Ser-492 as this residue is conserved in mouse and bovine thrombomodulin and lies within a sequence Ser-Gly-Ser-492-Gly-Glu-Pro, which has strong similarity to chondroitin sulphate attachment sites in other proteoglycans. Five peptides with N-linked carbohydrate were also isolated and contained glycosylation sites in the lectin-like domain (Asn-47, Asn-115, Asn-116) and in the fourth (Asn-382) and fifth (Asn-409) epidermal growth factor domains. The role of N-linked and simple O-linked carbohydrates in the functions of human thrombomodulin remain unclear. The present studies demonstrate, however, that the presence of chondroitin sulphate in human thrombomodulin has profound effects on all of the anticoagulant properties of this important anticoagulant thrombin receptor.
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: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:Glycopeptides have been isolated from tryptic digests of kappa-type light chains separated from human myeloma proteins obtained from the serum of two patients, Car and Rai. The glycopeptides are derived from the variable region of the chain in both cases, but from different sections. On the basis of homology it is deduced that glycopeptide from Car, kappaI type, is derived from position 25-31 whereas that from Rai, kappaII type, is from position 62-77, their sequences being respectively Ala-Ser-Gln-Asn-Ile-Ser and Phe-Ser-Gly-Ser-Gly-Ser-Gly(Thr,Asp)Phe-Thr-Leu-Asx-Ile-Ser-Arg. The significance of the results is discussed in connexion with the nature of the attachment site of carbohydrate to protein.
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: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:We report on amino acid substitutions in the quinolone resistance-determining region of type II topisomerases and the prevalence of reserpine-inhibited efflux for 70 clinical isolates of S. pneumoniae for which the ciprofloxacin MIC is >/=4 microgram/ml and 28 isolates for which the ciprofloxacin MIC is </=2 microgram/ml. The amino acid substitutions in ParC conferring low-level resistance (MICs, 4 to 8 microgram/ml) included Phe, Tyr, and Ala for Ser-79; Asn, Ala, Gly, Tyr, and Val for Asp-83; Asn for Asp-78; and Pro for Ala-115. Isolates with intermediate-level (MICs, 16 to 32 microgram/ml) and high-level (MICs, 64 microgram/ml) resistance harbored substitutions of Phe and Tyr for Ser-79 or Asn and Ala for Asp-83 in ParC and an additional substitution in GyrA which included either Glu-85-Lys (Gly) or Ser-81-Phe (Tyr). Glu-85-Lys was found exclusively in isolates with high-level resistance. Efflux contributed primarily to low-level resistance in isolates with or without an amino acid substitution in ParC. The impact of amino acid substitutions in ParE was minimal, and no substitutions in GyrB were identified.
Project description:BACKGROUND:Phomafungin is a recently reported broad spectrum antifungal compound but its biosynthetic pathway is unknown. We combed publicly available Phoma genomes but failed to find any putative biosynthetic gene cluster that could account for its biosynthesis. RESULTS:Therefore, we sequenced the genome of one of our Phoma strains (F3723) previously identified as having antifungal activity in a high-throughput screen. We found a biosynthetic gene cluster that was predicted to synthesize a cyclic lipodepsipeptide that differs in the amino acid composition compared to Phomafungin. Antifungal activity guided isolation yielded a new compound, BII-Rafflesfungin, the structure of which was determined. CONCLUSIONS:We describe the NRPS-t1PKS cluster 'BIIRfg' compatible with the synthesis of the cyclic lipodepsipeptide BII-Rafflesfungin [HMHDA-L-Ala-L-Glu-L-Asn-L-Ser-L-Ser-D-Ser-D-allo-Thr-Gly]. We report new Stachelhaus codes for Ala, Glu, Asn, Ser, Thr, and Gly. We propose a mechanism for BII-Rafflesfungin biosynthesis, which involves the formation of the lipid part by BIIRfg_PKS followed by activation and transfer of the lipid chain by a predicted AMP-ligase on to the first PCP domain of the BIIRfg_NRPS gene.