The solid-state catalytic synthesis of tritium labeled amino acids,peptides and proteins

Summary New catalytic reaction between a solid bioorganic compound and activated spillover tritium (ST), based on High-temperature Solid-state Catalytic Isotopic Exchange (HSCIE) was examined. The HSCIE mechanism and determination of the reactivity of hydrogen atoms in amino acids, peptides and proteins was investigated. Quantum mechanical calculations of the reactivity of hydrogen atoms in amino acids in the HSCIE reaction were done. The carbon atom with a greater proton affinity undergoes a greater exchange of hydrogen for tritium in HSCIE. The electrofilic nature of spillover hydrogen in the reaction of HSCIE was revealed. The isotope exchange between ST and the hydrogen of the solid organic compound proceeds with a high degree of configuration retention at the carbon atoms. The HSCIE reaction enables to synthesize tritium labeled proteins with a specific activity of 20–30 mCi/mg and kept biological activity.Presented at the 3rd International Congress on Amino Acids, Peptides and Analogues. Vienna, August, 23–27, 1993     

The use of thermally activated tritium atoms for structural-biological investigations: the topography of the TMV protein-accessible surface of the virus

Thermally activated tritium atoms were used for studying the topography of the TMV protein-accessible surface of the virus. The accessibility profile of amino acid residues in a protein polypeptide chain was determined from data on the intramolecular distribution of a tritium label in the TMV protein. It was shown that tryptic peptides T3, T4, T12, the N-terminal region of peptide T1 and the proximal tryptic peptide T8 (located 20 to 25 A (1 A = 0.1 nm) from the viral axis) are accessible to tritium labelling. The fact of tritiation of the viral RNA was detected as well. This evidence was compared with the high-resolution X-ray analysis data for the TMV. A model is suggested to explain the exposure of the buried sites of the virus to thermally activated tritium atoms. The possibilities and limitations of this method in studying the surface topography of proteins in supramolecular systems as well as for location of protein antigenic regions are discussed.     

Folding of immunogenic peptide fragments of proteins in water solution. I. Sequence requirements for the formation of a reverse turn

A systematic examination by 1H nuclear magnetic resonance of the population of beta-turn-containing conformers in several series of short linear peptides in water solution has demonstrated a dependence on amino acid sequence which has important implications for initiation of protein folding. The peptides consist of a number of variants of the sequence Tyr-Pro-Tyr-Asp, the trans isomer of which was previously shown to contain a reverse turn in water. Two-dimensional rotating-frame nuclear Overhauser effect spectroscopy provides unequivocal evidence that substantial populations of reverse turn conformations occur in water solutions of certain of these peptides. In the unfolded state, the peptides adopt predominantly extended chain (beta) conformations in water. It appears probable from the nuclear Overhauser effect connectivities observed that the reverse turns in the trans isomers are predominantly type II. The low temperature coefficient of the amide proton resonance of the residue at position 4 of the turn suggests the presence of an intramolecular hydrogen bond. The presence of the beta-turn conformation has been confirmed for certain peptides by circular dichroism measurements. Substitutions at positions 3 and 4 in the sequence Tyr-Pro-Tyr-Asp-Val can enhance or abolish the beta-turn population in the trans peptide isomers. The residue at position 3 of the turn is the primary determinant of its stability. A small amount of additional stabilization appears to result from an electrostatic interaction between the side-chain of residue 4 and the unblocked amino terminus. For peptides of the series Tyr-Pro-X-Asp-Val, where X represents all L-amino acid except Trp and Pro, the temperature coefficient of the Asp4 amide proton resonance provides a measure of the beta-turn population. The beta-turn populations in water solution measured in this way correlate with the beta-turn probabilities determined from protein crystal structures. This indicates that it is frequently the local amino acid sequence, rather than medium- to long-range interactions in the folded protein, that determines the beta-turn conformation in the folded state. Such sequences are excellent candidates for protein folding initiation sites. A high population of structured forms appears to be present in the cis isomer of certain of the peptides, as shown by a considerable increase in the proportion of the cis isomer and by measurement of nuclear Overhauser effects and 3JN alpha coupling constants.(ABSTRACT TRUNCATED AT 400 WORDS)     

Catalysis by human leukocyte elastase. Aminolysis of acyl-enzymes by amino acid amides and peptides

Acyl-enzymes of human leukocyte elastase (HLE) were generated in situ during the hydrolysis of peptide thiobenzyl esters and served as substrates for aminolysis by a variety of amino acid amides and short peptide nucleophiles. For amino acid amides, there is a positive correlation between nucleophilic reactivity toward N-methoxysuccinyl (MeOSuc)-Ala-Ala-Pro-Val-HLE and the hydrophobicity of the side chain. For peptides, nucleophilicity toward MeOSuc-Ala-Ala-Pro-Val-HLE decreases dramatically with increasing chain length. Combined, these results suggest that substrate specificity for the P1' residue may be more dependent on side chain hydrophobicity than on specific, structural features of the side chain and there may be no important binding interactions available past S1'. Kinetic parameters were also determined for the nucleophilic reactions of PheNH2 and TyrNH2 with MeOSuc-Pro-Val-HLE, MeOSuc-Ala-Pro-Val-HLE, MeOSuc-Ala-Ala-Pro-Val-HLE, and MeOSuc-Ala-Ala-Pro-Ala-HLE. Reactivity of these acyl-enzymes toward nucleophilic attack displays no dependence on peptide chain length but does increase significantly for the substrate with Ala at P1. This same correlation between reactivity and acyl-enzyme structure is also seen for nucleophilic attack by water.     

Relative reactivity of lysine and other peptide-bound amino acids to oxidation by hypochlorite

Antibacterial and inflammatory responses of neutrophils and macrophages produce hypochlorite as a major oxidant. Numerous side chains of amino acids found in extracellular proteins can be modified by hypochlorite, including His, Arg, Tyr, Lys, Trp, and Met. We studied the relative reactivity of each of these amino acid residues in short N-blocked peptides, where other residues in the peptide were highly resistant to hypochlorite attack. Hypochlorite treatment led to modified peptides in each case, which were detected by changes in retention on reversed-phase HPLC. A distinct single product, consuming two equivalents of hypochlorite per equivalent of peptide, was obtained from the Lys-containing peptides. UV spectroscopy, nuclear magnetic resonance (NMR), and electrospray/mass spectroscopy identified this product as the dichloramine at the epsilon-amino group of the Lys side chain. The dichloramine at Lys did not decompose to form a detectable amount of carbonyl reactive with dinitrophenylhydrazine. The dichloramine at Lys did however quantitatively revert back to Lys during HCl digestion of the tetrapeptide for amino acid analysis, with simultaneous modification of the adjacent Phe residue. The formation of the dichloramine at Lys was not blocked by peptides or acetylated amino acids that contained Tyr, His, or Arg. In contrast, the presence of equimolar Met-containing peptide, or N-Acetyl-Trp, both inhibited the formation of the dichloramine at Lys. Thus, Met and Trp side chains of proteins might be able to protect Lys from chloramine formation under some circumstances, but this interpretation must consider that Met and Trp are typically found in relatively inaccessible hydrophobic sites, whereas lysine is typically exposed on the protein surface. The hierarchy of amino acid reactivities examined here will aid in the prediction of residues in biological samples most likely to be modified by hypochlorite.     

Structural features of peptoid–peptide hybrids in lipid–water interfaces

The inclusion of peptoid monomers into antimicrobial peptides (AMPs) increases their proteolytic resistance, but introduces conformational flexibility (reduced hydrogen bonding ability and cis/trans isomerism). We here use NMR spectroscopy to answer how the insertion of a peptoid monomer influences the structure of a regular α-helical AMP upon interaction with a dodecyl phosphocholine (DPC) micelle. Insertion of [(2-methylpropyl)amino]acetic acid in maculatin-G15 shows that the structural change and conformational flexibility depends on the site of insertion. This is governed by the micelle interaction of the amphipathic helices flanking the peptoid monomer and the side chain properties of the peptoid and its preceding residue.     

Analysis of the antigen binding site of anti-deoxycholate monoclonal antibody using a novel affinity labeling reagent, acyl adenylate

Large-scale analysis of protein-protein interaction sites is especially needed in the postgenomic era. The combination of affinity labeling with mass spectrometry is a potentially useful high-throughput screening method for this purpose. However, reagents in current use are not ideal as some cause damage to the target molecule and others have poor solubility in physiologic aqueous buffers. In this paper, we describe a novel affinity labeling reagent, acyl adenylate, which is highly soluble in aqueous solutions and reacts in a pH-dependent manner. The adenylate of deoxycholic acid reacts with amino groups on the side chain of a lysine residue and at the N-terminus of proteins/peptides. The reactivity and stability of this reagent were investigated, and it was confirmed that, after formation of a reversible ligand-protein complex under weakly acidic conditions, derivatization with acyl adenylate occurred at the target site under weakly alkaline condition. We further demonstrated the utility of this reagent for affinity labeling using a monoclonal antibody with high affinity for deoxycholic acid. Competitive ELISA indicated that deoxycholic acid was labeled around the antibody ligand binding site, thus enabling the structural elucidation of the ligand-protein interaction. In addition, LC/ESI-MS/MS analysis of the labeled peptide obtained by enzymatic digestion and affinity extraction allowed the identification of the structure surrounding the antigen binding site.     

Study of solid-phase catalytic isotopic exchange of hydrogen in alpha-conotoxin G1 under the effect of spillover-tritium

Tritium-labeled alpha-conotoxin G1 with a molar radioactivity of 35 Ci/mmol and full biological activity (according to the binding to nicotinic acetylcholine receptor) was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE). The tritium distribution in the molecule of alpha-conotoxin G1 was revealed by 3H NMR spectroscopy. Tritium was found in all amino acid residues except for the Asn4-Pro5-Ala6 fragment. The data on the comparative reactivity of C-H bonds, the ab initio quantum-chemical calculation of the hydrogen exchange reaction, and the information on the spatial structures of alpha-conotoxin G1 in solution and in crystal state allowed us to establish that the reactivity of H atoms may be increased by their interaction with the electron donor O and N atoms at the transition state of the HSCIE reaction. A decrease in the rate of the HSCIE reaction could be caused by both a poor spatial accessibility of C-H bonds and a limited mobility of the peptide fragment containing these bonds.     

Stereospecific binding of diastereomeric peptides to salmon sperm deoxyribonucleic acid. Further evidence for partial intercalation

A series of diastereomeric dipeptide amides, containing an N-terminal L-lysyl residue and a C-terminal L- or D-amino acid with a derivatized aromatic ring on the side chain, was synthesized to determine the dependence of (1) the chirality of the N-terminal amino acid alpha-carbon and (2) the length of the N-terminal amino acid side chain for intercalation of the aromatic ring. The nature of the complex between the peptide and DNA (i.e., electrostatic, intercalative, or a combination of these) was determined by UV and CD studies, viscometric titrations, and 1H NMR studies. The results of these studies reveal distinct differences in the binding site of the aromatic rings of the various peptides. In particular, the results suggest that the alpha- and epsilon-amino groups of the lysyl residue bind electrostatically to adjacent phosphates on the DNA backbone in a stereospecific manner. As a result of this stereospecificity, the aromatic rings of the peptides with the L-L designation point toward the DNA helix, while those of the peptides of the L-D designation point away from the helix. This is completely consistent with previously reported work [Gabbay, E.J., Adawadkar, P. D., & Wilson, W. D. (1976) Biochemistry 15, 146; Gabbay, E. J., Adawadkar, P. D., Kapicak, L., Pearce, S., & Wilson, W. D. (1976) Biochemistry 15, 152]. The results also indicate a great dependence on the length of the side chain for intercalation of the aromatic ring. Specifically, if the side chain is long enough, and flexible enough, the aromatic ring can fully or partially intercalate, regardless of the chirality of the N-terminal amino acid alpha-carbon. However, if the side chain is too short, only partial intercalation is observed for peptides of the L-D designation, and no intercalation is observed for peptides of the L-D designation.     

Structure-wise discrimination of cytosine,thymine, and uracil by proteins in terms of their nonbonded interactions

The molecular recognition and discrimination of very similar ligand moieties by proteins are important subjects in protein–ligand interaction studies. Specificity in the recognition of molecules is determined by the arrangement of protein and ligand atoms in space. The three pyrimidine bases, viz. cytosine, thymine, and uracil, are structurally similar, but the proteins that bind to them are able to discriminate them and form interactions. Since nonbonded interactions are responsible for molecular recognition processes in biological systems, our work attempts to understand some of the underlying principles of such recognition of pyrimidine molecular structures by proteins. The preferences of the amino acid residues to contact the pyrimidine bases in terms of nonbonded interactions; amino acid residue–ligand atom preferences; main chain and side chain atom contributions of amino acid residues; and solvent-accessible surface area of ligand atoms when forming complexes are analyzed. Our analysis shows that the amino acid residues, tyrosine and phenyl alanine, are highly involved in the pyrimidine interactions. Arginine prefers contacts with the cytosine base. The similarities and differences that exist between the interactions of the amino acid residues with each of the three pyrimidine base atoms in our analysis provide insights that can be exploited in designing specific inhibitors competitive to the ligands.     

Residue contacts in protein structures and implications for protein folding

The preferential association of amino acid side groups with specific side chain atoms are examined in 44 known protein structures. The resulting association potentials among residue side groups are used to detect structural homology in proteins displaying little or no homology in their primary sequences. Suggestions are also made regarding the nature of the protein folding process. They are based on statistical observations that delineate the extent of short and long range interactions and that display side group bias in association with other side chain atoms on their N-terminal side.     

615小鼠血红蛋白α链的氨基酸组成及个别肽段的氨基酸序列

用CM-Cellulose-23柱层析分离纯化了615小鼠珠蛋白α链,测定其N端氨基酸残基为缬氨酸.615小鼠珠蛋白α链含有141个氨基酸残基,其中19个亮氨酸残基,10个组氨酸残基,9个缬氨酸残基,上述氨基酸残基的数目与文献中其亲本C₅₇BL不同.用胰蛋白酶水解615小鼠珠蛋白α链,发现有不溶性的‘核心’和可溶性的酶解片段.其中一个酶解肽段从N端数第8位氨基酸残基发生了突变,由亲本的缬氨酸变为亮氨酸.     

Amino acid sequence of a ferredoxin from thermoacidophilic archaebacterium, Sulfolobus acidocaldarius. Presence of an N6-monomethyllysine and phyletic consideration of archaebacteria

The amino acid sequence of a ferredoxin from a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius, was determined by a combination of various conventional methods to be as follows: Gly-Ile-Asp-Pro-Tyr-Arg-Thr-His-Lys-Pro-Val-Val-Gly-Asp-Ser-Ser-Gly-His- Lys-Ile -Tyr-Gly-Pro-Val-Glu-Ser-Pro-Lys(Me)-Val-Leu-Gly-Val-His-Gly-Thr-Ile-Val -Gly-Va l-Asp-Phe-Asp-Leu-Cys-Ile-Ala-Asp-Gly-Ser-Cys-Ile-Thr-Ala-Cys-Pro-Val-As n-Val-P he-Gln-Trp-Tyr-Glu-Thr-Pro-Gly-His-Pro-Ala-Ser-Glu-Lys-Lys-Ala-Asp-Pro-V al-Asn- Glu-Gln-Ala-Cys-Ile-Phe-Cys-Met-Ala-Cys-Val-Asn-Val-Cys-Pro-Val-Ala-Ala- Ile-Asp -Val-Lys-Pro-Pro. It was composed of 103 amino acid residues giving a molecular weight of 10,908 excluding Fe and S atoms. This ferredoxin contained an N6-monomethyllysine residue at position 29 which was determined by a comparison of the elution profile of the acid hydrolysates of the protein and peptides on an amino acid analyzer with three methyl derivatives of lysine and also by field desorption mass spectrometry of a purified peptide. The ferredoxin has only 7 cysteine residues, which probably participate in constructing the Fe-S clusters of this ferredoxin, indicating the presence of a unique chelate structure. Comparison of this ferredoxin with other archaebacterial ferredoxins indicated that the archaebacteria might have multiple origins in an evolutionary tree.     

Interaction of adrenocorticotropin peptides with microheterogeneous systems — A fluorescence study

Adrenocorticotropin (ACTH) and α-melanocyte stimulating hormone (α-MSH) are peptides which present many physiological effects related to pigmentation, motor and sexual behavior, learning and memory, analgesia, anti-inflammatory and antipyretic processes. The 13 amino acid residues of α-MSH are the same initial sequence of ACTH and due to the presence of a tryptophan residue in position 9 of the peptide chain, fluorescence techniques could be used to investigate the conformational properties of the hormones in different environments and the mechanisms of interaction with biomimetic systems like sodium dodecyl sulphate (SDS) micelles, sodium dodecyl sulphate-poly(ethylene oxide) (SDS-PEO) aggregates and neutral polymeric micelles. In buffer solution, fluorescence parameters were typical of peptides containing tryptophan exposed to the aqueous medium and upon addition of surfactant and polymer molecules, the gradual change of those parameters demonstrated the interaction of the peptides with the microheterogeneous systems. From time-resolved experiments it was shown that the interaction proceeded with conformational changes in both peptides, and further information was obtained from quenching of Trp fluorescence by a family of N-alkylpyridinium ions, which possess affinity to the microheterogeneous systems dependent on the length of the alkyl chain. The quenching of Trp fluorescence was enhanced in the presence of charged micelles, compared to the buffer solution and the accessibility of the fluorophore to the quencher was dependent on the peptide and the alkylpyridinium: in ACTH(1–21) highest collisional constants were obtained using ethylpyridinium as quencher, indicating a location of the residue in the surface of the micelle, while in α-MSH the best quencher was hexylpyridinium, indicating insertion of the residue into the non-polar region of the micelles. The results had shown that the interaction between the peptides and the biomimetic systems where driven by combined electrostatic and hydrophobic effects: in ACTH(1–24) the electrostatic interaction between highly positively charged C-terminal and negatively charged surface of micelles and aggregates predominates over hydrophobic interactions involving residues in the central region of the peptide; in α-MSH, which presents one residual positive charge, the hydrophobic interactions are relevant to position the Trp residue in the non-polar region of the microheterogeneous systems.     

Characterization of continuous B‐cell epitopes in the N‐terminus of glutamate decarboxylase67 using monoclonal antibodies

Glutamate decarboxylase (GAD) is an autoantigen associated with the autoimmune disorders Type‐1 diabetes (T1D) and stiff‐person syndrome (SPS). The protein, being an essential enzyme involved in the production of the inhibitory neurotransmitter γ‐aminobutyric acid, exists in two isoforms, GAD67 and GAD65. Both isoforms may be targeted by autoantibodies in SPS and T1D patients, although SPS primarily is associated with the presence of GAD67 autoantibodies, whereas T1D mainly is associated with the presence of GAD65 autoantibodies. In this study, we describe antibody reactivity to overlapping GAD67 peptides covering the complete protein sequence by modified peptide enzyme‐linked immunosorbent assay in order to identify potential GAD67 epitopes using two monoclonal antibodies (mAbs). Both GAD67 mAbs showed reactivity to linear epitopes located at the N‐terminal end of GAD67. The epitopes of GAD mAb 1 and 2 were identified as the amino acid sequences NAGADPNTTN and TETDFSNLF, respectively, corresponding to amino acids 14–23 and 91–99. Fine mapping of the epitopes revealed that antibody reactivity was related to amino acid side‐chain functionality, rather than amino acid side‐chain specificity. Additionally, results suggested that non‐contact amino acids in the epitope structure were essential for antibody reactivity. The exact role of these amino acids remains to be determined, but they are thought to be involved in backbone hydrogen bonds or stabilization of the epitope structure. As only limited knowledge is available in relation to antigenic regions of GAD67, this study contributes to characterization of GAD67 epitopes and may be a first step in the development of peptide‐based therapeutics against SPS. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

The solid-state catalytic isotope exchange of hydrogen in α-conotoxin G1 by the tritium spillover

Tritium-labeled α-conotoxin G1 with a molar radioactivity of 35 Ci/mmol and full biological activity (according to the binding to nicotinic acetylcholine receptor) was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE). The tritium distribution in the molecule of α-conotoxin G1 was revealed by³H NMR spectroscopy. Tritium was found in all amino acid residues except for the Asn4-Pro5-Ala6 fragment. The data on the comparative reactivity of C-H bonds, theab initio quantum-chemical calculation of the hydrogen exchange reaction, and the information on the spatial structures of α-conotoxin G1 in solution and in crystal state allowed us to establish that the reactivity of H atoms may be increased by their interaction with the electron donor O and N atoms at the transition state of the HSCIE reaction. A decrease in the rate of the HSCIE reaction could be caused by both a poor spatial accessibility of C-H bonds and a limited mobility of the peptide fragment containing these bonds.     

Isolation and characterization of the cyanogen bromide fragments of lobster arginine kinase (homarus vulgaris).

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10 percent) and subsequently submitted to gel-filtration. The large polypeptide subfractions were citraconylated and resubmitted to differnt gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C-terminal position because one methionyl residue is situated at the C-terminal position in the native protein. The polypeptide with 132 residues possessed N-acetylated residue at N-terminal position: therefore this polypeptide is located at the N-terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present results (37, 687).     

Proteolytically stable peptides by incorporation of α-Tfm amino acids

A series of model peptides containing α-trifluoromethyl-substituted amino acids in five different positions relative to the predominant cleavage site of the serine protease α-chymotrypsin was synthesized by solution methods to investigate the influence of α-Tfm substitution on the proteolytic stability of peptides. Proteolysis studies demonstrated absolute stability of peptides substituted in the P₁ position and still considerable proteolytic stability for peptides substituted at the P₂ and P′₂ positions compared with the corresponding unsubstituted model peptide. Comparison with peptides containing the fluorine-free disubstituted amino acid α-aminoisobutyric acid allowed to separate electronic from steric effects. Furthermore, the absolute configuration of the α-Tfm-substituted amino acid was found to exert considerable effects on the proteolytic stability, especially in P′₁ substituted peptides. Investigations of this phenomenon using empirical force field calculations revealed that in the (S,R,S)-diasteromer the steric constraints exhibited by the α-Tfm group can be outweighed by an advantageous interaction of the fluorine atoms with the serine side chain of the enzyme. In contrast, a favourable interaction between substrate and enzyme is impossible for the (S,S,S)-diastereomer. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes

Conformational aspects of N-glycosylation have been investigated with a series of proline-containing peptides as molecular probes. The results demonstrate that, depending on the position of the imino acid in the peptide chain, dramatic alterations of glycosylation rates are produced, pointing to a critical contribution of the amino acids framing the 'marker sequence' triplet Asn-Xaa-Thr(Ser) on the formation of a potential sugar-attachment site. No glycosyl transfer at all was detectable to those peptides containing a proline residue either in position Xaa or in the next position beyond the threonine of the Asn-sequon on the C-terminal side, whereas the hexapeptide Pro-Asn-Gly-Thr-Ala-Val was glycosylated at a high rate. (Emboldened residues denote the 'marker sequence' that is identical in all the peptides; italicized residues distinguish the positions of proline in the various peptides.) Studies with space-filling models reveal that the lack of glycosyl-acceptor capabilities of Ala(Pro)-Asn-Gly-Thr-Pro-Val might be directly related to their inability to adopt and/or stabilize a turn or loop conformation which permits the catalytically essential interaction between the hydroxy amino acid and the asparagine residue within the 'marker sequence' [Bause & Legler (1981) Biochem. J. 195, 639-644]. This conclusion is supported by circular-dichroism spectroscopic data, which suggest structure-forming potentials in this type of non-acceptor peptides dominating over those that favour the induction of an appropriate sugar-attachment site in the acceptor peptides. The lack of acceptor properties of Tyr-Asn-Pro-Thr-Ser-Val indicates that even small modifications in the 'recognition' pattern are not tolerated by the N-glycosyltransferases.     

Phosphatidylcholine liposomes containing cholesterol analogues with side chains of various lengths

The effect of the length of the side chain of sterols on their interaction with phosphatidylcholine was studied by measuring the permeability properties of liposomes constituted with sterol analogues with side chains of various lengths. The sensitivities of liposomes constituted with these sterol analogues toward digitonin and polyene antibiotics were also examined.The effects of sterols on phase transition of phosphatidylcholine were examined by measuring their effects on permeability increase due to perturbation of phase equilibrium and by differential scanning calorimetry. An analogue with a short side chain, isopropyl (C-22), had a very similar effect to cholesterol in suppressing the permeability increase, suggesting that the full length of the side chain is not necessary for this effect.The permeability of egg yolk phosphatidylcholine at 42°C was suppressed as much by the analogue C-22 as by cholesterol. Androstene-3-β-ol, an analogue without a side chain, however, had little suppressive effect. Thus it is concluded that the condensing effect of sterol requires a side chain, but not the full length of side chain.Liposomes constituted with analogues having a side chain with more than 5 carbon atoms showed maximum reactivity with a polyene antibiotic, amphotericin B, whereas those constituted with analogues having a side chain with less than 4 carbon atoms showed weaker reactivity. These findings indicate that a side chain with more than 5 carbon atoms is essential for the maximum interaction of liposomes with amphotericin B. Unlike amphotericin B, filipin reacted almost equally well with liposomes containing C-22 and with those containing cholesterol. Thus the chain length of the side chain of sterol is less important for interaction of liposomes with filipin than for their interaction with amphotericin B.Liposomes containing analogues having a side chain with more than 6 carbon atoms showed maximum reactivity with digitonin. Thus for the maximum interaction of liposomes with digitonin, the side chain of sterol should be longer than 6 carbon atoms.