Biomedical subjects

C Schnarrenberger

Publications and source records attributed to C Schnarrenberger.

At least 19 recordsLinked to original sources

PubMed2001

Induction of a peroxisomal malate dehydrogenase isoform in liver of starved rats.

The influence of starvation on malate dehydrogenase (MDH) in rat liver was investigated. Native electrophoresis revealed two MDH isoforms in non-starved rats and three isoenzymes in starved rats. After sucrose density gradient centrifugation of cell organelles from liver, MDH activity was detected in the mitochondrial and cytosolic fractions from non-starved rats. However, additional activity was found in the peroxisomal fraction from starved rats. The latter was identified as the electrophoretically new isoform in starved animals. The three isoforms of malate dehydrogenase from hepatocytes were separated and partially purified by chromatography on DEAE-Toyopearl. Several kinetic and regulatory properties of the three isoforms were rather similar. It is suggested that the newly expressed isoform of MDH operates in the glyoxylate cycle of liver peroxisomes of food-starved animals.

Animals↗

PubMed2001

Purification and cloning of chloroplast 6-phosphogluconate dehydrogenase from spinach. Cyanobacterial genes for chloroplast and cytosolic isoenzymes encoded in eukaryotic chromosomes.

Previous attempts to purify chloroplast 6-phosphogluconate dehydrogenase (cp6PGDH), a key enzyme of the oxidative pentose phosphate pathway, have been unsuccessful due to rapid activity loss. An efficient purification protocol was developed and the enzyme from spinach leaves was purified 1000-fold to apparent homogeneity with a specific activity of 60 U.mg-1. The enzyme is a homodimer with subunits of 50 kDa. Antibodies raised against the purified cp6PGDH detected a 53-kDa protein from a crude extract, indicating alterations during purification. Purified cp6PGDH was microsequenced and the corresponding spinach cDNA was cloned using PCR techniques and degenerate primers. The cDNA for cytosolic 6PGDH from spinach was cloned for comparison. Phylogenetic analysis in the context of available homologues from eukaryotes and eubacteria revealed that animal and fungal cytosolic 6PGDH sequences are more similar to their homologues from gamma-proteobacteria, whereas plant 6PGDH is more similar to its cyanobacterial homologues. The ancestral gene for higher plant 6PGDH was acquired from the antecedent of plastids through endosymbiosis and gene transfer to the nucleus. A subsequent gene duplication gave rise to higher plant cytosolic 6PGDH, which assumed the function of its pre-existing cytosolic homologue through endosymbiotic gene replacement. The protein phylogeny of both 6PGDH and of the first enzyme of the oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase, indicate a surprisingly close relationship between the plant and Trypanosoma brucei lineages, suggesting that T. brucei (a relative of Euglena gracilis) may be secondarily nonphotosynthetic.

Amino Acid Sequence↗

PubMed1999

Characterization, cloning, and evolutionary history of the chloroplast and cytosolic class I aldolases of the red alga Galdieria sulphuraria.

Two fructose-1,6-bisphosphate aldolases from the acido- and thermophilic red alga Galdieria sulphuraria were purified to apparent homogeneity and N-terminally microsequenced. Both aldolases had similar biochemical properties such as Km (FBP) (5.6-5.8 microM) and molecular masses of the native enzymes (165kDa) as determined by size exclusion chromatography. The subunit size of the purified aldolases, as determined by SDS-PAGE, was 42kDa for both aldolases. The isoenzymes were not inhibited by EDTA or affected by cysteine or potassium ions, implying that they belong to the class I group of aldolases, while other red algae are known to have one class I and one class II aldolase inhibited by EDTA. cDNA clones of the cytosolic and plastidic aldolases were isolated and sequenced. The gene for the cytosolic isoenzyme contained a 303bp untranslated leader sequence, while the gene for the plastidic isoenzyme exhibited a transit sequence of 56 amino-acid residues. Both isoenzymes showed about 48% homology in the deduced amino-acid sequences. A gene tree relates both aldolases to the basis of early eukaryotic class I aldolases. The phylogenetic relationship to other aldolases, particularly to cyanobacterial class II aldolases, is discussed.

Algal Proteins↗

PubMed1999

Chloroplast class I and class II aldolases are bifunctional for fructose-1,6-biphosphate and sedoheptulose-1,7-biphosphate cleavage in the Calvin cycle.

Class I and class II aldolases are products of two evolutionary non-related gene families. The cytosol and chloroplast enzymes of higher plants are of the class I type, the latter being bifunctional for fructose-1,6- and sedoheptulose-1,7-P2 in the Calvin cycle. Recently, class II aldolases were detected for the cytosol and chloroplasts of the lower alga Cyanophora paradoxa. The respective chloroplast enzyme has been shown here to be also bifunctional for fructose-1,6- and sedoheptulose-1,7-P2. Kinetics, also including fructose-1-P, were determined for all these enzymes. Apparently, aldolases are multifunctional enzymes, irrespective of their class I or class II type.

Cell Compartmentation↗

PubMed1998

Eubacterial origin of nuclear genes for chloroplast and cytosolic glucose-6-phosphate isomerase from spinach: sampling eubacterial gene diversity in eukaryotic chromosomes through symbiosis.

Higher plants possess two distinct nuclear-encoded glucose-6-phosphate isomerase (GPI) isoenzymes, a cytosolic enzmye of the Embden-Meyerhof pathway and a chloroplast enzyme essential to storage and mobilization of carbohydrate fixed by the Calvin cycle. We have purified spinach chloroplast GPI to homogeneity, determined amino acid sequences from the active enzyme, and cloned cDNAs for chloroplast and cytosolic GPI isoenzymes from spinach. Sequence comparisons reveal three distantly related families of GPI genes that are non-uniformly distributed among contemporary eubacteria and archaebacteria, suggesting that ancient gene diversity existed for this glycolytic enzyme. Spinach chloroplast GPI is much more similar to its homologue from the cyanobacterium Synechocystis PCC6803 than it is to the enzyme from any other source, providing strong evidence that the gene for chloroplast GPI was acquired by the nucleus via endosymbiotic gene transfer from the cyanobacterial antecedants of chloroplasts. Eukaryotic nuclear genes for cytosolic GPI are more similar to eubacterial than to archaebacterial homologues, suggesting that these too were acquired by eukaryotes from eubacteria, probably during the course of the endosymbiotic origin of mitochondria. Chloroplast and cytosolic GPI provide evidence for a eubacterial origin of yet another component of the eukaryotic glycolytic pathway.

Amino Acid Sequence↗

PubMed1997

The reaction mechanism of phosphomannomutase in plants.

The enzyme phosphomannomutase catalyzes the interconversion of mannose-1-phosphate (Man-1-P) and mannose-6-phosphate (Man-6-P). In mammalian cells the enzyme has to be activated by transfer of a phosphate group from a sugar-1.6-P2 (Guha, S.K. and Rose, Z.B. (1985) Arch. Biochem. Biophys. 243, 168). In contrast, in the red alga Galdieria sulphuraria the co-substrate (Man-1.6-P2 or Glc-1.6-P2) is converted to the corresponding sugar monophosphate while the substrate is converted to the sugar bisphosphate in each reaction cycle. Evidence is presented that the same reaction mechanism occurs in spinach and yeast.

Animals↗

PubMed1997

Multiple recruitment of class-I aldolase to chloroplasts and eubacterial origin of eukaryotic class-II aldolases revealed by cDNAs from Euglena gracilis.

The photosynthetic protist Euglena gracilis is one of few organisms known to possess both class-I and class-II fructose-1,6-bisphosphate aldolases (FBA). We have isolated cDNA clones encoding the precursor of chloroplast class-I FBA and cytosolic class-II FBA from Euglena. Chloroplast class-I FBA is encoded as a single subunit rather than as a polyprotein, its deduced transit peptide of 139 amino acids possesses structural motifs neccessary for precursor import across Euglena's three outer chloroplast membranes. Evolutionary analyses reveal that the class-I FBA of Euglena was recruited to the chloroplast independently from the chloroplast class-I FBA of chlorophytes and may derive from the cytosolic homologue of the secondary chlorophytic endosymbiont. Two distinct subfamilies of class-II FBA genes are shown to exist in eubacteria, which can be traced to an ancient gene duplication which occurred in the common ancestor of contemporary gram-positive and proteobacterial lineages. Subsequent duplications involving eubacterial class-II FBA genes resulted in functional specialization of the encoded products for substrates other than fructose-1,6-bisphosphate. Class-II FBA genes of Euglena and ascomycetes are shown to be of eubacterial origin, having been acquired via endosymbiotic gene transfer, probably from the antecedants of mitochondria. The data provide evidence for the chimaeric nature of eukaryotic genomes.

Amino Acid Sequence↗

PubMed1997

The evolution of the Calvin cycle from prokaryotic to eukaryotic chromosomes: a case study of functional redundancy in ancient pathways through endosymbiosis.

The evolutionary histories of the 12 enzymes that catalyze the reactions of the Calvin cycle in higher-plant chloroplasts are summarized. They are shown to be encoded by a mixture of nuclear genes of cyanobacterial and proteobacterial origin. Moreover, where cytosolic isoforms of these enzymes are found they are almost invariably encoded by genes of clearly endosymbiont origin. We infer that endosymbiosis resulted in functional redundancy that was eliminated through differential gene loss, with intruding eubacterial genes repeatedly replacing pre-existing nuclear counterparts to which they were either functionally or structurally homologous. Our findings fail to support the 'product-specificity corollary', which predicts re-targeting of nuclear-encoded gene products to the organelle from whose genome they originated. Rather it would appear that the enzymes of central carbohydrate metabolism have evolved novel targeting possibilities regardless of their origins. Our findings suggest a new hypothesis to explain organelle genome persistence, based on the testable idea that some organelle-encoded gene products might be toxic when present in the cytosol or other inappropriate cellular compartments.

Biological Evolution↗

PubMed1996

Induction of glyoxylate cycle enzymes in rat liver upon food starvation.

The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, have been detected in liver of food-starved rats. Activities became measurable 3 days and peaked 5 days after the beginning of starvation. Both enzymes were found in the peroxisomal cell fraction after organelle fractionation by isopycnic centrifugation. Isocitrate lyase was purified 112-fold by ammonium sulfate precipitation, and chromotography on DEAE-cellulose and Toyopearl HW-65. The specific activity of the purified enzyme was 9.0 units per mg protein. The K(m)(isocitrate) was 68 microM and the pH optimum was at pH 7.4. Malate synthase was enriched 4-fold by ammonium sulfate precipitation. The enzyme had a K(m)(acetyl-CoA) of 0.2 microM, a K(m)(glyoxylate) of 3 mM and a pH optimum of 7.6.

Animals↗

PubMed1996

Microsequencing and cDNA cloning of the Calvin cycle/OPPP enzyme ribose-5-phosphate isomerase (EC 5.3.1.6) from spinach chloroplasts.

Ribose-5-phosphate isomerase (RPI) catalyses the interconversion of ribose-5-phosphate and ribulose-5-phosphate in the reductive and oxidative pentose phosphate pathways in plants. RPI from spinach chloroplasts was purified and microsequenced. Via PCR with degenerate primers designed against microsequenced peptides, a hybridisation probe was obtained and used to isolate several cDNA clones which encode RPI. The nuclear-encoded 239 amino acid mature RPI subunit has a predicted size of 25.3 kDa and is translated as a cytosolic precursor possessing a 50 amino acid transit peptide. The processing site of the transit peptide was identified from protein sequence data. Spinach leaves possess only one type of homodimeric RPI enzyme which is localized in chloroplasts and is encoded by a single nuclear gene. Molecular characterization of RPI supports the view that a single amphibolic RPI enzyme functions in the oxidative and reductive pentose phosphate pathways of spinach plastids.

Aldose-Ketose Isomerases↗

PubMed1996

Molecular characterization of transketolase (EC 2.2.1.1) active in the Calvin cycle of spinach chloroplasts.

A cDNA encoding the Calvin cycle enzyme transketolase (TKL; EC 2.2.1.1) was isolated from Sorghum bicolor via subtractive differential hybridization, and used to isolate several full-length cDNA clones for this enzyme from spinach. Functional identity of the encoded mature subunit was shown by an 8.6-fold increase of TKL activity upon induction of Escherichia coli cells that overexpress the spinach TKL subunit under the control of the bacteriophage T7 promoter. Chloroplast localization of the cloned enzyme is shown by processing of the in vitro synthesized precursor upon uptake by isolated chloroplasts. Southern blot-analysis suggests that TKL is encoded by a single gene in the spinach genome. TKL proteins of both higher-plant chloroplasts and the cytosol of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a possible eubacterial origin of these nuclear genes. Chloroplast TKL is the last of the demonstrably chloroplast-localized Calvin cycle enzymes to have been cloned and thus completes the isolation of gene probes for all enzymes of the pathway in higher plants.

Amino Acid Sequence↗

PubMed1996

Higher-plant chloroplast and cytosolic fructose-1,6-bisphosphatase isoenzymes: origins via duplication rather than prokaryote-eukaryote divergence.

Full-size cDNAs encoding the precursors of chloroplast fructose-1,6-bisphosphatase (FBP), sedoheptulose-1,7-bisphosphatase (SBP), and the small subunit of Rubisco (RbcS) from spinach were cloned. These cDNAs complete the set of homologous probes for all nuclear-encoded enzymes of the Calvin cycle from spinach (Spinacia oleracea L.). FBP enzymes not only of higher plants but also of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a eubacterial origin of these eukaryotic nuclear genes. Chloroplast and cytosolic FBP isoenzymes of higher plants arose through a gene duplication event which occurred early in eukaryotic evolution. Both FBP and SBP of higher plant chloroplasts have acquired substrate specificity, i.e. have undergone functional specialization since their divergence from bifunctional FBP/SBP enzymes of free-living eubacteria.

Amino Acid Sequence↗

PubMed1995

Purification and characterization of a galactose-1-phosphate: UDP-glucose uridyltransferase from the red alga Galdieria sulphuraria.

The galactose-1-phosphate uridyltransferase of the red alga Galdieria sulphuraria has been purified about 1800-fold to a final specific activity of approximately 140 U/mg protein. The purification involved chromatography on DEAE-Fractogel, hydroxyapatite, decyl-agarose, and DEAE-Tentacle gel. After SDS/PAGE, the enzyme preparation showed only one protein band of 42 kDa. The enzyme is a homodimer with a molecular mass of 82 kDa as estimated from the sedimentation velocity or 60 kDa as estimated by gel filtration. It has a broad pH optimum between pH 7 and pH 9. The apparent Km values for the forward and backward reactions are Km(Glc1P) = 105 microM, Km(UDP-galactose) = 30 microM, Km(Gal1P) = 400 microM, and Km(UDP-Glc) = 20 microM. The activation energy of the reaction is 45 kJ mol-1. The enzyme is specific for the galactose 1-phosphate to UDP-galactose interconversion in the Leloir pathway while the alternate enzyme for the Isselbacher pathway, UDP-galactose pyrophosphorylase, could not be detected in G. sulphuraria.

Chromatography, Gel↗

PubMed1995

The plastid aldolase gene from Chlamydomonas reinhardtii: intron/exon organization, evolution, and promoter structure.

Genomic clones encoding the plastidic fructose-1,6-bisphosphate aldolase of Chlamydomonas reinhardtii were isolated and sequenced. The gene contains three introns which are located within the coding sequence for the mature protein. No introns are located within or near the sequence encoding the transit-peptide, in contrast to the genes for plastidic aldolases of higher plants. Neither the number nor the positions of the three introns of the C. reinhardtii aldolase gene are conserved in the plastidic or cytosolic aldolase genes of higher plants and animals. The 5' border sequences of introns in the aldolase gene of C. reinhardtii exhibit the conserved plant consensus sequence. The 3' acceptor splice sites for introns 1 and 3 show much less similarity to the eukaryotic consensus sequences than do those of intron 2. The plastidic aldolase gene has two tandemly repeated CAAT box motifs in the promoter region. Genomic Southern blots indicate that the gene is encoded by a single locus in the C. reinhardtii genome.

Amino Acid Sequence↗

PubMed1995

Cloning of the amphibolic Calvin cycle/OPPP enzyme D-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) from spinach chloroplasts: functional and evolutionary aspects.

Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme D-ribulose-5-phosphate 3-epimerase (R5P3E)(EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T7 promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.

Amino Acid Sequence↗

PubMed1994

Expression and sequence of the only detectable aldolase in Chlamydomonas reinhardtii.

Only one aldolase can be resolved from Chlamydomonas reinhardtii by anion-exchange chromatography on DEAE-cellulose. Western blots with specific antisera against plastid and cytosol aldolase of spinach and Northern blots with the respective cDNAs from spinach indicate that only one aldolase, the plastid enzyme, is expressed. Full-length cDNA clones for the plastidic aldolase were isolated from cDNA library constructed from poly(A)+ mRNA of C. reinhardtii with plastid-aldolase-specific probes from spinach. The clones contained a 1.7-kb insert with an open reading frame for 374 amino acid residues covering the mature chloroplast protein of 347 amino acids and a N-terminal transit peptide of 27 amino acids for chloroplast import. The calculated molecular mass of the mature protein is 37.6 kDa and that of the precursor protein is 40.3 kDa. The aldolase of C. reinhardtii shows amino acid similarity of 62 to 67% with the chloroplast aldolase and of 47 to 52% with the cytosolic enzymes of higher plants, respectively. An evolutionary tree with all known class I aldolases shows separate clusters for the chloroplast and for the cytosol aldolases in higher plants and green algae. The aldolase of C. reinhardtii connects at a basal position with the chloroplast aldolases of higher plants.

Amino Acid Sequence↗

PubMed1994

Chloroplast and cytosolic triosephosphate isomerases from spinach: purification, microsequencing and cDNA cloning of the chloroplast enzyme.

Chloroplast and cytosolic triosephosphate isomerases from spinach were separated and purified to homogeneity. Both enzymes were partially sequenced by Edman degradation. Using degenerate primers designed against the amino acid sequences, a homologous probe for the chloroplast enzyme was amplified and used to isolate several full-size cDNA clones. Chloroplast triosephosphate isomerase is encoded by a single gene in spinach. Analysis of the chloroplast cDNA sequence in the context of its homologues from eukaryotes and eubacteria reveals that the gene arose through duplication of its preexisting nuclear counterpart for the cytosolic enzyme during plant evolution.

Amino Acid Sequence↗

PubMed1994

Molecular characterization of a novel, nuclear-encoded, NAD(+)-dependent glyceraldehyde-3-phosphate dehydrogenase in plastids of the gymnosperm Pinus sylvestris L.

Angiosperms and algae possess two distinct glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes, an NAD(+)-dependent tetramer involved in cytosolic glycolysis and an NADP(+)-dependent enzyme of the Calvin cycle in chloroplasts. We have found that the gymnosperm Pinus sylvestris possesses, in addition to these, a nuclear-encoded, plastid-specific, NAD(+)-dependent GAPDH, designated GapCp, which has not previously been described from any plant. Several independent full-size cDNAs for this enzyme were isolated which encode a functional transit peptide and mature subunit very similar to that of cytosolic GAPDH of angiosperms and algae. A molecular phylogeny reveals that chloroplast GapCp and cytosolic GapC arose through gene duplication early in chlorophyte evolution. The GapCp gene is expressed as highly as that for GapC in light-grown pine seedlings. These findings suggest that aspects of compartmentalized sugar phosphate metabolism may differ in angiosperms and gymnosperms and furthermore underscore the contributions of endosymbiotic gene transfer and gene duplication to the nuclear complement of genes for enzymes of plant primary metabolism.

Amino Acid Sequence↗