Subunit Of RuBPCase Research Articles

Immunodetection of ribulose-1,5-bisphosphate carboxylase in mutants of Euglena gracilis impaired in photosynthesis

For the first time, an antibody is used to detect the presence and to measure the concentration of ribulose-1,5-biphosphate carboxylase (RuBPCase) in several mutants of Euglena gracilis impaired in photosynthesis. Where present, the enzyme is correlated to its activity and to the chlorophyll content and photosynthetic CO 2-fixation ability of the respective mutants. Dark-grown wild-type strains Z and bacillaris and mutants O 1BS, O 2BX, G 1BU and P 1BXL (which make normal sized chloroplasts with abnormal internal structure in the light) and Y 1BXD (which does not develop the plastid beyond the proplastid stage in the light) each has about 0.1 × 10 −11 μmol of active RuBPCase/cell. RuBPCase content increases in the light, but to a greater extent oin wild-type cells than in the above mutants, except possibly for mutant G 1BU. The specific activity of the RuBPCase in vivo is estimated from the data to be about 1 nmol CO 2-fixed min −1 (pmol RuBPCase) −1 in the light for both the wild-type and the above mutant strains and in the dark for the wild-type Z and some of the above mutant strains. RuBPCase was not detected in mutants Y 3BUD, W 34ZUD, SM-L1, W 37HeHL and W 3BUL, but was detected in mutant Y 9ZNa1L which has no detectable protochlorophyll(ide) or chlorophyll(ide). In addition to recognizing both the large and small subunits of RuBPCase, the antibody also recognizes a polypeptide of > 60 000 daltons whose function is unknown.

Encoding of both subunits of ribulose-1,5-bisphosphate carboxylase by organelle genome of Cyanaphora paradoxa

The intracellular photosynthetic entities (cyanelles) of the protozoan Cyanophora paradoxa (Fig. 1) have long been regarded as endosymbiotic cyanobacteria due to morphological and physiological similarities such as unstacked thylakoids1, a rudimentary yet lysozyme-sensitive cell wall2,3, and phycobilisomes as light-collecting apparatus4. However, recent investigations of cyanelle DNA cast doubt on this classification. The cyanelle genome has been found to resemble chloroplast DNA in size and structure5–7. In light of the endosymbiont hypothesis of eukaryotic cell evolution8 these paradoxical features of the cyanelles suggest that C. paradoxa is an evolutionary intermediate between cyanobacteria, the presumed plastidal ancestors, and modern photosynthetic organelles. The ribulose-1,5-bisphosphate carboxylase (RuBPCase) of C. paradoxa is composed of eight large (LSU) and eight small (SSU) subunits9. In higher plants and green algae the LSU of the enzyme is encoded by plastid DNA and the SSU is nucleus-encoded10. In cyanobacteria the LSU gene has been located on the chromosome11. No data are available on the SSU gene. Here we have investigated the cyanelles' evolutionary position by determining the location of the genes for the two subunits of RuBPcase in C. paradoxa. By heterologous hybridization the genes for both subunits of RuBPCase have been located on the cyanelle genome. Furthermore, LSU and SSU probes exhibit homology to the same BglII and HindIII fragment, suggesting a close proximity of the two genes.

Study on nuclear and cytoplasmic genome expression in wheat by two-dimensional gel electrophoresis

In this first analysis the protein patterns obtained by two-dimensional gel electrophoresis of 8 day-old leaves from 18 alloplasmic wheat lines are compared. From 440 spots retained on the basis of their reproducibility, 36 proteins were observed to vary in different cytoplasms, allowing us to distinguish the T. aestivum cytoplasm from 5 Aegilops cytoplasms. Twenty-four of the 36 variable proteins could be structurally related to the large subunit of RuBPCase. Nuclear variation between 3 wheat varieties was observed for 14 proteins.

Ploidy Effects in Isogenic Populations of Alfalfa (Medicago sativa L.)

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) was isolated from isogenic diploid-tetraploid and tetraploid-octoploid sets of alfalfa (Medicago sativa L.) leaves. Molecular weights of RuBPCase subunits were similar across ploidy levels of both isogenic sets with subunits of 52,000 and 14,000. Apparent K(m)(CO(2)) values and substrate specificity factors (V(c)K(o)/V(o)K(c)) of RuBPCase were similar across ploidy levels of both isogenic sets. These results indicate that ploidy had no effect on the kinetic properties of RuBPCase in alfalfa.

In-vitro synthesis of phycobiliproteids and ribulose-1,5-bisphosphate carboxylase by non-poly-adenylated-RNA of Cyanidium caldarium and Porphyridium aerugineum

Polyadenylated and nonpolyadenylated mRNa from the red algae Cyanidium caldarium Geitler and Porphyridium aerugineum Geitler were translated in a cell-free system. The α-and β-subunits of phycocyanin and allophycocyanin and also both subunits of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase) were identified with the help of specific antibodies as translation products of non-polyadenylated mRNA. Both subunits of RuBPCase were synthesized with molecular weights in the range of the mature forms. This is in contrast to the findings with green algae and higher plants where the small subunits of RuBPCase are always made by polyadenylated mRNA in the form of a larger precursor molecule. We discuss our findings with regard to the systematic position of the algae used and the evolution of plastids.

A number of different nuclear genes for the small subunit of RuBPCase are transcribed in petunia.

The sequences in the petunia genome which encode the small subunit polypeptides of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase have been characterized. Sequence analysis of four cDNA clones indicate that there are several distinct genes transcribed in leaf tissue. There is 8-9% nucleotide divergence between the transcripts however these changes do not alter the encoded amino acid sequence. Examination of nuclear DNA by Southern hybridization and analysis of cloned small subunit genes confirm that there are a number of different genes which encode this single protein.

Restriction endonuclease analysis of plastid DNA from tomato, potato and some of their somatic hybrids

The buoyant density and endonuclease restriction patterns of potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum) ptDNA were examined and compared with those of their somatic hybrids. The plastids from these plants, both of which belong to the family of Solanaceae, contain a single DNA species whose density of 1.697 gcm-3 and size of approximately 156 kbp are similar to those of ptDNA from other higher plants. The Sal I restriction patterns were indistinguishable; however, those obtained with Kpn I, Pst I, and Eco RI disclosed that each species possesses a unique ptDNA. These observations suggest a relatively recent divergence of both species. Of the twelve hybrid lines screened, eight contained exclusively potato ptDNa and four contained only tomato ptDNA at a 0.1–3% level of detection. Rearrangements of modifications of DNA were not detected. The plastid identities of three hybrid lines that had previously been analyzed by isoelectric focusing of RuBPcase subunits (Melchers et al. 1978) agreed with those determined by restriction endonuclease analysis.

Hydrolysis of Ribulose-1,5-bisphosphate Carboxylase by Endoproteinases from Senescing Barley Leaves

The hydrolysis of (14)C-labeled ribulose-1,5-bisphosphate carboxylase (RuBPCase) by two partially purified endoproteinases from senescing barley (Hordeum vulgare v. Numar) leaves is described. The major thiol proteinase, EP(1), exhibits biphasic kinetics which appear to be caused by a region of the large subunit of RuBPCase that is highly sensitive to attack by EP(1). This proteinase further hydrolyzes both the large and small subunit to smaller peptides. A second proteinase, EP(2), appears to convert the small subunit of RuBPCase rapidly to a 13.7-kilodalton fragment during initial stages of hydrolysis and then to degrade both this fragment and the large subunit. The presence of a third endoproteinase, EP(3), was discovered when [(14)C]RuBPCase, which appeared to be homogeneous by sodium dodecyl sulfate polyacrylamide electrophoresis, seemed to undergo very low but significant rates of "autolysis." The large molecular weight fragments produced by EP(3) were different from those of EP(1) and EP(2).

Synthesis of Chloroplast Proteins during Germination and Early Development of Cucumber

Cell-free protein synthesizing systems have been used to study the developmental changes in the synthesis of chloroplast proteins in the cotyledons of cucumber seedlings grown in the light or in the dark. Escherichia coli and wheat germ in vitro protein synthesizing systems have been used to assay the changes in the levels of the mRNA's coding for ribulose 1,5-bisphosphate carboxylase (RuBPCase). The large subunit of cucumber RuBPCase has been identified among the translation products of the E. coli system. The wheat germ system translates the cucumber mRNA coding for the small subunit of RuBPCase to produce a 25,000 molecular weight precursor polypeptide. Plastids isolated from light-grown cotyledons were used to study developmental changes in their capacity to synthesize protein. The data obtained indicate that in the light there is an initial 48-hour period of accumulation of the mRNA's coding for the large and small subunits of RuBPCase, coupled with an increase in the capacity of the isolated plastids to synthesize protein. This is followed by a decline. This decline is not reflected in the accumulation of RuBPCase in the cotyledons which remains constant over the period of study.

Autodigestion in Crude Extracts of Soybean Leaves and Isolated Chloroplasts as a Measure of Proteolytic Activity

Two methods of measuring protein breakdown resulting from self-digestion during incubation in extracts of soybean leaves were examined. The release of free alpha-amino-nitrogen was measured with ninhydrin, and the disappearance of the large subunit of ribulose bisphosphate carboxylase (RuBPcase) was followed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rates of protein breakdown were measured as a function of temperature, pH, and leaf developmental stage and in the presence of various proteinase inhibitors. These treatments had differential effects on apparent proteolysis, depending on the method used. Determination of the ratio of alpha-amino-nitrogen plus peptide bond-nitrogen to alpha-amino-nitrogen indicated that the ninhydrin method detected the activity of exopeptidases preferentially. The disappearance of the large subunit of RuBPCase as shown on gels was due primarily to the activity of endopeptidases. The sensitivity of the two types of proteolytic degradation to proteinase inhibitors differed.Determination of temporal changes in proteolytic activity during leaf development showed that total proteolytic activity, measured by either method, increased during leaf expansion and maturation and decreased during senescence. Incubation of intact isolated chloroplasts at 37 C resulted in the breakdown of the large subunit of RuBPcase, although the chloroplasts contained no measurable proteinase activity as determined by the release of alpha-amino-nitrogen during the incubation. No acid proteinase (pH 4.5) activity was detected in the chloroplasts when hemoglobin was used as a substrate. These results indicate that the proteinases which break down RuBPCase in isolated chloroplasts may not be detectable by conventional assay procedures.

Presence de sous-unites de la RubPcase dan les enveloppes des chloroplastes d'epinard

Electrophoretic analysis of the envelope of Spinach chloroplasts demonstrated the presence of two polypeptides with apparent molecular weight similar to that of the subunits of the ribulose-1,5-biphosphate-carboxylase (RubPcase). The identification of the small and large subunits of RubPcase, in the envelope fraction, was further established with serum anti-RubPcase. These molecules do not seem in relation with a stromatic contamination which could occur during the course of the envelopes preparation. Their presence could reflect an ‘in vivo’ binding with the chloroplastic envelope and perhaps a transient stage in the process of the transfer of the RubPcase small subunit into the chloroplast.