Specific detection of banana residue in processed foods using polymerase chain reaction.

Specific polymerase chain reaction (PCR) methods were developed for the detection of banana residue in processed foods. For high banana specificity, the primer set BAN-F/BAN-R was designed on the basis of the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) genes of chloroplasts and used to obtain amplified products specific to banana by both conventional and real-time PCR. To confirm the specificity of these methods, genomic DNA samples from 31 other species were examined; no amplification products were detected. Subsequently, eight kinds of processed foods containing banana were investigated using these methods to confirm the presence of banana DNA. Conventional PCR had a detection limit of 1 ppm (w/w) banana DNA spiked in 50 ng of salmon testis DNA, whereas SYBR Green I real-time semiquantitative PCR had a detection limit as low as 10 ppm banana DNA. Thus, both methods show high sensitivity and may be applicable as specific tools for the detection of trace amounts of banana in commercial food products.

Interleukin 4 increases the antibody response against Rubisco in mice.

The influence of interleukin 4 (IL-4) on antibody titer in serum and spleen culture supernatant in mice immunized with spinach (Spinacia oleracea L.) Rubisco was investigated. Therefore, we boosted one mouse additionally to the antigen with recombinant mouse IL-4. We found that the Rubisco-specific antibody titer in serum as well as in spleen cell culture supernatant was significantly enhanced in the IL-4 mouse. Most of the antibodies were of the IgG1 subclass. After hybridoma generation, Rubisco-specific antibodies were found in more than 95% of the wells tested compared to about 12% of the control mouse.

Preparation of polyclonal antibodies of Rubisco large and small subunits and their application in the functional analysis of the genes.

Spinach Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) large (rbcL) and small (rbcS) subunits were separated by SDS-PAGE, and protein amount and purity were determined by Bradford assay. Polyclonal antibodies against rbcL and rbcS subunit were generated in female BALB/c mice and had no cross-reaction with each other. A total of 81 microg antigens were used and 0.3 ml anti-sera with titer of 1:5000 were yielded. The antibodies were also applicable to study rbcL and rbcS in tobacco plant Nicotiana benthamiana. Potato virus X vector pGR107 induced silencing of rbcS gene by Agrobacterium in Nicotiana benthamiana was performed. The expression level of rbcL and rbcS was lower in rbcS silenced plants than that in control plants as detected by the corresponding antibodies. This implied that the expression of rbcL was regulated by rbcS.

Degradation of ribulose-bisphosphate carboxylase by vacuolar enzymes of senescing French bean leaves: immunocytochemical and ultrastructural observations.

The possible involvement of vacuolar cysteine proteinases in degradation of ribulose-bisphosphate carboxylase (Rubisco) in senescing French bean leaves was studied by ultrastructural and immunocytochemical analyses with antibodies raised against the large subunit (LSU) of Rubisco and SH-EP, a cysteine proteinase from Vigna mungo that is immunologically identical to one of the major proteinases of French bean plants. Primary leaves of 10-day-old plants were detached and placed at 25 degrees C in darkness for 0, 4, and 8 days to allow their senescence to proceed. The leaves at each senescence stage were subjected to the conventional electron microscopic and immunocytochemical studies. The results indicated that the chloroplasts of senescing French bean leaves were separated from the cytoplasm of the cell periphery and taken into the central vacuole and that the Rubisco LSU in the chloroplasts was degraded by vacuolar enzymes such as an SH-EP-related cysteine proteinase that developed in senescing leaves. The present results together with the results of previous biochemical studies using vacuolar lysates support the view that Rubisco is degraded through the association of chloroplasts with the central vacuole during the senescence of leaves that were detached and placed in darkness.

Production and characterization of a specific rubisco monoclonal antibody, and its use in rubisco quantification during Zantedeschia aethiopica spathe development.

Ribulose 1,5-bisphosphate carboxylase/oxygenase was purified from leaves of Zantedeschia aethiopica and used to immunize female Balb/c mice. Monoclonal antibodies (MAbs) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. A random selected IgG2a subclass MAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose CL-4B, with a recovery of 84.3% and it was apparently homogeneous on native PAGE. The monoclonality of the purified MAb was determined by IEF. The MAb was highly specific for Rubisco from leaves of Z. aethiopica as determined by Western blotting and was used to determine the concentration of Rubisco protein by enzyme-linked immunoadsorbent assay (ELISA), at three distinct stages of Z. aethiopica spathe development and in the leaf. The results suggest de novo synthesis of Rubisco during the spathe regreening, which could explain, at least in part, the increase of photosynthetic activity observed during regreening.

Quantitation of cat immunoglobulins in the hemolymph of cat fleas (Siphonaptera: Pulicidae) after feeding on blood.

Passage of ingested cat immunoglobulin G (IgG) into the hemocoel of cat fleas, Ctenocephalides felis (Bouché), was examined using antibody capture enzyme-linked immunosorbent assays (ELISA) and Western blotting. Fleas were fed heparinized cat blood via membrane feeders. Cat IgG was present in the hemolymph of engorged female fleas 1 h after ingestion at an estimated quantity of 35 +/- 14 micrograms/ml. The prevalence of fleas with demonstrable cat IgG in their hemolymph 1 h after feeding was 100% for both female and male fleas. Following a single blood meal, cat IgG was present in the hemolymph of all 15 fleas tested 1 h after ingestion but dissipated below detectable levels in 10 of 20 fleas examined 3 h after ingestion, and was detectable in only 1 of 10 fleas examined 18 h after ingestion. However, when fleas were provided with continual access to blood over a 72-h period, IgG content in hemolymph, as measured in excised, triturated legs of individual fleas, remained fairly constant (3-16 pg IgG per sample). Flea feeding studies using specific antisera indicated that IgG in flea hemolymph retained its binding activity, and that at least a portion of the IgG was intact. Passage of ingested host antibody from gut into hemocoel is a prerequisite for the possible development of antiflea vaccines that target antigens outside of the flea midgut lumen (e.g., key components of the flea endocrine system controlling oogenesis).

Light-dependent fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in chloroplasts isolated from wheat leaves.

The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) is degraded into an N-terminal side fragment of 37 kDa and a C-terminal side fragment of 16 kDa by the hydroxyl radical in the lysates of chloroplasts in light (H. Ishida et al. 1997, Plant Cell Physiol 38: 471-479). In the present study, we demonstrate that this fragmentation of the LSU also occurs in the same manner in intact chloroplasts, and discuss the mechanisms of the fragmentation. The fragmentation of the LSU was observed when intact chloroplasts from wheat leaves were incubated under illumination in the presence of KCN or NaN3, which is a potent inhibitor of active oxygenscavenging enzyme(s). The properties, such as molecular masses and cross-reactivities against the site-specific anti-LSU antibodies, of the fragments found in the chloroplasts were the same as those found in the lysates. These results indicate that, as in the lysates, the fragmentation of the LSU in the intact chloroplasts was also caused by the hydroxyl radical generated in light. The fragmentation of the LSU was completely inhibited by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU), and only partially inhibited by methyl viologen in the lysates. The addition of hydrogen peroxide to the lysates stimulated LSU fragmentation in light, but did not induce any fragmentation in darkness. Thus, we conclude that both production of hydrogen peroxide and generation of the reducing power at thylakoid membranes in light are essential requirements for fragmentation of the LSU.

A soluble protein is imported into Euglena chloroplasts as a membrane-bound precursor.

The Euglena precursor to the small subunit of ribulose-15-bisphosphate carboxylase/oxygenase (pSSU) is a polyprotein. To determine the transport route from cytoplasm to chloroplast, Euglena was pulse labeled with 35S-sulfate and the organelles were separated on sucrose gradients. After a pulse, pSSU was found in the endoplasmic reticulum (ER) and Golgi apparatus. During a chase, ER-and Golgi-localized pSSU decreased concomitant with the appearance of SSU in chloroplasts. SSU was not found in pSSU-containing ER and Golgi fractions. Na2CO3 did not remove pSSU from ER or Golgi membranes, indicating that it was an integral membrane protein. pSSU was inserted in vitro into canine microsomes, and Na2CO3 did not remove pSSU from the microsomal membrane. The in vivo and in vitro experiments show that Euglena pSSU is inserted into the ER membrane and transported as an integral membrane protein to the Golgi apparatus before chloroplast import and polyprotein processing.

Evidence that some dinoflagellates contain a ribulose-1,5-bisphosphate carboxylase/oxygenase related to that of the alpha-proteobacteria.

The ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit from several dinoflagellates has a structure similar to that of the Form II enzyme from Rhodospirillum and Rhodobacter species rather than the Form I Rubisco of eukaryotic algae and higher plants. The dinoflagellate Rubisco was identified on native polyacrylamide gels by autoradiographic detection of the stable Rubisco-[2'-14C]-2-carboxy-D-arabinitol 1,5-bisphosphate complex. The antibody to the Symbiodinium sp. large subunit cross reacts with both the Rhodospirillum rubrum and Rhodobacter sphaeroides Form II enzyme whereas antibodies to the R. rubrum Rubisco cross react with a range of dinoflagellate Rubisco large subunits. The N-terminal amino acid sequence of the large subunit from both Symbiodinium sp. and Amphidinium carterae confirmed this relation. The lack of inhibition of the dinoflagellate Rubisco by 6-phosphogluconate is consistent with this structure.

Five palmitoylated polypeptides in the 50 KDa range are not recognized by an antibody against ribulose-biphosphate-carboxylase-oxygenase in Chlamydomonas reinhardtii.

After incubation of Chlamydomonas reinhardtii cells with radioactive palmitic acid several labelled bands appeared after gel electrophoresis of delipidated protein extract. Among them, two bands (a major and a minor one) were detected in the 50 KDa range, which is the region where the LSU of the Rubisco (large sub-unit of the ribulose-biphosphate-carboxylase-oxygenase) was also found. Careful analyses by two-dimensional gel electrophoresis have shown that the five palmitate-labelled polypeptides detected in this region do not match with polypeptides immunoreacting with antibody against Rubisco. In addition, polypeptides labelled by palmitate cannot be immunoprecipitated with the same antibody further demonstrating that, in C. reinhardtii, the large sub-unit of Rubisco is not palmitoylated but unindentified proteins.

Effects of mutations at residue 309 of the large subunit of ribulosebisphosphate carboxylase from Synechococcus PCC 6301.

Previous studies [G. S. Hudson et al. (1989) J. Biol. Chem. 265, 808-814] showed that the faster turnover rates and lower affinities for CO2 of ribulosebisphosphate carboxylase/oxygenases from C4 plants, compared to C3 and C3/C4 plants, were specified by the chloroplast-encoded large subunits. In pairs of closely related C3 and C4 species from three genera, these kinetic changes were accompanied by only three to six amino acid residue substitutions, depending on the genus. None of these substitutions occurred near the active site and only one, 309Met (C3) to Ile (C4), was common to all three genera. Unlike the plant carboxylases, the highly homologous enzyme from the cyanobacterium Synechococcus PCC 6301 folds and assembles properly when its rbcL and rbcS genes are coexpressed in Escherichia coli. Furthermore, the cyanobacterial enzyme has Ile at position 309 of the large subunit, a high turnover number, and a poor affinity for CO2. 309Ile was replaced with Met and several other residues by site-directed mutagenesis of the cyanobacterial rbcL. Met and Leu were tolerated at this position with no alteration in the kinetic or structural properties of the assembled holoenzyme. However, substitution with Val, Gly, Trp, or Arg prevented the assembly of the subunits. The indifference to Met or Ile at this position, as well as the tolerance for Leu which is not observed with any natural ribulosebisphosphate carboxylase, leads to the conclusion that either the 309Met/Ile substitution has no effect on the kinetic properties of the plant enzyme, despite the correlation apparent in previous studies, or the cyanobacterial enzyme is sufficiently different from the plant enzyme in other respects that the influence of residue 309 is masked.

Alleged common antigenic determinant of tobacco mosaic virus coat protein and the host protein ribulose-1,5-bisphosphate carboxylase is an artifact of indirect ELISA and western blotting.

We have reported the detection of an antigenic determinant shared by the tobacco mosaic virus coat protein and the large subunit of ribulose-1,5-bisphosphate carboxylase, a host protein (R.G. Dietzgen and M. Zaitlin Virology 155, 262-266, 1986). This conclusion was questioned by D. Zimmermann and M.H.V. Van Regenmortel (Arch. Virol. 106, 15-22, 1989). Thus we have reinvestigated this unexpected serological cross-reaction in Western immunoblotting and indirect ELISA. We found that when skimmed milk instead of bovine serum albumin was used as a blocking agent and as a diluent for antibodies and alkaline phosphatase conjugates, the alleged cross-reaction was abolished. In light of these findings, we retract our previous conclusions.

Identification and characterization of receptors for protein import into chloroplasts and mitochondria.

An anti-idiotypic antibody approach was used to identify chloroplast and mitochondrial protein component(s) which interact with the corresponding signal sequence. The proteins thus identified can be operationally defined as receptor(s) for import of proteins into chloroplasts and mitochondria. The import receptor(s) was found in "contact sites" between the outer and inner membrane of chloroplast envelope or of mitochondria.

Distinct properties of Escherichia coli products of plant-type ribulose-1,5-bisphosphate carboxylase/oxygenase directed by two sets of genes from the photosynthetic bacterium Chromatium vinosum.

We have recently described the existence of two sets of genes encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase), rbcA-rbcB and rbcL-rbcS, in the photosynthetic purple sulfur bacterium Chromatium vinosum (Viale, A.M., Kobayashi, H., and Akazawa, T. (1989) J. Bacteriol. 171, 2391-2400). These genes were cloned in plasmid vectors, and their expression was studied in Escherichia coli. Expression of rbcA-rbcB in E. coli was obtained under the control of its own promoter. On the other hand, expression of rbcL-rbcS in this host was not observed unless these genes were cloned under the control of the tac promoter. Purified rbcA-rbcB and rbcL-rbcS products from E. coli consisted of large and small subunits in equimolar ratios. They also showed very close elution profiles to Rbu-P2 carboxylase isolated from C. vinosum in size-exclusion chromatography columns, thus suggesting hexadecameric (L8S8) structures. Vmax of Rbu-P2 carboxylase were very similar for both enzymes, but the Km values for CO2 and ribulose 1,5-bisphosphate showed some differences. Immunochemical and N-terminal amino acid sequence analyses of the large and small subunits encoded by rbcA-rbcB and rbcL-rbcS also differed, especially at the level of the small subunits. The comparisons described above as well as the analysis of C. vinosum crude extracts by anion-exchange chromatography indicated that Rbu-P2 carboxylase encoded by rbcA-rbcB was the only species detected in the photosynthetic bacterium.

Ribulose 1,5-bisphosphate carboxylase-oxygenase. I. Structural, immunochemical and catalytic properties.

Some structural, immunochemical and catalytic properties are examined for ribulose 1,5-bisphosphate carboxylase-oxygenase from various cellular organisms including bacteria, cyanobacteria, algae and higher plants. The native enzyme molecular masses and the subunit polypeptide compositions vary according to enzyme sources. The molecular masses of the large and small subunits from different cellular organisms, on the other hand, show a relatively high homology due to their well-conserved primary amino acid sequence, especially that of the large subunit. In higher plants, the native enzyme and the large subunit are recognized by the antibodies raised against either the native or large subunit, whereas the small subunit apparently cross-reacts only with the antibodies directed against itself. A wide diversity exists, however, in the serological response of the native enzyme and its subunits with antibodies directed against the native enzyme or its subunits from different cellular organisms. According to numerous kinetic studies, the carboxylase and oxygenase reactions of the enzyme with ribulose 1,5-bisphosphate and carbon dioxide or oxygen require activation by carbon dioxide and magnesium prior to catalysis with ribulose 1,5-bisphosphate and carbon dioxide or oxygen. The activation and catalysis are also under the regulation of other metal ions and a number of chloroplastic metabolites. Recent double-labeling experiments using radioactive ribulose 1,5-bisphosphate and 14CO2 have elucidated the carboxylase/oxygenase ratios of the enzymes from different organisms. Another approach, i.e., genetic experiments, has also been used to examine the modification of the carboxylase/oxygenase ratio.

Tobacco mosaic virus coat protein and the large subunit of the host protein ribulose-1,5-biphosphate carboxylase share a common antigenic determinant.

An immunological relationship was detected between the coat protein of the common (U1) strain of tobacco mosaic virus (TMV) and the large subunit of the ubiquitous CO2-fixing host enzyme, ribulose-1,5-biphosphate carboxylase (RuBisCo). When assayed by Western immunoblotting or indirect ELISA, polyclonal antisera to TMV coat protein and to RuBisCo reacted with both antigens. In addition, a monoclonal antibody specific for the C-terminal antigenic determinant of TMV coat protein reacted with RuBisCo. Conversely, several monoclonal antibodies generated to the large subunit of RuBisCo reacted with TMV coat protein. This cross-reactivity was verified by an examination of the amino acid sequences of both proteins. A region of homology was found between the carboxy proximal portion of coat protein and the sequence 60-73 residues from the amino terminus of RuBisCo large subunit. This homology was not mirrored at the nucleic acid level because of different codon usages for the two proteins.

Synthesis of large subunit of ribulosebisphosphate carboxylase by thylakoid-bound polyribosomes from spinach chloroplasts.

Intact chloroplasts were isolated from developing first leaves of spinach. The chloroplasts were broken and separated into an extensively washed membrane (thylakoid) fraction and a soluble (stroma) fraction. The membrane fraction contained polyribosomes with properties similar to those of thylakoid-bound polyribosomes of other organisms. The distribution of mRNA for large-subunit ribulosebisphosphate carboxylase (LS) was determined by translating RNA from chloroplasts, thylakoids, and stroma in a wheat germ cell-free translation system. LS translation product was identified by immunoprecipitation with antibody to LS from spinach, electrophoresis of the immunoprecipitated product, and fluorography. At least 44% of translatable chloroplast LS-mRNA was in the washed thylakoid fraction. Thylakoid-bound LS-mRNA was in polyribosomes since LS was produced by thylakoids in an Escherichia coli cell-free translation system under conditions where initiation did not take place. Our results demonstrate that membrane-bound polyribosomes can synthesize the stroma-localized polypeptide LS, and suggest that the thylakoids may be an important site of its synthesis.

Independent regulation of synthesis of form I and form II ribulose bisphosphate carboxylase-oxygenase in Rhodopseudomonas sphaeroides.

Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBPC-O) activity was greatly enhanced when Rhodopseudomonas sphaeroides was grown in a mineral salts medium supplied with 1.5% CO2 in hydrogen. Analysis of cell extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that cells growing on 1.5% CO2 in H2 specifically accumulated RuBPC-O polypeptides. Quantitative immunological determinations revealed that accumulation of form I and form II RuBPC-O closely correlates with the increase of specific activity. However, the two enzymes appeared to be derepressed at different levels. Upon transfer from heterotrophic to autotrophic (1.5% CO2) growth conditions, the intracellular form I RuBPC-O concentration was augmented 17-fold, whereas the form II RuBPC-O content increased only fourfold. As a result, the form I-form II ratio changed from 0.5 to about 2.0. Since this change in the RuBPC-O ratio occurred in the early stage of growth, it suggests that form I RuBPC-O is required for growth under drastic CO2 limitation. The difference in the extent of derepression of form I and form II RuBPC-O also indicates that the synthesis of each enzyme is regulated somewhat independently of the other.

Synthesis of ribulose biphosphate carboxylase in greening pea leaves. Coordination of mRNA level of two subunits.

Ribulose biphosphate carboxylase/oxygenase, with its large subunit encoded in the chloroplast and the small subunit in the nucleus, is induced by light. Accumulation of the enzyme, its two mRNA levels, and the synthesis rate of enzyme protein in pea leaves were followed during induction to understand the role of mRNA levels during greening subunit synthesis. The relative mRNA levels for the large and the small subunits increased coordinately up to 3-4 days, which almost corresponded to an inflection point of the accumulation profile of RuBisCO, and then the two mRNA levels gradually decreased. To obtain information of subunit synthesis, the extent of labelling of the two subunits were determined for both assembled and unassembled subunits using specific IgG. Unassembled subunits were found for both polypeptides, with a slight excess of the small one. The observed synthesis rates of the small and the large subunits were roughly coordinated without overproduction and almost stoichiometric amounts of the two polypeptides were found. The profiles of observed synthesis rate of the two subunits and the holoenzyme were similar to those of their mRNA levels. These results suggest that the synthesis of ribulose biphosphate carboxylase/oxygenase protein and its accumulation are dependent on the coordinated change of the two mRNA levels in greening pea leaves.