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C Schnarrenberger

Publications and source records attributed to C Schnarrenberger.

At least 37 records · Page 2Linked to original sources
PubMed1993

Plant aldolase: cDNA and deduced amino-acid sequences of the chloroplast and cytosol enzyme from spinach.

We report the sequences of full-length cDNAs for the nuclear genes encoding the chloroplastic and cytosolic fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) from spinach. A comparison of the deduced amino-acid sequences with one another and with published cytosolic aldolase sequences of other plants revealed that the two enzymes from spinach share only 54% homology on their amino acid level whereas the homology of the cytosolic enzyme of spinach with the known sequences of cytosolic aldolases of maize, rice and Arabidopsis range from 67 to 92%. The sequence of the chloroplastic enzyme includes a stroma-targeting N-terminal transit peptide of 46 amino acid residues for import into the chloroplast. The transit peptide exhibits essential features similar to other chloroplast transit peptides. Southern blot analysis implies that both spinach enzymes are encoded by single genes.

Amino Acid Sequence↗
PubMed1992

Repression of the Plastidic Isoenzymes of Aldolase, 3-Phosphoglycerate Kinase, and Triosephosphate Isomerase in the Barley Mutant "albostrians".

White leaves of the mutant line albostrians and green leaves of the wild-type cultivar Salome of barley (Hordeum vulgare L.) were screened for the presence of plastidic and cytosolic isoenzymes of sugar-phosphate metabolism. Isoenzyme separation was achieved by anion-exchange chromatography on Fractogel TSK DEAE-650(S). The mutant tissue had a markedly reduced level of plastidic 3-phosphoglycerate kinase, triosephosphate isomerase, and aldolase activity. In contrast, the activity of plastidic glucosephosphate isomerase, fructose 1,6-bisphosphatase, 6-phosphogluconate dehydrogenase, starch phosphorylase, and ADP-glucose pyrophosphorylase was in the same range as in wild-type leaf tissue. The activity of the corresponding cytosolic isoenzymes (including UDP-glucose pyrophosphorylase) showed essentially no differences in mutant and wild type. The same trend was observed in dark-grown mutant and wild-type leaves. Interestingly, the total activity levels of all isoenzymes were about the same when comparing dark-grown and light-grown mutant or wild-type plants. From these data, it is concluded that mutant leaves exhibit a selective decrease of a subgroup of plastidic isoenzymes associated with the Calvin cycle.

Journal Article↗
PubMed1990

Purification by Immunoadsorption and Immunochemical Properties of NADP-Dependent Malic Enzymes from Leaves of C(3), C(4), and Crassulacean Acid Metabolism Plants.

NADP:malic enzyme from corn (Zea mays L.) leaves was purified by conventional techniques to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies raised against this protein in rabbits were purified, coupled covalently to protein A-Sepharose CL-4B, and used as an immunoaffinity resin to purify the NADP:malic enzymes of the C(3) plants spinach (Spinacia oleracea L.) and wheat (Triticum aestivum L.), of the Crassulacean acid metabolism (CAM) plant Bryophyllum daigremontianum R. Hamed et Perr. de la Bathie and the C(4) plants corn, sugarcane (Saccharum officinarum L.), and Portulaca grandiflora L. Such procedures yielded homogeneous protein preparations with a single protein band, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, except for P. grandiflora L. with two bands. The specific activities of the purified proteins ranged between 56 and 91 units (milligrams per protein). NADP:malic enzyme represented up to 1% of the total soluble protein in C(4) plants, 0.5% in the CAM plant, and less than 0.01% in C(3) plants. In immunotitration tests involving immunoprecipitation and immunoinhibition of activity by an antiserum against the corn leaf enzyme, the NADP:malic enzymes of corn and sugarcane showed virtually full identity of epitopes, while the NADP:malic enzymes of the C(3) and CAM plants exhibited a cross-reaction of one-twentieth and one-fourth by these tests, respectively. The NADP:malic enzyme of P. grandiflora exhibited characteristics more closely related to the enzymes of C(3) and CAM plants than to those of C(4) plants.

Journal Article↗
PubMed1990

Isolation and characterization of the cytosolic and chloroplastic 3-phosphoglycerate kinase from spinach leaves.

The cytosol and chloroplast 3-phosphoglycerate kinases (3-PGK) from spinach (Spinacia oleracea L.) were purifled to apparent homogeneity. The procedure included a conventional anion-exchange chromatography on DEAE-cellulose and mainly a series of HPLC columns. The charge differences of the two isoenzymes were so small that separation was only successful by anion-exchange chromatography on a HPLC SynChropak AX 300 column. The portion of the two isoenzmyes in leaf tissue was estimated as 5% and 95%. The major 3-PGK was associated with isolated chloroplasts while the other 3-PGK was only found in the soluble cell fraction. The specific activity of the purified enzymes were in the order of 800 units (per milligram of protein). The molecular weight for the two 3-PGKs under nondenaturing (size exclusion chromatography) and denaturing (SDS-PAGE) conditions were in the order of 40 kilodaltons, with the cytosolic 3-PGK being slightly smaller than the chloroplastic 3-PGK. An antiserum against the chloroplastic 3-PGK showed only 4.6% cross-reaction of the chloroplastic 3-PGK with the cytosolic 3-PGK. The kinetics for glycerate-3-phosphate and MgATP(2-) were biphasic. The presence of Na(2)SO(4) changed the MgATP(2-) dependence to linearity but not the glycerate-3-phosphate dependence.

Journal Article↗
PubMed1988

Purification and Properties of Mesophyll and Bundle Sheath Cell alpha-Glucan Phosphorylases from Zea mays L. : Equivalence of the Enzymes with the Cytosol and Plastid Phosphorylases from Spinach.

Two major alpha-glucan phosphorylases (I and II) from leaves of the C(4) plant corn (Zea mays L.) were previously shown to be compartmented in mesophyll and bundle sheath cells, respectively (C Mateyka, C Schnarrenberger 1984 Plant Sci Lett 36: 119-123). The two enzymes were separated by chromatography on DEAE-cellulose and purified to homogeneity by affinity chromatography on immobilized starch, according to published procedures, as developed for the cytosol and chloroplast phosphorylase from the C(3) plant spinach. The two alpha-glucan phosphorylases have their pH optimum at pH 7. The specificity for polyglucans was similar for soluble starch and amylopectin, however, differed for glycogen (K(m) = 16 micrograms per milliliter for the mesophyll cell and 250 micrograms per milliliter for the bundle sheath cell phosphorylase). Maltose, maltotriose, and maltotetraose were not cleaved by either phosphorylase. If maltopentaose was used as substrate, the rate was about twice as high with the bundle sheath cell phosphorylase, than with the mesophyll cell phosphorylase. The phosphorylase I showed a molecular mass of 174 kilodaltons and the phosphorylase II of 195 kilodaltons for the native enzyme and of 87 and of 53 kilodaltons for the SDS-treated proteins, respectively. Specific antisera raised against mesophyll cell phosphorylase from corn leaves and against chloroplast phosphorylase from spinach leaves implied high similarity for the cytosol phosphorylase of the C(3) plant spinach with mesophyll cell phosphorylase of the C(4) plant corn and of chloroplast phosphorylase of spinach with the bundle sheath cell phosphorylase of corn.

Journal Article↗
PubMed1988

Two Class I Aldolases in the Green Alga Chara foetida (Charophyceae).

Aldolase activity of Chara foetida (Braun) could be separated into a minor (peak I) and a major peak (peak II) by ion-exchange chromatography on DEAE-cellulose. Affinity chromatography on P-cellulose resulted in highly purified aldolase preparations with specific activities of 3.2 and 4.8 units per milligram protein and molecular subunit masses of 37 and 35 kilodalton, as shown by SDS-PAGE, for the aldolase of peak I and peak II, respectively. Both aldolases belong to class I aldolase since the activity is not inhibited by 1 millimolar EDTA. The K(m) (fructose-1,6-bisphosphate) values were 0.64 and 13.4 micromolar, respectively. The aldolase of peak I showed a 6.7 times stronger crossreaction with a specific antiserum against the cytosol aldolase of spinach than with an antiserum against the chloroplast aldolase of spinach. On the other hand the aldolase of peak II showed a 5.1 times stronger cross-reaction with the alpha-plastidaldolase antiserum than with the alpha-cytosol-aldolase antiserum. For algae this is the first separation of two class I aldolases. They are similar to the cytosol and chloroplast aldolases in higher plants, but different from a reported class I (Me(2+) independent) and class II (Me(2+) dependent) aldolase in other algae.

Journal Article↗
PubMed1986

Distinction between Cytosol and Chloroplast Fructose-Bisphosphate Aldolases from Pea, Wheat, and Corn Leaves.

A reinvestigation of cytosol and chloroplast fructose-1,6-bisphosphate (FBP) aldolases from pea (Pisum sativum L.), wheat (Triticum aestivum L.) and corn leaves (Zea mays L.) revealed that the two isoenzymes can be separated by chromatography on diethylaminoethyl (DEAE)-cellulose although the separation was often less clear-cut than for the two aldolases from spinach leaves. Definite distinction was achieved by immunoprecipitation of the two isoenzymes with antisera raised against the respective isoenzymes from spinach leaves. The proportion of cytosol aldolase as part of total aldolase activity was 8, 9, 14, and 4.5% in spinach (Spinacia oleracea L.), pea, wheat, and corn leaves, respectively. For corn leaves we also obtained values of up to 15%. The K(m) (FBP) values were about 5-fold lower for the cytosol (1.1-2.3 micromolar concentration) than for the chloroplast enzymes (8.0-10.5 micromolar concentration). The respective K(m) (fructose-1-phosphate, F1P) values were about equal for the cytosol (1.0-2.3 millimolar concentration) and for the chloroplast aldolase (0.6-1.7 millimolar concentration). The ratio V (FIP)/V (FBP) was 0.20 to 0.27 for the cytosol and 0.07 to 0.145 for the chloroplast aldolase. Thus, cytosol and chloroplast aldolases from spinach, pea, wheat, and corn leaves differ quite considerably in the elution pattern from DEAE-cellulose, in immunoprecipitability with antisera against the respective isoenzymes from spinach leaves, and in the affinity to FBP.

Journal Article↗
PubMed1983

Purification, subunit structure and immunological comparison of fructose-bisphosphate aldolases from spinach and corn leaves.

The cytosol and chloroplast fructose-bisphosphate aldolases from spinach leaves were separated by ion-exchange chromatography on DEAE-cellulose, and were purified by subsequent affinity chromatography on phosphocellulose to apparent homogeneity as judged from polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two aldolases had specific activities of 7.2 and 7.8 units mg protein-1. Molecular weight determinations by electrophoresis in sodium dodecyl sulfate gels and by sedimentation velocity centrifugation in sucrose gradients showed that the aldolases contained four subunits of Mr 38 000 and 35 000, respectively. Antibodies against the cytosol and chloroplast aldolase from spinach leaves were raised in a guinea pig and in a rabbit, respectively. In the Ouchterlony double-diffusion test, the two aldolases did not cross-react. A small degree of cross-reaction was observed by a test in which immune complexes were adsorbed to a solid-phase support (Staphylococcus aureus Cowan I cells) and nonbound enzyme activity was determined after centrifugation. These results imply major structural differences between the two spinach leaf aldolases. Only one major aldolase could be resolved on DEAE-cellulose from corn leaves. The aldolase was purified and had a specific activity of 6.4 units X mg protein-1. The corn leaf aldolase cross-reacted with the antiserum raised against the chloroplast enzyme from spinach leaves, but not with the other antiserum. Thus, the corn leaf aldolase could be identified as a chloroplast enzyme. Since aldolase activity is mostly restricted to the bundle sheath cells of corn leaf, it was concluded that it is compartmentalized in the chloroplasts of these cells but not in chloroplasts of the mesophyll cells.

Centrifugation, Density Gradient↗
PubMed1983

Intracellular compartmentation of isozymes of sugar phosphate metabolism in green leaves.

The present paper has summarized evidence for the presence of two isozymes for many enzyme activities of sugar phosphate metabolism in plant leaves. These two isozymes are clearly compartmentalized in the chloroplasts and in the cytosol of plant leaf cells. In C4 plants there exists an additional isozyme in the mesophyll cells of these leaves in addition to the two isozymes in the bundle sheath cells. Such cell-compartment-specific and cell-specific isozymes provide duplicate (and possibly triplicate) enzyme systems for complete or almost complete pathways (ie, glycolysis, gluconeogenesis, and the oxidative pentose phosphate pathway). They provide a basis for the understanding as to how many isozymes one may expect in plants. They also provide a challenge to determine what their function is particularly in the differential regulation of metabolic pathways in different cell compartments. Based on his genetic analyses Weeden [1981] has recently proposed a model for the evolution of chloroplast-specific isozymes of sugar phosphate metabolism. This model rests on the endosymbiotic theory for the origin of chloroplasts. It still is highly speculative. However, cell-compartment specific isozymes may eventually provide a means of studying plant evolution, especially if we succeed in analyzing their primary structure.

Antigen-Antibody Complex↗
PubMed1976

Lectins as membrane components of mitochondria from Ricinus communis.

1. Mitochondria were isolated from developing endosperm of Ricinus communis and were fractionated into outer membrane and inner membrane. The relative purity of the two membrane fractions was determined by marker enzymes. The fractions were also examined by negative-stain electron microscopy. 2. Membrane fractions were sequentially extracted in the following way. (a) Suspension in 0.5M-potassium phosphate, pH7.1; (b)suspension in 0.1M-EDTA (disodium salt)/0.05M-potassium phosphate, pH7.1; (c) sonication in 0.05M-potassium phosphate, pH7.1;(d)sonication in aq. Triton X-100 (0.1%). The membranes were pelleted by centrifugation at 100 000g for 15 min, between each step. Agglutination activity in the extracts was investigated by using trypsin-treated rabbit erythrocytes. 3. The addition of lactose to inner mitochondrial membrane resulted in the solubilization of part of the lectin activity, indicating that the protein was attached to the membrane via its carbohydrate-binding site. Pretreatment of the membranes with lactose before tha usual extraction procedure showed that lactose could extract lectins that normally required more harsh treatment of the membrane for solubilization. 4. Lectins extracted from inner membranes were purified by affinity chromatography on agarose gel. Polyacrylamide-gel electrophoresis of purified samples in sodium dodecyl sulphate indicated that at least part of the lectin present in inner mitochondrial membrane was identical with the R. communis agglutinin of mol.wt. 120 000.

Agglutination Tests↗
PubMed1975

Development and intracellular distribution of enzymes of the oxidative pentose phosphate cycle in radish cotyledons.

Developmental and compartmentation studies were used to evaluate the relative roles of the oxidative pentose phosphate cycle, the Calvin cycle, and the glycolysis in cotyledons of radish (Raphanus sativus L.).Glucose-6-P dehydrogenase, 6-P-gluconate dehydrogenase, glucose-6-P isomerase, and the NAD-dependent glyceraldehyde-3-P dehydrogenase were present in high activity in ungerminated seeds, increased about 2-fold during germination in the dark, and were slightly enhanced by light. In contrast, NADP-dependent glyceraldehyde-3-P dehydrogenase was developed to only a small degree in the dark, but increased severalfold in continuous white or far red light. The activity of phosphofructokinase was low throughout germination.The separation of cell compartment-specific isoenzymes showed that, except in ungerminated seeds, the plastid enzyme accounted for 40 to 45% of the total activity of 6-P-gluconate dehydrogenase and for 15 to 20% of glucose-6-P isomerase. The remaining activity was due to the cytosolic isoenzymes. The presence of glucose-6-P dehydrogenase and 6-P-gluconate dehydrogenase in plastids was also established by their presence in the isolated organelle. The NAD-dependent glyceraldehyde-3-P dehydrogenase was mostly due to the cytosolic isoenzyme, whereas the NAD-dependent activity associated with the NADP-dependent glyceraldehyde-3-P dehydrogenase was very small.The data indicate that the enzymes of the oxidative pentose phosphate cycle are present in the cytosol throughout germination. In the plastids these enzymes already became fully developed during early germination in the dark, whereas enzymes of the Calvin cycle increased only in the light. Glycolysis seemed to be of minor importance.

Journal Article↗