Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts

In previous communications we have demonstrated that the subunits of normal human erythrocyte purine nucleoside phosphorylase can be resolved into four major (1–4) and two minor (1p and 2p) components with the same molecular weight but different apparent isoelectric points (and net ionic charge). The existence of subunits with different charge results in a complex isoelectric focusing pattern of the native erythrocytic enzyme. In contrast, the isoelectric focusing pattern of the native enzyme obtained from cultured human fibroblasts is simpler. The multiple native isoenzymes obtained from human erythrocytes and human brain have isoelectric points ranging from 5.0 to 6.4 and from 5.2 to 5.8, respectively, whereas cultured human fibroblasts have two major native isoenzymes with apparent isoelectric points of 5.1 and 5.6.Purine nucleoside phosphorylase has been purified at least a hundredfold from ³⁵S-labeled cultured human fibroblasts. A two-dimensional electrophoretic analysis of the denatured purified normal fibroblast enzyme revealed that it consists mainly of subunit 1 (90%) with small amounts of subunits 2 (10%) and 3 (1%). This accounts for the observed differences between the native isoelectric focusing and the electrophoretic patterns of the erythrocyte and fibroblast enzymes. The purine nucleoside phosphorylase subunit 1 is detectable in the autoradiogram from a two-dimensional electrophoretic analysis of a crude, unpurified extract of ³⁵S-labeled cultured normal human fibroblasts. The fibroblast phosphorylase coincides with the erythrocytic subunit 1 of the same enzyme, and the cultured fibroblasts of a purine nucleoside phosphorylase deficient patient (patient I) lack this protein component, genetically confirming the identity of the purine nucleoside phosphorylase subunit in cultured fibroblasts.This work was supported by a grant from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, United States Public Health Service. L. J. G. is supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

A Chlamydomonas reinhardii mutant with catalytically and structurally altered ribulose-5-phosphate kinase

The biochemical lesion in a light-sensitive, acetate-requiring Chlamydomonas mutant was identified. This strain, designated rpk, exhibited photosynthetic rates less than 3% of the wild-type. Analysis of photosynthetic products by high-performance liquid chromatography demonstrated an accumulation of ¹⁴C label in pentose and hexose monophosphates. After 1 min of photosynthesis in ¹⁴CO₂ these intermediates comprised 27.5% of the label in the mutant compared with 8% in the wild-type. The mutant pheno-type was caused by a 20-fold reduction in ribulose-5-phosphate (Ru5P)-kinase (EC 2.7.1.19) activity. The mutant exhibited wild-type levels of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) and transketolase (EC 2.2.1.1) indicating that the mutation specifically affected Ru5P kinase. In a cross of the mutant with the wild-type, tetrad progeny segregated in a Mendelian fashion (1:1) and light-sensitivity cosegregated with reduced Ru5P-kinase activity and an acetate requirement for growth. Almost normal levels of Ru5P-kinase protein were detected in the mutant by probing nitrocellulose replicas of sodium dodecylsulfate-polyacrylamide gels with anti-Ru5P-kinase antibody. The subunit size of the mutant enzyme, 42 kDa, was identical to that of the wild-type. Isoelectric focusing of the native protein determined that the mutant protein was altered, exhibiting a more acidic isoelectric point than the wild-type protein. Thus, the molecular basis for the lesion affecting Ru5P-kinase activity in mutant rpk is a charge alteration which results in a partially impaired enzyme.Abbreviations Chl chlorophyll - Da dalton - FCCP carbonylcyanide-p-trifluorophenylhydrazone - RuBP ribulose-1,5-bisphosphate - Ru5P ribulose-5-phosphate     

Crystal structure of the ternary complex of ribulose-1,5-bisphosphate carboxylase, Mg(II), and activator CO2 at 2.3-A resolution

The activated ternary complex, enzyme-CO2-Mg(II), of the dimeric ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum can be prepared in the same crystal form that was used for the crystallographic structure determination of the native nonactivated enzyme (Schneider, G., Br?nden, C.-I., & Lorimer, G. (1986) J. Mol. Biol. 187, 141-143). The three-dimensional structure of the activated enzyme has been determined to a nominal resolution of 2.3 A by protein crystallographic methods. The activator CO2 forms a carbamate with Lys191, located at the bottom of the funnel-shaped active site. In both subunits, this labile adduct is stabilized by a Mg(II) ion, bound to the carbamate and the side chains of Asp193 and Glu194. One solvent molecule was found within the first coordination sphere of the metal ion. The metal-binding site in ribulose-1,5-bisphosphate carboxylase consists thus of at least three protein ligands, all located on loop 2 of the beta/alpha barrel. One additional metal ligand, the side chain of the conserved Asn111, was observed close to the Mg(II) ion in the B-subunit. Other structural differences at the active site between the activated and nonactivated enzyme are limited to side-chain positions. Nevertheless, it is obvious that the hydrogen-bonding pattern in the vicinity of the activator site is completely altered.     

Modulation of the tight binding of carboxyarabinitol 1,5-bisphosphate to the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase

The large subunit (L) of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) from Synechococcus PCC 6301 was expressed in Escherichia coli, purified as the octamer L8, and analyzed for its ability to tightly bind the transition state analog, 2-carboxyarabinitol 1,5-bisphosphate (CABP). [14C]CABP remained tightly bound to L8 after challenging with [12C]CABP and gel filtration, indicating that L8 alone without the small subunit (S) could tightly bind CABP. Binding of CABP to L8 induced a shift in the gel filtration profile due to apparent aggregation of L8. Aggregation did not occur with the L8S8-CABP complex nor with L8-CABP in the presence of 150 mM MgCl2. If ionic strength was increased with either KCl or MgCl2 during or after the binding of [14C]CABP to L8, [14C]CABP in the complex exchanged with [12C]CABP and was lost from the protein. Ionic strength strongly affected the rate constant (k4) for [14C]CABP dissociation from the L8-[14C]CABP complex, but had little effect on k4 for the L8S8-CABP complex. The differences in CABP binding characteristics between the L8-CABP and L8S8-CABP complexes demonstrate that S is intimately involved in maintaining the stability of the tight binding of CABP to the active site. These are the same interactions stabilizing the intermediate, 3-keto-2-carboxyarabinitol 1,5-bisphosphate, to native rubisco during CO2 fixation.     

Purification and characterization of phosphoinositide 3-kinase from rat liver

Phosphoinositide 3-kinase was purified 27,000-fold from rat liver. The enzyme was purified by acid precipitation of the cytosol followed by chromatography on DEAE-Sepharose, S-Sepharose, hydroxylapatite, Mono-Q, and Mono-S columns. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified phosphoinositide 3-kinase preparation contained an 85-kDa protein and a protein doublet of approximately 110 kDa. The 85- and 110-kDa proteins focus together on native isoelectric focusing gels and are cross-linked by dithiobis(succinylamide propionate), showing that the 110- and 85-kDa proteins are a complex. The apparent size of the native enzyme, as determined by gel filtration, is 190 kDa. The 85-kDa subunit is the same protein previously shown to associate with polyoma virus middle T antigen and the platelet-derived growth factor receptor (Kaplan, D. R., Whitman, M., Schaffhausen, B., Pallas, D. C., White, M., Cantley, L., and Roberts, T. M. (1987) Cell 50, 1021-1029). The two proteins co-migrate on two-dimensional gels; and, using a Western blotting procedure, 32P-labeled middle T antigen specifically blots the 85-kDa protein. The purified enzyme phosphorylates phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. The apparent Km values for ATP were found to be 60 microM with phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate as the substrate. The apparent Km for phosphatidyinositol is 60 microM, for phosphatidylinositol 4-phosphate is 9 microM, and for phosphatidylinositol 4,5-bisphosphate is 4 microM. The maximum specific activity using phosphatidylinositol as the substrate is 0.8 mumol/mg/min. The enzyme requires Mg2+ with an optimum of 5 mM. Substitution of Mn2+ for Mg2+ results in only approximately 10% of the Mg2(+)-dependent activity. Physiological calcium concentrations have no effect on the enzyme activity. Phosphoinositide 3-kinase has a broad pH optimum around 7.     

Activation and regulation of ribulose bisphosphate carboxylase-oxygenase in the absence of small subunits.

Ribulose 1,5-bisphosphate carboxylase from Rhodospirillum rubrum requires CO2 and Mg2+ for activation of both CO2, both the carboxylase and oxygenase activities are stimulated by 6-phoshpo-D-gluconate, fructose 1,6-bisphosphate, 2-phosphoglycolate, 3-phosphoglycerate, NADPH, and fructose 6-phosphate. The carboxylase activity is not activated by ribose 5-phosphate. The substrate, ribulose bisphosphate, neither activates nor inhibits the CO2 and Mg2+ activation of this enzyme. Activation by CO2 and Mg2+ is rapid and results in increased susceptibility to active-site-directed protein modification reagents. Because the R. rubrum carboxylase-oxygenase is a dimer of large subunits and contains no small subunits, these results suggest that the effector binding sites of the higher plant enzyme may also be found on the large subunit.     

2-Bromoacetylaminopentitol 1,5-bisphosphate as an affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

2-Bromoacetylaminopentitol 1,5-bisphosphate (BrAcNH-pentitol-P2) (an epimeric mixture of 2-bromoacetylamino-2-deoxy-D-ribitol bisphosphate and 2-bromoacetylamino-2-deoxy-D-arabinitol 1,5-bisphosphate) has been synthesized from D-ribulose 1,5-bisphosphate by reductive amination with sodium cyanoborohydride followed by bromoacetylation of the resultant amine with bromoacetyl bromide. Under conditions that favor full activation of the enzyme, ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum is completely inactivated by BrAcNH-pentitol-P2 in a pseudo-first order process. A rate saturation is observed with a minimal inactivation half-life of 38 min and Kinact for reagent of 0.38 mM. The competitive inhibitor 2-carboxyribitol 1,5-bisphosphate reduces the rate of inactivation, and kinetic analyses are consistent with the protection reflecting true competition of inhibitor and reagent for the same site. As shown with isotopically labeled reagent, complete inactivation is associated with covalent incorporation of 1.1 mol of reagent/mol of subunit. Based on reversibility of inactivation by thiolysis and based on analysis of labeled products in acid hydrolysates of the modified enzyme, a methionyl sulfonium salt is the reaction product. In the absence of CO2 and Mg2+ (ligands required for activation), the enzyme is resistant to BrAcNH-pentitol-P2, which suggests that the site-specific modification of a methionyl residue requires a fully functional catalytic center.     

A Purified Zinc Protease of Pea Chloroplasts,EP1, Degrades the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

A previously reported endopeptidase (EP1) from pea chloroplasts was purified over 11,000-fold using a four-step protocol involving ultrafiltration, sucrose gradient centrifugation, isoelectric focusing, and high performance liquid chromatography gel filtration. The enzyme was determined to be a metalloprotease requiring bound Zn2+ and added Mg2+ or Ca2+ for proper activity. Its localization in the stroma of pea chloroplasts was confirmed by demonstrating its insensitivity to thermolysin when the envelope was intact. A contaminating serine protease that attacks EP1 was found. The contaminating protease was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride, but not by o-phenanthroline, whereas EP1 sensitivities were the reverse. EP1 is able to hydrolyze the large subunit of native ribulose-1,5-bisphosphate carboxylase/oxygenase under physiological conditions.     

Protein composition of the carboxysomes of Thiobacillus neapolitanus

For purifying carboxysomes of Thiobacillus neapolitanus an isolation procedure was developed which resulted in carboxysomes free from whole cells, protoplasts and cell fragments. These purified carboxysomes are composed of 8 proteins and at the most of 13 polypeptides. The two most abundant proteins which make up more than 60% of the carboxysomes, are ribulose-1,5-bisphosphate carboxylase and a glycoprotein with a molecular weight of 54,000. The shell of the carboxysomes consists of four glycoproteins, one also with a molecular weight of 54,000. The other proteins are present in minor quantities. Ribulose-1,5-bisphosphate carboxylase is the only enzyme which could be detected in the carboxysomes and 3-phosphoglycerate was the only product formed during incubation with ribulose-1,5-diphosphate and bicarbonate. The supernatant of a broken and centrifuged carboxysome suspension contained the large subunit of ribulose-1,5-bisphosphate carboxylase. The small subunit of ribulose-1,5-bisphosphate carboxylase was found in the pellet together with the shell proteins which indicates that the small subunit of ribulose-1,5-bisphosphate carboxylase is connected to the shell.Abbreviations RuBisCO ribulose-1,5-bisphosphate carboxylase - PMSF phenylmethylsulfonyl fluoride - PAA gelectrophoresis, polyacrylamide gelelectrophoresis - SDS sodium dodecyl sulphate - CIE crossed immunoelectrophoresis - IEF isoelectric focusing     

Formation of a carboxyarabinitol bisphosphate complex with ribulose bisphosphate carboxylase/oxygenase and theoretical specific activity of the enzyme

¹⁴C-Labeled 2-carboxyarabinitol-1,5-bisphosphate was bound to both nonactivated and CO₂and Mg²⁺ activated forms of ribulose bisphosphate carboxylase/oxygenase. The complex could be precipitated with 20% polyethylene glycol and 20 mm MgCl₂ for quantitation of the moles of the affinity label bound per mole of enzyme. The [¹⁴C]carboxyarabinitol-P₂ bound to the nonactivated enzyme could be exchanged with a 100-fold excess of the unlabeled compound. With the activated enzyme the binding of [¹⁴C]carboxyarabinitol-P₂ was so tight that it did not exchange with the unlabeled compound and a binding stoichiometry of one molecule per active site was assumed. This tight binding was dependent upon pretreatment of the enzyme with both CO₂ and MgCl₂ in the same manner that enzyme activation depended on CO₂ and Mg²⁺ concentrations. Various enzyme preparations from spinach leaves tightly bound [¹⁴C]carboxyarabinitol-P₂ in proportion to their specific activities. By extrapolating to a maximum binding of 8 mol of [¹⁴C]carboxyarabinitol-P₂ per mole of this A₈B₈ enzyme a theoretical specific activity of 2.8 μmol · min^?1 · mg protein^?1 was indicated. Enzyme preparations purified from spinach leaves generally have a specific activity in the range of 1.0 to 2.3.     

Cooperative effect of fructose bisphosphate and glyceraldehyde-3-phosphate dehydrogenase on aldolase action

The combination of binding and kinetic approaches is suggested to study (i) the mechanism of substrate-modulated dynamic enzyme associations; (ii) the specificity of enzyme interactions. The effect of complex formation between aldolase and glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) on aldolase catalysis was investigated under pseudo-first-order conditions. No change in kcat but a significant increase in KM of fructose 1,6-bisphosphate for aldolase was found when both enzymes were obtained from muscle. In contrast, kcat rather than KM changed if dehydrogenase was isolated from yeast. Next, the conversion of fructose 1-phosphate was not affected by interactions between enzyme couples isolated from muscle. The influence of fructose phosphates on the enzyme-complex formation was studied by means of covalently attached fluorescent probe. We found that the interaction ws not perturbed by the presence of fructose 1-phosphate; however, fructose 1,6-bisphosphate altered the dissociation constant of the enzyme complex. A molecular model for fructose 1,6-bisphosphate-modulated enzyme interaction has been evaluated which suggests that high levels of fructose bisphosphate would drive the formation of the 'channelling' complex between aldolase and glyceraldehyde-3-phosphate dehydrogenase.     

Determination of ligand-binding constants by isoelectric focusing. Generalization and extension of theory

The theory of a proposed isoelectric focusing procedure for the determination of ligand binding constants has been generalized (via numerical calculations) to non-linear, electrophoretic velocity profiles and systems for which the change in velocity of the protein accompanying ligand binding depends upon pH. The theory has also been extended to include binding of ligand to heterogeneous sites. Determination of accurate values of the binding parameters depends upon extrapolation of apparent parameters to infinite dilution of protein.     

Interactions between ribulose-1,5-bisphosphate carboxylase and stromal metabolites. II. Corroboration of the role of this enzyme as a metabolite buffer

The capacity of ribulose-1,5-bisphosphate carboxylase to bind reversibly chloroplast metabolites which are the substrates for both thylakoid and stromal enzymes was assessed using spinach chloroplasts and chloroplast extracts and with pure wheat ribulose-1,5-bisphosphate carboxylase. Measurements of the rate of coupled electron flow to methyl viologen in ‘leaky’ chloroplasts (which retained the chloroplast envelope and stromal enzymes but which were permeable to metabolites) and also with broken chloroplasts and washed thylakoids were used to study the effects of binding ADP and inorganic phopshate to ribulose-1,5-bisphosphate carboxylase. The presence of ribulose-1,5-bisphosphate carboxylase significantly altered the values obtained for apparent K_m for inorganic phosphate and ADP of coupled electron transport. The K_m (P_i) in washed thylakoids was 60–80 μM, in ‘leaky’ chloroplasts it was increased to 180–200 μM, while in ‘leaky’ chloroplasts preincubated with KCN and ribulose 1,5-bisphosphate the value was decreased to 40–50 μM. Similarly, the K_m (ADP) of coupled electron transport in washed thylakoids was 60–70 μM, in ‘leaky’ chloroplasts it was 130–150 μM and with ‘leaky’ chloroplasts incubated in the presence of KCN and ribulose 1,5-bisphosphate a value of 45–50 μM was obtained. The ability of ribulose 1,5-bisphosphate carboxylase to reduce the levels of free glycerate 3-phosphate in the absence of ribulose 1,5-bisphosphate was examined using a chloroplast extract system by varying the concentrations of stromal protein or purified ribulose 1,5-bisphosphate carboxylase. The effect of binding glycerate 3-phosphate to ribulose-1,5-bisphosphate carboxylase on glycerate 3-phosphate reduction was to reduce both the rate an the amount of NADPH oxidation for a given amount of glycerate 3-phosphate added. The addition of ribulose 1,5-bisphosphate reinitiated NADPH oxidation but ATP or NADPH did not. Incubation of purified ribulose-1,5-bisphosphate carboxylase with carboxyarabinitolbisphosphate completely inhibited the catalytic activity of the enzyme and decreased inhibition of glycerate-3-phosphate reduction. Two binding sites with different affinities for glycerate 3-phosphate were observed with pure ribulose-1,5-bisphosphate carboxylase.     

An effect of enzyme and ligand concentration on the state of aggregation of aspartate transcarbamylase of E. coli: I. The binding of CTP and ATP to the enzyme.

Detailed binding studies of the inhibitor, cytidine triphosphate (CTP), to native Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) reveal significant changes in subunit interaction when enzyme concentration is altered. In contrast, similar binding studies of the activator, adenosine triphosphate (ATP), do not reveal such changes, but do indicate more complex subunit interactions than previously reported. Equilibrium dialysis studies of 4 degrees C are consistent with six binding sites for CTP and ATP per enzyme molecule of molecular weight 310 000, at all enzyme concentrations. CTP binding studies reveal a progressive change from apparent positive to negative cooperativity as the enzyme concentration is decreased. ATP binding studies reveal complex subunit interactions involving a mixture of apparent negative and positive cooperativity. Sucrose gradient studies indicate the presence of at least three enzymatically active polymeric forms of the enzyme. The preliminary sedimentation studies indicate possible ligand and enzyme concentration perturbations of a preexisting association equilibrium in the aspartate transcarbamylase system. The binding data are therefore interpreted in terms of an association model.     

DIABLA: a new screening method for the discovery of protein targets

Dynamic isoelectric/anisotropy binding ligand assay (DIABLA) is a new method to identify proteins in a complex sample that bind to a molecule of interest. This is accomplished by first using capillary isoelectric focusing (cIEF) to separate the proteins in a capillary based on their isoelectric point. This separation is performed while the compound being tested is present in the separation buffer. When the proteins are focused, the entire capillary is scanned to identify regions of nonzero anisotropy, which are locations where the test compound is interacting with a focused protein band. DIABLA was demonstrated by observing the binding of fluorescein-tagged progesterone to an MCF-7 breast cancer cell lysate. The proteins were tagged with rhodamine to permit their observation and then focused in the presence of the tagged progesterone. Anisotropy measurements show that progesterone binds to six different proteins bands in the sample.     

Differential expression of ribulose-1,5-bisphosphate carboxylase in reciprocal F1 hybrids of a C3 and a C4-likeFlaveria species

Stable reciprocal hybrids between Flaveria pringlei (C3) and F. brownii (C4-like) have been produced by standard breeding techniques. There are no differences in the isoelectric focusing patterns of the catalytic subunits of the ribulose-1,5-bisphosphate carboxylase/oxygenase from F. pringlei, F. brownii, or the reciprocal hybrids. The enzyme from both species also contains an identical noncatalytic subunit polypeptide. However, the carboxylase enzyme from F. brownii contains another isomeric form of noncatalytic subunit polypeptide which is resolveable by isoelectric focusing. This isomeric form constitutes about 50% of the total noncatalytic subunits in this species. It comprises only about 10% of the total noncatalytic subunit population in the C3 x C4 plants, but about 42% of the noncatalytic subunits in the reciprocal cross. The concentrations of the holoenzyme in the reciprocal hybrids are comparable to those of the respective maternal parent. We hypothesize that a differential inheritance of parental chloroplasts by the reciprocal hybrids may be associated with this apparent maternal influence on the expression of the noncatalytic polypeptides and the holoenzyme concentration.     

Crystallization of the activated ternary complex of ribulose-1,5-bisphosphate carboxylase-oxygenase isolated from Rhodospirillum rubrum and from an Escherichia coli clone

Ribulose-1,5-bisphosphate carboxylase-oxygenase was purified from the photosynthetic bacterium Rhodospirillum rubrum as well as from an Escherichia coli clone overproducing the enzyme. Although the latter enzyme contains 25 additional amino acid residues at the N terminus, both preparations yielded isomorphous tetragonal, bipyramidal crystals of the ternary complex of the enzyme with CO2 and Mg2+. Crystallization is sensitive to variation in pH and to the addition of the transition state analog, 2-carboxyarabinitol-1,5-bisphosphate. The systematic absences in the X-ray diffraction photographs suggest a tetragonal space group P4(3)2(1)2 or the enantiomorph P4(1)2(1)2 with cell dimensions a = b = 83 A, c = 290 A. There is one molecule per asymmetric unit. The resolution on still photographs is 3 A. The crystals are comparable to some of those already published but differ from others.     

The transition between the open and closed states of rubisco is triggered by the inter-phosphate distance of the bound bisphosphate

d-Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) catalyses the central CO(2)-fixing reaction of photosynthesis in a complex, multiple-step process. Several structures of rubisco complexed with substrate analogues, inhibitors and products have been determined by X-ray crystallography. The structures fall into two well-defined and distinct states. The active site is either "open" or "closed". The timing and mechanism of the transition between these two states have been uncertain. We solved the crystal structure of unactivated (metal-free) rubisco from tobacco with only inorganic phosphate bound and conclude that phosphate binding per se does not trigger closure, as it does in the similarly structured enzyme, triosephosphate isomerase. Comparison of all available rubisco structures suggests that, instead, the distance between the terminal phosphates (P1 and P2) of the bisphosphate ligand is the trigger: if that distance is less than 9.1 A, then the active site closes; if it is greater than 9.4 A then the enzyme remains open. Shortening of the inter-phosphate distance results from the ligand binding in a more curved conformation when O atoms of the ligand's sugar backbone interact either with the metal, if it is present, or with charged groups in the metal-binding site, if the metal is absent. This shortening brings the P1 phosphate into hydrogen bonding contact with Thr65. Thr65 exists in two discrete states related by a rotation of the backbone psi torsion angle. This rotation is coupled to domain rotation and hence to active site closure. Rotation of the side-chain of Thr65 also affects the C-terminal strand of large subunit which packs against Loop 6 after closure. The position of the C-terminal strand in the closed state is stabilised by multiple polar interactions with a distinctive highly-charged latch site involving the side-chain of Asp473. In the open state, this latch site may be occupied instead by phosphorylated anions.     

Inhibition of ribulose bisphosphate carboxylase assembly by antibody to a binding protein

We have developed an assay to monitor in vitro the posttranslational assembly of the chloroplast protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Most of the newly synthesized 55-kD catalytic ("large") subunits of this enzyme occur in a 29S complex together with 60- and 61-kD "binding" proteins. When the 29S complex is incubated with ATP and MgCl2 it dissociates into subunits, and the formerly bound large subunits now sediment at 7S (still faster than expected for a monomer). Upon incubation at 24 degrees C, these large subunits assemble into RuBisCO. The minority of newly made large subunits which are not bound to the 29S complex also sediment at 7S. When endogenous ATP was removed by addition of hexokinase and glucose, the dissociation of the 29S complex was inhibited. Nevertheless, the 7S large subunits assembled into RuBisCO, and did so to a greater extent than in controls retaining endogenous ATP. Thus the 7S large subunits are also assembly competent, at least when ATP is removed. Apparently, in chloroplast extracts, ATP can have a dual effect on the assembly of RuBisCO: on the one hand, even at low concentrations it can inhibit incorporation of 7S large subunits RuBisCO; on the other hand, at higher concentrations it can lead to substantial buildup of the 7S large subunit pool by causing dissociation of the 29S complex, and stimulate overall assembly. At both high and zero concentrations of ATP, however, antibody to the binding protein inhibited the assembly of endogenous large subunits into RuBisCO. Thus it appears that all assembly-competent large subunits are associated with the binding protein, either in the 7S complex or in the 29S complex. The involvement of the binding protein in RuBisCO assembly may represent the first example of non-autonomous protein assembly in higher plants and may pose problems for the genetic engineering of RuBisCO from these organisms.