Evidence supporting lysine 166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase as the essential base which initiates catalysis

The epsilon-amino group of Lys-166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase was postulated as the essential base which initiates catalysis by abstracting the proton at C-3 of ribulose 1,5-bisphosphate (Hartman, F. C., Soper, T. S., Niyogi, S. K., Mural, R. J., Foote, R. S., Mitra, S., Lee, E. H., Machanoff, R., and Larimer, F. W. (1987) J. Biol. Chem. 262, 3496-3501). To scrutinize this possibility, the site-directed Gly-166 mutant, totally devoid of ribulosebisphosphate carboxylase activity, was examined for its ability to catalyze each of three partial reactions. When carbamylated at Lys-191 (i.e. activated with CO2 and Mg2+), wild-type enzyme catalyzed the hydrolysis of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate, the six-carbon reaction intermediate of the carboxylase reaction (Pierce, J., Andrews, T. J., and Lorimer, G. H. (1986a) J. Biol. Chem. 261, 10248-10256). Likewise, when carbamylated at Lys-191, the Gly-166 mutant also catalyzed the hydrolysis of this reaction intermediate. The carbamylated wild type catalyzed the enolization of ribulose 1,5-bisphosphate as indicated by the transfer of 3H radioactivity from [3-3H]ribulose, 1,5-bisphosphate to the medium. However, even when carbamylated at Lys-191, the mutant protein did not catalyze the enolization of ribulose 1,5-bisphosphate. Additionally, unlike the decarbamylated wild-type enzyme, which catalyzed the decarboxylation of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate in the absence of Mg2+, the mutant protein was inactive in this partial reaction. These properties exclude the epsilon-amino group of Lys-166 as an obligatory participant in the hydrolysis of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate. In contrast, these properties are consistent with the epsilon-amino group of Lys-166 functioning as an acid-base catalyst in the enolization of ribulose 1,5-bisphosphate (when the enzyme is carbamylated) and in the decarboxylation of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate (when the enzyme is decarbamylated). Alternatively, Lys-166 may stabilize the transition states of these two partial reactions.     

Dynamic, ligand-dependent conformational change triggers reaction of ribose-1,5-bisphosphate isomerase from Thermococcus kodakarensis KOD1

Ribose-1,5-bisphosphate isomerase (R15Pi) is a novel enzyme recently identified as a member of an AMP metabolic pathway in archaea. The enzyme converts d-ribose 1,5-bisphosphate into ribulose 1,5-bisphosphate, providing the substrate for archaeal ribulose-1,5-bisphosphate carboxylase/oxygenases. We here report the crystal structures of R15Pi from Thermococcus kodakarensis KOD1 (Tk-R15Pi) with and without its substrate or product. Tk-R15Pi is a hexameric enzyme formed by the trimerization of dimer units. Biochemical analyses show that Tk-R15Pi only accepts the α-anomer of d-ribose 1,5-bisphosphate and that Cys(133) and Asp(202) residues are essential for ribulose 1,5-bisphosphate production. Comparison of the determined structures reveals that the unliganded and product-binding structures are in an open form, whereas the substrate-binding structure adopts a closed form, indicating domain movement upon substrate binding. The conformational change to the closed form optimizes active site configuration and also isolates the active site from the solvent, which may allow deprotonation of Cys(133) and protonation of Asp(202) to occur. The structural features of the substrate-binding form and biochemical evidence lead us to propose that the isomerase reaction proceeds via a cis-phosphoenolate intermediate.     

Inhibition and inactivation of liver aldolase

Substrate analogs xylulose 1,5-bisphosphate, glucitol 1,6-bisphosphate, α-2,5-anhydroglucitol 1,6-bisphosphate, α-, β-methyl fructofuranoside 1,6-bisphosphate, ribulose 1,5-bisphosphate, ribulose 5-phosphate, and ribose 5-phosphate and inactivating agents 1-chloro-2, 4-dinitrobenzene, 4-hydroxymercuribenzoate, and pyridoxal phosphate were examined for their effects on liver aldolase. These studies support the use of the β-anomer and acyclic form as substrate. They also suggest that the liver enzyme active site is similar to the muscle enzyme but with a much weaker 6-phosphate binding site.     

Isolation and partial characterization of Rhodopseudomonas sphaeroides mutants defective in the regulation of ribulose bisphosphate carboxylase/oxygenase.

Several mutants of Rhodopseudomonas sphaeroides defective in the derepression of the enzyme ribulose 1,5-bisphosphate carboxylase have been isolated by using the unstable Tn5 vectors pJB4JI and pRK340. Transpositional insertion mutants obtained with pJB4JI were demonstrated to be incapable of increasing ribulose 1,5-bisphosphate carboxylase/oxygenase levels when grown on butyrate-bicarbonate medium or under conditions of carbon starvation, whereas the wild-type strain increased activity four- to eightfold. When the wild-type strain was starved for carbon in the presence of chloramphenicol, no derepression was observed. Crude extracts from mutant and wild-type strains had distinct and consistent differences in protein content as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chromatographic evidence indicated that mutants were defective in the regulation of only one of the two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase synthesized by R. sphaeroides.     

Ribulose 1,5-bisphosphate carboxylase from the thermophilic, acidophilic alga, Cyanidium caldarium (Geitler). Purification, characterisation and thermostability of the enzyme.

An an initial stage in the study of proteins from thermophilic algae, the enzyme ribulose 1,5-bisphosphate carboxylase 2-phospho-D-glycerate carboxylyase (dimerizing, EC 4.1.1.39) was purified 11-fold from the thermophilic alga Cyandium caldarium, with a 24% recovery. This purified enzyme appeared homogeneous on polyacrylamide gels and could be dissociated into two subunit types of molecular weights 55,000 and 14,900. The optimal assay temperature was 42.5 degrees C, whilst enzyme purified from Chlorella spp. showed maximum activity at 35 degrees C. The thermostability of Cyanidium ribulose 1,5-bisphosphate carboxylase was considerably greater than that of the Chlorella enzyme, and the presence of Mg2+ and HCO-3 further enhanced this heat stability. A break in the Arrhenius plot occured at 20 degrees C for Chlorella ribulose 1,5-bisphosphate carboxylase and 36 degrees C for the enzyme from Cyanidium. It is suggested that the thermostability of Cyanidium ribulose 1,5-bisphosphate carboxylase is a result of an inherent stability of the enzyme molecule which permits efficient CO2 fixation at high temperatures but results in low activity in the mesophilic temperature range.     

Characterization of antiserum directed against form II ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides.

Antiserum directed against form II ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides showed no cross-reactivity towards the form I enzyme as evidenced by a lack of immunopreciptation. In addition, this antiserum failed to inhibit form I enzymatic activity.     

A site-specific mutation within the active site of ribulose-1,5-bisphosphate carboxylase of Rhodospirillum rubrum

In vitro mutagenic techniques have generated an asp→glu substitution at residue 198 adjacent to the carbamate-divalent metal ion binding site of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase. A single C→A nucleotide change in the coding strand created the mutant and introduced a new EcoRI restriction site on the expression plasmid pRR2119. Although the carboxylase:oxygenase ratio remained the same, the mutant enzyme had slightly altered kinetic properties. The e.p.r. spectra of the quaternary complexes enzyme.activator carbamate.Mn²⁺.2-carboxyarabinitol 1,5-bisphosphate and enzyme.activator carbamate.Mn²⁺.4-carboxyarabinitol 1,5-bisphosphate for mutant and wild-type enzymes were different, indicating that the metal ion was in a slightly altered environment. These findings are consistent with the hypothesis that, besides the carbamate at lys 201, the carboxyl group of asp 198 contributes to the formation of the divalent metal ion binding site.     

Retention of the oxygens at C-2 and C-3 of D-ribulose 1,5-bisphosphate in the reaction catalyzed by ribulose-1,5-bisphosphate carboxylase.

Ribulose-1,5-bisphosphate carboxylase catalyzes the conversion of D ribulose 1,5-bisphosphate and CO2 to 3-phospho-D-glycerate, with retention of the oxygen atoms at both C-2 and C-3 of the substrate. This observation is consistent with mechanistic pathways involving an enediol intermediate and eliminates suggested mechanisms that involve covalent intermediates between the enzyme and ribulose 1,5-bisphosphate in which the substrate oxygen at C-2 or C-3 is compulsorily lost.     

Posttranscriptional Regulation of Ribulose 1,5-bisphosphate Carboxylase Small Subunit Accumulation in Chlamydomonas reinhardtii

The coordinated synthesis of the subunits of ribulose 1,5-bisphosphate carboxylase was examined by analysis of the chloroplast ribosome-deficient mutant of Chlamydomonas reinhardtii, ac20crl. The absence of the chloroplast-synthesized large subunit of this enzyme from cells of this strain is a direct consequence of the lack of chloroplast ribosomes. In contrast, the absence of the cytoplasmically-synthesized small subunit of ribulose 1,5-bisphosphate carboxylase from this mutant is not understood. To discern the cause of this absence, we have compared results of in vivo radioactive labeling experiments with those of cell-free translations of RNA from ac20crl. Protein products from these experiments were identified by one-and two-dimensional electrophoretic analyses. Neither subunit, revealed either as a stained band or by fluorography of proteins radioactively labeled in vivo for 2 hours, was detected in ac20crl. Cell-free translation of polyadenylated RNA obtained from ac20crl, however, revealed wild-type levels of mRNA for the precursor to the small subunit. This messenger was found to be associated with subribosomal RNP and polysomes. We conclude that the absence of the small subunit of ribulose 1,5-bisphosphate carboxylase from ac20crl is the result of a translational or posttranslational event.     

The Activation State of Ribulose 1,5-bisphosphate Carboxylase in Maize Leaves in Dark and Light

A spectrophotometric procedure for assay of initial and totalactivity of ribulose 1,5-bisphosphate carboxylase in maize leaveswas established. The extraction of the crude enzyme from maizeleaf tissue, which was prefrozen in liquid nitrogen, desaltingof the extract, and assay of the enzyme was completed within3 min. From experiments adding deactivated ribulose 1,5-bisphosphatecarboxylase to the leaf tissue prior to extraction it was estimatedthat the maximum extent of activation during extraction, desaltingand assay was 8%. In predarkened leaves the enzyme showed 67to 84% of maximal activation while in preilluminated leavesthe enzyme showed 89 to 98% of maximal activation. These resultsindicate that deactivation of the enzyme in the dark is nota reason for the previous finding of a transient peak of ribulose1,5-bisphosphate in maize leaves during induction of photosynthesis[Usuda (1985) Plant Physiol. 78: 859–864]. This transientincrease in the substrate level upon illumination might be explainedby the presence of an unknown negative effector for ribulose1,5-bisphosphate carboxylase in vivo in leaf tissue in the dark,or limiting CO₂ supply to the enzyme during the induction period. (Received May 30, 1985; Accepted August 16, 1985)     

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.     

Derepression of the synthesis of D-ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

The synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase in Rhodospirillum rubrum was greatly influenced by the conditions of culture. When grown photolithotrophically in an atmosphere containing low levels of CO2 (1.5 to 2%), enzyme synthesis was derepressed, with the result that the enzyme comprised up to 50% of the soluble protein of the cells as determined by immunological quantitation. This response was not observed when R. rubrum was grown photolithotrophically in an atmosphere of 5% CO2 in hydrogen. Similarly, the derepression of ribulose 1,5-bisphosphate carboxylase/oxygenase was observed in photoheterotrophically (butyrate)-grown cultures only after the HCO3- supply was nearly exhausted. The increase in enzyme activity observed in derepressed cultures was not paralleled by an increase in the in vivo CO2 fixation rate. Apparently, R. rubrum derepresses the synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase when exposed to low CO2 concentrations to scavenge the limited CO2 available to such cultures.     

Ribulose 1,5-bisphosphate carboxylase-oxygenase. I. Structural, immunochemical and catalytic properties

Some structural, immunochemical and catalytic properties are examined for ribulose 1,5-bisphosphate carboxylase-oxygenase from various cellular organisms including bacteria, cyanobacteria, algae and higher plants. The native enzyme molecular masses and the subunit polypeptide compositions vary according to enzyme sources. The molecular masses of the large and small subunits from different cellular organisms, on the other hand, show a relatively high homology due to their well-conserved primary amino acid sequence, especially that of the large subunit. In higher plants, the native enzyme and the large subunit are recognized by the antibodies raised against either the native or large subunit, whereas the small subunit apparently cross-reacts only with the antibodies directed against itself. A wide diversity exists, however, in the serological response of the native enzyme and its subunits with antibodies directed against the native enzyme or its subunits from different cellular organisms. According to numerous kinetic studies, the carboxylase and oxygenase reactions of the enzyme with ribulose 1,5-bisphosphate and carbon dioxide or oxygen require activation by carbon dioxide and magnesium prior to catalysis with ribulose 1,5-bisphosphate and carbon dioxide or oxygen. The activation and catalysis are also under the regulation of other metal ions and a number of chloroplastic metabolites. Recent double-labeling experiments using radioactive ribulose 1,5-bisphosphate and 14CO2 have elucidated the carboxylase/oxygenase ratios of the enzymes from different organisms. Another approach, i.e., genetic experiments, has also been used to examine the modification of the carboxylase/oxygenase ratio.     

Control of CO2 fixation. Regulation of spinach ribulose-5-phosphate kinase by stromal metabolite levels

The effect of stromal metabolites on the light-activated form of ribulose-5-phosphate kinase was studied with the enzyme rapidly extracted from illuminated spinach chlorplasts. In some instances, the effect of metabolites on the dark-inactivated enzyme extracted from darkened chloroplasts was also investigated. (1) The light-activated form of the enzyme is competitively inhibited with respect to ribulose 5-phosphate by 6-phosphogluconate, ribulose 1,5-bisphosphate, 3-phosphoglycerate and phosphate. Also, fructose 1,6-bisphosphate is inhibitory. All these compounds, except ribulose 1,5-bisphosphate, show an increasing inhibitory effect at lower pH values. Therefore, in the presence of these inhibitors, ribulose-5-phosphate kinase becomes strongly pH dependent. These compounds also exert an inhibitory effect on the dark-inactivated enzyme. (2) The assay of stromal levels of 6-phosphogluconate showed that this compound increased dramatically during a light-dark transient. (3) The dark-inactivated form of ribulose-5-phosphate kinase is strongly inhibited by ADP, the inhibition being competitive with respect to ATP. (4) A simulation of stromal metabolite levels in the enzyme activity assay indicates that in illuminated chloroplasts ribulose-5-phosphate kinase attains only about 4% of its maximal activity. When the fully light-activated enzyme is assayed under conditions occurring in the stroma in the dark, the activity is further decreased by a factor of 20. The same assay with the dark-inactivated enzyme yields an activity of virtually zero. (5) These results demonstrate that in the chloroplasts ribulose-5-phosphate kinase can not only be very efficiently switched off in the dark, but also be subjected to fine control during the illuminated state through the action of stromal metabolites.     

Ribulose-1,5-bisphosphate carboxylase: enzyme-catalyzed appearance of solvent tritium at carbon 3 of ribulose 1,5-bisphosphate reisolated after partial reaction

When ribulose 1,5-bisphosphate is allowed to react with carbon dioxide in tritiated water in the carboxylation reaction catalyzed by ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum, the ribulose 1,5-bisphosphate reisolated after partial reaction is found to be labeled. The specific radioactivity of the remaining substrate pool rises during the course of the reaction. Experiments in deuterium oxide show that the isotopic label resides on carbon 3. Earlier failures to detect this exchange process probably derive from the use of enzyme that was, in the absence of carbon dioxide, inactive. The present results provide direct evidence for the intermediacy of the enediol between C-2 and C-3 of ribulose 1,5-bisphosphate and show that the enolization step is at least partially rate limiting in the overall carboxylase reaction. The specific radioactivity of the product 3-phospho-D-glycerate remains constant throughout the course of the reaction at about one-sixth that of the solvent. This strengthens the argument against the involvement of "sticky" protons in the reaction.     

Facile assay of enzymes unique to the Calvin cycle in intact cells, with special reference to ribulose 1,5-bisphosphate carboxylase.

A procedure for the facile measurement, in intact cells, of two enzymes unique to the Calvin cycle, ribulose 1,5-bisphosphate carboxylase and phosphoribulokinase, is described. The procedure involved a simple toluene treatment to render phototrophic cells permeable to the necessary substrates, effectors, and cofactors. Whole-cell ribulose 1,5-bisphosphate carboxylase activity quantitatively approximates the activity obtained in cell-free extracts. In addition, the activity measured with toluene-treated whole cells results in a stoichiometric carboxylation of ribulose 1,5-bisphosphate to phosphoglyceric acid. The assay procedures described are most convenient for determining enzyme levels as a function of growth. Moreover, such an assay should open the way to further studies on the regulation of CO₂ assimilation by direct measurement of the enzymes concerned within the cell.     

Regulation of photosynthesis in nitrogen deficient wheat seedlings

Nitrogen effects on the regulation of photosynthesis in wheat (Triticum aestivum L., cv Remia) seedlings were examined. Ribulose 1,5-bisphosphate carboxylase/oxygenase was rapidly extracted and tested for initial activity and for activity after incubation in presence of CO₂ and Mg²⁺. Freeze clamped leaf segments were extracted for determinations of foliar steady state levels of ribulose 1,5-bisphosphate, triose phosphate, 3-phosphoglycerate, ATP, and ADP. Nitrogen deficient leaves showed increased ATP/ADP and triose phosphate/3-phosphoglycerate ratios suggesting increased assimilatory power. Ribulose 1,5-bisphosphate levels were decreased due to reduced pentose phosphate reductive cycle activity. Nevertheless, photosynthesis appeared to be limited by ribulose 1,5-bisphosphate carboxylase/oxygenase, independent of nitrogen nutrition. Its degree of activation was increased in nitrogen deficient plants and provided for maximum photosynthesis at decreased enzyme protein levels. It is suggested that ribulose 1,5-bisphosphate carboxylase/oxygenase activity is regulated according to the amount of assimilatory power.     

Rhizobium japonicum mutant strains unable to grow chemoautotrophically with H2

Rhizobium japonicum strain SR grows chemoautotrophically on a mineral salts medium when incubated in an H2- and CO2-containing atmosphere. Mutant strains unable to grow or that grow very poorly chemoautotrophically with H2 have been isolated from strain SR. The mutant isolation procedure involved mutagenesis with ethyl methane sulfonate, penicillin selection under chemoautotrophic growth conditions, and plating of the survivors onto medium containing carbon. The resulting colonies were replica plated onto medium that did not contain carbon, and the plates were incubated in an H2- and CO2-containing atmosphere. Mutant strains unable to grow under these conditions were chosen. Over 100 mutant strains with defects in chemoautotrophic metabolism were obtained. The phenotypes of the mutants fall into various classes. These include strains unable to oxidize H2 and strains deficient in CO2 uptake. Some of the mutant strains were capable of oxidizing H2 only when artificial electron acceptors were provided. Two mutant strains specifically lack activity of the key CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase. Other mutant strains lack both H2-oxidizing ability and ribulose 1,5-bisphosphate carboxylase activity.     

The orientation of substrate and reaction intermediates in the active site of ribulose-1,5-bisphosphate carboxylase

There are four possible orientations of the substrate ribulose 1,5-bisphosphate in the active site of ribulose-1,5-bisphosphate carboxylase. Distinction between these four possible orientations has been made on the basis of 31P NMR and borohydride-trapping experiments. The orientation of the reaction-intermediate analog, 2'-carboxy-D-arabinitol 1,5-bisphosphate with respect to the divalent metal ion was determined by 31P NMR studies of the quaternary complex, enzyme.CO2.Ni2+.2'-carboxyarabinitol 1,5-bisphosphate. Assignment of the phosphorus resonances of this complex was made by labeling the phosphoryl group at either C-1 or C-5 with 17O. The phosphorus atom closer to the paramagnetic metal ion, Ni2+, to which the broader of the phosphorus resonances is attributed, has been identified as that attached to C-1. When bound to the active site of carbaminated enzyme, D-ribulose 1,5-bisphosphate was reduced by sodium borohydride with absolute stereospecificity to D-arabinitol 1,5-bisphosphate. The reduction of the enzyme-bound substrate thus occurred on the Si face of the C-2 carbonyl group. These two results together establish that ribulose 1,5-bisphosphate is oriented within the active site so that 1) the phosphoryl group at C-1 is closer to the divalent metal ion than that at C-5 and 2) the Si face of the carbonyl group points to the "outside world."     

Loose association of ribulose 1,5-bisphosphate carboxylase/oxygenase with chloroplast thylakoid membranes

The intra-chloroplastic distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) between thylakoid membranes and stroma was studied by determining the enzyme activities in the two fractions, obtained by the rapid centrifugation of hypotonically disrupted chloroplast preparations of spinach and pea leaf tissues. The membrane-associated form of RuBisCO was found to increase in proportion to the concentration of MgCl₂ in the disrupting medium; with 20 mM MgCl₂ approximately 20% of the total RuBisCO of spinach chloroplasts and 10% of that of pea chloroplasts became associated with thylakoid membranes. Once released from membranes in the absence of MgCl₂, addition of MgCl₂ did not cause reassociation of the enzyme. The inclusion of KCl in the hypotonic disruption buffer also caused the association of RuBisCO with membranes; however, up to 30 mM KCl, only minimal enzyme activities could be detected in the membranes, whereas above 40 mM KCl there was a sharp increase in the membrane-associated form of the enzyme.Higher concentrations of chloroplasts during the hypotonic disruption, as well as addition of purified preparations of RuBisCO to the hypotonic buffer, resulted in an increase of membrane-associated activity. Therefore, the association of the enzyme with thylakoid membranes appears to be dependent on the concentration of RuBisCO. P-glycerate kinase and aldolase also associated to the thylakoid membranes but NADP-linked glyceraldehyde-3-P dehydrogenase did not. The optimal conditions for enzyme association with the thylakoid membranes were examined; maximal association occurred at pH 8.0. The association was temperature-insensitive in the range of 4° to 25° C. RuBisCO associated with the thylakoid membranes could be gradually liberated to the soluble form upon shaking in a Vortex mixer at maximal speed, indicating that the association is loose.Abbreviations DTT dithiothreitol - RuBP ribulose 1,5-bisphosphate - RuBisCO ribulose 1,5-bisphosphate carboxylase/oxygenase - MES 2-(N-morpholino) ethane sulfonic acid