Drosophila cell fusion induced by wheat germ agglutinin.

The effects of wheat germ agglutinin on Drosophila embryonic cell lines growing on cover-glasses was examined by scanning electron microscopy. At low concentrations of the lectin (5-10 mug/ml), cells spread against the glass surface and fused to form syncytia. At high concentration, damage to the cell surface was evidenced as extensive membrane shrivelling and loss of surface microfilaments. Fusion also occurred under these conditions. There was some indication that the morphology of cells in division remains undisturbed by wheat germ agglutinin. The coalescence of cells and morphologic disotrtion induced by wheat germ agglutinin were not inhibited by N-acetylglucosamine, the hapten inhibitor of the lectin, under the conditions utilized in this study.     

A wheat germ cell-free system is a novel way to screen protein folding and function

For high-throughput protein structural analysis, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as that involving Escherichia coli cells, have been developed, the number of overexpressed proteins showing the same biological activities as those of the native proteins is limited. A novel wheat germ cell-free protein synthesis system was developed recently, and most of the proteins functioning in solution were synthesized as soluble forms. This suggests the applicability of this protein synthesis method to determination of the solution structures of functional proteins. To examine this possibility, we have synthesized two (15)N-labeled proteins and obtained (1)H-(15)N HSQC spectra for them. The structural analysis of these proteins has already progressed with an E. coli overexpression system, and (1)H-(15)N HSQC spectra for biologically active proteins have already been obtained. Comparing the spectra, we have shown that proteins synthesized with a wheat germ cell-free system have the proper protein folding and enough biological activity. This is the first experimental evidence of the applicability of the wheat germ cell-free protein synthesis system to high-throughput protein structural analysis.     

Control of protein synthesis in a wheat germ cell-free system

Thermostable low molecular weight translational inhibitor was found in wheat germ cell-free extract. The inhibitor was formed during preincubation of wheat S-23 fraction with components of the energy-supplying system (ATP, GTP, phosphoenolpyruvate) in the absence of exogenous mRNA. The inhibitor effectively and irreversibly blocks protein synthesis in both wheat germ and rabbit reticulocyte systems. Our results seem to suggest that the inhibitor can activate wheat endogenous mRNA, which under the standard conditions does not reveal template activity but, once activated, can effectively compete with exogenous mRNA.     

A novel protein programmed by the mRNA conserved in dry wheat embryos. The principal site of cysteine incorporation during early germination

If bulk mRNA from dry wheat embryos (wheat germ) is used to direct cell-free incorporation of [35S]cysteine into proteins, a striking proportion of the total radioactivity is channeled into a single protein. During early postimbibition development, when protein synthesis is directed by the mRNA conserved in dry embryos, incorporation of cysteine is preponderantly (20-25%) directed into synthesis of this one protein: the 'early' cysteine-labeled protein (Ec). When conserved mRNA from the dry embryos has been fully degraded, as when cellular or cell-free protein synthesis is directed by the mRNA in germinated embryos, synthesis of Ec is not detected. Reliable detection of Ec requires prior alkylation of wheat embryo proteins, and it was especially interesting to find that when wheat embryo proteins are alkylated by iodo[14C]acetamide, two proteins co-dominate the distribution of radioalkylated products in dodecylsulphate/polyacrylamide gels: Ec and wheat germ agglutinin. Using co-electrophoresis with the isotopically labeled protein to detect a dye-staining counterpart, Ec has been purified by combined cation-exchange and gel-filtration chromatography of alkylated wheat germ proteins. The purified protein can be recovered in milligram quantity (5-10 mg/100 g wheat germ) and compositional analysis shows that it is unusually rich in cysteine (approx. 15%) and glycine (approx. 17%), as is wheat germ agglutinin.     

Characterization of a wheat germ agglutinin-like lectin from adult wheat plants

Radioimmuno-and enzyme-linked immunosorbent assays show that a substantial amount of wheat germ agglutinin(WGA)-like protein is present at the base of the shoot and in the roots of adult wheat (Triticum aestivum L.) plants. The protein can be purified by hapten-and antibody-mediated affinity procedures. It forms an arc of identity with the embryo lectin upon Ouchterlony double-diffusion and is an active lectin that agglutinates trypsinized erythrocytes in an N-acetylglucosamine-and chitin-inhibitable manner. Reduced and carboxyamidated protein comigrates with the 18-kdalton subunits of embryo lectin on sodium dodecyl sulfate-polyacrylamide gels. Invivo labeling of 9-d-old, hydroponically grown plants with ³⁵S-labeled sulfate demonstrates that at least some of the WGA-like protein is synthesized de novo. Immunocytochemistry with rabbit anti-WGA and colloidal-gold-conjugated second antibody shows that cross-reactive protein is present at the tips of new adventitious roots. In reactive cells, the lectin is localized near the inner surface of the vacuole membrane. Wheat plants contain up to 100 ng of WGA-like protein after the first week of growth, but the level fluctuates thereafter. Since most of the lectin is present at the base of the shoot and much less is found in older roots, these fluctuations may be the consequence of changes in the initiation of new advantitious roots.Abbreviations ELISA enzyme-linked immunosorbent assay - GlcNAc N-acetylglucosamine - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - WGA wheat germ agglutinin     

Evidence for the regulation of protein synthesis by a wheat germ phosphoprotein factor

A 48-kilodalton phosphoprotein, termed T-protein or pT, isolated from wheat germ and purified to homogeneity is found to inhibit the translation of tobacco mosaic virus (TMV) RNA in both wheat germ and reticulocyte lysates. The translation of TMV RNA in both systems was inhibited over 80% by 8 microM pT. There was no evidence to indicate that the reticulocyte lysate also contained a pT-like protein. pT was rapidly phosphorylated in the wheat germ and reticulocyte lysates. Although the relationship between pT phosphorylation and inhibition of protein synthesis is not known, there is evidence to indicate that complete phosphorylation of pT is not required for inhibition. Furthermore, no significant differences in the kinetics of inhibition of protein synthesis between prephosphorylated and unmodified pT were observed. Investigation of the mechanism of inhibition indicated that neither the aminoacylation of tRNA nor the elongation of nascent polypeptide chains was affected by pT. On the other hand, pT was found to prevent the formation of the 80S initiation complex. This action of pT was not due to the binding of pT to the ribosomes. However, the effect of pT was found to vary with the concentrations and types of mRNA used in the translational system. These results suggest that pT may interact with specific region(s) of the mRNA and prevent its translation. Alternatively, pT could block the translation of mRNA by binding to one or more of the initiation factors that interact with mRNA to facilitate mRNA binding to the 43S preinitiation complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel way of amino acid-specific assignment in 1H-15N HSQC spectra with a wheat germ cell-free protein synthesis system

For high-throughput protein structural analyses, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones utilizing E. coli cells, have been developed, a lot of proteins functioning in solution still were synthesized as insoluble forms. Recently, a novel wheat germ cell-free protein synthesis system was developed, and many of such proteins were synthesized as soluble forms. This means that the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we synthesized (15)N-labeled proteins with this wheat germ cell-free system, and confirmed this applicability on the basis of the strong similarity between the (1)H-(15)N HSQC spectra for native proteins and the corresponding ones for synthesized ones.In this study, we developed a convenient and reliable method for amino acid selective assignment in (1)H-(15)N HSQC spectra of proteins, using several inhibitors for transaminases and glutamine synthase in the process of protein synthesis. Amino acid selective assignment in (1)H-(15)N HSQC spectra is a powerful means to monitor the features of proteins, such as folding, intermolecular interactions and so on. This is also the first direct experimental evidence of the presence of active transaminases and glutamine synthase in wheat germ extracts.     

A specific wheat germ agglutinin-immunoreactive protein in human placenta

In this study we examined human placenta for the presence of molecules antigenically related to a plant lectin, wheat germ agglutinin. The initial results of immunolocalization using polyclonal antibodies against wheat germ agglutinin showed that human placenta contains protein(s) recognized specifically. Staining of syncytiotrophoblast brush border and cytotrophoblast, granular in appearance was observed in first trimester human placenta. Specific binding was also seen in trophoblast-derived JAr and BeWo carcinoma cells. Isolation of wheat germ agglutinin-immunoreactive material from human placenta was achieved by ion-exchange- and affinity-chromatography on anti-wheat germ agglutinin-immunoglobulin G-Sepharose. The placental protein having molecular mass of 66 kD was identified as specific. The protein of 66 kD was characterized as a calcium-dependent, asialofetuin-binding molecule.     

Initiation factors that bind mRNA. A comparison of mammalian factors with wheat germ factors

Three mammalian eukaryotic initiation factors (eIF) are required for the ATP-dependent binding of mRNA to the 40 S ribosomal subunit. These three factors, eIF-4A, eIF-4B, and eIF-4F, have also been isolated from wheat germ. Three assays were used to measure the ability of the wheat germ factors to interact with and/or substitute for the mammalian factors. Two assay systems were used to measure partial reactions involving the interaction of the three factors, ATP, and mRNA: 1) RNA-dependent ATP hydrolysis and 2) cross-linking of the factors to the 5' cap of oxidized mRNA. A third assay system was used to measure the ability of the factors to support initiation of protein synthesis. The results of the ATP hydrolysis and cross-linking experiments indicate that the wheat germ factors can interact with or substitute for the mammalian factors. Wheat germ eIF-4A appears to be functionally equivalent to mammalian eIF-4A. Wheat germ eIF-4B and eIF-4F appear to be isozymes possessing functions similar to mammalian eIF-4F. Wheat germ eIF-4B does not appear to be a functional equivalent to the mammalian eIF-4B. In a complete translation system from wheat germ, mammalian factors partially substitute for wheat germ factors, whereas the wheat germ factors are ineffective in the mammalian system.     

Removal of wheat-germ agglutinin increases protein synthesis in wheat-germ extracts

Affinity chromatography of wheat germ extracts on a chitin column increased the rate and extent of protein synthesis, programmed by rabbit globin mRNA. Addition of purified wheat germ agglutinin to the chitin-treated extract reduced the rate of protein synthesis to about the levels seen in the untreated extracts. Experiments where the ratio of messenger to extract and the ratio of supernatant to ribosomes were varied, indicated that addition of wheat germ agglutinin reduced the amount of available ribosomes. Reduced and carboxymethylated wheat germ agglutinin failed to inhibit protein synthesis and was unable to bind to the ribosomes. However, labelled intact agglutinin was found to be bound to ribosomes. The bound agglutinin was not released by acid treatment. The inhibiting effect of wheat germ, agglutinin on protein synthesis could not be counteracted by addition of N-acetyl-D-glucosamine or sialic acid, whereas thiols partially diminished the inhibition. The data indicate that wheat germ agglutinin binds reversibly to ribosomes, probably through mixed disulfide formation, and that chitin treatment increases the ability of wheat germ extracts to support protein synthesis, at least in part, by removing the wheat germ agglutinin. The possibility that chitin treatment also removed other inhibitors of protein synthesis cannot be excluded.     

Determination of the amounts of the protein synthesis initiation and elongation factors in wheat germ

Previous work by Browning et al. (Browning, K. S., Lax, S. R., Humphreys, J., Ravel, J. M., Jobling, S. A., and Gehrke, L. (1988) J. Biol. Chem. 263, 9630-9634) indicated that wheat germ extracts do not contain sufficient amounts of some of the protein synthesis initiation factors to obtain optimal translation of all mRNAs. In this investigation, a quantitative enzyme-linked immunosorbent assay was used to determine the amounts of eukaryotic initiation factors (eIF) 2, 3, 4A, 4F, and (iso)4F as well as the amounts of 40 S ribosomal subunits and elongation factors (EF) 1 alpha and 2 present in wheat germ extracts. EF-1 alpha is present in the highest amount (approximately 5% of the total protein), and eIF-4F is present in the lowest amount (approximately 0.03% of the total protein). The micromolar amounts of the factors and ribosomes are as follows: EF-1 alpha, 34; EF-2, 5.2; eIF-2, 1.5; eIF-3, 0.7; eIF-4A, 3.0, eIF-4F, 0.09; eIF-(iso)4F, 0.8; and 40 S ribosomal subunits, 3.2. The molar ratios of the factors to 40 S ribosomal subunits are approximately 11:1 for EF-1 alpha, 1.6:1 for EF-2, 0.45:1 for eIF-2, 0.2:1 for eIF-3, 0.9:1 for eIF-4A, 0.03:1 for eIF-4F, and 0.25:1 for eIF-(iso)4F. These findings strongly suggest that the concentrations of the initiation factors, particularly those factors required for the binding of mRNA to ribosomes, may play a major role in regulating the translation of mRNAs within the cell.     

Comparative studies on the increase by polyamines of fidelity of protein synthesis in Escherichia coli and wheat germ cell-free systems.

In the presence of polyamines, the fidelity of protein synthesis in a wheat germ cell-free system was increased significantly, while it was increased slightly in an $\text{E. coli}$ cell-free system. The effective concentration of polyamines for the increase in fidelity of protein synthesis was nearly equal to that for the stimulation of protein synthesis in a wheat germ cell-free system.     

Efficient synthesis of a disulfide-containing protein through a batch cell-free system from wheat germ.

We have developed a highly productive cell-free protein synthesis system from wheat germ, which is expected to become an important tool for postgenomic research. However, this system has not been optimized for the synthesis of disulfide-containing proteins. Thus, we searched here for translation conditions under which a model protein, a single-chain antibody variable fragment (scFv), could be synthesized into its active form. Before the start of translation, the reducing agent dithiothreitol, which normally is added to the wheat germ extract but which inhibits disulfide formation during translation, was removed by gel filtration. When the scFv mRNA was incubated with this dithiothreitol-deficient extract, more than half of the synthesized polypeptide was recovered in the soluble fraction. By addition of protein disulfide isomerase in the translation solution, the solubility of the product was further improved, and nearly half of the soluble polypeptides strongly bound to the antigen immobilized on an agarose support. This strong binding component had a high affinity as shown by surface-plasmon resonance analysis. These results show that the wheat germ cell-free system can produce a functional scFv with a simple change of the reaction ingredients. We also discuss protein folding in this system and suggest that the disulfide bridges are formed cotranslationally. Finally, we show that biotinylated scFv could be synthesized in similar fashion and immobilized on a solid surface to which streptavidin is bound. SPR measurements for detection of antigens were also possible with the use of this immobilized surface.     

A novel way to express proline-selectively labeled proteins with a wheat germ cell-free protein synthesis system

For high-throughput protein structural analyses, it is essential to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones involving Escherichia coli cells, have been developed, the number of overexpressed proteins exhibiting the same biological activities as those of the native ones is limited. A novel wheat germ cell-free protein synthesis system was developed recently, and most of the synthesized proteins that should function in solution were found to be in soluble forms. This suggests the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we developed a selective labeling technique for amino acids having amide functional groups (other than proline residues) involving the use of several inhibitors for transaminases. This paper in turn describes a proline-selective labeling technique. Based on our results, we have succeeded in constructing a complete amino acid selective labeling technique for the wheat germ cell-free protein synthesis system.     

Purification and characterisation of adenosine-3',5'-phosphate-independent protein kinase from wheat germ

cAMP-independent protein kinase was isolated from the wheat germ and purified to electrophoretic homogeneity. The molecular weight of enzyme was approximately 20,000, Km for ATP was (1 +/- 0.2) x 10(-5) M. V was 215 nmol phosphate mg enzyme-1 min-1, and the isoelectric point was at pH 9.2. The enzyme promotes phosphorylation of casein and crude wheat germ ribosomes.     

Distribution of wheat germ agglutinin in young wheat plants

A liquid phase, competition-binding radioimmunoassay for wheat germ agglutinin, with a detection limit of 10 nanograms, was developed in order to determine the distribution of this lectin in young wheat plants. Affinity columns for wheat germ agglutinin removed all antigenically detectable activity from crude extracts of wheat tissue; thus, the antigenic cross-reactivity detected by the assay possesses sugar-binding specificity similar to the wheat germ-derived lectin. The amount of lectin per dry grain is approximately 1 microgram, all associated with the embryo. At 34 days of growth, the level of lectin per plant was reduced by about 50%, with approximately one-third in the roots and two-thirds in the shoot. The data also indicate that actively growing regions of the plant (the bases of the leaves and rapidly growing adventitious roots) contain the highest levels of lectin. Half of the lectin associated with the roots could be solubilized by washing intact roots in buffer containing oligomers of N-acetylglucosamine, whereas the remainder is liberated only upon homogenization of the tissue.     

The molecular basis for the differential translation of TMV RNA in tobacco protoplasts and wheat germ extracts

Translation of tobacco mosaic virus (TMV) RNA in tobacco protoplasts yields the 17.5-K coat protein, a 126-K protein and a 183-K protein which is generated by an efficient readthrough over the UAG termination codon at the end of the 126-K cistron. In wheat germ extracts, however, only the 5'-proximal 126-K cistron is translated whereas the 183-K readthrough protein is not synthesized. Purification and sequence analysis of the endogenous tyrosine tRNAs revealed that the uninfected tobacco plant contains two tRNAs^Tyr, both with GΨA anticodons which stimulate the UAG readthrough in vitro and presumably in vivo. In contrast, ˜85% of the tRNA^Tyr from wheat germ contains a QΨA anticodon and ˜15% has a GΨA anticodon. Otherwise the sequences of tRNAs^Tyr from wheat germ and tobacco are identical. UAG readthrough and hence synthesis of the 183-K protein is only stimulated by tRNA^Tyr_GΨA and not at all by tRNA^Tyr_QΨA. The tRNAs^Tyr from wheat leaves were also sequenced. This revealed that adult wheat contains tRNA^Tyr_GΨA only. This is very much in contrast to the situation in animals, where Q-containing tRNAs are characteristic for adult tissues whereas Q deficiency is typical for the neoplastic and embryonic state.