Computed three-dimensional structures for theras-binding domain of theraf-p74 protein complexed withras-p21 and with its suppressor protein, rap-1A

The three-dimensional structures of theras-p21 protein and its protein inhibitor, rap-1A, have been computed bound to theras-binding domain, RBD (residues 55–131), of theraf-p74 protein, a critical target protein ofras-p21 in theras-induced mitogenic signal transduction pathway. The coordinates of RBD have been reconstructed from the stereoview of an X-ray crystal structure of this domain bound to rap-1A and have been subjected to energy minimization. The energy-minimized structures of bothras- p21 and rap-1A, obtained in previous studies, have been docked against RBD, using the stereo figure of the RBD-rap-1A complex, based on a six-step procedure. The final energy-minimized structure of rap-1A-RBD is identical to the X-ray crystal structure. Comparison of theras-p21- and rap-1A-RBD complexes reveals differences in the structures of effector domains ofras-p21 and rap-1a, including residues 32–47, a domain that directly interacts with RBD, 60–66, 96–110, involved in the interaction ofras-p21 withjun kinase (JNK) andjun protein, and 115–126, involved in the interaction of p21 with JNK. The structure of the RBD remained the same in both complexes with the exception of small deviations in its-2 binding loop (residues 63–71) and residues 89–91, also involved in binding to rap-1A. The results suggest that the binding of these two proteins to RBD may allow them to interact with other cellular target proteins such as JNK andjun.     

Microbial α-mannosidases

Microbial α-mannosidases are used in the analysis of glycopeptides and the developmental regulation of lysosomal enzymes. This survey presents comparison of properties of this high molecular weight, oligomeric protein from a number of microbial sources.     

Engineering the lac permease for purification and crystallization

The lactose permease is being used as a model system for the rational redesign of a membrane protein with the goal of increasing the likelihood of crystallization. Various modifications to the protein have been added for the purposes of purification, stability, and potential for crystallization. The addition of six consecutive histidines at the C-terminus of the protein allows for the rapid purification by nickel-chelate chromatography, and the insertion of an entire protein domain into one of the inner cytoplasmic loops of the permease gives the resulting protein a larger hydrophilic surface area. The increase in polar surface area makes the fusion protein easier to handle and more likely to crystallize. In particular, the introduction of cytochromeb562 ofE. coli into the central hydrophilic domain of the lac permease results in a fusion protein with the transport activity of the permease and the visible absorbance spectrum of the cytochrome. The red permease is very easy to monitor through the steps of expression, purification, concentration, and crystallization.     

A novel plasma membrane-bound thioredoxin from soybean

Two thioredoxin cDNAs from soybean were isolated by screening an expression library using an anti-(plasma membrane) serum. The nucleotide sequences of the two cDNAs were found to be 89% identical. The polypeptides encoded by the two cDNAs, designated TRX1 and TRX2, contain a disulfide active site, as found in other thioredoxins. TRX1 was expressed as a fusion protein in Escherichia coli and shown to possess thiol-disufide interchange activity. Unlike other eukaryotic thioredoxins, these two soybean thioredoxins contain a putative transmembrane domain in their N-terminal regions. To determine subcellular location, the TRX1 was fused with a reporter epitope at its C-terminus and expressed in transgenic tobacco plants. The fusion protein was co-purified with plasma membrane markers 1,3-glucan synthase and vanadate-sensitive ATPase, indicating the plasma membrane location of TRX1. When the reporter epitope was inserted between the start codon and the transmembrane domain in the N-terminus, the fusion protein was found in the soluble fraction, possibly due to disruption of the transmembrane domain by the highly hydrophilic epitope sequence. Taken together, our results demonstrate that soybean TRX1 is a plasma membrane-bound thioredoxin, which is most likely anchored to the membrane through the N-terminal transmembrane domain. It is known that plant plasma membranes contain various proteins with thiol-disulfide interchange activity. The soybean thioredoxins reported here are the first group of such proteins to be characterized at the molecular level. However, the biological function of the plasma membrane-bound thioredoxin remains to be determined.     

Substrate subsite recognition of the xyloglucan endo-transglycosylase or xyloglucan-specific endo-(1→4)-β-d-glucanase from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds

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.     

Determination of base specificity of multiple ribonucleases from crude samples

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     

Substrate and inhibitor specificities of the thermostable alcohol dehydrogenase allozymes ADH-71k and ADH-FCh.D. ofDrosophila melanogaster

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.     

HOST SPECIFICITY IN THE RED ALGAL PARASITES BOSTRYCHIOCOLAX AUSTRALIS AND DAWSONIOCOLAX BOSTRYCHIAE (CHOREOCOLACACEAE,RHODOPHYTA)1

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.