Purification and properties of ribulosebisphosphate carboxylase large subunit binding protein

The large subunit binding protein, an abundant plastid protein implicated in the assembly of ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO), has been highly purified from leaves of Pisum sativum. The 720 kilodaltons purified binding protein is composed of two types of subunits of 60 and 61 kilodaltons. Highly specific polyclonal antibodies have been raised against the binding protein. The antibodies do not cross-react with the large subunit nor do anti-RubisCO antibodies cross-react with the binding protein. A higher molecular weight form of the binding protein is immunoprecipitated from products of P. sativum polysomes translated in a wheat-germ system, indicating that the binding protein is synthesized by cytoplasmic ribosomes. Immunoblotting reveals the presence of binding protein in extracts of tobacco, wheat and barley leaves and castor bean endosperm.

The previously reported dissociation of the binding protein-large subunit complex upon addition of ATP in vitro has been confirmed and the fates of the dissociated subunits further investigated. The dissociated binding protein subunits are not phosphorylated or adenylated in vitro by added ATP.

     

Brain pyridoxal kinase dissociation of the dimeric structure and catalytic activity of the monomeric species

Reversible dissociation of the dimeric structure of brain pyridoxal kinase into subunits was attained by addition of guanidinium HCl (2 M). The molecular mass of the subunits (40 kDa) was determined by HPLC chromatography. Separation of the processes of refolding and association of the monomeric species was achieved by attaching the protein subunits to a rigid matrix (Affi-gel 15). The matrix-bound monomer is catalytically competent. The reaction of the crosslinking reagent 4,4'-dimaleimidestilbene 2,2'-disulfonate (DMDS), a derivatized stilbene, with the dimeric structure of pyridoxal kinase resulted in the formation of an oligomeric species of 80 kDa detectable by SDS-PAGE. The crosslinked subunits exhibit the same catalytic parameters as the native enzyme. The presence of two nucleotide-binding sites per dimer was determined by fluorimetric titrations using pyridoxyl-ATP, a strong competitive inhibitor with respect to ATP. The ATP analog binds with a Kd = 5 microM to each nucleotide site of the dimeric enzyme. The mode of binding pyridoxyl-ATP to the kinase is discussed in reference to a model which assumes the presence of two binding domains per subunit.     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver.

A cyclic AMP-adenosine binding protein from mouse liver has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate and by analytical ultracentrifugation. The binding protein had a Stokes radium of 48 A based on gel chromatography. Both the purified binding protein and the binding activity in fresh cytosol sedimented as 9 S on sucrose gradient centrifugation. The homogeneous protein had a sedimentation coefficient (S20, w) of 8.8 x 10-13 s, as calculated from sedimentation velocity experiments. By use of the Stokes radius and S20, w', the molecular weight was calculated to be 180,000. The protein was composed of polypeptides having the same molecular weight of 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thus appeared to consist of four subunits of equal size. The isoelectric point, pI = 5.7. The binding capacity for cyclic AMP increased by preincubating the receptor protein in the presence of Mg2+ ATP. This process, tentatively termed activation, was studied in some detail and was shown not be be be accompanied by dissociation, aggregation, or phosphorylation of the binding protein. Cyclic AMP was bound to the protein with an apparent dissociation constant (Kd) of 1.5 x 10-7 M. The binding of cyclic AMP was competitively inhibited by adenosine, AMP, ADP, and ATP whose inhibition constants were 8 x 10-7 M, 1.2X 10-6 M, 1.5 X 10-6 M, and higher than 5 x 10-6 M respectively. A hyperbolic Scatchard plot was obtained for the binding of adenosine to the activated binding protein, indicating more than one site for adenosine. The binding of adenosine to the site with the highest affinity (Kd=2 x 10-7 M) for this nucleoside was not suppressed by excess cyclic AMP and was thus different from the aforementioned cyclic AMP binding site. Cyclic GMP, GMP, guanosine, cyclic IMP, IMP, and inosine did not inhibit the binding of either cyclic AMP or adenosine. The binding protein had no cyclic AMP phosphodiesterase, adenosine deaminase, phosphofructokinase, or protein kinase activities, nor does it inhibit the catalytic subunit of the cyclic AMP-dependent protein kinase.     

Binding proteins for adenosine 3′:5′-cyclic monophosphate in bovine adrenal cortex

Five peaks of cyclic AMP-binding activity could be resolved by DEAE-cellulose chromatography of bovine adrenal-cortex cytosol. Two of the binding peaks co-chromatographed with the catalytic activities of cyclic AMP-dependent protein kinases (ATP-protein phosphotransferase, EC 2.7.1.37) of type I or type II respectively. A third binding protein was eluted between the two kinases, and appeared to be the free regulatory moiety of protein kinase I. Two of the binding proteins for cyclic AMP, sedimenting at 9S in sucrose gradients, could also bind adenosine. They bound cyclic AMP with an apparent equilibrium dissociation constant (K(d)) of about 0.1mum, and showed an increased binding capacity for cyclic AMP after preincubation in the presence of K(+), Mg(2+) and ATP. The two binding proteins differed in their apparent affinities for adenosine. The isolated regulatory moiety of protein kinase I had a very high affinity for cyclic AMP (K(d)<0.1nm). At low ionic strength or in the presence of MgATP, the high-affinity binding of cyclic AMP to the regulatory subunit of protein kinase I was decreased by the catalytic subunit. At high ionic strength and in the absence of MgATP the high-affinity binding to the regulatory subunit was not affected by the presence of catalytic subunit. Under all experimental conditions tested, dissociation of protein kinase I was accompanied by an increased affinity for cyclic AMP. To gain some insight into the mechanism by which cyclic AMP activates protein kinase, the interaction between basic proteins, salt and the cyclic nucleotide in activating the kinase was studied.     

Adenine nucleotides promote dissociation of pertussis toxin subunits

Pertussis toxin is composed of an enzymatically active A subunit and a binding component (B oligomer). Both the holotoxin and the isolated A subunit have previously been shown to exhibit NAD glycohydrolase activity although the A subunit is more active on a molar basis than the holotoxin. We have investigated the mechanism by which ATP stimulates the activity of this toxin. Since dissociation of pertussis toxin subunits would result in increased NAD glycohydrolase activity, the ability of ATP to promote release of the A subunit from the B oligomer was examined. In the presence of the zwitterionic detergent 3-(3-cholamidopropyldimethyl)-1-ammonio)-propanesulfonate, concentrations of ATP as low as 1 microM promoted subunit dissociation. The concentration of ATP required for release of the A subunit was similar to that required for stimulation of NAD glycohydrolase activity. Both ATP and ADP promoted subunit dissociation and stimulated NAD glycohydrolase activity. In contrast, AMP and adenosine did not alter NAD glycohydrolase activity or affect subunit structure. The ability of ATP to decrease the affinity of the A subunit for the B oligomer may play a role in nucleotide stimulation of pertussis toxin activity.     

Purification and some properties of rabbit skeletal muscle fructose 6-phosphate kinase inhibitor.

The loss of activity of rabbit skeletal muscle FPK on storage and its restoration by ATP, AMP and cyclic AMP has prompted us to look for an inhibitory unit of the enzyme. We have purified this inhibitory factor from the crude muscle extract and isolated from crystalline FPK; both proteins have the same Mw of about 68,000 (SDS). Carboxymethylation revealed species of lower molecular weight. It is suggested that two different kinds of FPK exist, one composed only of "active" subunits and another composed only of "inactive" (inhibitor) subunits. States of intermediate activity exist, created by dissociation, reassociation and exchange of subunits, because the inhibitor and FPK share several subunits. A model is proposed where one or several inhibitors of molecular mass 68,000 replace the corresponding number of active subunits of 93,000 daltons, the structure of the native molecule remaining tetrameric. It is shown that cyclic AMP exerts its activation function on FPK only in the presence of the inhibitory protein, probably by displacing the exchange of the subunit in favor of the active tetrameric species of 360,000. Ammonium chloride plays probably an opposite role in this exchange. The inhibitor coverts the Michaelian behavior with respect to F-6-P into a cooperative response (sigmoidal shape of the curve) characterized by a Hill coefficient of 2. The Michaelian response with respect to ATP is preserved, the corresponding constant being only slightly affected. In the presence of subsaturating concentration of inhibitor, mixed species are detected. As a first approximation one can propose that a reversible equilibrium exists between free and complex FPK subunits. The dissociation constant of this equilibrium being equal to 4 X 10(-8) M in moles of FPK protomers.     

The use of affinity chromatography in purification of cyclic nucleotide receptor proteins.

Biospecific affinity chromatography has been used to purify specific cyclic AMP and cyclic GMP receptor proteins. Several variables are important for successful purification of the cyclic AMP receptor protein, the most critical being the length of the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to agarose specifically retains the cyclic AMP receptor protein by interaction with the immobilized nucleotide. Binding of the cyclic AMP receptor subunit of cyclic AMP-dependent protein kinase to the immobilized nucleotide results in dissociation of the catalytic protein phosphokinase subunit which is not retained. The retained cyclic AMP receptor protein is subsequently eluted by cyclic AMP. Homogeneous cyclic AMP receptor protein prepared from rabbit skeletal muscle by affinity chromatography has been characterized. The molecular weight of the native protein as determined by analytical ultracentrifugation and polyacrylamide gel electrophoresis at varying acrylamide concentrations is 76 800 and 82 000, respectively. The protein is asymmetric with frictional and axial ratios of 1.64 and 12. SDS and urea polyacrylamide gel electrophoresis indicate that the native cyclic AMP receptor is composed of two identical subunits of 42 700 molecular weight. The native protein dimer binds 2 moles of cyclic AMP per mole of protein and is active in suppressing activity of isolated catalytic subunits of cyclic AMP-dependent protein kinase. Cyclic GMP receptor protein from bovine lung has been purified using the same affinity chromatography media. Since cyclic nucleotide binding to cyclic GMP-dependent protein kinase does not result in dissociation of regulatory receptor and catalytic phosphotransferase subunits, the cyclic GMP-dependent protein kinase holoenzyme is retained on the column and can be subsequently specifically eluted with cyclic GMP.     

Comparison of adenosine 3':5'-monophosphate-dependent protein kinases from rabbit skeletal and bovine heart muscle.

Homogeneous preparations of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase from rabbit skeletal (Peak I) and bovine heart muscle have been compared. Each enzyme has an S20,w value of 7.0. Each enzyme binds 2 mol of cyclic AMP per mol of enzyme and is dissociated in the presence of saturating concentrations of cyclic AMP into a demeric regulatory subunit-cyclic AMP complex and two catalytic subunits. The isolated subunits recombine, resulting in the formation of the original holoenzyme in each case. Several differences between the two enzymes were found. Different salt concentrations are necessary for elution of the respective enzyme from DEAE-cellulose. Their regulatory subunits differ with respect to their sedimentation constants and mobility on sodium dodecyl sulfate gel electrophoresis. The regulatory subunit of the heart enzyme is rapidly phosphorylated by MgATP but this does not occur with the skeletal muscle enzyme. MgATP is bound with high affinity only to the skeletal muscle enzyme. The enzymes have different apparent dissociation constants and Hill coefficients for cyclic AMP binding. With the skeletal muscle enzyme MgATP increases the dissociation constants for cyclic AMP about 10-fold and decreases the Hill coefficient, while with the heart enzyme phosphorylation decreases the cissociation constant for cyclic AMP 5- to 6-fold and increases the Hill coefficient. Different concentrations of cyclic AMP are required to dissociate the skeletal and heart muscle enzymes. The presence of MgATP increases the concentration of cyclic AMP required to dissociate the skeletal muscle enzyme but decreases the concentration necessary to dissociate the heart enzyme.     

Reconstitution of types I and II adenosine cyclic 3',5'-phosphate dependent protein kinase

Fluorescence intensity and anisotropy measurements using the fluorescent adenosine cyclic 3',5'-phosphate (cAMP) analogue 1,N6-ethenoadenosine cyclic 3',5'-phosphate (epsilon-cAMP) are sensitive to the dissociation of epsilon-cAMP which occurs when either the type I or the type II regulatory subunit (RI or RII) of cAMP-dependent protein kinase associates with the catalytic subunit. Studies using epsilon-cAMP show that MgATP has opposite effects on the reconstitution of both types of protein kinase: MgATP strongly stabilizes the type I holoenzyme while it slightly destabilizes the type II holoenzyme. The synthetic substrate Kemptide has a small inhibitory effect on the reconstitution of both holoenzymes when tested at 10 microM concentration. The protein kinase inhibitor has a larger effect which is especially pronounced in the reassociation of the type I enzyme. The diminished relative ability of the type I regulatory subunit to compete with the protein kinase inhibitor suggests that the combined effects of the two opposing equilibria (epsilon-cAMP and catalytic subunit binding) are different for the two types of regulatory subunits. Displacement experiments show that cAMP and epsilon-cAMP bind about equally well to the type I subunit. Slow conformational changes accompanying the binding of epsilon-cAMP by both regulatory subunits are greatly accelerated with the holoenzymes, suggesting that dissociation of the holoenzymes occurs via ternary complexes. The time courses of epsilon-cAMP binding also show the heterogeneity of binding characteristics of RII. The 37 000-dalton fragment of type II subunit retains the epsilon-cAMP binding properties of the native subunit. However, only a fraction of the fragment preparation (approximately 32% estimated from sedimentation measurements) binds the catalytic subunit well, suggesting heterogeneity of cleavage.     

The separation, properties and possible subunit composition of adenosine 3',5'-monophosphate-dependent protein kinases in brown adipose tissue.

Two 8.5-S protein kinases (ATP : protein phosphotransferase EC 2.7.1.37) and one 6.6-S protein kinase were purified 500--1000-fold from the acid-soluble fraction of brown adipose tissue. The catalytic properties of the kinases were similar. Each kinase was activated by cyclic AMP and had two components of cyclic AMP binding. In the presence of 200 nM cyclic AMP, undissociated kinase activity sedimented at 7.7 or 5.5 S. Free catalytic activity (3.2 S) could be detected but was unstable. Free regulatory units could not be detected. The 8.5-S protein kinase was dissociated by freezing and thawing to a 7.7-S variety with loss of the higher affinity component of binding. The 7.7-S kinase was sedimented through linear gradients of sucrose containing different concentrations of cyclic AMP. At each concentration, kinase activity lost from the holoenzyme peak (% of original) was identical with the amount of cyclic AMP bound at equilibrium (% oof maximum). Similar experiments on the 8.5-S kinase showed that the binding component with higher affinity was not associated with the release of catalytic activity. The results were consistent with the propostal that the kinases isolated contained one more cyclic AMP binding subunit than catalytic subunit (3 : 2 for 8.5 S and 2 : 1 for 6.6 S) and that this extra subunit was released to give an equal number of subunits of each type before catalytic activity was liberated.     

The Rubisco subunit binding protein

Chloroplasts contain an abundant soluble protein that binds non-covalently newly synthesized large and small subunits of the enzyme ribulose bisphosphate carboxylase-oxygenase. This binding protein has been purified from Pisum sativum and Hordeum vulgare in the form of a dodecamer consisting of equal amounts of two types of subunit. These subunits are synthesized as higher molecular mass precursors by cytoplasmic ribosomes before import into the chloroplast. Antibodies raised against the purified binding protein from Pisum sativum detect polypeptides not only in extracts of plastids from several plant species but also in cell extracts of several bacterial species. The oligomeric binding protein dissociates reversibly into monomeric subunits in the presence of 1–5 mmol/liter MgATP. For one type of subunit the cDNA sequence has been isolated and determined and reveals homology with certain bacterial proteins.These observations are discussed in relation to the idea that the binding protein is an example of a general class of proteins termed "molecular chaperones" which are required for the correct assembly of certain oligomeric proteins such as the carboxylase from their subunits.Abbreviations BP Binding protein - Rubisco Ribulose bisphosphate carboxylase-oxygenase     

Assembly of the stator in Escherichia coli ATP synthase. Complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha

Alpha subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure alpha was monomeric, was competent in nucleotide binding, and had normal N-terminal sequence. In F1 subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F1-depleted membranes with restoration of ATP-driven proton pumping. Therefore interaction between the stator delta subunit and the N-terminal residue 1-22 region of alpha occurred normally when pure alpha was complexed with other F1 subunits. On the other hand, three different types of experiments showed that no interaction occurred between pure delta and isolated alpha subunit. Unlike in F1, the N-terminal region of isolated alpha was not susceptible to trypsin cleavage. Therefore, during assembly of ATP synthase, complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha. We suggest that the N-terminal 1-22 residues of alpha are sequestered in isolated alpha until released by binding of beta to alpha subunit. This prevents 1/1 delta/alpha complexes from forming and provides a satisfactory explanation of the stoichiometry of one delta per three alpha seen in the F1 sector of ATP synthase, assuming that steric hindrance prevents binding of more than one delta to the alpha3/beta3 hexagon. The cytoplasmic fragment of the b subunit (bsol) did not bind to isolated alpha. It might also be that complexation of alpha with beta subunits is prerequisite for direct binding of stator b subunit to the F1-sector.     

ATPase activity of magnesium chelatase subunit I is required to maintain subunit D in vivo.

During biosynthesis of chlorophyll, Mg(2+) is inserted into protoporphyrin IX by magnesium chelatase. This enzyme consists of three different subunits of approximately 40, 70 and 140 kDa. Seven barley mutants deficient in the 40 kDa magnesium chelatase subunit were analysed and it was found that this subunit is essential for the maintenance of the 70 kDa subunit, but not the 140 kDa subunit. The 40 kDa subunit has been shown to belong to the family of proteins called "ATPases associated with various cellular activities", known to form ring-shaped oligomeric complexes working as molecular chaperones. Three of the seven barley mutants are semidominant mis-sense mutations leading to changes of conserved amino acid residues in the 40 kDa protein. Using the Rhodobacter capsulatus 40 and 70 kDa magnesium chelatase subunits we have analysed the effect of these mutations. Although having no ATPase activity, the deficient 40 kDa subunit could still associate with the 70 kDa protein. The binding was dependent on Mg(2+) and ATP or ADP. Our study demonstrates that the 40 kDa subunit functions as a chaperon that is essential for the survival of the 70 kDa subunit in vivo. We conclude that the ATPase activity of the 40 kDa subunit is essential for this function and that binding between the two subunits is not sufficient to maintain the 70 kDa subunit in the cell. The ATPase deficient 40 kDa proteins fail to participate in chelation in a step after the association of the 40 and 70 kDa subunits. This step presumably involves a conformational change of the complex in response to ATP hydrolysis.     

The binding of aurovertin to isolated beta subunit of F1 (mitochondrial ATPase). Stoicheiometry of beta subunit in F1.

1. Beef-heart mitochondrial ATPase (F1) is inactivated and dissociated by incubation with 0.85 M LiCl. ATP partly protects against inactivation. Three dissociation products could be identified after chromatography on diethylaminoethylcellulose: the delta subunit which is not adsorbed, the beta subunit which may be eluted from the column, and the alpha and gamma subunits which remain bound to the column. 2. Aurovertin binds to dissociated F1 with a fluorescence enhancement equal to about 30% that found with F1. Unlike intact F1 which shows two kinetically separated phases of fluorescence enhancement, only a fast phase is found with dissociated enzyme. 3. Fluorescence measurements at varying aurovertin and protein concentrations indicate that aurovertin binds to dissociated F1 in a simple 3-component reaction with dissociation constant 0.4 muM. There are two indistinguishable binding sites, calculated on the basis of the initial F1 concentration before dissociation. 4. The beta subunit was isolated from dissociated F1 by DEAE-cellulose chromatography. It has no ATPase activity but reacts with aurovertin with a fluorescence enhancement similar to that of dissociated F1. 5. The isolated beta subunit contains one aurovertin binding site with a dissociation constant of 0.56 muM. 6. It is concluded that F1 contains two beta subunits.     

Tyrosine modification of glucose dehydrogenase from Bacillus megaterium. Effect of tetranitromethane on the enzyme in the tetrameric and monomeric state

The active tetrameric glucose dehydrogenase from Bacillus megaterium is rapidly inactivated upon reaction with tetranitromethane. The inactivation is correlated with the nitration of a single tyrosine residue/subunit. The nitration does not influence the dissociation-reassociation process of the enzyme. The inactivation is prevented by the presence of NAD, AMP, ATP. The sequence around the nitrated tyrosine residue was determined and the residue was identified as Tyr-254 in the covalent structure of the enzyme. After dissociation of the enzyme into its monomers two tyrosine residues become susceptible to nitration. The nitrated subunits are unable to reassociate to the tetramer. Isolation and sequence analysis of the peptides containing nitrotyrosine indicated that two different tyrosine residues are predominantly modified. One residue is Tyr-254 which is essential for the catalytic activity and the other one is Tyr-160 which seems to be located in the subunit binding area.     

Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site

AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.     

Adenosine cyclic 3',5'-monophosphate-dependent protein kinase from human platelets.

A single cyclic AMP-dependent protein kinase (EC 2.7.1.37) has been isolated from human platelets by using DEAE-cellulose ion-exchange chromatography and Sephadex G-150 gel filtration. The molecular weight of the protein kinase was estimated to be 86 490. In the presence of cyclic AMP, the protein kinase could be dissociated into a catalytic subunit of molecular weight 50 000, and either one regulatory subunit of molecular weight 110 000 or two regulatory subunits of molecular weights 110 000 and 38 100, depending on the pH used. Recombination of either of the regulatory subunits with the catalytic subunit restored cyclic AMP-dependency in the catalytic subunit. The apparent Km for ATP in the presence of 10 muM Mg2+ was 4 muM (plus cyclic AMP) and 4.3 muM (minus cyclic AMP). The concentration of cyclic AMP needed for half-maximal stimulation of the protein kinase was 0.172 muM and apparent dissociation constants of 3.7 nM (absence of MgATP) and 0.18 muM (presence of MgATP) were exhibited by the "protein kinase-cyclic AMP complex". The enzyme required Mg2+ for maximum activity and showed a pH optimum of 6.2 with histone as substrate. In addition to four major endogenous platelet protein acceptors of apparent molecular weights 45 000, 28000, 18 500, and 11 100, the platelet protein kinase also phosphorylated the exogenous acceptor proteins thrombin, collagen and histone, all capable of inducing platelet aggregation. Prothrombin, a nonaggregating agent, was not phosphorylated.     

ATP-released large subunits participate in the assembly of RuBP carboxylase

Preincubation of 35S-methionine-labeled chloroplast extracts with ATP at 0 degree C potentiates the subsequent assembly of labeled large subunits into RuBPCase . This is correlated with the dissociation of newly synthesized large subunits from the 29S large subunit binding protein complex. These released large subunits then assemble into RuBPCase in a second, nucleotide-stimulated reaction. The data demonstrate that the 29S complex can play an active role in the assembly of RuBPCase .     

Structure of catabolite gene activator protein at 2.9-A resolution. Incorporation of amino acid sequence and interactions with cyclic AMP

The amino acid sequence of the Escherichia coli catabolite gene activator protein has been fit into a 2.9-A resolution electron density map. Each subunit of the dimer consists of two structurally distinct domains. The larger NH2-terminal domain is seen to bind cyclic AMP and forms all of the contacts between the subunits. The cyclic AMP is completely buried between the interior of the "beta roll" structure of the large domain and a long alpha helix; it makes important hydrogen-bonding interactions with residues from both subunits. The guanidinium group of a buried Arg makes an internal salt link with the phosphate of cyclic AMP. The 6-amino group of adenine interacts simultaneously with both subunits. This interaction with both subunits and the fact that cyclic GMP and cyclic IMP do not activate catabolite gene activator protein suggest that the binding of cyclic AMP may alter the relative orientation of the two subunits, which in turn would change the structure of a DNA binding site that is presumed to span the two smaller domains. The distribution and nature of side chains in the small domain do not rule out the possibility that catabolite gene activator protein binds to left-handed B-DNA.