tRNA is the source of low-level trans-zeatin production in Methylobacterium spp

Pink-pigmented facultatively methylotrophic bacteria (PPFMs), classified as Methylobacterium spp., are persistent colonizers of plant leaf surfaces. Reports of PPFM-plant dialogue led us to examine cytokinin production by PPFMs. Using immunoaffinity and high-performance liquid chromatography (HPLC) purification, we obtained 22 to 111 ng of trans-zeatin per liter from culture filtrates of four PPFM leaf isolates (from Arabidopsis, barley, maize, and soybean) and of a Methylobacterium extorquens type culture originally recovered as a soil isolate. We identified the zeatin isolated as the trans isomer by HPLC and by a radioimmunoassay in which monoclonal antibodies specific for trans-hydroxylated cytokinins were used. Smaller and variable amounts of trans-zeatin riboside were also recovered. trans-Zeatin was recovered from tRNA hydrolysates in addition to the culture filtrates, suggesting that secreted trans-zeatin resulted from tRNA turnover rather than from de novo synthesis. The product of the miaA gene is responsible for isopentenylation of a specific adenine in some tRNAs. To confirm that the secreted zeatin originated from tRNA, we mutated the miaA gene of M. extorquens by single exchange of an internal miaA fragment into the chromosomal gene. Mutant exconjugants, confirmed by PCR, did not contain zeatin in their tRNAs and did not secrete zeatin into the medium, findings which are consistent with the hypothesis that all zeatin is tRNA derived rather than synthesized de novo. In germination studies performed with heat-treated soybean seeds, cytokinin-null (miaA) mutants stimulated germination as well as wild-type bacteria. While cytokinin production may play a role in the plant-PPFM interaction, it is not responsible for stimulation of germination by PPFMs.     

Evidence for a lactose-mediated association between two nuclear carbohydrate-binding proteins

Nuclear proteins were extracted in 2 M NaCl from membrane-depletednuclei isolated from HL60 cells. Extracted proteins were submittedto affinity chromatography columns containing immobilized glucose,galactose or lactose. The polypeptides present in the differenteluted fractions were resolved by SDS—PAGE and were eithersilver stained or analysed by immunoblotting with monoclonalor polyclonal antibodies, respectively, raised against the glucose-bindingprotein CBP67 and the galactose-binding proteins CBP35 and L14.The results presented here show that HL60 cell nuclei containCBP35 and a glucose-binding lectin of 70 kDa (CBP70). Thesedata account for the previously reported binding of neoglyco-proteinscontaining glucosyl and galactosyl residues to HL60 cell nuclei.Furthermore, the present study provides the new informationthat CBP35 can associate with CBP70 by interactions dependenton the binding of CBP35 to lactose, and the results of someaffinity chromatography experiments strongly suggest that CBP35and CBP70 associate by protein—protein interactions. Thepotential function of this lactose-mediated interaction is discussedwith respect to data recently reported by others showing thatCBP35 is involved in in vitro mRNA splicing and that lactoseinhibits the processing of the pre-RNA substrate. HL60 lectins nucleus protein—protein interactions     

小麦叶绿体中细胞分裂素结合蛋白的功能

采用ＢＡ－Ｓｅｐｈａｒｏｓｅ６Ｂ亲和层析法从小麦叶绿体中纯化了ＣＴＫ结合蛋白并制备了抗体。小麦叶绿体经ＣＴＫ结合蛋白的抗体处理后,表现出：希尔反应活力降低;ＭＶ（包括光系统Ｉ和Ⅱ）和ＰＭＳ（包括光系统Ｉ）系统的毫秒延迟发光的慢相强度下降;光合磷酸化活力受到抑制。说明小麦叶绿体中的ＣＴＫ结合蛋白存在状态的改变会影响叶绿体的能量转换过程。     

Cytokinin oxidase activity and cytokinin content in roots of sunflower under water stress

Contents of trans-zeatin riboside (ZR), dihydrozeatin riboside (DZR) and N6-(delta2-isopentenyl) adenosine (iPA) was quantified by an indirect ELISA using polyclonal antibodies, in the roots, xylem sap and leaves of pot grown sunflower plants subjected to water stress (RWC of leaves approximately 65 per cent). The delivery rates of all three cytokinins decreased significantly under stress. Cytokinin levels also decreased in roots and in leaves of stressed plants. Three-fold increase in cytokinin oxidase activity was observed in stressed roots after polymin P-ammonium sulphate fractionation. Further purification using Con A agarose resulted in elution of protein with cytokinin oxidase activity and was found to be 30 kDa protein on SDS-PAGE.     

Cytokinin-binding proteins from tobacco callus share homology with osmotin-like protein and an endochitinase

To study the signal transduction of cytokinins, we characterized cytokinin-binding proteins (CBPs) isolated from tobacco callus Nicotiana tabacum. Two high-affinity CBPs, CBP1 and CBP2, were isolated from the soluble fraction of tobacco callus BY-2 cells by anion exchange chromatography on a DEAE-cellulose column and affinity chromatography on a benzyladenine (BA)-linked Sepharose 4B column. Cytokinin-binding activity was determined by the equilibrium dialysis method. The degree of purification of CBP1 and CBP2 was 270 and 600-fold, respectively. These proteins had molecular masses of 34 kDa and 26 kDa, and to bind benzyladenine (BA) with dissociation constants (Kd) of 8.9 x 10(-6) M and 1.1 x 10(-6) M, respectively. Binding of BA to CBP2 was inhibited by zeatin and kinetin but not by adenine, adenosine, ATP or IAA. The optimum pH for binding of BA to CBP1 and CBP2 was approximately pH 6.5 and 7.5, respectively. CBP1 showed significant homology (90%) with endochitinase and CBP2 with osmotin-like protein (OLP). These findings and the results of immunoblotting analysis and cytokinin-binding assay of recombinant OLP indicated that CBP2 is OLP, a stress protein.     

Epitope mapping of the human biliary amphipathic, anionic polypeptide: similarity with a calcium-binding protein isolated from gallstones and bile, and immunologic cross-reactivity with apolipoprotein A-I.

Biliary amphipathic anionic polypeptide (APF) the major protein of the pigment-lipoprotein complex in bile, and calcium-binding protein (CBP) from gallstones are both small (less than 10 kDa), highly acidic, amphipathic proteins present in bile and closely associated also with pigmented areas in human gallstones. Polyclonal antibodies against APF have shown cross-reactivity with plasma high density lipoproteins (HDL). This study examines the hypothesis that APF and CBP might be closely related or even identical, and might also share common epitopes with the larger apoA-I (23 kDa). To assess this, immunoreactivity of the three delipidated, highly purified proteins was determined against a panel of 12 monoclonal antibodies (MAbs) prepared against APF and a panel of 4 MAbs against apoA-I. APF was isolated from bile by zonal ultracentrifugation. CBP was isolated from proteins precipitated from bile by CaCl2, as well as from the calcium bilirubinate shells of cholesterol gallstones, by extraction successively with methyl-t-butyl ether, methanol, and Na2EDTA, followed by Sephadex G-25 chromatography and two-stage preparative SDS-PAGE. ApoA-I was prepared by two types of chromatography: Sephacryl S200 chromatography and heparin-chromatographic immunoaffinity. Specific polyclonal antibodies to APF and apoA-I were prepared from immunized rabbits. MAbs to APF and apoA-I were prepared by immunization of mice, using standard hybridoma technique. Western blotting of APF and CBP in 15% SDS-PAGE yielded one band with an apparent molecular weight of 6.5 kDa, which, along with apoA-I, was immunostained by polyclonal antibodies to APF and apoA-I. Using 12 MAbs against APF with three types of ELISA (direct antigen binding, competitive antigen displacement, and epitope competition between antibodies), it was shown that APF and delipidated apoA-I shared six epitopes, three of which were detected also on the surface of intact HDL particles. Six other epitopes were present in APF but not apoA-I, four of which were exposed on the surface of HDL. Four MAbs against apoA-I reacted with APF and CBP. Amino acid analyses of APF and CBP were similar with 20-23% acidic and 7-11% basic amino acids and low contents of cysteine, methionine, and tyrosine; both differed from apoA-I in containing isoleucine and cysteine. Using ELISA and one MAb (no. 32) against APF, this polypeptide was detected in human plasma HDL, the pigment-lipoprotein complex in the bile of humans, dogs, and rats, and in both pigment and cholesterol gallstones.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunolocalization of 1-<Emphasis Type="Italic">O</Emphasis>-sinapoylglucose:malate sinapoyltransferase in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The serine carboxypeptidase-like protein 1- O-sinapoylglucose:malate sinapoyltransferase (SMT) catalyzes the transfer of the sinapoyl moiety of 1- O-sinapoylglucose to malate in the formation of sinapoylmalate in some members of the Brassicaceae. Rabbit polyclonal monospecific antibodies were raised against the recombinant SMT produced in Escherichia coli from the corresponding Arabidopsis thaliana (L.) Heynh. cDNA. Immunoblot analysis of protein from different Arabidopsis tissues showed that the SMT is produced in all plant organs, except in the seeds and young seedlings. The enzyme was most abundant in older seedlings as well as in rosette leaves and the flowering stem of the plant. Minor amounts were found in the cauline leaves, flower buds and siliques. Traces were detected in the root and flowers. Arabidopsis and transgenic tobacco ( Nicotiana tabacum L.) plants expressing the full-length Arabidopsis SMT containing an N-terminal signal peptide showed apparent molecular masses of the protein of 52-55 kDa. The difference of ca. 8 kDa compared to the recombinant protein produced in E. coli was shown to be due to post-translational N-glycosylation of SMT in plants. Immunofluorescent labeling of Arabidopsis leaf sections localized SMT to the central vacuoles of mesophyll and epidermal cells. Comparable leaf sections of an SMT deletion mutant showed no vacuolar immunofluorescent labeling. We conclude that Arabidopsis SMT is synthesized as a precursor protein that is targeted to the endoplasmic reticulum where the signal peptide is removed. The correct N-terminus of the recombinantly produced SMT protein lacking the signal peptide was confirmed by Edman degradation. The protein is probably glycosylated in the Golgi apparatus from where it is subsequently routed to the vacuole.     

Purification and characterization of mRNA cap-binding protein from Drosophila melanogaster embryos.

A protein with specific affinity for the mRNA cap structure was purified both from the postribosomal supernatant and from the ribosomal high-salt wash of Drosophila melanogaster embryos by m7GTP-Sepharose chromatography. This protein had an apparent molecular mass of 35 kilodaltons (kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size very different from those of the cap-binding proteins that have been characterized thus far. Drosophila 35-kDa cap-binding protein (CBP) could also be isolated from the ribosomal high-salt wash as part of a salt-stable protein complex consisting of polypeptides of 35, 72, and 140 to 180 kDa. Polyclonal antibodies against Drosophila 35-kDa CBP neither reacted with eucaryotic initiation factor 4E from rabbit reticulocytes nor affected mRNA translation in a rabbit reticulocyte cell-free system. However, in a cell-free system from Drosophila embryos, mRNA translation was specifically inhibited by these antibodies. The requirement of 35-kDa CBP for mRNA translation in Drosophila was diminished under ionic conditions in which the importance of mRNA cap structure recognition was reduced. Despite the structural differences between Drosophila 35-kDa CBP and mammalian initiation factor 4E, both proteins were functionally interchangeable in the in vitro translation system from Drosophila embryos.     

Changes in Gene Expression from Diploid to Autotetraploid Status of Lycopersicon esculentum

The cytokinin activity of the root exudate, the leaves, and the apices of vegetative and flowering white lupin plants (Lupinus albus L.) was investigated. The level of cytokinin activity in the root exudate decreased over the 11-week experimental period. Four peaks of cytokinin activity were recorded in the root exudate of 8-week-old plants after fractionation on Sephadex LH-20. Two of these peaks co-eluted with zeatin and zeatin riboside. It is suggested that the remaining peaks represent nucleotide and glucoside cytokinins. The cytokinin levels in extracts of the mature leaves fluctuated slightly over the experimental period. Three peaks of activity co-eluting with zeatin, zeatin riboside and the glucoside cytokinins were recorded in extracts of mature leaves of 8-week-old plants. In the apices cytokinin activity could only be detected in the inflorescences of flowering plants. It would appear that cytokinins produced by the roots accumulate in the fully expanded mature leaves, but are utilized in the rapidly growing apical region and in young expanding leaves.