Subunits Of Ribulose Diphosphate Carboxylase Research Articles

Isolation and Enzymic Characterization of Euglena Proplastids

Organelles were isolated from dark-grown Euglena gracilis Klebs by sucrose density gradient centrifugation. Plastids, identified by triosephosphate isomerase and NADP glyoxylate reductase were present at an equilibrium density of 1.24 grams per cubic centimeter clearly separated from mitochondria at an equilibrium density of 1.22 grams per cubic centimeter. Assay for choline phosphotransferase and glucose-6-phosphatase showed that endoplasmic reticulum membranes were present at a density of 1.12 grams per cubic centimeter. The plastid fraction contained phosphofructokinase, pyruvate kinase, triosephosphate isomerase and aldolase indicating the operation of a glycolytic pathway. During regreening pyruvate kinase and phosphofructokinase in the developing proplastid decreased, neither enzyme being present in the mature chloroplast. However, plastids were present in the photosynthetic cell as shown by a peak of glycolysis enzymes at an equilibrium density of 1.24 grams per cubic centimeter.The integrity of isolated plastids was demonstrated by their capacity for protein synthesis. Plastids isolated from dark-grown cells rapidly incorporated [(35)S]methionine into protein with an absolute dependence on added ATP. The large subunit of ribulose diphosphate carboxylase was the major polypeptide synthesized by these isolated plastids.

On the nature of the polymorphism of the small subunit of ribulose-1,5-diphosphate carboxylase in the amphidiploid Nicotiana tabacum

The N-terminal amino acid sequence of the small subunit of ribulose-1,5=disphosphate carboxylase from the amphidiploidNicotiana tabacum contains two polymorphisms. From examination of the equivalent sequences in the putative parent speciesNicotiana sylvestris andtomentosiformis it is concluded that the amphidiploidNicotiana tabacum has inherited two alleles for the small subunit of ribulose diphosphate carboxylase, one from each parent species. The alleles continue to be retained and expressed. The relevance of these findings is discussed in relation to the successful adaption of the amphidiploidNicotiana tabacum to a wide range of environments.

Differential Localization of Fraction I Protein between Chloroplast Types

The soluble proteins of C(3) and C(4) mesophyll chloroplasts and C(4) bundle sheath extracts have been analyzed by gel electrophoresis for fraction I protein. Gel scans of soluble protein from C(4) bundle sheath extracts and C(3) mesophyll chloroplasts showed typical fraction I protein peaks that could be identified by ribulose diphosphate carboxylase activity. No such peak was observed for C(4) mesophyll chloroplasts, which also lacked both large and small subunits of ribulose diphosphate carboxylase on sodium dodecyl sulfate gels. The absence of fraction I protein in these chloroplasts was reflected in the soluble protein to chlorophyll ratios, which were roughly 3-fold lower than the ratio obtained for C(3) chloroplasts. The carboxylating enzyme in C(4) mesophyll cells, phosphoenolpyruvate carboxylase, was found to be a major protein in the cytoplasm of C(4) mesophyll protoplasts, and had higher mobility than fraction I protein.

In vitro synthesis of the large subunit of ribulose diphosphate carboxylase on 70 S ribosomes

Polyribosomes isolated from greening barley leaves were active in directing protein synthesis, using soluble components isolated from Escherichia coli. A peptide of 55,000 molecular weight was a major product of translation activity. This peptide was precipitated by antibody to ribulose 1,5-diphosphate carboxylase (RuDPCase) and comigrated with the large subunit of RuDPCase on sodium dodecyl sulfate-polyacrylamide gels. Cyanogen bromide peptides of the peptide of 55,000 molecular weight also corresponded to the peptides prepared from authentic RuDPCase large subunit. The peptides synthesized were shown by sucrose density gradient sedimentation to be largely associated with 70 S ribosomes.

Synthesis of soluble, thylakoid, and envelope membrane proteins by spinach chloroplasts purified from gradients

Intact spinach chloroplasts that had been purified on gradients of silica sol incorporated [ 35S]methionine into soluble and membrane-bound products, using light as the sole energy source. The labeled chloroplasts were lysed osmotically and fractionated on a discontinuous gradient of sucrose into the soluble fraction and the thylakoid and envelope membranes. About 29% of the radioactivity in the chloroplast was recovered in the soluble fraction, 59% in the thylakoid membranes, and 0.1% in the envelope membranes. The products of protein synthesis in the different fractions, as well as in the whole chloroplast, were analyzed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. There were two zones of radioactivity in the gels of the soluble fraction, the major zone coincident with the large subunit of ribulose diphosphate carboxylase at a molecular weight of about 50,000. The thylakoid membranes contained five labeled polypeptides, the most active having a molecular weight of about 31,000. The envelope membranes contained a major radioactive component of a molecular weight of about 50,000 and two other minor components.

Ribulose Diphosphate Carboxylase Synthesis in Euglena

The transfer of dark-grown cultures of Euglena gracilis Klebs strain Z regreening in the light back into darkness resulted in a dramatic increase in ribulose diphosphate carboxylase activity. On a culture volume basis activity increased 4-fold over a 24-hour dark period, although on a protein basis activity declined because of rapid cell division. Mixed assays with light- and dark-growing cell extracts provided no evidence for the removal of an inhibitor of ribulose diphosphate carboxylase upon transferring regreening cells back to darkness. Although ribulose diphosphate carboxylase activity increased over a 24-hour dark period, there was no concomitant increase in the potential of the cells for photosynthetic carbon dioxide fixation.Higher light intensities than the optimum for ribulose diphosphate carboxylase synthesis during regreening resulted in a greater relative rate of synthesis on transfer to darkness so that the maximum activity of ribulose diphosphate carboxylase reached in the dark was constant, regardless of light intensity during regreening. A tentative hypothesis to explain these results is that the synthesis of the large and small subunits of ribulose diphosphate carboxylase occur at different stages of cell development, light being necessary for the synthesis of the large subunit and also for regulating the synthesis of the small subunit.

Separation and Characterization of Subunits of Ribulose Diphosphate Carboxylase

Phenolic compounds covalently bound to macromolecules occur in wheat flour and are released by acid or alkaline hydrolysis. It is concluded that changes in phenolic compounds may partly account for the aging effect in flours