Ferredoxin Cross-Links to a 22 kD Subunit of Photosystem I

We have used a cross-linking approach to study the interaction of ferredoxin (Fd) with photosystem I (PSI). The cross-linking reagent N-ethyl-3-(3-dimethylaminopropyl) carbodiimide was found to cross-link spinach Fd to a 22 kilodalton subunit of PSI in both isolated spinach (Spinacia oleracea) PSI complexes and spinach thylakoid membranes. The product had an apparent molecular weight of 38 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified as a cross-linked product using specific antibodies to Fd and the 22 kilodalton subunit. In both a native PSI complex (200 Chl/P700) and a PSI core complex (100 Chl/P700), a second cross-linked product at 36 kilodaltons was seen. The latter cross-reacted with an antibody to Fd but did not cross-react with antibodies directed against the 24.3, 22, 19, 17.3 or 8.5 kilodalton, or psaC subunits of PSI. Its composition remains to be determined. In thylakoids only the 38 kilodalton product was observed along with a cross-linked complex of Fd and Fd:NADP⁺ reductase.     

Purification and Properties of Nonproteolytic Degraded ADPglucose Pyrophosphorylase from Maize Endosperm

ADPglucose pyrophosphorylase from developing endosperm tissue of starchy maize (Zea mays) was purified 88-fold to a specific activity of 34 micromoles α-glucose-1-P produced per minute per milligram protein. Rabbit antiserum to purified spinach leaf ADPglucose pyrophosphorylase was able to inhibit pyrophosphorolysis activity of the purified enzyme by up to 90%. The final preparation yielded four major protein staining bands following sodium dodecyl sulfate polyacrylamide gel electrophoresis. When analyzed by Western blot hybridization only the fastest migrating, 54 kilodaltons, protein staining band cross-reacted with affinity purified rabbit antispinach leaf ADPglucose pyrophosphorylase immunoglobulin. The molecular mass of the native enzyme was estimated to be 230 kilodaltons. Thus, maize endosperm ADPglucose pyrophosphorylase appears to be comprised of four subunits. This is in contrast to the respective subunit and native molecular masses of 96 and 400 kilodaltons reported for a preparation of maize endosperm ADPglucose pyrophosphorylase (Fuchs RL and JO Smith 1979 Biochim Biophys Acta 556: 40-48). Proteolytic degradation of maize endosperm ADPglucose pyrophosphorylase appears to occur during incubation of crude extracts at 30°C or during the partial purification of the enzyme according to a previously reported procedure (DB Dickinson, J Preiss 1969 Arch Biochem Biophys 130: 119-128). The progressive appearance of a 53 kilodalton antigenic peptide suggested the loss of a 1 kilodalton proteolytic fragment from the 54 kilodalton subunit. The complete conservation of the 54 kilodalton subunit structure following extraction of the enzyme in the presence of phenylmethylsulfonyl fluoride and/or chymostain was observed. The allosteric and catalytic properties of the partially purified proteolytic degraded versus nondegraded enzyme were compared. The major effect of proteolysis was to enhance enzyme activity in the absence of added activator while greatly decreasing its sensitivity to the allosteric effectors 3-P-glycerate and inorganic phosphate.     

Polymorphism of a photosystem I subunit caused by alloploidy in Nicotiana

The photosystem I complex from Nicotiana tabacum, which has an alloploid genome, contains subunits of 17.5 and 18.5 kilodaltons whose N-terminal amino acid sequences are highly homologous. Comparative analysis of photosystem I subunits among N. tabacum and its ancestral plants, N. tomentosiformis and N. sylvestris, revealed that the 17.5 kilodalton subunit of N. tabacum derives from N. sylvestris, and the 18.5 kilodalton subunit from N. tomentosiformis.     

Chlamydomonas reinhardtii Phosphoribulokinase : Sequence, Purification, and Kinetics

The sequence and kinetic properties of phosphoribulokinase purified from Chlamydomonas reinhardtii were determined and compared with the spinach (Spinacea oleracea) enzyme. Chlamydomonas phosphoribulokinase was purified to apparent homogeneity, with a specific activity of 410 micromoles per minute per milligram. Polyclonal antibodies to the purified protein were used to isolate a Chlamydomonas cDNA clone, which, upon sequencing, was found to contain the entire coding region. The transit peptide cleavage site was determined by Edman analysis of the mature protein. The precursor protein consists of a 31 amino acid transit peptide and a 344 amino acid mature polypeptide. The mature polypeptide has a calculated molecular weight of 38.5 kilodaltons and a pl of 5.75. The V_max of the purified enzyme was 465 micromoles per minute per milligram, with apparent K_m values of 62 micromolar ATP and 56 micromolar ribulose 5-phosphate. Immunoblot analysis indicated antigenic similarity and a similar subunit size for the enzyme from five higher plant species and Chlamydomonas. Southern blot analysis of Chlamydomonas genomic DNA indicated the presence of a single phosphoribulokinase gene. Comparison of the mature proteins from Chlamydomonas and spinach revealed 86 amino acid differences in primary structure (25% of the total) without a major difference in kinetic properties. The transit peptides of the spinach and Chlamydomonas proteins possessed little sequence homology.     

Organelle-bound malate dehydrogenase isoenzymes are synthesized as higher molecular weight precursors

Biosynthesis of malate dehydrogenase isoenzymes was studied in cotyledons of watermelons (Citrullus vulgaris Schrad., var. Stone Mountain). The glyoxysomal and mitochondrial isoenzymes are synthesized as higher molecular weight precursors which can be immunoprecipitated by mono-specific antibodies from the products of in vitro translation in reticulocyte lysates programed with cotyledonary mRNA and with the same size from enzyme extracts of pulse-labeled cotyledons. During translocation from the cytosol into the organelles, processing takes place. An 8 kilodalton extra sequence is cleaved from the glyoxysomal precursor and a 3.3 kilodalton extra sequence from the mitochondrial precursor producing the native subunits of 33 and 38 kilodaltons, respectively. The data support a post-translational translocation of the organelle-destined malate dehydrogenase isoenzymes. The in vitro translation of the cytosolic malate dehydrogenase I yields a product which has the same molecular weight as the subunit of the native isoenzyme (39.5 kilodaltons).     

Pea chloroplast glutathione reductase: purification and characterization

Glutathione reductase (EC 1.6.4.2) was purified from intact pea (Pisum sativum) chloroplasts by a method which includes affinity chromatography on ADP-agarose. Fractions from the affinity column which had glutathione reductase activity consisted of polypeptides of 60 and 32 kilodaltons. Separation of the proteins by electrophoresis on native gels showed that glutathione reductase activity was associated with 60 kilodalton polypeptides and not with the 32 kilodalton polypeptides. Antibodies to spinach whole leaf glutathione reductase (60 kilodaltons) cross-react with the chloroplast 60 kilodalton glutathione reductase but not the 32 kilodalton polypeptides. In the absence of dithiothreitol the 60 kilodalton polypeptides showed a shift in apparent molecular weight on sodium dodecyl sulfate gels to 72 kilodaltons. Dithiothreitol did not alter the activity of the chloroplast enzyme. Chloroplast glutathione reductase is relatively insensitive to NADPH.     

Purification and preliminary characterization of sucrose-phosphate synthase using monoclonal antibodies

Monoclonal antibodies specific for sucrose phosphate synthase (SPS; EC 2.4.1.14) have been obtained for the first time. Three independent clones have been isolated which inhibited spinach (Spinacia oleracea L.) leaf SPS activity and facilitated the enzyme purification by immunoprecipitation. All three clones were specific for the spinach enzyme but neither inhibited nor precipitated the SPS present in tissue extracts of maize (Zea mays L.), barley (Hordeum vulgare L.), soybean (Glycine max L.), and sugar beet (Beta vulgaris L.). The inhibition of SPS activity by all three clones was reversible in the presence of UDPG, suggesting the presence of an epitope at the substrate-binding site. Immunoprecipitates of active enzyme preparations consistently revealed the presence of a 120 kilodalton polypeptide, indicating that the enzyme may be a homotetramer with a native molecular weight of about 480 kilodaltons. The occasional appearance of a 52 kilodalton polypeptide in the immunoprecipitates of some enzyme preparations was not the result of proteolysis, was not necessary for enzyme activity, and did not contain an antigenic site as revealed by Western blotting experiments. All three antibodies bind weakly to the SDS denatured 120 kilodalton subunit bound to nitrocellulose. The specific activity of the purified spinach enzyme was determined for the first time to be approximately 150 units per milligram SPS protein (pH 7.5 and 25°C) based on quantitative immunoprecipitation of the enzyme.     

Analysis of Chromophytic and Rhodophytic Ribulose-1,5-Bisphosphate Carboxylase Indicates Extensive Structural and Functional Similarities among Evolutionarily Diverse Algae

Ribulose-1,5-bisphosphate carboxylase (Rubisco) from the algae Olisthodiscus luteus (chromophyte) and Griffithsia pacifica (rhodophyte) are remarkably similar to each other. However, both enzymes differ significantly in the structure and function when compared to Rubisco from green algae and land plants. Analysis of purified Rubisco from O. luteus and G. pacifica indicates that the size of the holoenzyme and stoichiometry of the 55 and 15 kilodalton subunit polypeptides are approximately 550 kilodaltons and eight:eight for both algae. Antigenic determinants are highly conserved between the O. luteus and G. pacifica enzymes and differ from those of the spinach subunit polypeptides. Sequence similarity between the two algal large subunits has been further confirmed by one-dimensional peptide mapping. Substrate ribulose bisphosphate has no effect on the rate of CO₂/Mg²⁺ activation of O. luteus and G. pacifica enzymes which contrasts to the extensive inhibition of spinach Rubisco activation at similar concentrations of this compound. In addition, the Michaelis constant for CO₂ and the inhibition constant for 6-phosphogluconate are similar for the O. luteus and G. pacifica catalyzed carboxylation reaction. Both values are intermediate to those observed for the tight binding spinach enzyme and weak binding prokaryotic (Rhodospirillum rubrum) enzyme. The biochemical similarities documented between O. luteus and G. pacifica may be due to a common evolutionary origin on the chromophytic and rhodophytic chloroplast but could also result from the fact that both subunit polypeptides are chloroplast DNA encoded in these algal taxa.     

The Subunit Structure of Potato Tuber ADPglucose Pyrophosphorylase

ADPglucose pyrophosphorylase has been extensively purified from potato (Solanum tuberosum L.) tuber tissue to study its structure. By employing a modified published procedure (JR Sowokinos, J Preiss [1982] Plant Physiol 69: 1459-1466) together with Mono Q chromatography, a near homogeneous enzyme preparation was obtained with substantial improvement in enzyme yield and specific activity. In single dimensional sodium dodecyl sulfate polyacrylamide gels, the enzyme migrated as a single polypeptide band with a mobility of about 50,000 daltons. Analysis by two-dimensional polyacrylamide gel electrophoresis, however, revealed the presence of two types of subunits which could be distinguished by their slight differences in net charge and molecular weight. The smaller potato tuber subunit was recognized by antiserum prepared against the smaller spinach leaf 51 kilodalton ADPglucose pyrophosphorylase subunit. In contrast, the anti-54 kilodalton raised against the spinach leaf subunit did not significantly react to the tuber enzyme subunits. The results are consistent with the hypothesis that the potato tuber ADPglucose pyrophosphorylase is not composed of a simple homotetramer as previously suggested, but is a product of two separate and distinct subunits as observed for the spinach leaf and maize enzymes.     

Isolation, Purification, and Subcellular Localization of Isozymes of Superoxide Dismutase from Scots Pine (Pinus sylvestris L.) Needles

Two of four isozymes of superoxide dismutase (SOD) (EC 1.15.1.1) were purified from Scots pine (Pinus sylvestris L.) needles. One form was cytosolic (SOD-1) and the other was associated with chloroplasts (SOD-3). The holoenzyme molecular masses was estimated at approximately 35 kilodaltons by gel filtration. The subunit molecular weight of the dimeric enzymes was estimated to 16.5 kilodaltons (SOD-1) and 20.4 kilodaltons (SOD-3) on sodium dodecyl sulfatepolyacrylamide gels. The NH₂-terminal sequence of the pine enzymes showed similarities to other purified superoxide dismutases located in the corresponding compartment. The cytosolic form revealed two additional amino acids at position 1 and 2 at the NH₂-terminal. Both forms were cyanide- and hydrogenperoxide-sensitive and SOD-3 was found to contain approximately one copper atom per subunit, indicating that they belong to the cupro-zinc SODs. The isoelectric point was 4.9 and 4.5 for SOD-1 and SOD-3, respectively.     

Bovine liver monoamine oxidase: A modified purification procedure and preliminary evidence for two subunits and one FAD

Bovine liver mitochondrial monoamine oxidase was isolated in a more active state and in higher yields by an improved purification method which utilized β-mercaptoethanol and which contained several other important modifications. The subunit structure of the purified enzyme components was investigated by chemical and enzymatic methods. The subunit molecular weight of the three enzyme components isolated was estimated to be 52,000 by sodium dodecyl sulfate disc electrophoresis and by exclusion-diffusion chromatography on Biogel A-5m with 6 m guanidine HCl as the solvent. The number of peptides observed in the peptide map of the tryptic digest of the S-β-carboxymethylcysteine derivative of the enzyme also showed that the subunit molecular weight was about 52,000. Since it was previously reported that the monomer molecular weight of the enzyme was about 110,000, the active enzyme is made up of two subunits. The NH₂-terminus of the enzyme of both subunits is blocked since Edman degradation and aminopeptidase failed to release an NH₂-terminal amino acid. The COOH-terminal amino acid of both subunits was shown to be leucine by carboxypeptidase digestion of the enzyme since it was liberated quantitatively. From the FAD content of the enzyme and the subunit data, it is proposed that the enzyme probably consists of two subunits which differ possibly in that only one subunit contains 8-α-cysteinyl FAD.     

Regulatory and Structural Properties of the Cyanobacterial ADPglucose Pyrophosphorylases

ADPglucose pyrophosphorylase (EC 2.7.7.27) has been purified from two cyanobacteria: the filamentous, heterocystic, Anabaena PCC 7120 and the unicellular Synechocystis PCC 6803. The purification procedure gave highly purified enzymes from both cynobacteria with specific activities of 134 (Synechocystis) and 111 (Anabaena) units per milligram protein. The purified enzymes migrated as a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular mass corresponding to 53 (Synechocystis) and 50 (Anabaena) kilodaltons. Tetrameric structures were determined for the native enzymes by analysis of gel filtrations. Kinetic and regulatory properties were characterized for the cyanobacterial ADPglucose pyrophosphorylases. Inorganic phosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. The Synechocystis enzyme was activated 126-fold by 3-phosphoglycerate, with saturation curves exhibiting sigmoidicity (A_0.5 = 0.81 millimolar; n_H = 2.0). Activation by 3-phosphoglycerate of the enzyme from Anabaena demonstrated hyperbolic kinetics (A_0.5 = 0.12 millimolar; n_H = 1.0), having a maximal stimulation of 17-fold. I_0.5 values of 95 and 44 micromolar were calculated for the inhibition by inorganic phosphate of the Synechocystis and Anabaena enzyme, respectively. Pyridoxal-phosphate behaved as an activator of the cyanobacterial enzyme. It activated the enzyme from Synechocystis nearly 10-fold with high apparent affinity (A_0.5 = 10 micromolar; n_H = 1.8). Phenylglyoxal modified the cyanobacterial enzyme by inactivating the activity in the presence of 3-phosphoglycerate. Antibody neutralization experiments showed that anti-spinach leaf (but not anti-Escherichia coli) ADPglucose pyrophosphorylase serum inactivated the enzyme from cyanobacteria. When the cyanobacterial enzymes were resolved on sodium dodecyl sulfate- and two-dimensional polyacrylamide gel electrophoresis and probed with Western blots, only one protein band was recognized by the anti-spinach leaf serum. The same polypeptide strongly reacted with antiserum prepared against the smaller spinach leaf 51 kilodalton subunit, whereas the anti-54 kilodalton antibody raised against the spinach subunit reacted weakly to the cyanobacterial subunit. Regulatory and immunological properties of the cyanobacterial enzyme are more related to the higher plant than the bacterial enzyme. Despite this, results suggest that the ADPglucose pyrophosphorylase from cyanobacteria is homotetrameric in structure, in contrast to the reported heterotetrameric structures of the higher plant ADPglucose pyrophosphorylase.     

Fructose 2,6-bisphosphate, carbohydrate partitioning, and crassulacean Acid metabolism

To initiate structural studies of the ADPglucose pyrophosphorylase from spinach an improved purification procedure was devised. The modified purification scheme allowed the isolation of 20 to 30 milligrams pure enzyme from 10 kilogram of spinach leaves. Electrophoresis of the purified enzyme confirmed an earlier study which showed that the enzyme was putatively composed of two subunits (Copeland L, J Preiss 1981 Plant Physiol 68: 996-1001). The two subunits migrate as 51 and 54 kilodalton proteins upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both proteins can be detected on Western blots of leaf homogenates prepared under denaturing conditions suggesting that both subunits exist in vivo. Anion-exchange chromatography in the presence of urea allowed resolution of the 51 and 54 kilodalton proteins. They possess different N-terminal amino acid sequences and tryptic peptide maps. Western blot analysis reveals that the 51 and 54 kilodalton proteins are antigenically dissimilar. The 51 but not the 54 kilodalton protein is immunologically related to the ADPglucose pyrophosphorylase from maize endosperm and potato tuber.     

Active Glucose Transport and Proton Pumping in Tonoplast Membrane of Zea mays L. Coleoptiles Are Inhibited by Anti-H-ATPase Antibodies

A tonoplast enriched fraction was obtained from Zea mays L. coleoptiles by isopycnic centrifugation of microsomal membranes in a sucrose step gradient. At the 18/26% interface chloride-stimulated and nitrate-inhibited proton pumping activity coincided with a Mg²⁺-ATP dependent accumulation of 3-O-methyl-d-glucose (OMG) as determined by a membrane filtration technique using ¹⁴C-labeled substrate. OMG transport showed an apparently saturable component with a K_m of 110 micromolar, and was completely inhibited by 10 micromolar carbonyl cyanide m-chlorophenylhydrazone. Polyclonal antibodies against solubilized native tonoplast H⁺-ATPase and its 62 and 72 kilodalton subunits were assayed for their ability to inhibit proton pumping and OMG accumulation. Antibodies against both the native enzyme and the putative catalytic subunit (72 kilodalton) strongly inhibited proton pumping and OMG transport whereas antibodies against the 62 kilodalton subunit had only a slight effect on both processes.     

The aggregation states of spinach phosphoribulokinase

Phosphoribulokinase (PRK; EC 2.1.7.19) is active in illuminated chloroplasts and inactive in darkened chloroplasts. This regulatory mechanism is mediated by thioredoxin-dependent reduction of a kinase disulfide in vivo. Extracts of spinach (Spinacia oleracea L.) leaves in the presence of 10 mM dithiothreitol contain a single 80-kDa form of PRK as judged by gel filtration. Gel filtration of thiol-free extracts of light-harvested tissue shows the presence of two inactive forms of PRK, the 80-kDa form and an aggregate (> 550 kDa) form, but treatment of both forms with dithiothreitol restores kinase activity. Gel filtration following extraction of dark-harvested tissue in the absence of dithiotreitol demonstrates the presence of only the heavier form. Inclusion of 400 mM (NH₄)₂SO₄ in the homogenization buffer during extraction of light-harvested tissue suppresses the formation of the high-M _r form of PRK, but does not eliminate the aggregate form observed in extracts of dark-harvested leaves. However, prolonged treatment of extracts from dark-harvested tissue with 400 mM (NH₄)₂SO₄ results in conversion of the high-M _r form of phosphoribulokinase to the low-M _r form. The data are consistent with the heavier form of phosphoribulokinase being the normal in-vivo aggregation state in the dark, while the lighter form is the normal aggregation state in the light.This research was sponsored jointly by the science and education administration of the U.S. Department of Agriculture under Grant No. 88-37130-3722 from the Competitive Research Grants Office and by the Office of Health and Environmental Research, U.S. Department of Energy under Contract DE-AC05-84OR21400 with Martin Marietta Energy Systems Inc., Oak Ridge, Tenn., USA. The author is Postdoctoral Investigator supported by the U.S. Department of Agriculture through Subcontract No. 88-37130-3722 from the Biology Division of Oak Ridge National Laboratory to the University of Tennessee.     

Chromosomal and plasmid locations for phosphoribulokinase genes in Alcaligenes eutrophus.

Genes coding for phosphoribulokinase (PRK), a key enzyme of the Calvin cycle, were localized in the genome of the chemoautotroph Alcaligenes eutrophus. The NH2-terminal sequence of the PRK subunit was determined. With a synthetic oligodeoxynucleotide probe complementary to a portion of this sequence, hybridization analysis revealed PRK genes to be located on both the chromosome and the megaplasmid pHG1 of A. eutrophus H16.     

Subunit Composition and Organization of the Vacuolar H-ATPase from Oat Roots

The vacuolar H⁺-translocating ATPase (H⁺-ATPase), originally reported to consist of three major subunits, has been further purified from oat roots (Avena sativa var Lang) to determine the complete subunit composition. Triton-solubilized ATPase activity was purified by gel filtration on Sephacryl S400 and ion-exchange chromatography (Q-Sepharose). ATP hydrolysis activity of purified preparations was inhibited by 100 nanomolar bafilomycin A₁, a specific vacuolar-type ATPase inhibitor. The purified oat H⁺-ATPase (relative molecular weight = 650,000) was composed of polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. To analyze the organization of the H⁺-ATPase subunits, native vacuolar membranes were treated with KI and MgATP to dissociate peripheral proteins. Release of 70, 60, 44, 42, 36, and 29 kilodalton polypeptides from the membrane was accompanied by a loss of ATP hydrolysis and ATP-dependent H⁺-pumping activities. Five of the peripheral subunits were released from the membrane as a large complex of 540 kilodaltons. Vesicles that had lost the peripheral sector of the ATPase could hold a pH gradient generated by the proton-translocating pyrophosphatase, suggesting that the integral sector of the ATPase did not form a H⁺-conducting pathway. Negative staining of native vesicles revealed knob-like structures of 10 to 12 nanometers in dense patches on the surface of vacuolar membranes. These structures were removed by MgATP and KI, which suggested that they were the peripheral sectors of the H⁺-ATPase. These results demonstrate that the vacuolar H⁺-ATPase from oat roots has 10 different subunits. The oat vacuolar ATPase is organized as a large peripheral sector and an integral sector with a subunit composition similar, although not identical to, other eukaryotic vacuolar ATPases. Variations in subunit composition observed among several ATPases support the idea that distinct types of vacuolar H⁺-ATPases exist in plants.     

NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase

Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b₅ reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b₅ reductase).     

Isolation and characterization of wheat ribulose-1,5-diphosphate carboxylase

Wheat ribulose-1,5-diphosphate carboxylase purified to homogeneity had a MW of 540 000, sedimentation coefficient (S_{20, W}) of 18.5 S, apparent diffusion constant (D_app) of 3.07 × 10^?7 cm²/sec, Stoke's radius 5.44 nm, and fractional ratio of 1.17. Electron microscopy revealed particles of 10–12 nm diameter. The enzyme was dissociated by sodium dodecyl sulphate into two subunits of MW 53 000 (S_{20, W} = 3.0 S) and 13 500 (S_{20, W} = 1.7 S). The total amino acid residues in the large and small subunits were 481 and 117, respectively. Tryptic peptide maps of the two subunits confirmed the estimated numbers of Arg and Lys residues. Although the amino acid pattern of the large subunit closely resembled that from barley, rather than that for spinach, beet or tobacco, the pattern of the small subunit was markedly different from those of all the other species.