伸展素在黄瓜胞壁中的转化及其与羟脯氨酸和异联酪氨酸的关系

用~(14)C-Pro和~3H_2-Tyr离体暗培养黄瓜子叶,发现细胞质、SECW和RCW中的Hyp/Pro和Idt/Tyr都随时间呈线性增加。这两种比值后两者高于前者,而两种比值增加速度之比在胞质部分最大、RCW中最小。表明在胞质和胞壁中都有Pro羟化和Tyr异联化过程,但羟化作用主要发生在胞质中,异联化主要在RCW中。 理化处理(高渗溶液和CHM)和放射性示踪证明胞质中存在HRGP库;它被分泌到胞壁后,先以离子键与壁结合,后转变为共价键与壁结合的伸展素。     

An intramolecular linkage involving isodityrosine in extensin

We isolated isodityrosine, a diphenyl ether linked amino acid, from cell wall hydrolysates and from two tryptic peptides of extensin. Determination of the molecular weights, net charges and composition of the peptides indicated that isodityrosine (IDT) can form a short intramolecular linkage in sequences consisting of:

     

Smart peptide libraries are accessible via enzymatic modifications

The minireview summarizes the recent preparation of thefollowing unusually modified combinatorial peptide collectionsuseful for diagnostics and screening in drug finding. Tissuetransglutaminase catalyzes cross couplings with transamidationbetween Gln and Lys peptide chains resulting in libraries withisopeptide bonds. The enzyme is involved in the triggering ofautoantigenic B- and T-cell epitopes of coeliac disease. Themicrobial enzyme EpiD involved in lantibiotic biosynthesiscatalyzes oxidative decarboxylation of C-terminal cysteineresidues in peptide libraries transforming peptidyl-cysteinesto peptide (2-mercaptovinyl)amides. Novel backbone modifiedpeptide libraries are prepared using oxazole and thiazolebuilding blocks carrying amino acid side chains. These aminoacids have been found in many biologically active naturalproducts from marine and microbial organisms such as microcinB17. Dityrosine and isodityrosine linked peptide dimerlibraries are accessible by oxidative phenol coupling usinghorseradish peroxidase. Such structural elements are found forexample in the polycyclic glycopeptide antibiotics of thevancomycin type. Microstructured layers of linear and cyclicpeptide libraries are generated on transducer surfaces forcellular assays, sensor developments and even chiralrecognition. Examples include a light-directed andmicrostructured electrochemical polymerization of phenollabelled peptides.     

Isolation of extensin precursors by direct elution of intact tomato cell suspension cultures

Dilute salt solutions eluted peroxidase and hydroxyproline-rich glycoproteins (HRGP's) very rapidly (60 % within 10s) from the surface of intact tomato cells grown in suspension culture. Further purification of the HRGPs based on (a) their solubility in 10% trichloroacetic acid and (b) chromatography on carboxymethyl cellulose, gave two components (P1 and P2) rich in serine, tyrosine, lysine and arabinosylated hydroxyproline. The sum of the hydroxyproline arabinoside profiles of P1 and P2 approximated that of the wall. P1, unlike P2, was histidine-rich and also contained proline. Significantly, isodityrosine (IDT) was absent from P1 and P2 but present in cell wall hydrolysates where, the Hyp:IDT molar ratio was ca 15: 1. In cells 4 days after subculture, ³H-proline pulse-chase data indicated turnover of P1 and P2 presumably resulting from covalent attachment to the wall as neither P1 nor P2 appeared in the growth medium. At day four the cell mean generation time (MGT) was 4.6 days, the cell hydr oxyproline content was 0.7 % (w/w), the half lives of P1 and P2 were both ca 12 hr, and the combined CaCl₂ elutable P1 and P2 precursor pools contained ca 400 μg Hyp/g cells (dry weight). Calculated from the MGT and Hyp content, the cell demand was 44.μg Hyp/g cells (dry weight)/hr. The precursor pool size was therefore sufficient for 9 hours growth. However the pool turnover calculated from half life and pool size was 5.6 %/hr or 22.4μg Hyp/g cells (dry weight)/hr. Thus the supply of P1 and P2 precursors met > 50 % of the cell wall demand. Corroborative experiments showed that after depletion of the P1 and P2 pools by salt elution, washed cells resuspended in growth medium repleted the precursor pools at a rate corresponding to a synthesis of 43μg Hyp/g cells (dry weight)/hr, or 98 % of the demand. These data allow us to make the following suggestions: P1 and P2 represent monomeric extensin precursor subunits. Salt elution of P1 and P2 indicates their ionic binding by pectic carboxyl groups. The rapidity of elution indicates a high diffusivity of these extended rodlike macromolecules through the cell wall. This may imply a preferred orientation for P1 and P2 perpendicular rather than parallel to the plane of the wall. The lack of IDT in P1 and P2 implies that IDT forms in muro, possibly via peroxidase. We speculate that some of these IDT residues may crosslink an extensin precursor ‘tweft’ around a cellulose microfibrillar ‘twarp’. Such formation of heteromultimeric extensin interpenetrated by microfibrils would create a mechanically coupled extensin-cellulose network.     

Extensin—a major cell wall glycoprotein

Abstract The structure of extensin is described in detail. It has a hydroxyproline-rich backbone, which contains repeating peptides glycosylated by short side chains and it adopts a polyproline II helical conformation. The glycoprotein is synthesized intracellularly and soluble precursors are secreted to the wall, where they are bound, perhaps, by the formation of isodityrosine cross-links. The various hypotheses, including the most recent ‘warp and weft’ model, which have been suggested to explain the attachment of extensin to the other wall polymers are discussed. The possible functions of extensin in defence and in the control of extension growth are described in addition to its probable structural role. Other glycoproteins which resemble extensin are also mentioned.     

Smart peptide libraries are accessible via enzymatic modifications

Summary The minireview summarizes the recent preparation of the following unusually modified combinatorial peptide collections useful for diagnostics and screening in drug finding. Tissue transglutaminase catalyzes cross couplings with transamidation between Gln and Lys peptide chains resulting in libraries with isopeptide bonds. The enzyme is involved in the triggering of autoantigenic B- and T-cell epitopes of coeliac disease. The microbial enzyme EpiD involved in lantibiotic biosynthesis catalyzes oxidative decarboxylation of C-terminal cysteine residues in peptide libraries transforming peptidyl-cysteines to peptide (2-mercaptovinyl)amides. Novel backbone modified peptide libraries are prepared using oxazole and thiazole building blocks carrying amino acid side chains. These amino acids have been found in many biologically active natural products from marine and microbial organisms such as microcin B17. Dityrosine and isodityrosine linked peptide dimer libraries are accessible by oxidative phenol coupling using horseradish peroxidase. Such structural elements are found for example in the polycyclic glycopeptide antibiotics of the vancomycin type. Microstructured layers of linear and cyclic peptide libraries are generated on transducer surfaces for cellular assays, sensor developments and even chiral recognition. Examples include a light-directed and microstructured electrochemical polymerization of phenol labelled peptides.