Further proof for the catalytic role of the larger subunit in the spinach leaf ribulose-1,5-diphosphate carboxylase

The catalytically active oligomeric form of the larger subunit, A_m, obtained from spinach leaf ribulose-1,5-diphosphate carboxylase by pretreatment with p-mercuribenzoate at pH 7.5 followed by incubation at pH 9.0, was free of the smaller subunit based on C-terminal amino acid analyses. Valine was the predominant C-terminus of the A_m preparations, the release of tyrosine being negligibly small [cf. Sugiyama and Akazawa, $\text{Biochemistry}$ 9 (1970) 4499]. The pH optimum of the ribulose-1,5-diphosphate carboxylase reaction by A_m was about 8.5, in comparison to the native enzyme which showed an alkaline pH optimum only in the absence of Mg²⁺. The substrate saturation curve of the catalytic subunit with respect to bicarbonate followed the Michaelis-Menten equation, as contrasted to the anomalous reaction kinetics of the native ribulose-1,5-diphosphate carboxylase molecule reported previously. These overall results indicate that the allosteric properties of spinach ribulose-1,5-diphosphate carboxylase are possibly conveyed by a unique structural conformation that requires the presence of the smaller subunit in association with the larger catalytic subunit component of the enzyme molecule.     

Neutron small angle scattering of the Mo-Fe protein (nitrogenase) from Clostridium pasteurianum

Neutron small angle scattering measurements of solutions of the Mo-Fe protein from $\text{C. pasteurianum}$ have yielded the following results. The molecular weight of the protein is 208,000 ± 10,000, in agreement with figures obtained by other methods. The radius of gyration is 39.8 ± 0.7 Å in H₂O, and 37.6 ± 0.3 Å in D₂O. The experimental scattering curves have been compared with the calculated scattering curves of simple homogeneous bodies. It is concluded that the MoFe protein from $\text{C. pasteurianum}$ is a non spherical particle having an axial ratio of 2:1, and that it probably has little, if any, solvent containing cavities.     

D-Ribulose 1, 5-diphosphate carboxylase from blue-green algae

D-Ribulose 1, 5-diphosphate carboxylase has been purified to a state of homogeneity from the marine blue-green alga $\text{Agmenellum}$$\text{quadruplicatum}$ strain PR-6. The enzyme has been found to be easily separated from the bulk soluble protein by means of centrifugation into a sucrose gradient. RuDP carboxylase from $\text{Agmenellum}$, upon chromatography using a calibrated Sephadex G-200 column, exhibits a molecular weight of 456,000 daltons, considerably smaller than the protein from eucaryotic algae. Only one polypeptide of approximately 56,000 daltons was obtained upon dissociation in sodium dodecylsulfate.     

Enzyme levels in relation to obligate phototrophy in chlamydobotrys

During the transition from photoheterotrophic growth on acetate to phototrophic growth on carbon dioxide, there is a decrease in isocitrate lyase and increase in ribulose-1,5-diphosphate carboxylase activity in Chlamydobotrys stellata cultures. The increase in ribulose-1,5-diphosphate carboxylase activity is the result of protein synthesis, there being a close correlation between increase in enzyme activity and protein precipitated by antibody to ribulose-1,5-diphosphate carboxylase. The purified ribulose-1,5-diphosphate carboxylase was similar to the constitutive enzyme from other green algae having a molecular weight of 530,000 and composed of two types of subunit of molecular weight 53,000 and 14,000.     

Ribulose diphosphate oxygenase. V. Presence in ribulose diphosphate carboxylase from Rhodospirillum rubrum

Ribulose-1,5-diphosphate carboxylase was purified fifteenfold from Rhodospirillum rubrum grown autotrophically under H₂ and CO₂. There was RuDP oxygenase activity associated with the carboxylase. The oxygenase had maximal activity at pH 9.4. Although these bacterial RuDP oxygenase and carboxylase activities were cold labile, activity could not be restored by treatment at 50° in the presence of Mg⁺⁺ and a sulfhydryl reagent, in contrast to results with the enzyme from eukaryotes.     

Preliminary X-ray studies on Pseudomonas isoamylase

Single crystals of Pseudomonas isoamylase (M_r 95,000) belong to space group P2₁2₁2₁ with unit cell dimensions of $\text{a = 137.9}\text{A}\text{?}\text{, b = 52.9}\text{A}\text{?}\text{, c = 151.2}\text{A}\text{?}$. A Guinier plot of the X-ray small-angle scattering of the protein solution gave the radius of gyration of the molecule as 27.5 Å.     

A simplified purification and some properties of ribulose 1,5-diphosphate carboxylase from barley

A rapid procedure was developed for purifying ribulose 1,5-diphosphate carboxylase from barley leaves. After (NH₄)₂SO₄ fractionation, the unique sedimentation properties of the enzyme were exploited to effect a single step purification to 90% homogeneity. High speed centrifugation pelleted the enzyme with complete recovery of activity. Residual impurities were then removed by diethylaminoethyl cellulose chromatography and density gradient centrifugation. The purified protein exhibited size heterogeneity due to polymerization. The polymerization products were enzymatically active aggregates of ribulose 1,5-diphosphate carboxylase and were precipitated by an antibody specific for the enzyme.     

Ribulose Diphosphate Carboxylase Synthesis in Euglena: III. Serological Relationships of the Intact Enzyme and its Subunits

Ribulose 1,5-diphosphate carboxylase was isolated from Euglena gracilis Klebs strain Z Pringsheim, Chlorella fusca var. vacuolata, and Chlamydobotrys stellata, and the subunits from each enzyme were separated and purified by gel filtration on Sephadex G-200 in the presence of sodium dodecyl sulfate. Rabbit antibody was elicited against purified Euglena ribulose 1,5-diphosphate carboxylase whole enzyme and the isolated large and small subunits. Euglena ribulose 1,5-diphosphate carboxylase showed partial immunological identity on Ouchterlony gels with the Chlorella and Chlamydobotrys carboxylases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates between antibody to the Euglena large subunit and the isolated large subunits of the Chlorella and Chlamydobotrys enzymes showed this was due to determinants on the large subunit. There was no serological affinity between the small subunits of the Euglena, Chlorella, and Chlamydobotrys carboxylases, and NH₂-terminal amino acid analyses provided further evidence of variability in the structure of the small subunits.     

Heterogeneity of large subunits of ribulose-1,5-bisphosphate carboxylase from Hydrogenomonas eutropha

Homogeneous ribulose-1,5-bisphosphate carboxylase purified from autotrophically grown $\text{Hydrogenomonas eutropha}$ can be dissociated with sodium dodecylsulfate into small 15,000-dalton subunits and large 56,000- and 52,000-dalton subunits (the latter in a mole ratio of 5:3). The overal mole ratio of small to large subunits is 1.08. Considering the molecular weight of the native enzyme (516,000), the simplest quarternary structure of this enzyme consists of 8 large (mixed) and 8 small subunits. Isolation of the enzyme from cells under conditions that should minimize proteolysis has no effect upon the observed heterogeneity of the large subunits.     

Inactivation of ribulosebisphosphate carboxylase/oxygenase from rhodospirillum rubrum and spinach with the new affinity label 2-bromo-1,5-dihydroxy-3-pentanone 1,5-bisphosphate

In an attempt to identify the active-site base believed to initiate catalysis by ribulosebisphosphate carboxylase, we have synthesized 2-bromo-1, 5-dihydroxy-3-pentanone 1,5-bisphosphate, a reactive analogue of a postulated intermediate of carboxylation. Although highly unstable, this compound can be shown to inactivate the carboxylases from both $\text{Rhodospirillum}\text{,}\text{rubrum}$ and spinach rapidly and irreversibly. Inactivation follows pseudo first-order kinetics, shows rate saturation and is greatly reduced by saturating amounts of the competitive inhibitor, 2-carboxyribitol 1,5-bisphosphate. The incorporation of reagent, quantified by reducing the modified carboxylases with [³H]NaBH₄, shows that inactivation results from the modification of approximately one residue per catalytic subunit of the $\text{Rhodospirillum}\text{,}\text{rubrum}$ enzyme and less than one residue per protomeric unit of the spinach enzyme.     

Small-angle x-ray scattering study of lysine transfer RNA ligase from yeast

The scattered X-ray intensities from dilute solutions of lysine transfer RNA ligase, in 0.1 m-phosphate buffer at pH 7.0, have been measured at 21 °. The radius of gyration R (37.5 Å), the molecular weight M (114,000), and the volume V (295,000 ų) were determined.A comparison between the scattering curves obtained from the enzyme and the theoretical scattering curves of different triaxial bodies shows that the shape of the molecule can be represented by an oblate ellipsoid with the semiaxes A = 62.7, B = 50.1 and $\text{C = 23.5}\text{A}\text{?}$.     

Regulation of Soybean Net Photosynthetic CO(2) Fixation by the Interaction of CO(2), O(2), and Ribulose 1,5-Diphosphate Carboxylase

Kinetic properties of soybean net photosynthetic CO₂ fixation and of the carboxylase and oxygenase activities of purified soybean (Glycine max [L.] Merr.) ribulose 1, 5-diphosphate carboxylase (EC 4.1.1.39) were examined as functions of temperature, CO₂ concentration, and O₂ concentration. With leaves, O₂ inhibition of net photosynthetic CO₂ fixation increased when the ambient leaf temperature was increased. The increased inhibition of CO₂ fixation at higher temperatures was caused by a reduced affinity of the leaf for CO₂ and an increased affinity of the leaf for O₂. With purified ribulose 1,5-diphosphate carboxylase, O₂ inhibition of CO₂ incorporation and the ratio of oxygenase activity to carboxylase activity increased with increased temperature. The increased O₂ sensitivity of the enzyme at higher temperature was caused by a reduced affinity of the enzyme for CO₂ and a slightly increased affinity of the enzyme for O₂. The similarity of the effect of temperature on the affinity of intact leaves and of ribulose 1,5-diphosphate carboxylase for CO₂ and O₂ provides further evidence that the carboxylase regulates the O₂ response of photosynthetic CO₂ fixation in soybean leaves. Based on results reported here and in the literature, a scheme outlining the stoichiometry between CO₂ and O₂ fixation in vivo is proposed.     

Tagatose-1,6-diphosphate activation of lactate dehydrogenase from Streptococcus cremoris

Tagatose-1,6-diphosphate was an effective substitute for fructose-1,6-diphosphate in the activation of lactate dehydrogenase (EC 1.1.1.27) from $\text{Streptococcus cremoris}$ AM2. The K_m for pyruvate, V_max and 0.5 values (activator concentration at half-maximal velocity) were similar with each activator. Of the other sugar phosphates and glycolytic intermediates tested only glucose-1,6-diphosphate activated the enzyme although the 0.5 value was 200 times that for the ketohexose diphosphates. Lactate dehydrogenases from several other organisms belonging to the Lactobacillaceae were equally stimulated by fructose-1,6-diphosphate and tagatose-1,6-diphosphate.     

Purification,some properties and quaternary structure of thed-ribulose 1,5-diphosphate carboxylase ofAlcaligenes eutrophus

d-Ribulose 1,5-diphosphate carboxylase has been purified from autotrophically grown cells of the facultative chemolithotrophic hydrogen bacteriumAlcaligenes eutrophus. The enzyme was homogeneous by the criteria of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 505000 determined by gel filtration and sucrose density gradient centrifugation, and a sedimentation coefficient of 18.2 S was obtained. It was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis that the enzyme consists of two types of subunits of molecular weight 52000 and 13000.Electron microscopy on the intact and the partially dissociated enzyme lead to the construction of a model for the quaternary structure of the enzyme which is composed of 8 large and 8 small subunits. The most probable symmetry of the enzyme molecule is 4:2:2.Michaelis constant (K _m ) values for ribulose 1,5-diphosphate, Mg^2-, and CO₂ were 0.59 mM, 0.33 mM, and 0.066 mM measured under air. Oxygen was a competitive inhibitor with respect to CO₂ suggesting that the enzyme also exhibits an oxygenase activity. The oxygenolytic cleavage of ribulose 1,5-diphosphate was shown and a 1:1 stoichiometry between oxygen consumption and 3-phosphoglycerate formation observed.Abbreviations DTE dithioerythritol - EDTA ethylenediamine tetraacetate - RuDP d-ribulose 1,5-diphosphate     

Crystallographic data for a penicillin receptor : exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61.

A pencillin-sensitive enzyme, the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R61, has been crystallized from polyethylene glycol (M_r = 6000 to 7500) solution at pH 7·6. X-ray examination of the orthorhombic crystals shows the space group is P2₁2₁2₁, with unit cell dimensions $\text{a = 51·1}\text{A}\text{?}\text{, b = 67·4}\text{A}\text{?}\text{,}\text{and}\text{c = 102·9}\text{A}\text{?}$. With four molecules of molecular weight 38,000, the $\text{A}\text{?}{}^3\text{/}\text{dalton}$ ratio for the cell is 2·33. The crystals are stable to irradiation for 75 hours and are suitable for structure analysis to at least 2·4 Å resolution. The radius of gyration of the molecule in solution at pH 6.8 is 20.8 Å.     

Inhibition of D-ribulose 1,5-bisphosphate carboxylase by pyridoxal 5′-phosphate

Homogeneous D-ribulose 1,5-bisphosphate carboxylase from $\text{Rhodospirillum rubrum}$, $\text{Chlamydomonas reinhardtii}$, and $\text{Hydrogenomonas eutropha}$ are inhibited by low concentrations of pyridoxal 5′-phosphate. In the case of the enzyme from $\text{Rhodospirillum rubrum}$, this inhibition is strongly antagonized by the substrate, D-ribulose 1,5-bisphosphate. These results suggest that pyridoxal 5′-phosphate may act close to or at the ribulose 1,5-bisphosphate binding site of the enzyme from $\text{Rhodospirillum rubrum}$.     

An investigation of the structure of Alfalfa mosaic virus by small-angle neutron scattering

Small-angle neutron scattering experiments have been performed on the tubular bottom component of Alfalfa mosaic virus (AMV) and the “30 S” particle (a quasispherical reassembled AMV coat protein particle) with the aim of determining the internal structure of the virus. Scattering curves were obtained out to a resolution of $\text{1}\text{50}\text{A}\text{?}{}^{\mathrm{?1}}$ at a number of H₂O/²H₂O ratios and were analysed using a model fitting technique. This involves calculating the scattering intensity due to a parameterised distribution of scattering density representing the particle and comparing this to the experimental data after taking into account the effect of instrumental smearing. The use of the contrast variation method enables the internal consistency of the model to be well tested.Three models are used in an attempt to explain the scattering curve of the 30 S particle. A single homogeneous shell is shown to be inadequate and two other models introducing the presumed T = 1 icosahedral symmetry of the particle are presented and discussed. The most satisfactory of these consists of 60 spherical monomers of radius 19 Å symmetrically placed in pairs about the 2-fold icosahedral positions.The analysis of the bottom component data has yielded a low resolution model for the virus, which is shown to be consistent with its composition as given by earlier physico-chemical measurements. In the model the RNA is uniformly packed throughout the interior of the capsid (which is cylindrical with hemispherical ends) out to a radius of about 65 Å and with a packing fraction of 20%. Within the limitations of an homogeneous shell model, the protein capsid has an outer radius of 94 Å and thickness of 23 Å, but arguments are presented based on the marked lattice structure of the cylindrical capsid and the analysis of the scattering data of the 30 S particle, that this model underestimates the thickness of the protein shell and that it in fact makes contact with the RNA at about 65 Å.     

Localization and properties of ribulose diphosphate carboxylase from castor bean endosperm

A substantial portion of the ribulose 1,5-diphosphate carboxylase activity in the endosperm of germinating castor beans (Ricinus communis var. Hale) is recovered in the proplastid fraction. The partially purified enzyme shows homology with the enzyme from spinach (Spinacia oleracea) leaves, as evidenced by its reaction against antibodies to the native spinach enzyme and to its catalytic subunit. The enzyme from the endosperm of castor beans has a molecular weight of about 500,000 and, with the exception of a higher affinity for ribulose 1,5-diphosphate, has similar kinetic properties to the spinach enzyme. The castor bean carboxylase is inhibited by oxygen and also displays ribulose 1,5-diphosphate oxygenase activity with an optimum at pH 7.5.     

In Vitro Protein Synthesis by Plastids of Phaseolus vulgaris: V. Incorporation of C-Leucine into a Protein Fraction Containing Ribulose 1,5-Diphosphate Carboxylase

A crude chloroplast preparation of primary leaves of Phaseolus vulgaris was allowed to incorporate ¹⁴C-leucine into protein. A chloroplast extract was prepared and purified for ribulose 1,5-diphosphate carboxylase by ammonium sulfate precipitation, chromatography on Sephadex G-200, and chromatography on Sepharose 4B. The distribution of radioactive protein and enzyme in fractions eluted from Sepharose 4B was nearly the same. The radioactivity in the product was in peptide linkage, since it was digested to a trichloroacetic acid-soluble product by Pronase. Whole cells in the plastid preparation were not involved in the incorporation of amino acid into the fraction containing ribulose 1,5-diphosphate carboxylase, since incorporation still occurred after removal of cells. The incorporation into the fraction containing ribulose 1,5-diphosphate carboxylase occurs on ribosomes of plastids, since this incorporation is inhibited by chloramphenicol. These plastid preparations may be incorporating amino acid into ribulose 1,5-diphosphate carboxylase, but the results are not conclusive on this point.