Uptake and degradation of iodine-labelled chylomicron remnant particles by monolayers of rat hepatocytes.

1. Rat chylomicrons were labelled with 125I with 69--72% of the iodine in the protein moiety. Less than 1 nmol of iodine was incorporated per nmol of protein. Of the peptide radioactivity 44--56% was in apolipoprotein A-1, 30--40% in the C peptides and 11--15% in apolipoprotine B. The arginine-rich peptide, which accounted for about 14% of the chylomicron protein mass as determined by scanning of sodium dodecyl sulphate-polyacrylamide gels, contained very little radioactivity. 2. Chylomicron remnants generated with postheparin plasma from iodine-labelled chylomicrons showed a relative increase in the percentage of the arginine-rich peptide (76--90% of the apolipoprotein mass according to gel scanning). The major portion of the peptide iodine label was present in apolipoprotein A-1 (43--57%), B (22--32%) and C peptides (17--35%). 3. When iodine-labelled chylomicron remnants were added to rat hepatocytes in primary culture, labelled peptides were taken up and degraded by the hepatocytes by a saturable process. The Vmax. for the uptake was calculated to the 300ng of protein/h per mg of cell protein and the apparent Km as 7.7 microgram of protein/mg of cell protein. A larger proportion of the 125I-labelled lipids of the remnants (mainly polar lipids) was taken up. This suggest that these may also enter the cells by a mechanism that does not involve particulate uptake, such as phospholipid exchange. 4. The degradation of labelled peptides was inhibited by colchicine, concanavalin A, chloroquine and NH4Cl, which also inhibit degradation of the cholesteryl ester portion. All these drugs exerted their inhibition mainly after the uptake of labelled peptide. No degradation occurred at 4 degrees C, and also the uptake was markedly decreased. 5. The uptake of labelled chylomicron remnant peptide was 77 times as effective as that of labelled sucrose, which is likely to be taken up randomly by pinocytosis.     

Apolipoprotein A-II: chemical synthesis and biophysical properties of three peptides corresponding to fragments in the amino-terminal half.

Three peptide fragments of apolipoprotein A-II corresponding to residues 17--31, 12--31, and 7--31 have been synthesized by solid-phase techniques and purified to apparent homogeneity. Each of these fragments contains residues 18--30, a region previously proposed to possess potential amphipathic helical properties. Secondary structural changes of these synthetic fragments accompanying their interaction with phospholipid have been studied by circular dichroism. The magnitude of this interaction has been evaluated from the yields and stoichiometry of lipid-protein complexes isolated by density gradient ultracentrifugation. Fragment 17--31, the smallest peptide containing the proposed amphipathic helix, did not interact with dimyristoylphosphatidylcholine (DMPC) single bilayer vesicles at 24 degrees C; upon addition of DMPC, no ellipticity change could be detected nor could a stable lipid-peptide complex be isolated. However, fragments 12--31 and 7--31 did interact with phosphilipid; in the absence of lipid, both fragments had primarily disordered structures, but when isolated as DMPC-peptide complexes, both fragments possessed increased helical structure. The phospholipid:peptide molar ratio was 14:1 for fragment 12--31 and 27:1 for fragment 7--31. Studies of space-filling models of these fragments suggest that hydrophobicity and/or length are important properties of phospholipid binding apoproteins.     

In vitro lipid metabolism in the rat pancreas. II. Effects of secretagogues on fatty acid metabolism, net lipolysis and ATP levels.

1. The concentration of carbamylcholine, bombesin, pancreozymin, pentagastrin and secretin evoking a similar 4--5-fold maximal increase in amylase secretion from rat pancreatic fragments were 3.10(-6), 10(-7), 10(-8), 3.10(-6), and 3.10(-6) M, respectively. The maximal concentration of vasoactive intestinal peptide tested (3.10(-6) M) increased amylase secretion by 250%. The six secretagogues could be separated into two groups according to their effects on lipid metabolism and ATP levels. 2. When used at their optimal concentrations, carbamylcholine, bombesin, pancreozymin, and pentagastrin lowered pancreatic ATP levels by 18-26% and increased net release of free fatty acids by 68-105%. 3. The effects of 3.10(-6) M carbamylcholine and 10(-8) M pancreozymin on the metabolism of 3H2O, D-[U-14C]glucose and [1-14C]acetate were similar; the incorporation of radioactivity in the fatty acid moiety of glycerolipids decreased by 20--50% whereas the incorporation of 3H from 3H2O and of 14C from [U-14C]glucose increased by 20--35% in the glycerol moiety. In addition, the oxidation of [U-14C]glucose, [1-14C]acetate and [1-14C]palmitate to 14CO2 increased by 15--32% while the esterification of [1-14C]palmitate, [1-14C]-linoleate, and [1-14C]arachidonate was inhibited by 14--23%. The spectrum of fatty acids labeled with [1-14C]acetate indicated an inhibition of the malonic acid pathway whereas the elongation of polyenoic fatty acids was unaltered.     

Location of the antigenic determinants of bovine pancreatic ribonuclease.

Four antigenic regions of native bovine pancreatic ribonuclease have been located by using antibodies that react specifically with segments 1--13, 31--79, and 80--124. These antibodies were purified by affinity chromatography on columns to which these peptide segments were bound. Analysis of precipitin curves indicates that there are at least three antigenic determinants to which antibody molecules can bind simultaneously in the presence of excess antibodies. Analysis of binding data, however, for each purified specific antibody preparation, carried out by the method of Berzofsky et al. [Berzofsky, J. A., Curd, J. G., & Schechter, A. N. (1976) Biochemistry, 15, 2113], leads to an estimate of four for the number of antigenic determinants in ribonuclease; this estimate had also been made earlier by Stelos et al. [Stelos, P., Fothergill, J. E., & Singer, S. J. (1960) J. Am. Chem. Soc. 82, 6034]. We find that one determinant is associated with each of segments 1--13 and 80--124 and two with segment 31--79. No antigenic activity could be detected for segment 14--29 either in native ribonuclease or in the free fragment. These conclusions are based on (1) the use of specific peptides to isolate purified antibodies by affinity chromatography, (2) immunoprecipitation of an antigenic peptide from the peptic digest of ribonuclease, (3) competitive inhibition studies with various peptide and protein fragments [cyanogen bromide fragments 1--13, 31--79, and 80--124, the tryptic peptides 40--61 and 105--224, S-peptide, S-protein, and des(121--124)-RNase], and (4) comparison and evaluation of the published effects on antigenicity of chemical and enzymatic modifications and changes in sequence among homologous ribonucleases. These approaches provide evidence that the four antigenic determinants are localized around the alpha-helical portion of segment 1--10, somewhere in segment 40--61, at the beta bend in segment 63--75, and either at the beta bend or beta sheet in segment 87--104 of native ribonuclease.     

Conformational analysis of a potent SSTR3-selective somatostatin analogue by NMR in water solution.

The three-dimensional structure of a potent SSTR3-selective analogue of somatostatin, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-Agl(8)(N(beta) Me, 2-naphthoyl)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-Agl(8)(N(beta) Me, 2-naphthoyl)]-SRIF) (peptide 1) has been determined by (1)H NMR in water and molecular dynamics (MD) simulations. The peptide exists in two conformational isomers differing mainly by the cis/trans isomerization of the side chain in residue 8. The structure of 1 is compared with the consensus structural motifs of other somatostatin analogues that bind predominantly to SSTR1, SSTR2/SSTR5 and SSTR4 receptors, and to the 3D structure of a non-selective SRIF analogue, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-2Nal(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-2Nal(8)]-SRIF) (peptide 2). The structural determinant factors that could explain selectivity of peptide 1 for SSTR3 receptors are discussed.     

Formation of complement subcomponent C1q-immunoglobulin G complex. Thermodynamic and chemical-modification studies.

The interaction between the complement subcomponent C1q and immunoglobulin G was investigated under a variety of experimental conditions. Formation of the subcomponent C1q--immunoglobulin G complex was shown to be an equilibrium process. Thermodynamic studies of the effect of varying the ionic strength indicate that over the salt range 0.15--0.225 M-NaCl the binding of subcomponent C1q to immunoglobulin aggregates releases 9--12 salt ions (Na+ and/or Cl-), illustrating the importance of ionic interactions for the formation of the complex. The effects of small peptide and organic ion inhibitors support this conclusion. Chemical modifications of carboxylate residues on immunoglobulin G by glycine ethyl ester/water-soluble carbodi-imide (up to 12 residues modified per whole molecule of immunoglobulin G) and of lysine residues by acetic anhydride (3 residues per whole molecule of immunoglobulin G) or methyl acetimidate (19 residues per whole molecule of immunoglobulin G) lowered the binding affinity of immunoglobulin for subcomponent C1q. Modification of arginine residues by cyclohexane-1,2-dione-1,2 (14 residues per whole molecule of immunoglobulin G) and of tryptophan by hydroxynitrobenzyl bromide (2 residues per whole molecule of immunoglobulin G), however, had little or no effect. The results are consistent with the proposal that the subcomponent-C1q-binding site on immunoglobulin G is to be found on the last two beta-strands of the Cv2 domain [Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek (1980) Nature (London) 288, 338--344].     

Processing and presentation of insulin. II. Evidence for intracellular, plasma membrane-associated and extracellular degradation of human insulin by antigen-presenting B cells

To study the biochemistry of processing of a soluble protein Ag by an APC, we investigated how 125I-labeled human insulin (HI) is processed in situ by TA3 mouse hybridoma B cells. Fractionation of TA3 cells into their extracellular, plasma membrane-associated and intracellular compartments coupled with the use of HPLC enabled us to analyze several peptides derived from each compartment. One HI peptide found in all three compartments is composed of residues A1-A14 disulfide-linked to B7-B26 (A1-A14/B7-B26). The presence of this peptide in the extracellular compartment likely resulted from digestion of HI by an enzyme(s) released from the APC. Extracellular processing of radiolabeled HI was inhibited completely by unlabeled HI and N-ethylmaleimide, an inhibitor of a previously described insulin-specific protease, partially by lysozyme but not by BSA or OVA. This suggests that the enzyme involved in the extracellular processing of insulin is relatively insulin-specific and gives rise to the A1-A14/B7-B26 peptide. The processing of HI both at the plasma membrane and intracellularly was inhibited by chloroquine, monensin, and NH4Cl, suggesting that both intracellular pH changes and endocytic and exocytic events may be required for these compartments to process insulin. Kinetic analyses revealed that the processing of insulin into the A1-A14/B7-B26 peptide is first detected at the plasma membrane then intracellularly and finally in the extracellular compartment. This unlabeled A1-A14/B7-B26 peptide was purified from the extracellular compartment of TA3 APC by HPLC; when presented by TA3 APC this peptide effectively stimulated pork insulin (PI/I-Ad) specific Th cells to secrete IL-2. These data, taken together with the identification of another processed insulin peptide, A7-A11/B7-B26, have enabled us to elucidate the first steps in the biochemical pathway(s) of processing of insulin as an Ag in a B cell APC.     

Determination of the structure of a membrane-incorporated ion channel. Solid-state nuclear magnetic resonance studies of gramicidin A.

Solid-state nuclear magnetic resonance (NMR) measurements on 13C-labeled analogues of the ion channel-forming peptide, gramicidin A, have been used to directly determine the structure of this peptide in lipid membranes. Seven gramicidin analogues, each labeled in a single carbonyl group of gly2, L-ala3, D-leu4, L-val7, D-leu10, D-leu12, or D-leu14 were synthesized by the solid-phase method. These gramicidin analogues were incorporated into aligned multilayers of dimyristoylphosphatidylcholine, or diether lipid bearing 14- or 16-carbon chains, at a 1:15 peptide:lipid mole ratio. Proton-enhanced, 13C, solid-state spectra were obtained at several temperatures and over a range of sample orientations with respect to the spectrometer magnetic field to permit accurate measurement of the chemical shift anisotropies. The observed anisotropies indicate that all of the labeled carbonyl bonds are oriented almost parallel to the molecular long axis and perpendicular to the lipid bilayer plane. These orientations are consistent with gramicidin forming a beta 6.3 single-strand helix that is oriented parallel to the methylene chains of the lipid molecules. Comparison of the linewidths from labeled residues that are in the innermost turn of the helix (gly2, ala3, and D-leu4), in the center of the molecule (val7), and in the turn nearest the lipid bilayer surface (D-leu10, D-leu12, and D-leu14) suggests that although the peptide behaves largely as a rigid barrel, segments of the peptide close to the membrane surface possess greater motional freedom.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ab initio modeling of small, medium, and large loops in proteins.

This study presents different procedures for ab initio modeling of peptide loops of different sizes in proteins. Small loops (up to 8--12 residues) were generated by a straightforward procedure with subsequent "averaging" over all the low-energy conformers obtained. The averaged conformer fairly represents the entire set of low-energy conformers, root mean square deviation (RMSD) values being from 1.01 A for a 4-residue loop to 1.94 A for an 8-residue loop. Three-dimensional (3D) structures for several medium loops (20--30 residues) and for two large loops (54 and 61 residues) were predicted using residue-residue contact matrices divided into variable parts corresponding to the loops, and into a constant part corresponding to the known core of the protein. For each medium loop, a very limited number of sterically reasonable C(alpha) traces (from 1 to 3) was found; RMSD values ranged from 2.4 to 5.9 A. Single C(alpha) traces predicted for each of the large loops possessed RMSD values of 4.5 A. Generally, ab initio loop modeling presented in this work combines elements of computational procedures developed both for protein folding and for peptide conformational analysis.     

The role of the angiotensin AT2 receptor on the diurnal variations of nociception and motor coordination in rats

Phasic pain demonstrates significant diurnal variation in rats. Angiotensin II modulates pain transmission and the diurnal variation in nociception in several rodent pain models. The participation of AT2 receptors in the diurnal regulation of nociception is not yet elucidated. In the present study we investigated the effects of selective peptide AT2 agonist CGP 42112A and the nonpeptide AT2 receptor antagonist PD 123319 on the nociception, motor coordination and arterial blood pressure. Male Wistar 12 weeks old rats were used. CGP 42112A was injected at single doses of 1 and 5 μg/rat intracerebroventricularly (ICV) and infused chronically ICV at a dose of 12 μg/rat/day during 14 days by osmotic minipumps. PD123319 was injected at single doses of 1 and 5 μg/rat, ICV and chronically subcutaneously at a dose of 10 mg/kg/day/14 days. Nociception was assessed by an analgesimeter, arterial blood pressure (ABP) was measured by tail cuff method, and motor coordination by Rota-rod method. Single doses of CGP 42112A (1 and 5 μg/rat) provoked a short lasting antinociception. Unlike acute injection, chronic CGP 42112A infusion increased nociception at the beginning and the end of light phase thus attenuating the diurnal variations observed in the controls. Moreover, it produced an increase of ABP and improved motor coordination. Both acute (1 μg/rat) and chronic PD 123319 treatment resulted in a decrease of pain threshold and chronic treatment attenuated its diurnal fluctuation. Our data support a role for Ang II type 2 receptors in the control of diurnal variations of nociception in rats.     

Semisynthetic analogs of cytochrome c reconstructed from natural and synthetic peptides.

A biologically active semisynthetic hybrid of horse heart cytochrome c has been prepared by combining the heme peptide 1 through 65 (HP 1-65), prepared by CNBr cleavage of natural cytochrome c, with a semisynthetic peptide corresponding to positions 66 through 104. A fully protected synthetic peptide 66--79 was prepared by a modified solid phase peptide synthesis procedure and was converted to its N-hydroxysuccinimide ester. A peptide corresponding to residues 81--104 of cytochrome c was also isolated from the CNBr cleavage mixture and its epsilon-amino groups and tyrosyl hydroxyl group were protected selectively with the t-butyloxycarbonyl group. This partially protected peptide was reacted with t-butyloxycarbonyl methionine N-hydroxysuccinimide ester to give a derivative having methionine at position 80. This product was deprotected, purified and then t-butyloxycarbonyl groups were again introduced specifically on the epsilon-amino groups to give the peptide, Boc(Lys,Tyr)80--104. A semisynthetic peptide corresponding to residues 66 through 104 of cytochrome c was prepared by condensing the synthetic peptide 66--79 N-hydroxysuccinimide ester with t-butyloxycarbonyl (Lys,Tyr)80--104. The semisynthetic product was deprotected, purified and combined under anaerobic conditions with a heme peptide, HP 1-65, that was isolated from the products of CNBr cleavage of native cytochrome c. The reconstituted semisynthetic cytochrome c was purified by ion exchange chromatography and was shown to have the same oxygen uptake as native cytochrome c when assayed in the succinate oxidase system.     

Sendai virus N-terminal fusion peptide consists of two similar repeats, both of which contribute to membrane fusion.

The N-terminal fusion peptide of Sendai virus F1 envelope glycoprotein is a stretch of 14 amino acids, most of which are hydrophobic. Following this region, we detected a segment of 11 residues that are strikingly similar to the N-terminal fusion peptide. We found that, when anchored to the membrane by palmitoylation of its N-terminus, this segment (WT-palm-19-33) induces membrane fusion of large unilamellar liposomes to almost the same extent as a segment that includes the N-terminal fusion peptide. The activity of WT-palm-19-33 was dependent on its specific sequence, as a palmitoylated peptide with the same amino-acid composition but a scrambled sequence was inactive. Interestingly, two mutations (G7A and G12A) known to increase F1- induced cell-cell fusion, also increased the homology between the N-terminal fusion peptide and WT-palm-19-33. The role of the amino-acid sequence on the fusogenicity, secondary structure, and mechanism of membrane fusion was analyzed by comparing a peptide comprising both homologous segments (WT 1-33), a G12A mutant (G12A 1-33), a G7A-G12A double mutant (G7A-G12A 1-33), and a peptide with a scrambled sequence (SC 1-33). Based on these experiments, we postulate that replacement of Gly 7 and Gly12 by Ala increases the alpha helical content of the N-terminal region, with a concomitant increase in its fusogenic activity. Furthermore, the dissimilar abilities of the different peptides to induce membrane negative curvature as well as to promote isotropic 31P NMR signals, suggest that these mutations might also alter the extent of membrane penetration of the 33-residue peptide. Interestingly, our results serve to explain the effect of the G7A and G12A mutations on the fusogenic activity of the parent F1 protein in vivo.     

Structure of iturine A, a peptidolipid antibiotic from Bacillus subtilis.

A mixture of iturines extracted from Bacillus subtilis gave, on column chromatography, iturine A, iturine B, and iturine C. Iturine A has the entire antifungal activity. It is a mixture of two homologous peptidolipids C48,H74N12O14 and C49H76N12O14 (mp 177 degrees C, [alpha]D-1.7 degrees in methanol (c 0.05 g/mL); mol wt 1042 and 1056). The lipid moiety is a mixture of 3-amino-12-methyltridecanoic acid and 3-amino-12-methyltetradecanoic acid. The peptide moiety contains 7 mol of amino acids: D-Asn2, L-Asn, L-Gln, L-Pro, L-Ser, and D-Tyr. A cyclic structure for iturine A with the serine residue linked to the fatty amino acids through a peptide bond has been domonstrated. By mild HCl hydrolysis, lipid-soluble and water-soluble peptides were obtained. They were analyzed by chemical methods and by mass spectrometry. Permethylated and perdeuteriomethylated derivatives of iturine A were also subjected to mass spectrometric analysis. Both chemical analysis and mass spectrometry led to the cyclic structure I for iturine A.     

Affinity labeling of 3 alpha-hydroxysteroid dehydrogenase with 3 alpha-bromoacetoxyandrosterone and 11 alpha-bromoacetoxyprogesterone. Isolation and sequence of active site peptides containing reactive cysteines; sequence confirmation using nucleotide sequence from a cDNA clone

Homogeneous 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, EC 1.1.1.50) of rat liver cytosol is potently inhibited at its active site by nonsteroidal anti-inflammatory drugs (NSAIDs). Using 3 alpha-bromoacetoxy-5 alpha-androstan-17-one (BrAnd, a substrate analog) and 11 alpha-bromoacetoxyprogesterone (Br11P, a glucocorticoid analog) as affinity-labeling agents, kinetic evidence was obtained that these agents alkylate this site. Inactivation of 3 alpha-HSD with either [14C]BrAnd or [14C] Br11P led to the incorporation of 1 mol of affinity-labeling agent per enzyme monomer. Complete acid hydrolysis of 3 alpha-HSD radiolabeled with either agent followed by amino acid analysis led to the identification of [14C]carboxymethylcysteine indicating that [14C]BrAnd and [14C]Br11P covalently tag discrete reactive cysteine(s) at the enzyme active site. Trypsin digestion of [14C]BrAnd-inactivated 3 alpha-HSD followed by peptide mapping led to the purification of a single radiolabeled peptide (3A1) which gave the following sequence: H2N-Ser-Ile-Gly-Val-Ser-Asn-Phe-Asn-X-Arg-CO2H. Identical experiments on [14C] Br11P-inactivated 3 alpha-HSD led to the purification of three radiolabeled peptides (11P1-11P3). The major radiolabeled peptide (11P1) had an identical sequence to 3A1 which was tagged with [14C]BrAnd. The minor radiolabeled peptides had the following sequences: H2N-Ser-Lys-Asp-Ile-Ile-Leu-Val-Ser-Tyr-X-Thr-Leu-Gly-Ser-Ser-Arg-CO2H (11P2) and H2N-Ser-Pro-Val-Leu-Leu-Asp-Asp-Pro-Val-Leu-X-Ala-Ile-Ala-Lys-CO2H (11P3). In each peptide group X was identified as carboxymethylcysteine. Alignment of the peptide sequences with the primary structure of 3 alpha-HSD, deduced from its cDNA clone, assigned peptide 11P1 to residues 162-171, peptide 11P2 to residues 208-223, and peptide 11P3 to residues 232-246 of the amino acid sequence. The reactive cysteines correspond to Cys170, Cys217, and Cys242. We propose that Cys170 labeled by BrAnd may lie within the catalytic pocket of the enzyme. By contrast the 11 alpha-bromoacetoxy group in Br11P labeled several reactive cysteines which may be involved in the binding of glucocorticoids and NSAIDs.     

The synthesis of glycopeptide fragments of human plasma alpha1-acid glycoproteins by sequential elongation at the terminal-amino group.

Glycopeptides corresponding to sequences 27--28, 48--49, and 58--59 of human plasma alpha1-acid glycoproteins have been synthesized by sequential elongation of the peptide chain at the terminal amino group. 2-Acetamido-3,4,6-tri-O-acetyl-1-N-(L-aspart-4-oyl)-2-deoxy-beta-D-glucopyranosylamine was condensed with the p-nitrophenyl esters of protected amino acids to give the corresponding protected glycodipeptides having the sequences Gly-(GlcNAc-4-)Asn, Pro-(GlcNAc-4-)Asn, Val-(GlcNAc-4-)Asn, Leu-(GlcNAc-4-)Asn, Glu-(GlcNAc-4-)Asn, Tyr-(GlcNAc-4-)Asn, Ser-(GlcNAc-4-)Asn, and Cys-(GlcNAc-4-)Asn. Deprotection of the carbohydrate and of the peptide residues of these compounds was achieved, except for those having N-tert-butyloxycarbonyl protective groups, to give the corresponding free glycopeptides. The glycotripeptide 2-acetamido-1-N-(2-N-[N-(tert-butyloxycarbonyl)-L-glutam-1-oyl-L-tyrosyl]-L-aspart-4-oxy)-2-deoxy-beta-D-glucopyranosylamine, having the amino acid sequence 10--12 of human plasma alpha1-acid glycoprotein, was prepared by condensation of 2-acetamido 3,4,6-tri-O-acetyl-2-deoxy-1-N-[2-N-(L-tyrosyl)-L-aspart-4-oyl[-beta-D-glucopyranosylamine with 5-benzyl 1-p-nitrophenyl N-(tert-butyloxycarbonyl)-L-glutamate, followed by removal of the ester groups.     

Characterization of a synthetic peptide corresponding to a receptor binding domain of mouse interferon gamma.

A receptor binding region of mouse interferon gamma (IFN gamma) has previously been localized to the N-terminal 39 amino acids of the molecule by use of synthetic peptides and monoclonal antibodies. In this report, a detailed analysis of the synthetic peptide corresponding to this region, IFN gamma (1-39), is presented. Circular dichroism (CD) spectroscopy indicated that the peptide has stable secondary structure under aqueous conditions and adopts a combination of alpha-helical and random structure. A peptide lacking two N-terminal amino acids, IFN gamma (3-39), had similar secondary structure and equivalent ability to compete for receptor binding, while peptides lacking four or more N-terminal residues had reduced alpha-helical structure and did not inhibit 125I-IFN gamma binding. Substitution of proline, a helix-destabilizing amino acid, for leucine (residue 8) of a predicted amphipathic alpha-helix (residues 3-12), IFN gamma (1-39) [Pro]8, resulted in a substantial reduction in the helical content of the peptide, supporting the presence of helical structure in this region. However, destabilization of the helix did not reduce the competitive ability of the peptide. A peptide lacking eight C-terminal residues, IFN gamma (1-31), did not block 125I-IFN gamma binding and had no detectable alpha-helical structure, suggesting a requirement of the predicted second alpha-helix (residues 20-34) for receptor interaction and helix stabilization. Substitution of phenylalanine for tyrosine at position 14, IFN gamma (1-39) [Phe]14, a central location of a predicted omega-loop structure, did not affect the secondary structure associated with the region yet resulted in a 30-fold increase in receptor competition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Importance of the release of strand 1C to the polymerization mechanism of inhibitory serpins.

Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emphysema, liver cirrhosis, and angioedema. Understanding the structure of the polymers and the mechanism of polymerization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12-m34 peptide (homologous to P14-P3 of antithrombin reactive loop), representing the entire length of s4A, prevented polymerization totally. A 6-mer peptide (homologous to P14-P9 of antithrombin) not only allowed polymerization to occur, but induced it. This effect could be blocked by the addition of a 5-mer peptide with s1C sequence of antithrombin or by an unrelated peptide representing residues 26-31 of cholecystokinin. The s1C or cholecystokinin peptide alone was unable to form a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 361-->Cys, Ser 283-->Cys, was engineered for the purpose of forming a disulfide bond between s1C and s2C to prevent movement of s1C. This mutant was resistant to polymerization if the disulfide bridge was intact, but, under reducing conditions, it regained the potential to polymerize. We have also modeled long-chain serpin polymers with acceptable stereochemistry using two previously proposed loop-A-sheet and loop-C-sheet polymerization mechanisms and have shown both to be sterically feasible, as are "mixed" linear polymers. We therefore conclude that the release of strand 1C must be an element of the mechanism of serpin polymerization.     

Protoplasts from Aspergillus nidulans.

A very effective lytic enzyme system for massive micro/macro-scale production of protoplasts from the filamentous fungus Aspergillus nidulans is described. A striking coincidence was observed between maximal lytic activity towards Aspergillus mycelium and the presece of both chitinase and alpha-(1 leads to 3)-glucanase activities. The release of protoplasts was greatly enhanced by preincubating the mycelium with 2-deoxy-D-glucose. Furthermore, protoplast formation was influenced by fungal age, culture conditions, pH of incubation and the osmotic stabilizer used. From 40 mg of fresh mycelium, grown for 14--16 h on 1% glucose in a low phosphate-citrate medium, preincubated with 2-deoxy-D-glucose for 45 min, and then incubated with the lytic enzyme mixture at pH 6.5 in the presence of 0.3--0.4 M (NH4) SO4, 2.5 x 10(8) stable protoplasts were produced within 3 h of incubation at 30 degrees C. Comparable results were obtained with 40--50 g of mycelium. At low osmotic stabilizer concentrations a peculiar type of regeneration was observed in the presence of the lytic enzyme system; within 12 h of incubation aberrant hyphal structure emerged from the large vacuolated protoplasts.     

Modeling of non-covalent complexes of the cell-penetrating peptide CADY and its siRNA cargo

CADY is a cell-penetrating peptide spontaneously making non-covalent complexes with Short interfering RNAs (siRNAs) in water. Neither the structure of CADY nor that of the complexes is resolved. We have calculated and analyzed 3D models of CADY and of the non-covalent CADY–siRNA complexes in order to understand their formation and stabilization. Data from the ab initio calculations and molecular dynamics support that, in agreement with the experimental data, CADY is a polymorphic peptide partly helical. Taking into consideration the polymorphism of CADY, we calculated and compared several complexes with peptide/siRNA ratios of up to 40. Four complexes were run by using molecular dynamics. The initial binding of CADYs is essentially due to the electrostatic interactions of the arginines with siRNA phosphates. Due to a repetitive arginine motif (XLWR(K)) in CADY and to the numerous phosphate moieties in the siRNA, CADYs can adopt multiple positions at the siRNA surface leading to numerous possibilities of complexes. Nevertheless, several complex properties are common: an average of 14 ± 1 CADYs is required to saturate a siRNA as compared to the 12 ± 2 CADYs experimentally described. The 40 CADYs/siRNA that is the optimal ratio for vector stability always corresponds to two layers of CADYs per siRNA. When siRNA is covered by the first layer of CADYs, the peptides still bind despite the electrostatic repulsion. The peptide cage is stabilized by hydrophobic CADY–CADY contacts thanks to CADY polymorphism. The analysis demonstrates that the hydrophobicity, the presence of several positive charges and the disorder of CADY are mandatory to make stable the CADY–siRNA complexes.     

Synthesis of Tc-99m labeled 1,2,3-triazole-4-yl c-met binding peptide as a potential c-met receptor kinase positive tumor imaging agent

The mesenchymal-epithelial transition factor (c-Met), which is related to tumor cell growth, angiogenesis and metastases, is known to be overexpressed in several tumor types. In this study, we synthesized technetium-99m labeled 1,2,3-triazole-4-yl c-Met binding peptide (cMBP) derivatives, prepared by solid phase peptide synthesis and the ‘click-to-chelate’ protocol for the introduction of tricarbonyl technetium-99m, as a potential c-Met receptor kinase positive tumor imaging agent, and evaluated their in vitro c-Met binding affinity, cellular uptake, and stability. The ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12, [^99mTc(CO)₃]13, and [^99mTc(CO)₃]14) were prepared in 85-90% radiochemical yields. The cold surrogate cMBP derivatives, [Re(CO)₃]12, [Re(CO)₃]13, and [Re(CO)₃]14, were shown to have high binding affinities (0.13 μM, 0.06 μM, and 0.16 μM, respectively) to a purified cMet/Fc chimeric recombinant protein. In addition, the in vitro cellular uptake and inhibition studies demonstrated the high specific binding of these ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12–14) to c-Met receptor positive U87MG cells.