The motilin pharmacophore in CHO cells expressing the human motilin receptor

We performed a structure-activity study with the human motilin receptor, which was recently cloned from thyroid tissue. N-terminal fragments, Ala-analogs of motilin, and motilides were tested in a cell line that expresses the cloned human motilin receptor and apoaequorin. Full potency to induce calcium fluxes was obtained with N-terminal fragments of 14 amino acids. Motilin fragments 1-14 in which residues 1 (Phe), 4 (Ile), and 7 (Tyr) were replaced by Ala showed the largest reduction in potency. Only motilides with an enol configuration had markedly higher potencies compared to erythromycin A. The potencies to induce Ca(2+) fluxes correlated strongly with rabbit binding and contractility data, suggesting that the cloned receptor is indeed the motilin receptor, responsible for contractile effects. Conservation of the motilin pharmacophore in evolution indicates an important physiological role of motilin.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

Identification of amino acid residues that direct differential ligand selectivity of mammalian and nonmammalian V1a type receptors for arginine vasopressin and vasotocin. Insights into molecular coevolution of V1a type receptors and their ligands

Arginine vasotocin (VT) is the ortholog in all nonmammalian vertebrates of arginine vasopressin (AVP) in mammals. We have previously cloned an amphibian V1atype vasotocin receptor (VT1R) that exhibited higher sensitivity for VT than AVP, while the mammalian V1a type receptor (V1aR) responded better to AVP than VT. In the present study, we identified the amino acid residues that confer differential ligand selectivity for AVP and VT between rat V1aR and bullfrog VT1R (bfVT1R). A chimeric rat V1aR having transmembrane domain (TMD) VI to the carboxyl-terminal tail (C-tail) of bfVT1R showed a reverse ligand preference for AVP and VT, whereas a chimeric VT1R with TMD VI to the C-tail of rat V1aR showed a great increase in sensitivity for AVP. A single mutation (Ile(315(6.53)) to Thr) in TMD VI of V1aR increased the sensitivity for VT, while a single mutation (Phe(313(6.51)) to Tyr or Pro(334(7.33)) to Thr) reduced sensitivity toward AVP. Interestingly the triple mutation (Phe(313(6.51)) to Tyr, Ile(6.53) to Thr, and Pro(7.33) to Thr) of V1aR increased sensitivity to VT but greatly reduced sensitivity to AVP, behaving like bfVT1R. Further, like V1aR, a double mutant (Tyr(306(6.51)) to Phe and Thr(327(7.33)) to Pro) of bfVT1R showed an increased sensitivity to AVP. These results suggest that Phe/Tyr(6.51), Ile/Thr(6.53), and Pro/Thr(7.33) are responsible for the differential ligand selectivity between rat V1aR and bfVT1R. This information regarding the molecular interaction of VT/AVP with their receptors may have important implications for the development of novel AVP analogs.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Express analysis of protein amino acid sequences. Primary structure of Penicillium chrysogenum 152A guanyl-specific ribonuclease

The complete amino acid sequence of Penicillium chrysogenum 152A guanyl-specific RNase has been established using automated Edman degradation of two non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the protein. The RNase contains 102 amino acid residues: His2, Arg3, Asp7, Asn8, Thr5, Ser11, Glu4, Gln2, Pro4, Gly11, Ala13, Cys4, Val8, Ile3, Leu3, Tyr9, Phe5 (Mr 10 747).     

Identification of mouse MD-2 residues important for forming the cell surface TLR4-MD-2 complex recognized by anti-TLR4-MD-2 antibodies,and for conferring LPS and taxol responsiveness on mouse TLR4 by alanine-scanning mutagenesis

The expression of MD-2, which associates with Toll-like receptor (TLR) 4 on the cell surface, confers LPS and LPS-mimetic Taxol responsiveness on TLR4. Alanine-scanning mutagenesis was performed to identify the mouse MD-2 residues important for conferring LPS and Taxol responsiveness on mouse TLR4, and for forming the cell surface TLR4-MD-2 complex recognized by anti-TLR4-MD-2 Ab MTS510. Single alanine mutations were introduced into mouse MD-2 (residues 17-160), and the mutants were expressed in a human cell line expressing mouse TLR4. Mouse MD-2 mutants, in which a single alanine mutation was introduced at Cys37, Leu71, Leu78, Cys95, Tyr102, Cys105, Glu111, Val113, Ile117, Pro118, Phe119, Glu136, Ile138, Leu146, Cys148, or Thr152, showed dramatically reduced ability to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510, and the mutants also showed reduced ability to confer LPS and Taxol responsiveness. In contrast, mouse MD-2 mutants, in which a single alanine mutation was introduced at Tyr34, Tyr36, Gly59, Val82, Ile85, Phe126, Pro127, Gly129, Ile153, Ile154, and His155 showed normal ability to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510, but their ability to confer LPS and Taxol responsiveness was apparently reduced. These results suggest that the ability of MD-2 to form the cell surface mouse TLR4-mouse MD-2 complex recognized by MTS510 is essential for conferring LPS and Taxol responsiveness on TLR4, but not sufficient. In addition, the required residues at codon numbers 34, 85, 101, 122, and 153 for the ability of mouse MD-2 to confer LPS responsiveness are partly different from those for Taxol responsiveness.     

Amino acid sequence of human plasma apolipoprotein C-II from normal and hyperlipoproteinemic subjects

The complete amino acid sequence of human plasma apolipoprotein C-II isolated from normal individuals and a subject with type V hyperlipoproteinemia has been determined. Apo-C-II contains 79 amino acids with the following amino acid composition: Asp4, Asn1, Thr9, Ser9, Glu7, Gln7, Pro4, Gly2, Ala6, Val4, Met2, Ile1, Leu8, Tyr5, Phe2, Lys6, Arg1, Trp1. Cleavage of apo-C-II by cyanogen bromide produced three peptides designated as CB-1 (9 residues), CB-2 (51 residues), and CB-3 (19 residues). Two peptides, CT-1 (50 residues) and CT-2 (29 residues), which overlapped the cyanogen bromide peptides, were obtained by tryptic digestion of citraconylated apo-C-II at the single arginine residue. The amino acid sequence of apo-C-II was obtained by the automated phenyl isothiocyanate degradation of intact apo-C-II and the peptides produced by cleavage of apo-C-II by cyanogen bromide and trypsin. Phenylthiohydantoins were identified by high performance liquid chromatography and chemical ionization-mass spectrometry. The amino acid sequence of apo-C-II from the normal subject was identical with the apo-C-II isolated from the hyperlipoproteinemic subject.     

Localization of the HIV-1 gp120 Conformational Epitope Recognized by Virus-Neutralizing Monoclonal Antibodies 2G12

A phage peptide library was used to select peptides interacting with virus-neutralizing monoclonal antibodies (mAb) 2G12 which recognize a discontinuous surface epitope of HIV-1 gp120. With the published X-ray data, gp120 regions involved in the antigenic determinant were predicted. Binding with mAb 2G12 was ascribed to Thr297, Phe383, Tyr384, Arg419, Ile240, Thr415, Leu416, Pro417, Lys421, and Trp112. Though distant in the gp120 sequence, these residues are close in space and form the 2G12 epitope on the gp120 surface.     

The erythromycin derivative EM-523 is a potent motilin agonist in man and in rabbit

Erythromycin may stimulate gastrointestinal motor activity via its effect upon motilin receptors. We have studied the ability of the derivative EM-523 [de(N-methyl)-N-ethyl-8,9-anhydroerythromycin A 6,9-hemiacetal] to induce contractions in duodenal smooth muscle strips and to displace labeled motilin bound to antral smooth muscle, in man and in rabbit. In both species EM-523 approached the potency of motilin for inducing contractions. Thus pED50 values were 7.84 +/- 0.11 and 8.69 +/- 0.12 for motilin in, respectively, man and rabbit, against 6.08 +/- 0.13 and 8.19 +/- 0.10 for EM-523. In rabbit the efficacy of both compounds decreased in parallel aborally, the responses to EM-523 could not be blocked by atropine (10(-7) M) or TTX (10(-7) M), and both compounds were unable to further enhance the maximum effect to the other compound. In binding studies the order of potency was the same as in the contraction studies. The pIC50 values were: motilin (8.84 +/- 0.31, 9.17 +/- 0.20) greater than EM-523 (7.89 +/- 0.1, 8.40 +/- 0.10). A Schild plot revealed that EM-523 was a competitive inhibitor of motilin receptor binding in man and in rabbit. We conclude that EM-523 is a potent motilin agonist.     

Isolation of Tryptic Peptides from the lie Chain of Ricin D and the Sequences of Some Tryptic Peptides Including N- and C-Terminal Peptides

Twenty tryptic peptides were isolated from the performic acid-oxidized He chain of ricin D by Dowex 1 × 2 column chromatography followed by paper chromatography. The amino acids contained in these peptides accounted for 218 out of 266 residues in the whole protein. The amino acid sequences of nine peptides were determined by manual liquid phase or automatic solid phase Edman degradation, and N- and C-terminal sequences of the He chain of ricin D were established to be NH₂–Ile–Phe–Pro–Lys–Gln–Tyr–Pro–Ile–Ile– and Cys–Ala–Pro–Pro–Pro–Ser–Ser–Gln–Phe, respectively.     

Comparison of spantide II and CP-96,345 for blockade of tachykinin-evoked contractions of smooth muscle.

CP-96,345, a quinuclidine, is a potent inhibitor of substance P for the NK1 receptor of bovine brain, but has reduced potency for the corresponding receptor of the rat and mouse, and none for NK2 or NK3 receptors. A related quinuclidine showed similar but lower potency than CP-96,345 for NK1. CP-96,345 was more potent than the spantide I of 1984, D-Arg1,Pro2,Lys3,Pro4,Gln5,Gln6,D-Trp7,Phe8,D-Trp9, Leu10,Leu11,NH2. Our continued designs for antagonists of substance P led to spantide II in 1990 which is: D-NicLys1,Pro2,3-Pal3,Pro4,D-Cl2Phe5,Asn6,D-Trp7 ,Phe8,D-Trp9,Leu10,Nle11-NH2. The pA2 values of spantide II and CP-96,345 for guinea pig taenia coli were 7.6 and 6.8, respectively. The pIC50 values for blockade of tachykinin-mediated neurotransmission in the rabbit iris sphincter were 6.1 and 5.4, respectively. Spantide II was nearly 10 times more potent than CP-96,345 in these two assays.     

Conformational transitions of human serum albumin depending on pH. Study using tritium markers]

Accessible surfaces of the HSA molecule in N-, F- and B-forms were studied in the present work by tritium labelling method which allowed to obtain detailed information on N-F- and N-B-transitions. In was shown that the F-form in comparison top the N-form is characterized by more high accessibility of Ser, Ala, Ile, Tyr, Phe, His, Arg, Pro, Val and Phe residues and in the B-form Tyr, Ser, Arg, Gly, Ile, Phe and Pro residues turn to be highly accessible. Full accessible surfaces of protein molecule at N-F- and N-B-transitions increase respectively from 39,000 to 70,400 A2 and from 39,000 to 47,000 A2. Basing on the prevailing increase of hydrophobic residues accessibility it is supposed that the molecule expansion testifies the separation of the subunits forming the molecule.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

Evaluation of essential and variable residues of nukacin ISK-1 by NNK scanning

Nukacin ISK-1, a type-A(II) lantibiotic, comprises 27 amino acids with a distinct linear N-terminal and a globular C-terminal region. To identify the positional importance or redundancy of individual residues responsible for nukacin ISK-1 antimicrobial activity, we replaced the native codons of the parent peptide with NNK triplet oligonucleotides in order to generate a bank of nukacin ISK-1 variants. The bioactivity of each peptide variant was evaluated by colony overlay assay, and hence we identified three Lys residues (Lys1, Lys2 and Lys3) that provided electrostatic interactions with the target membrane and were significantly variable. The ring structure of nukacin ISK-1 was found to be crucially important as replacing the ring-forming residues caused a complete loss of bioactivity. In addition to the ring-forming residues, Gly5, His12, Asp13, Met16, Asn17 and Gln20 residues were found to be essential for antimicrobial activity; Val6, Ile7, Val10, Phe19, Phe21, Val22, Phe23 and Thr24 were relatively variable; and Ser4, Pro8, His15 and Ser27 were extensively variable relative to their positions. We obtained two variants, Asp13Glu and Val22Ile, which exhibited a twofold higher specific activity compared with the wild-type and are the first reported type-A(II) lantibiotic mutant peptides with increased potency.     

Effects of orally administrated amino acids on myofibrillar proteolysis in chicks

We examined the effects of orally administrated amino acids on myfibrillar proteolysis in food-deprived chicks. Plasma N(tau)-methylhistidine concentration, as an index of myofibrillar proteolysis, was decreased by the administration of Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg but not by Asp, Val, Phe, Tyr or His to chicks. Orally administrated Cys was fatal to chicks. These results indicate that oral Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg administration suppressed myofibrillar proteolysis in chicks.     

Restricted rotation in chiral peptide nucleic acid (PNA) monomers--influence of substituents studied by means of 1H NMR

The influence of amino acid side chains [derived from: Ala, Val, Leu, Ile, Phe, Tyr(Bzl), Ser(Bzl), Thr(Bzl), Pro, Trp], incorporated into "aminoalkyl" part of PNA monomers, on the temperature-dependent distributions of rotamers about the tertiary amide bond was studied by means of 1H NMR at 0, 25 and 40 degrees C in CDCl3. The delta G0 values of the energy differences between individual rotamers were calculated. The results may be helpful in the designing of monomers with desirable properties.     

In vitro evolution of amphioxus insulin-like peptide to mammalian insulin

By site-directed mutagenesis, six insulin residues related to the insulin-receptor interaction were grafted, partially or fully, onto the corresponding position of a recombinant amphioxus insulin-like peptide (ILP) that contained the A- and B-domains of the deduced amphioxus ILP. After fermentation, purification, and enzymatic cleavage, six insulin-like double-chain ILP analogues were obtained: [A2Ile]ILP, [B12Val, B16Tyr]ILP, [B25Phe]ILP, [A2Ile, B12Val, B16Tyr, B25Phe]ILP (four-mutated ILP), [A2Ile, B12Val, B16Tyr, B24Phe, B25Phe]ILP (five-mutated ILP), and [A2Ile, B12Val, B16Tyr, B24Phe, B25Phe, B26Tyr]ILP (six-mutated ILP). Circular dichroism analysis showed that such replacement did not significantly affect their secondary and tertiary structure compared with that of the wild-type ILP. The insulin-receptor-binding activity of the four-, five-, and six-mutated ILP was 0.14%, 11%, and 11% of native insulin, respectively; the other three ILP analogues acquired none of the detectable insulin-receptor-binding potency. The growth-promoting activities of the five- and six-mutated ILP were both about 50% of native insulin, while that of the wild-type ILP was not detectable. By structure-function-based mutagenesis, the completely inactive amphioxus ILP was converted into a molecule with moderate mammalian insulin activity. These results indicated the following: first, the grafted as well as those inborn insulin-receptor-binding related residues can form an insulin-receptor-binding patch on the ILP analogues; second, the ILP can be used as a scaffold molecule to investigate the role of the insulin residues; third, the natural evolution of amphioxus ILP to mammalian insulin is a possible process and can be mimicked in the laboratory.     

Lysozyme modification by the fenton reaction and gamma radiation

A comparative study was performed on lysozyme modification after exposure to Fenton reagent (Fe(II)/H2 O2) or hydroxyl radicals produced by y radiation. The conditions were adjusted to obtain, with both systems, a 50% loss of activity of the modified ensemble. Gamma radiation modified almost all types of amino acid residues in the enzyme, with little specificity. The modification order was Tyr > Met = Cys > Lys > Ile + Leu > Gly > Pro = Phe > Thr + Ala > Trp = Ser > Arg > Asp + Glu, with 42 mol of modified residues per initial mole of native enzyme. In contrast, when the enzyme was exposed to the Fenton reaction, only some types of amino acids were modified. Furthermore, a smaller number of residues (13.5) were damaged per initial mole of enzyme. The order of the modified residues was Tyr > Cys > Trp > Met His > Ile + Leu > Val > Arg. These results demonstrate that the modifications elicited by these two free radical sources follow different mechanisms. An intramolecular free radical chain reaction is proposed to play a dominant role in the oxidative modification of the protein promoted by gamma radiation.