Use of Reduced Sulfur Compounds by Beggiatoa spp.: Enzymology and Physiology of Marine and Freshwater Strains in Homogeneous and Gradient Cultures

The marine Beggiatoa strains MS-81-6 and MS-81-1c are filamentous, gliding, colorless sulfur bacteria. They have traditionally been cultured in very limited quantities in sulfide gradient media, where they grow as chemolithoautotrophs, forming a thin horizontal plate well below the air-agar interface. There, the facultatively chemolithoautotrophic strain MS-81-6 quantitatively harvests the flux of sulfide diffusing from below and oxidizes it to sulfate by using oxygen as the electron acceptor. Only recently have these strains been cultivated in bulk in defined liquid media (K. D. Hagen and D. C. Nelson, Appl. Environ. Microbiol. 62:947-953, 1996). In the current study, the obligately chemolithoautotrophic strain MS-81-1c was shown to have, despite much greater storage of elemental sulfur, an apparent Y(infH)(inf(inf2))(infS) twice that of MS-81-6 when the two strains were grown in identical sulfide-limited gradient media. While the basis of this difference in energy conservation has not been established, differences in sulfur oxidation enzymes were noted. Strain MS-81-1c appeared to be able to oxidize sulfite by using either the adenosine phosphosulfate (APS) pathway or a sulfite:acceptor oxidoreductase. APS pathway enzymes (ATP sulfurylase and APS reductase) were present at relatively high and constant levels regardless of growth conditions, while the sulfite:acceptor oxidoreductase activity varied at least eightfold, with the highest activity produced in sulfide gradient medium. By contrast, strain MS-81-6 showed no detectable activity of the APS pathway enzymes and possessed a sulfite:acceptor oxidoreductase activity just sufficient to account for its observed rate of growth in sulfide gradient medium. Freshwater strain OH-75-2a showed activity and regulation of sulfite:acceptor oxidoreductase consistent with lithotrophic energy conservation, a feature not yet proven for any freshwater Beggiatoa strain.     

Carbon isotope fractionation by thermophilic phototrophic sulfur bacteria: evidence for autotrophic growth in natural populations.

Purple phototrophic bacteria of the genus Chromatium can grow as either photoautotrophs or photoheterotrophs. To determine the growth mode of the thermophilic Chromatium species, Chromatium tepidum, under in situ conditions, we have examined the carbon isotope fractionation patterns in laboratory cultures of this organism and in mats of C. tepidum which develop in sulfide thermal springs in Yellowstone National Park. Isotopic analysis (13C/12C) of total carbon, carotenoid pigments, and bacteriochlorophyll from photoautotrophically grown cultures of C. tepidum yielded 13C fractionation factors near -20%. Cells of C. tepidum grown on excess acetate, wherein synthesis of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase ribulose bisphosphate carboxylase) was greatly repressed, were isotopically heavier, fractionation factors of ca. -7% being observed. Fractionation factors determined by isotopic analyses of cells and pigment fractions of natural populations of C. tepidum growing in three different sulfide thermal springs in Yellowstone National Park were approximately -20%, indicating that this purple sulfur bacterium grows as a photoautotroph in nature.     

Carbon isotope fractionation by thermophilic phototrophic sulfur bacteria: evidence for autotrophic growth in natural populations

Purple phototrophic bacteria of the genus Chromatium can grow as either photoautotrophs or photoheterotrophs. To determine the growth mode of the thermophilic Chromatium species, Chromatium tepidum, under in situ conditions, we have examined the carbon isotope fractionation patterns in laboratory cultures of this organism and in mats of C. tepidum which develop in sulfide thermal springs in Yellowstone National Park. Isotopic analysis (13C/12C) of total carbon, carotenoid pigments, and bacteriochlorophyll from photoautotrophically grown cultures of C. tepidum yielded 13C fractionation factors near -20%. Cells of C. tepidum grown on excess acetate, wherein synthesis of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase ribulose bisphosphate carboxylase) was greatly repressed, were isotopically heavier, fractionation factors of ca. -7% being observed. Fractionation factors determined by isotopic analyses of cells and pigment fractions of natural populations of C. tepidum growing in three different sulfide thermal springs in Yellowstone National Park were approximately -20%, indicating that this purple sulfur bacterium grows as a photoautotroph in nature.     

香蕉rbcS基因启动子的克隆及序列分析

以巴西香蕉为材料,根据已经获得的香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的全长cDNA序列设计1对专一引物,通过PCR扩增得到了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基的基因组全长,序列长811 bp,含有2个内含子。根据其基因组序列设计引物,采用SEFA-PCR方法,以总DNA为模板克隆了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的启动子序列,长1 681 bp。用PLACE软件分析发现该序列具有启动子的基本元件TATA-box、CAAT-box,包含多个胁迫诱导元件,如光诱导元件、赤霉素、低温诱导元件、昼夜节律调控元件等。该序列的克隆与分析为进一步研究香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的表达调控奠定了基础。     

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.     

The Association of d-Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase with Phosphoribulokinase

When Ribulose- 1,5-bisphosphate carboxylase/oxygenase was purified from spinach leaves (Spinacia oleracea) using precipitation with polyethylene glycol and MgCl₂ followed by DEAE cellulose chromatography, 75% of phosphoribulokinase and 7% of phosphoriboisomerase activities copurified with ribulose- 1,5-bisphosphate carboxylase/oxygenase. This enzyme preparation showed ribose-5-phosphate and ribulose-5-phosphate dependent carboxylase and oxygenase activities which were nearly equivalent to its corresponding ribulose- 1,5-bisphosphate dependent activity. The ribose-5-phosphate and ribulose-5-phosphate dependent reaction rates were stable and linear for much longer time periods than the ribulose- 1,5-bisphosphate dependent rates. When sucrose gradients were used to purify ribulose- 1,5-bisphosphate carboxylase/oxygenase from crude stromal extracts, phosphoribulokinase was found to cosediment with ribulose- 1,5-bisphosphate carboxylase. Under these conditions most of the phosphoriboisomerase activity remained with the slower sedimenting proteins. Ammonium sulfate precipitation resulted in separation of the ribulose- 1,5-bisphosphate carboxylase peak from phosphoribulokinase peak. Crude extracts of peas Pisum sativum and spinach contained 0.725 to 0.730 milligram of phosphoribulokinase per milligram of chlorophyll, respectively, based on an enzyme-linked immunosorbent assay.     

Characterization of Xanthobacter strains H4-14 and 25a and enzyme profiles after growth under autotrophic and heterotrophic conditions

All Xanthobacter strains studied are versatile autotrophic bacteria, able to grow on methanol and other substrates. Strain 25a, a yellow-pigmented, pleomorphic, Gram-negative bacterium, capable of autotrophic growth on methanol, formate, thiosulfate, and molecular hydrogen, was isolated from an enrichment culture inoculated with soil from a subtropical greenhouse. Subsequent studies showed that the organism also grows on a wide range of multicarbon substrates. Ammonia, nitrate and molecular nitrogen were used as nitrogen sources. The taxonomic relationship of strains H4-14 and 25a with previously described Xanthobacter strains was studied by numerical classification. Strain H4-14 was identified as a X. flavus strain, but the precise position of strain 25a remained uncertain. It probably belongs to a new species of the genus Xanthobacter. The levels of various enzymes involved in autotrophic and heterotrophic metabolism were determined following growth of strains H4-14 and 25a in batch and continuous cultures. The mechanisms involved in controlling ribulose-1,5-bisphosphate carboxylase/oxygenase synthesis in Xanthobacter strains appear to be comparable to those observed for other autotrophic bacteria, namely repression by organic compounds and derepression by autotrophic energy sources, such as methanol and hydrogen.Abbreviations API appareils et procédés d'identification - CS citrate synthase - ED Entner-Doudoroff pathway - FBP fructose-1,6-bisphosphate - FDH formate dehydrogenase - HPS hexulose-6-phosphate synthase - ICDH isocitrate dehydrogenase - KDPG 2-keto-3-deoxy-6-phosphogluconate - MDH methanol dehydrogenase - PRK phosphoribulokinase - PQQ pyrrolo quinoline quinone - RuBisC/O ribulose-1,5-bisphosphate carboxylase/oxygenase - RuMP ribulose monophosphate     

Developmental and Photosynthetic Characteristics of a Photoautotrophic Chrysanthemum Culture

Chrysanthemum plantlets were cultivated in vitro on media with 2.0, 0.3, or 0 % sucrose, or photoautotrophically without an organic carbon source but with supplementation of the culture vessel atmosphere with 2 % CO₂. The photoautotrophically cultivated plantlets showed a better growth and multiplication, higher contents of chlorophyll (Chl) and carotenoids, higher Chl a/b ratio, net photosynthetic rate and ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase activities than plantlets grown on the medium with sucrose. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cotranscription, deduced primary structure, and expression of the chloroplast-encoded rbcL and rbcS genes of the marine diatom Cylindrotheca sp. strain N1.

The primary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from the marine diatom Cylindrotheca sp. strain N1 has been determined. Unlike higher plants and green algae, the genes encoding the large and the small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase are chloroplast-encoded and closely associated (Hwang and Tabita, 1989). The rbcL and rbcS genes in strain N1 are cotranscribed and are separated by an intergenic region of 46 nucleotide base pairs. Ribosome binding sites and a potential promoter sequence were highly homologous to previously determined chloroplast sequences. Comparison of the deduced primary structure of the diatom large and small subunits indicated significant homology to previously determined sequences from bacteria; there was much less homology to large and small subunits from cyanobacteria, green algae, and higher plants. Although high levels of recombinant diatom large subunits could be expressed in Escherichia coli, the protein synthesized was primarily insoluble and incapable of forming an active hexadecameric enzyme. Edman degradation studies indicated that the amino terminus of the large subunit isolated from strain N1 was blocked, suggesting that the mechanism responsible for processing and subsequent assembly of large and small subunits resembles the situation found with other eucaryotic ribulose-1,5-bisphosphate carboxylase/oxygenase proteins, despite the distinctive procaryotic gene arrangement and sequence homology.     

Characterization of the pyrenoid isolated from unicellular green algaChlamydomonas reinhardtii: Particulate form of RuBisCO protein

Summary The pyrenoid is a protein complex in the chloroplast stroma of eukaryotic algae. After the treatment with mercury chloride, pyrenoids were isolated by sucrose density gradient centrifugation from cell-wall less mutant cells, CW-15, as well as wild type cells, C-9, of unicellular green algaChlamydomonas reinhardtii. Pyrenoids were characterized as a fraction whose protein/chlorophyll ratio was very high, and also examined by Nomarski differential interference microscopy. Most of the components consisted of 55 kDa and 16 kDa polypeptides (11) which were immunologically identified as the large and small subunit of RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) protein, respectively. Some minor polypeptides were also detected. Substantial amount of RuBisCO protein is present as a particulate form in the pyrenoid in addition to the soluble form in algal chloroplast stroma.Abbreviations BPB bromophenol blue - DAB 3,3-diaminobenzidine - DTT dithiothreitol - ELISA enzyme-linked immunosorbent assay - High-CO₂ cells cells grown under air enriched with 4% CO₂ - Low-CO₂ cells cells grown under ordinary air (containing 0.04% CO₂) - NP-40 nonionic detergent (Nonidet) P-40 - PAGE polyacrylamide gel electrophoresis - PAP peroxidase-antiperoxidase conjugate - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SDS sodium dodecylsulfate     

Isolation and some properties of ribulose-1, 5-bisphosphate carboxylase-oxygenase from red kidney bean primary leaves

Purification of ribulose-1,5-bisphosphate carboxylase from primary leaves of Phaseolus vulgaris var. Red Kidney with ammonium sulfate precipitation, ion exchange chromatography, and gel filtration resulted in the complete loss of detectable oxygenase activity and the retention of a low velocity and a high K(m) form of both the carboxylase and oxygenase. The polyethylene glycol-6000-purified ribulose-1, 5-bisphosphate oxygenase displayed a broad pH optimum (7.9-9.4) and a high K(m) for O(2) and ribulose 1,5-bisphosphate (0.90 mm and 0.25 mm, respectively). Initiation of the oxygenase reaction with protein rather than ribulose 1,5-bisphosphate resulted in reduced activity. The enzymes prepared by the two purification procedures were electrophoretically different.Etiolated primary leaf tissue exhibited low rates of both carboxylase and oxygenase. Similar developmental kinetic activity was observed for both reactions during greening. Photosynthetic (14)CO(2) fixation was inhibited 95% by 100% N(2) gas during the first 24 hours of greening, but the inhibition was rapidly overcome by 48 to 72 hours of light exposure.     

An enzyme and13C-NMR study of carbon metabolism in heliobacteria

Heliobacteria are a group of anoxygenic phototrophs that can grow photoheterotrophically in defined minimal media on only a limited range of organic substrates as carbon sources. In this study the mechanisms which operate to assimilate carbon and the routes employed for the biosynthesis of cellular intermediates were investigated in a newHeliobacterium strain, HY-3. This was achieved using two approaches (1) by measuring the activities of key enzymes in cell-free extracts and (2) by the use of¹³C nuclear magnetic resonance (NMR) spectroscopy to analyze in detail the labelling pattern of amino-acids of cells grown on [¹³C] pyruvate and [¹³C] acetate.Heliobacterium strain HY-3 was unable to grow autotrophically on CO₂/H₂ and neither (ATP)-citrate lyase nor ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPcase) were detectable in cell-free extracts. The enzyme profile of pyruvate grown cells indicated the presence of a pyruvate:acceptor oxidoreductase at high specific activity which could convert pyruvate to acetyl-Coenzyme A. No pyridine nucleotide dependent pyruvate dehydrogenase complex activity was detected. Of the citric-acid cycle enzymes, malate dehydrogenase, fumarase, fumarate reductase and an NADP-specific isocitrate dehydrogenase were readily detectable but no aconitase or citrate synthase activity was found. However, the labelling pattern of glutamate in long-term 2-[¹³C] acetate incorporation experiments indicated that a mechanism exists for the conversion of carbon from acetyl-CoA into 2-oxoglutarate. A 2-oxoglutarate:acceptor oxidoreductase activity was present which was also assayable by isotope exchange, but no 2-oxoglutarate dehydrogenase complex activity could be detected. Heliobacteria appear to use a type of incomplete reductive carboxylic acid pathway for the conversion of pyruvate to 2-oxoglutarate but are unable to grow autotrophically using this metabolic route due to the absence of ATP-citrate lyase.     

Increased susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase to proteolytic degradation caused by oxidative treatments

The susceptibility of the chloroplastic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase to proteolysis by trypsin, chymotrypsin, proteinase K, and papain is enhanced by oxidative treatments including spontaneous oxidation of cysteines. Proteinases exhibit a high specificity for the oxidized inactive form of the carboxylase, cleaving its large subunit. Treatment of the inactive enzyme with dithiothreitol results in partial recovery of both carboxylase activity and resistance to proteolysis. This behavior may explain the specific degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase that occurs in vivo during leaf senescence.     

In situ assay of ribulose-1,5-bisphosphate carboxylase/oxygenase in Thiobacillus neapolitanus.

Cells permeabilized with chloroform yielded ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) activities nearly equal to those of cell extracts, thus indicating that both cytoplasmic and carboxysomal RuBisCO are functional in situ. The carboxysomal and cytoplasmic RuBisCO both form the CO2-Mg2(+)-enzyme ternary complex, as evidenced by stabilization with 2-C-carboxy-D-arabinitol-1,5-bisphosphate (CABP), a potent competitive inhibitor of RuBisCO. The data are consistent with the hypothesis that the carboxysome is functional in carbon dioxide fixation.     

Phosphorylation in vitro of the large subunit of the ribulose-1,5-bisphosphate carboxylase and of the glyceraldehyde-3-phosphate dehydrogenase

A protein kinase activity responsible for the in vitro phosphorylation of at least six endogenous polypeptides including the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is present in the stroma (3000 X g supernatant, S30) of spinach chloroplasts. The phosphorylation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit is strongly enhanced when sodium fluorure is used as a protein phosphatase inhibitor. Phosphorylation occurs on threonine and serine residues. The protein kinase involved is not Ca2+-dependent. There is also evidence for a protein phosphatase activity which suggests a coupled regulation by a phosphorylation-dephosphorylation process. The phosphorylating activity is drastically reduced when S30 is prepared from leaves harvested after a dark period. Phosphorylation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit is not related to its own synthesis. The in vitro phosphorylation of the glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) is also demonstrated.     

Differences in amino acid sequence of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from two species of Dasycladaceae

In contrast to other plants the plastid genome of Acetabularia is larger in size and shows a high degree of variability. This study on the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase demonstrates that strongly conserved areas also exist in the plastid genome of the Dasycladaceae. Searching for differences in the amino acid sequence of the large subunit from Acetabularia mediterranea and Acicularia schenckii, proteolytic peptides which differ in their elution behaviour in reverse-phase high-performance liquid chromatography were sequenced. Only six amino acids were found to be exchanged in the large subunit from these two species. Since these two species diverged approx. 150 million years ago, these results imply that 0.84 amino-acid exchanges per 100 amino acids have occurred in 10⁸ years, underlining the strong conservatism of the large subunit.Abbreviations A Acetabularia mediterranea - Ac. Acicularia schenckii - HPLC high-performance liquid chromatography - LSU large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - PAGE polyacrylamide gel electrophoresis - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate     

Reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase efficiency by the presence of sucrose during the tissue culture of strawberry plantlets

Summary In order to identify the physiological and biochemical events leading to the negative effects of the presence of sucrose in culture medium on the photosynthetic capacity of plantlets cultivated in vitro, time course in photosynthesis, metabolite pool sizes, and ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) activity were investigated in strawberry (Fragaria x ananassa Duch. cv. Kent) plantlets following their transfer to medium with or without sucrose. When the plantlets grown in medium without sucrose were transferred to a similar medium with 30 g liter⁻¹ sucrose, their net photosynthesis decreased and their level of phosphorylated compounds increased with time. In addition, initial catalytic turnover, total catalytic turnover, and the activation state of ribulose-1,5-bisphosphate carboxylase decreased in these plantlets. Conversely, when the plantlets grown in medium with 30 g liter⁻¹ sucrose were transferred to a similar medium without sucrose, their net photosynthesis slowly increased with time and their level of phosphorylated compounds slowly decreased. A slow increase with time of initial catalytic turnover, total catalytic turnover, and the activation state of ribulose-1,5-bisphosphate carboxylase was also observed in these plantlets. The results of the present paper suggest that the reduced photosynthetic capacity of strawberry plantlets cultivated in vitro in the presence of sucrose is the consequence of a reduction in the efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase due to its deactivation and the possible presence of putative inhibitors of carboxylation sites.     

A point mutation in the N-terminus of ribulose-1,5-bisphosphate carboxylase affects ribulose-1,5-bisphosphate binding

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans was used to generate novel enzymes. Two conserved residues, threonine 4 and lysine 11 in the N-terminus were changed. The substitution of threonine 4 with serine or valine had little effect on the kinetic parameters. The substitution of lysine 11 with leucine, which is non-polar, increased the K _m for ribulose-1,5-bisphosphate from 82 to 190 M but its replacement with glutamine, which has polar properties, had no appreciable effect.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - LSU large sub-unit of Rubisco - SSU small subunit of Rubisco We thank Dr. S. Gutteridge (DuPont, Wilmington, USA) for structural information and for his comments on the results described. The technical assistance of Mr. A. Cowland and Mr. I. Major was invaluable.     

The drelationship between CO2-assimilation rate,Rubisco carbamylation and Rubisco activase content in activase-deficient transgenic tobacco suggests a simple model of activase action

Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) activase were used to examine the relationship between CO₂-assimilation rate, Rubisco carbamylation and activase content. Plants used were those members of the r₁ progeny of a primary transformant with two independent T-DNA inserts that could be grown without CO₂ supplementation. These plants had from < 1% to 20% of the activase content of control plants. Severe suppression of activase to amounts below 5% of those present in the controls was required before reductions in CO₂-assimilation rate and Rubisco carbamylation were observed, indicating that one activase tetramer is able to service as many as 200 Rubisco hexadecamers and maintain wild-type carbamylation levels in vivo. The reduction in CO₂-assimilation rate was correlated with the reduction in Rubisco carbamylation. The anti-activase plants had similar ribulose-1,5-bisphosphate pool sizes but reduced 3-phosphoglycerate pool sizes compared to those of control plants. Stomatal conductance was not affected by reduced activase content or CO₂-assimilation rate. A mathematical model of activase action is used to explain the observed hyperbolic dependence of Rubisco carbamylation on activase content.Abbreviations CA1P 2-carboxyarabinitol-1-phosphate - P_ip_a intercellular, ambient partial pressure of CO₂ - PGA 3-phospho-glycerate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SSU small subunit of Rubisco     

Solubilization of ribulose-1,5-bisphosphate carboxylase from the membrane fraction of pea leaves

The solubilization of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the membrane fraction was studied in whole leaf extracts and chloroplasts from pea. The amount of membrane-bound Rubisco was dependent on the pH of the chloroplastic lysate buffer. Maximum binding was found at pH 8.0, with about 8% of total leaf Rubisco being bound. The binding of Rubisco to the membranes was strong, and it was not released by repeated washing with hypotonic buffer or by changing ionic strength. Detergents such as Triton X-100, Tween 20, deoxycholate and dodecylsulfate were effective in solubilizing the membrane-bound Rubisco. Triton X-100 was most effective in the range of 0.04% to 0.2% and it solubilized Rubisco from the membrane without any decrease in enzyme activity.Abbreviations BSA bovine serum albumin - CABP carboxyarabinitol-1,5-bisphosphate - DTT dithiothreitol - LDS lithium dodecylsulfate - LHC light-harvesting chlorophyll protein complex - RuBP ribulose-1,5-bisphosphate - Rubisco RuBP carboxylase/oxygenase - SDS sodium dodecylsulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis