蛋白质中二硫键附近肽段识别规律研究

本文对蛋白质中二硫键附近的残基进行了计算机统计分析,结果发现平行和反平行残基间存在着特异的配对规律。这种残基间的相互作用或识别,可能与蛋白质折叠过程中正确地形成二硫键有关。该结果有助于蛋白质工程设计。     

Reactions Catalyzed by Peg-Modified α-Chymotrypsin in Organic Solvents. Influence of Water Content and Degree of Modification

-Chymotrypsin was modified with cyanuric chloride activated monomethoxypolyethylene glycol (MPEG) with molecular weights 1900 and 5000. Using the higher molecular weight MPEG a product that was soluble in benzene at moderate levels of modification was obtained, whereas with MPEG 1900 almost all the enzyme's amino groups had to be modified for dissolving the conjugate. The catalytic activity decreased with increasing degree of substitution. Apparent V_max was considerably higher for the less modified enzyme preparation than for the more modified one, while K_m,app stayed almost constant. The modified enzyme was used for peptide synthesis. The reaction was dependent on the content of dissolved water. Both V_max,app and K_m,app increased with increasing water content. It was possible to achieve a process with complete conversion of substrate to dipeptide.     

Elevated serum α-fetoprotein level of nude mice bearing hepatoma cells by treatment with monoclonal antibodies to α-fetoprotein

This study was carried out to clarify the reason for elevation of serum α-fetoprotein (AFP) level of nude mice bearing hepatoma cells after treatment with monoclonal antibodies (MoAbs) to AFP. MoAbs to AFP showed no effect on the cumulative amounts of AFP secreted from human hepatoma cell line, HuH-7, in vitro. However, the treatment of nude mice bearing HuH-7N cells (HuH-7 xenograft) with MoAbs to AFP led to elevation of the serum AFP level in spite of the fact that the growth curve of HuH-7N cells was similar to that for PBS treatment. This apparent elevation of the serum AFP level is thought to be due to the slow elimination of AFP-MoAb immune complexes with little lattice structure from circulation, but not the enhancement of AFP secretion of HuH-7N cells. Thus, when using a MoAb alone or MoAb-drug conjugate, the serum AFP level should only be cautiously used as a tumor marker for evaluating the targeting immunotherapy.     

不同一级结构寡肽的α-羰酰及喹喔啉衍生物的荧光性质

通过对寡肽N-末端α-羰酰及喹喔啉衍生物和荧光在碘化钾、乙二醇、盐酸胍和氯化钠溶液中的变化测定结果表明:不同一级结构寡肽的羰酰荧光物的碘化钾淬灭过程彼此有差异.在不同浓度的乙二醇和盐酸胍溶液中,第三位氨基酸残基的种类和构型不同的寡肽羰酰衍生物的荧光变化亦有不同.乙二醇引起羰酰甘氨寡肽荧先发射峰位红移.丙氨寡肽的发射峰位蓝移;并且不同链长的甘氨短肽及丙氨短肽羰酰衍生物在不同浓度的乙二醇或盐酸胍溶液中各自的荧光变化有差异,但这种差异随肽链的延长逐渐减小.以上结果提示:尽管(含有2-6个氨基酸残基的)寡肽在溶液中难以形成二级结构,但它们的空间构象可能不是随机的;寡肽链越长,结构相对稳定.     

MONOCLONAL ANTIBODIES AS MOLECULAR MARKERS FOR THE INTRACELLULAR AND CELL WALL DISTRIBUTION OF CARRAGEENAN EPITOPES IN KAPPAPHYCUS (RHODOPHYTA) DURING TISSUE DEVELOPMENT1

Carrageenan, the major cell wall carbohydrate of certain red algae, is variable in structure and gelling properties. Sequence types include gelling (kappa and iota) and nongelling (lambda) types in addition to precursors, often in hybrid molecules containing more than one precursor and/or sequence type. Molecular markers to subunits were needed to study carrageenan synthesis, cell wall organization, and the relationship between structure and function. Monoclonal antibodies were produced to carrageenan, and their specificities were determined by competitive enzyme immunoassay. Antibodies were identified with specificities related to kappa, iota, and lambda carrageenan. The patterns of immunofluorescence localization on Kappaphycus alvarezii = Eucheuma alvarezii var. tambalang (Doty) sections were distinctive for each antibody. The antibody to a kappa-related epitope labeled mature tissue strongly; antibodies to an iota-related epitope and a lambda-related epitope labeled weakly, consistent with the kappa-enriched carrageenan produced by this alga. Kappa-related epitopes were distributed throughout the wall and matrix, whereas iota-related epitopes were concentrated in the middle lamella. Lambda-related epitopes were localized primarily at the plant cuticle where kappa and iota antigens were lacking. An antibody appeared to be specific for a precursor of the gelling subunits because it showed maximal wall and intracellular labeling at the youngest developmental stage. All antibodies labeled intracellular inclusions in the transition zone between the epidermis and medulla during the development of medullary cells from the peripheral meristem in young branches. The results demonstrate the intracellular synthesis of epitopes related to all major carrageenan subunits and their differential extracellular distribution.     

High-resolution solution structure of two members of a conformationally homogeneous combinatorial peptide library based on the classical zinc-finger motif

Summary We describe the high-resolution structure by NMR of two peptides that belong to a combinatorial library based on the zinc-finger motif. The library represents, to the best of our knowledge, the first example of a conformationally homogeneous peptide library and was obtained by introducing random residues in five positions of the -helical portion of a 26-residue consensus peptide (CP1) belonging to the Cys²-Hys² zinc-finger family. The result was shown to be a highly homogeneous -helical library (Bianchi et al., 1995). The structures of the parent compound (CP1) and of a representative member (CP1m) that was selected by screening the library with a monoclonal antibody are compared in detail as an example of the very high stability of the zinc-finger scaffold upon sequence variability. The two peptides exhibit an extremely high degree of structural similarity. The use of this type of conformationally constrained combinatorial library might represent a step forward in the design of peptidomimetics, as it considerably accelerates the process of the identification of the spatial relationship among the pharmacophoric groups.Abbreviations t-Bu tert-butyloxycarbonyl - Fmoc 9-fluorenylmethoxycarbonyl     

Development of a selective pseudosubstrate-based peptide inhibitor of pp60c-src protein tyrosine kinase

Summary Using a combinatorial peptide library method, we identified YIYGSFK as an efficient and specific peptide substrate for pp60^c-src protein tyrosine kinase (PTK) [Lam et al., Int. J. Pept. Protein Res., 45 (1995) 587]. Employing YIYGSFK as a template, we synthesized and evaluated a series of pseudosubstrate-based inhibitors for pp60^c-src. We found that the efficiency of a given inhibitor was highly dependent on the specific tyrosine analog used at the phosphorylation site of the substrate. One of these pseudosubstrate inhibitors, YI(2-Nal)GSFK, selectively inhibited the kinase activity of pp60^c-src, with a K_i of 24 M. This peptide inhibitor exhibited selectivity for pp60^c-src as compared to other PTKs tested, such as c-Abl and Bcr-Abl. Our results suggest that selective inhibitors for a specific PTK can be developed when the structure of a specific and efficient small peptide substrate for this PTK can be used as a template for structure modification.Abbreviations 1-Nal l-1-naphthylalanine - 2-Nal l-2-naphthylalanine - BOP benzotriazolyl-N-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate - BSA bovine serum albumin - cAPK cyclic AMP-dependent protein kinase - DIEA diisopropylethylamine - EGFR epidermal growth factor receptor - Fmoc fluorenylmethoxycarbonyl - HOBt 1-hydroxybenzotriazole - MES 2-[N-morpholino]ethanesulfonic acid - PBS phosphate-buffered salts - pCl l-p-chlorophenylalanine - pF l-p-fluorophenylalanine - PTK protein tyrosine kinase - TLC thin-layer chromatography     

Solid-phase synthesis of triple-helical collagen-model peptides

Summary The triple-helical conformation of collagen has been proposed to be important for mediation of cellular activities, such as adhesion and activation, extracellular matrix assembly, and enzyme function. We have developed synthetic protocols that allow for the study of biological activities of specific collagen sequences in triple-helical conformation. These methods primarily involve solid-phase assembly and covalent linkage of three peptide chains. The resultant triple-helical peptides have sufficient thermal stabilities to permit structural and biological characterization under physiological conditions. The present article critically reviews the various approaches for constructing synthetic triple-helices.This paper is based on a presentation given at the Symposium on Peptide Structure and Design as part of the 31st Annual ACS Western Regional Meeting held in San Diego, CA, USA, October 18–21, 1995.     

Studies on epimerization-free methods for the preparation of aminosuccinyl peptides

Summary We have found that guanidine acetate catalyses the transformation of a -benzyl-aspartyl peptide (Boc-Asp-(OBzl)-Leu-Trp-OMe) to an aminosuccinyl peptide (Boc-Asu-Leu-Trp-OMe). The reaction was accompanied by partial epimerization. However, not even a small amount of epimerization could be detected when the aminosuccinyl peptide was synthesised from Boc-Asp-Leu-Trp-OMe with the addition of DIC, HOPfp and guanidine acetate (as a catalyst). This reaction seems to be suitable for the epimerization-free solid phase synthesis of aminosuccinyl peptides, e.g. Asu⁶-Lamprey-III-GnRH (Glp-His-Trp-Ser-His-Asu-Trp-Lys-Pro-Gly-NH₂).     

Processing of Procarboxypeptidase E into Carboxypeptidase E Occurs in Secretory Vesicles

Abstract: Carboxypeptidase E (CPE) functions in the posttranslational processing of bioactive peptides. Like other peptide processing enzymes, CPE is initially produced as a precursor ("proCPE") that undergoes posttranslational processing at a site containing five adjacent Arg residues near the N-terminus and at other sites near the C-terminus of proCPE. The time course of the N-terminal processing step suggests that this conversion occurs in either the Golgi apparatus or the secretory vesicles. To delineate further the site of proCPE processing, pulse/chase analysis was performed under conditions that block transit out of the Golgi apparatus (brefeldin A, carbonyl cyanide m -chlorophenylhydrazone, or 20°C) or that block acidification of vesicles (chloroquine, monensin, or ammonium chloride). The results of these analysis suggest that efficient proCPE processing requires an acidic post-Golgi compartment. To test whether known processing enzymes can perform this cleavage, purified proCPE was incubated with furin, prohormone convertase 1, or a dynorphin converting enzyme, and the products were analyzed on denaturing polyacrylamide gels. Furin cleaves proCPE within the N-terminal region, although the reaction is not very efficient, requiring relatively large amounts of furin or long incubation times. The other two peptide processing enzymes did not cleave proCPE, whereas a relatively small amount of secretory granule extract was able to convert proCPE into CPE. Taken together, these findings suggest that the conversion of proCPE into CPE occurs primarily in secretory vesicles.