Small Subunit Of Ribulose Bisphosphate Carboxylase Research Articles

Ribulosebisphosphate carboxylase small subunit functions as a thioredoxin in the dithiothreitol-dependent activation of nadp-malate dehydrogenase

When subjected to an acidic treatment at pH 5.4 a homogeneous preparation of spinach ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) was partially depleted in its small subunit content. The small subunits were separated from the partially depleted holoenzyme by gel filtration on Sephadex G-50. Under these condition, approximately one out of eight small subunits per mol enzyme was released. The isolated small subunits could replace thioredoxin in the dithiothreitol activation of corn leaf NADP-malate dehydrogenase (EC 1.1.1.82), but not in the dithiothreitol activation of fructose 1,6-bisphosphatase. When present in the native or partially depleted holoenzymes the small subunits were not able to activate NADP-malate dehydrogenase. These results suggest: (i) that the dithiothreitol-NADP-malate dehydrogenase test is not specific for thioredoxin, since ribulose 1,5-bisphosphate carboxylase small subunit does not possess a thioredoxin structure; (ii) that the ribulose 1,5-bisphosphate carboxylase small subunit apparently participate in redox reactions.

Expression of the gene coding for the small subunit of ribulosebisphosphate carboxylase during differentiation of tobacco plant protoplasts.

A hybridization probe was used to study the regulation of expression of the gene coding for the small subunit of ribulose 1,5-bisphosphate carboxylase, during functional differentiation of protoplasts. A library of cDNA from poly(A)-containing RNA extracted from specially treated tobacco leaves was constructed in the plasmid pBR322 by blunt-end ligation. This library was screened by colony hybridization with 32P-labelled cDNA prepared from mRNA coding for the precursor of the small subunit. A positive colony was identified containing recombinant plasmids with a nucleotide sequence homologous to this mRNA. These plasmids, bound to diazobenzyloxymethylated cellulose paper, were then used as a hybridization probe. The results showed unambiguously that the small subunit was not transcribed in protoplasts but was transcribed in undifferentiated white and chlorophyll-containing green callus cultures derived from protoplasts. The discrepancy between these results and those obtained with classical techniques is discussed.

Blue light-induced synthesis of ribulosebisphosphate carboxylase in cultured plant cells

Upon illumination with blue light (350-550 nm) of suspension cultured cells (Nicotiana tabacum var. Samsun) the transition of leucoplasts to functional chloroplasts is induced. During the subsequent greening period chlorophylls as well as membrane and enzyme proteins are synthesized. Thus the amount of ribulosebisphosphate carboxylase (EC. 4.1.1.39) being small in leucoplasts increases dramatically due to de novo synthesis. This change is also reflected in the level of translatable messenger RNA specific for the small subunit of ribulosebisphosphate carboxylase which accumulates only in blue-irradiated cells; its in vitro translation product isolated by immunoprecipitation corresponds mainly to the precursor protein (Mr ≈ 20 000) of the small subunit. In contrast, red light (600-700 nm) does not induce synthesis of ribulosebisphosphate carboxylase. According to these findings it is proposed that blue light exerts its influence on ribulosebisphosphate carboxylase in cultured tobacco cells at a level below translation.

Regulation of the Synthesis of Ribulose-l,5-bisphosphate Carboxylase and Its Subunits in the Flagellate Chlorogonium elongatum. II. Coordinated Synthesis of the Large and Small Subunits

Abstract Transfer of heterotrophically grown cells of the unicellular green alga Chlorogonium elongatum to autotrophic growth conditions causes a 10 -15 fold increase in the amount of the chloroplastic enzyme ribulose-1,5-bisphosphate carboxylase. This increase was found to be due to de novo synthesis. The relative proportions of large and small subunits of the enzyme do not change. Their ratio is close to 3.4, the proportions in weight of the two subunits in the holoenzyme. Continous labelling with [35S]sulfate reveals that the ratios of incorporation into large and small subunits are essentially the same in autotrophic and heterotrophic cells. Pulse-chase experiments show that the subunits are degraded synchronously. The coordinated subunit synthesis cannot be uncoupled using inhibitors of protein and RNA synthesis or high temperature of cultivation of the alga. The results suggests a very tightly coordinated synthesis of the large and small subunits of ribulosebisphosphate carboxylase.

Glyphosine, a plant growth regulator, affects chloroplast membrane proteins

Glyphosine ( N, N-bis(phosphonomethyl)glycine) is known to increase sucrose levels in sugarcane and to cause chlorosis in maize and other plants. It has been suggested (Crofts, S.M., Arntzen, C.J., Vanderhoef, L.N. and Zettinger, C.S. (1974) Biochim. Biophys. Acta 335, 211–217) that its primary mode of action is to inhibit the synthesis of plastid rRNA. Growth of Lemna gibba L. G-3 on 5 · 10 −4M glyphosine causes the plants to produce fronds lacking chlorophyll. The plastids in these white fronds contain only a few internal membrane structures, some of which are stacked. Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows an accumulation of substantial amounts of both the large and small subunits of ribulosebisphosphate carboxylase by the white fronds. The membrane fraction from these fronds contains only traces of the light-harvesting chlorophyll a b apoprotein in comparison to control plants. In vivo labeling and immunoprecipitation show that the large subunit of ribulose-bisphosphate carboxylase is actively synthesized by the white fronds. However, labeling of the chlorophyll a b apoprotein and a 32000 dalton protein in the membrane fraction is extremely low compared to control plants. We conclude that in Lemna, glyphosine differentially affects the synthesis and/or processing of soluble proteins and some membrane chloroplast proteins, and could be useful in understanding the biogenesis of chloroplast membranes.

Peptide mapping of the ribulose bisphosphate carboxylase small subunit from the somatic hybrid of tomato and potato

Five somatic hybrids of potato and tomato obtained by protoplast fusion were identified using the isoelectric focusing patterns of ribulose bisphosphate carboxylase as phenotypic markers of the nuclear and chloroplast genomes. Together with the previously analyzed hybrids (14) this brings the number of verified somatic hybrids between the two species to nine. The products of both tomato and potato nuclear genomes are present in the ribulose bisphosphate carboxylase oligomer from all nine hybrids. Four of the hybrids contain the tomato large subunit and hence the tomato chloroplast DNA is functional, whereas in five hybrids the potato ribulose bisphosphate carboxylase large subunit and thereby the potato chloroplast DNA is present. The former are designated topatoes and the latter pomatoes (11).Mapping of chymotryptic peptides of the small subunit of tomato and potato revealed a tyrosine containing peptide with species specific mobility. Peptide maps from an analyzed somatic hybrid contained both the tyrosine peptide specific for potato and that specific for tomato. Maps of tryptic peptides of the small subunit of tomato differed from those of the potato in 2 peptides. The tryptic peptide maps of one analyzed hybrid contained the two peptides unique to potato as well as the two peptides unique to tomato. The tomato-potato hybrids are aneuploid with chromosome numbers ranging from 48 to 74, the expected allotetraploid number being 48 and the expected allohexaploid number 72.

Amino Acid Sequence of the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase from Spinach

The complete amino acid sequence of the small subunit of ribulosebisphosphate carboxylase from spinach has been determined. There are 120 amino acids. The N-terminus of the protein is frequently blocked. There is a tyrosine-proline substitution at position 91 and the average molecular weight of the two forms is 13 897. The interest of the sequence data for students of physiology and evolution is discussed.

Regulation of Glyoxysomal Enzymes during Germination of Cucumber

Developmental patterns of glyoxylate cycle and photosynthetic activities have been correlated with electrophoretic profiles of cotyledonary RNA and protein in both light- and dark-grown cucumber seedlings (Cucumis sativus L.) Cytoplasmic rRNA increases 10-fold between days 0 and 5, and the steepest increase coincides with the most rapid rise in activities of the glyoxysomal enzymes, isocitrate lyase and malate synthase. Chloroplast rRNA and ribulose bisphosphate (RuBP) carboxylase begin rising at day 3, followed about a day later by increases in glyoxylate reductase activity and chlorophyll content. Of these phototrophic indicators, only chlorophyll requires light for its initial appearance. Sodium dodecyl sulfate gel electrophoresis of total and soluble cotyledonary protein showed several developmental patterns, including: (a) progressive disappearance of storage protein present initially in particulate form; (b appearance and subsequent disappearance of a family of polypeptides identified by molecular weight, developmental profile, and density gradient centrifugation as subunits of glyoxysomal enzymes; and (c) appearance and progressive increase (in both light- and dark-grown cotyledons) of the large and small subunits of RuBP carboxylase, as well as other polypeptides presumably of chloroplast and peroxisomal origin.

Formation of the small subunit in the absence of the large subunit of ribulose 1,5-bisphosphate carboxylase in 70 S ribosome-deficient rye leaves

Immunological tests with monospecific antisera to ribulosebisphosphate carboxylase (EC 4.1.1.39) and to its large and small subunits indicated the presence of a protein with antigenic properties of the small subunit in the absence of the large subunit in the leaves of young rye plants ( Secale cereale L.) with a high-temperature-induced (32 °C) deficiency of 70 S plastid ribosomes. The small subunit-like protein was isolated from crude extracts of plastid ribosome-deficient 32 °C-grown leaf tissue by the use of columns with immobilized antibody. The main polypeptide retained by the immobilized antibodies had the same mobility after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels as the small subunit of ribulosebisphosphate carboxylase and was also immunologically identical to the small subunit. The small subunit-like protein was present in the supernatant as well as in the membrane fraction of isolated 70 S ribosome-deficient plastids. At very young stages of normal leaves grown at a permissive temperature (22 °C) an excess of small subunit was observed that was also not integrated into the complete ribulosebisphosphate carboxylase molecule. From the results, we conclude that the synthesis of the small subunit occurs on cytoplasmic ribosomes and is not strictly coordinated with the translation of the large subunit in the chloroplast. During early leaf development, the formation of the large subunit seems to be the ratelimiting step in the synthesis of ribulosebisphosphate carboxylase.

Identification of the small subunit of ribulose 1,5-bisphosphate carboxylase as a product of wheat leaf cytoplasmic ribosomes

Polyribosomes from greening wheat seedlings ( Triticum vulgaris) were allowed to incorporate [ 3H]leucine into proteins in the presence of a wheat germ supernatant fraction under conditions permitting the completion and release of polypeptides by cytoplasmic polyribosomes. The released proteins were analyzed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Discrete proteins, as well as a variety of poorly resolved proteins, were observed to have been labeled. The molecular weight distribution of the labeled proteins correlated well with the distribution of polyribosome size classes present in the samples. Neither the large nor the small subunit of ribulosebisphosphate carboxylase were detected as labeled peaks by this procedure. Immune precipitates formed by the addition of carrier small subunit, detergent, and anti-small subunit serum to the released proteins contained a substantial proportion of nonspecifically precipitated material resembling the population of released proteins, but they also contained two discrete peaks not resolved previously, one having mobility slightly faster than the light chain of immunoglobulin (20,000 daltons) and the other having mobility identical to that of small subunit carrier (ca. 12,000 daltons). Samples containing the latter material were shown to contain labeled tryptic peptides corresponding to those of the small subunit carrier. The results establish that the small subunit of ribulose bisphosphate carboxylase is the product of a small proportion of cytoplasmic polyribosomes during greening of etiolated wheat seedlings.