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71.
72.
We have investigated the substrate subsite recognition requirement of the xyloglucan endo-transglycosylase/xyloglucan-specific endo-(14)--d-glucanase (NXET) from the cotyledons of nasturtium seedlings. Seed xyloglucans are composed almost entirely of the Glc4 subunits XXXG, XLXG, XXLG and XLLG, where G represents an unsubstituted glucose residue, X a xylose-substituted glucose residue and L a galactosyl-xylose-substituted glucose residue. Thus in the xyloglucan sequence shown below, the xylose (Xyl) residues at the backbone glucose (Glc) residues numbered — 3,— 2, + 2 and + 3 may be galactose-substituted, and NXET cleaves between the unsubstituted glucose at — 1 and the xylose-substituted glucose at + 1, which never carries a galactosyl substituent. We have isolated the xyloglucan oligosaccharides XXXGXXXG and XLLGXLLG from NXET digests of tamarind seed xyloglucan, have modified them enzymatically using a pure xyloglucan oligosaccharide-specific -xylosidase from nasturtium seeds to give GXXGXXXG and GLLGXLLG, and have identified and compared the products of NXET action on XXXGXXXG, GXXGXXXG, XLLGXLLG and GLLGXLLG. We have also compared the molar proportions of XXXG, XLXG, XXLG and XLLG in native tamarind and nasturtium seed xyloglucans with those in NXET digests of these polysaccharides. Using these and existing data we have demonstrated that NXET action does not require xylosesubstitution at glucose residues — 4, — 2, + 1 and + 3 and that xylose substitution at + 2, is a requirement. There may also be a requirement for xylose substitution at — 3. We have demonstrated also that galactosyl substitution of a xylose residue at + 1 prevents, and at — 2 modifies, chain-cleavage. A partial model for the minimum substrate binding requirement of NXET is proposed.Abbreviations G
unsubstituted glucose residue
- X
xylose-substituted glucose residue
- L
galactosylxylose-substituted glucose residue
- F
fucosyl-galactosylxylose-substituted glucose residue
- Gal
galactose
- Glc
glucose
- HPAE
high-performance anion-exchange chromatography
- NXET
nasturtium xyloglucan endo-transglycosylase or xyloglucan-specific endo-(14)--d-glucanase
- Xyl
xylose
This work was funded jointly by Unilever UK and the Department of Trade and Industry (UK) via the LINK initiative Agro-Food Quality. 相似文献
73.
You-Di Liao 《Molecular biology reports》1994,20(3):149-154
Ribonucleases are widely found in the tissues of living organisms, but the functions of individual ribonucleases are not clear. To facilitate characterization of individual ribonucleases, I have developed a rapid method to separate and identify each ribonuclease from a crude sample by gel electrophoresis instead of by time-consuming purification steps. The ribonucleases in a crude sample are first separated by RNA-cast SDS-polyacrylamide gel electrophoresis and then eluted from the gel after ethidium bromide staining. To determine the base specificity of each ribonuclease, a 5 labelled oligonucleotide with known sequence is added to the enzyme eluate and the digested products are analyzed by denaturing gel electrophoresis. The base specificity of bovine pancreatic ribonuclease (RNase A), bullfrog oocyte-specific ribonuclease (RC-RNase), human serum ribonucleases and sweet potato leaf ribonucleases were determined by this method. Other properties of individual ribonucleases, e.g. substrate preference, may also be determined from crude samples by this method without further purification steps.Abbreviations RNase
ribonuclease
- SDS
sodium dodecyl sulfate 相似文献
74.
Purified thermostable alcohol dehydrogenase allozymes ADH-71k and ADH-FCh.D. ofDrosophila melanogaster have been compared with the two common enzyme forms ADH-F and ADH-S. Enzyme kinetic parameters for various primary and secondary alcohols were determined under standard conditions used previously. Both ADH-71k and ADH-FCh.D. show ADH-S-like reaction kinetics andK
m values, due to retrograde evolution at site 214, Pro Ser. Inhibition studies with alcohol dehydrogenase inhibitors pyrazole, 4-methylpyrazole, and cibacron blue 3GA were also performed. Activity measurements on crude extracts of larvae and flies from isogenic lines of ADH-FCh.D. revealed a consistently higher activity than in ADH-71k-containing strains, in contrast to the original strains.K.Th.E is indebted to the Royal Norwegian Council for Technological and Scientific Research for their postdoctoral fellowship. Prof. J. S. McKinley-McKee gave me the opportunity to work in his laboratory. I thank Dr. Knut Sletten of the Biochemical Institute for the kind gift of 2-methoxyethanol and amino acid analysis of some samples. The Biological Institute, Oslo, Section of General Genetics, is gratefully acknowledged for enabling me to use their fly-breeding facilities. Dr. John B. Gibson provided us with a sample of FCh.D. flies for the construction of isogenic lines in which Dr. Johan Hageman participated, owing to Postdoctoral Grant 436-931-P from the Foundation of Biological Research (BION), which is subsidized by the Netherlands Organization for Scientific Research (NWO). J. H. and Paula Truyens were involved in the measurements on the crude extracts. Work at Victoria University was supported by the VUW Internal Grant Committee. 相似文献
75.
76.
The determinants of host specificity, which are poorly understood in red algal parasites, were studied in the red algal parasites Bostrychiocolax australis Zuccarello et West and Dawsoniocolax bostrychiae (Joly et Yamaguishi-Tomita) Joly et Yamaguishi-Tomita. Culture studies were performed to determine host range, sites of host resistance, and genetics of transmission of resistance. Both species parasitize Bostrychia radicans (Montagne) Montagne, whereas Bostrychiocolax australis also parasitizes Bostrychia moritziana (Sonder ex Kützing) J. Agardh and Stictosiphonia kelanensis (Grunow ex Post) R. J. King et Puttock. Isolates of B. radicans resistant to both parasites were found worldwide, often within the same population as susceptible isolates. On resistant Bostrychia species and isolates, specificity was manifested at three stages: 1) host penetration, in which the spore germ peg failed to penetrate the host cuticle/wall; 2) parasite–host cell fusion, in which the fusion cell died and the parasite died; and 3) growth, in which parasites grew but soon died; parasites rarely reproduced and infections did not continue in culture. Resistance to parasite infection was usually transmitted as a dominant trait and did not segregate as a single locus during meiosis. In certain crosses, transmission of resistance was non-mendelian. 相似文献
77.
Mare K. Saba-El-Leil Sylvain Rivard David Morse Mario Cappadocia 《Plant molecular biology》1994,24(4):571-583
A genomic clone of the S11 allele from the self-incompatibility locus (S locus) in Solanum chacoense Bitt. has been isolated by cross-hybridization to the S. chacoense S13 allele and sequenced. The sequence of the S11 allele contains all the features expected for S genes of the Solanaceae, and S11 expression, as assessed by northern blots and RNA-PCR, was similar to that of other S. chacoense S alleles. The S11 protein sequence shares 95% identity with the phenotypically distinct S13 protein of S. chacoense and is the gametophytic S allele with the highest similarity to an existing allele so far discovered. Only 10 amino acid changes differentiate the mature proteins from these two alleles, which sets a new lower limit to the number of changes that can produce an altered S allele specificity. The amino acid substitutions are not clustered, suggesting that an accumulation of random point mutations can generate S allele diversity. The S11 intron is unusual in that it could be translated in frame with the coding sequence, thus suggesting an additional mechanism for the generation of new S alleles. 相似文献
78.
79.
Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide. 总被引:25,自引:5,他引:20
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E. A. Merritt S. Sarfaty F. van den Akker C. L'Hoir J. A. Martial W. G. Hol 《Protein science : a publication of the Protein Society》1994,3(2):166-175
Cholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B-pentamer complexed with the GM1 pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue. The binding of GM1 to cholera toxin thus resembles a 2-fingered grip: the Gal(beta 1-3)GalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue. The CTB5:GM1 pentasaccharide complex described here provides a detailed view of a protein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in GM1-mediated signal transduction. 相似文献
80.
Nucleotide sequence and style-specific expression of a novel proline-rich protein gene from Nicotiana alata 总被引:4,自引:0,他引:4
cDNA clones encoding a novel proline-rich protein (NaPRP4) have been isolated from a Nicotiana alata stylar cDNA library. The N-terminal part of the derived protein is highly rich in proline (32.2%) and contains several repeats such as Lys-Pro-Pro (7 times) and Pro-Thr-Lys-Pro-Pro-Thr-Tyr-Ser-Pro-Ser-Lys-Pro-Pro (twice); the C-terminal part, on the other hand, has a lower proline content (9.9%) and contains two potential N-glycosylation sites and all the six cysteine residues. Northern blot and in situ hybridisation analyses indicate that expression of the NaPRP4 gene is restricted to cells of the transmitting tract of the style. 相似文献