The presence of glycollate oxidase and dehydrogenase in Dunaliella primolecta

Homogenates of Dunaliella primolecta, D. salina and D. tertiolecta were assayed for glycollate oxidase and glycollate dehydrogenase. Both D. primolecta and D. salina but not D. tertiolecta showed substantial glycollate-dependent O₂-uptake which is characteristic of glycollate oxidase. L-Lactate was an alternative substrate and both glycollate- and L-lactate-dependent O₂ uptake were insensitive to 2 mM cyanide. Glycollate dehydrogenase, measured by following the glycollate-dependent reduction of 2,6-dichlorophenolindophenol under aerobic conditions, was present in D. primolecta, D. salina and D. tertiolecta. In the presence of glycollate and D-lactate, rates were additive so both glycollate and D-lactate dehydrogenases are present in the homogenates. Glycollate and D-lactate oxidation were both inhibited by 2 mM cyanide. Organelles released from phototrophically grown cells of D. primolecta were separated by isopycnic centrifugation on sucrose gradients. Glycollate oxidase was present in the peroxisome fraction at an equilibrium density of 1.25 g/cm³, while the major peak of glycollate dehydrogenase activity was in the mitochondrial fraction at an equilibirium density of 1.22 g/cm³.     

Glycollate oxidation by thylakoids of the cyanobacteria Anabaena cylindrica,Nostoc muscorum and Chlorogloea fritschii

The intracellular distribution of glycollate dehydrogenase EC 1.2.1.17 has been investigated in extracts of the cyanobacteria (blue-green algae) Anabaena cylindrica, Nostoc muscorum and Chlorogloea fritschii. Most of the enzyme activity was associated with a chlorophyll-containing cell-free pellet, which also exhibited Photosystem I and II activities. Sucrose density gradient centrifugation of this washed pellet resulted in the formation of a green band within which maximal chlorophyll concentration and enzymic glycollate oxidation coincided. Antiserum raised to this fraction obtained from A. cylindrica inhibited glycollate dehydrogenase and Photosystem II activity. The data indicate that most of the cyanobacterial glycollate dehydrogenase is associated with the thylakoids and thus provide evidence for the dual role of these membranes in photosynthetic and respiratory processes.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolindophenol - DPC diphenylcarbazide     

Localization of the enzyme 3-deoxy-d-manno-2-octulosonic acid aldolase

Summary Several strains of Gram-negative microorganisms were screened for maximum 3-deoxy-d-manno-2-octulosonic acid (KDO) aldolase (EC 4.1.2.23) activity. Although this enzyme has been noted to be inducible on special medium, no induction was found. By centrifugation studies the KDO aldolase was found to be localized in the cell wall or membrane fraction. The enzyme activity was very susceptible to small amounts of detergent in solution. Offprint requests to: M.-R. Kula     

Cell organelles from crassulacean-acid-metabolism (CAM) plants

Cell organelles were isolated from the CAM plants Crassula lycopodioides Lam., Bryophyllum calycinum Salisb. and Sedum rubrotinctum R.T. Clausen by isopycnic centrifugation in sucrose gradients. The inclusion of 2.5% Ficoll in the grinding medium proved to be essential for a satisfactory separation of cell organelles during the subsequent centrifugation. Peroxisomes, mitochondria, and whole and broken chloroplasts were at least partially resolved as judged by marker-enzyme-activity profiles. The isolated peroxisomes contained activities of glycollate oxidase, catalase, hydroxypyruvate reductase, glycine aminotransferase, serine-glyoxylate aminotransferase, and aspartate aminotransferase, comparable to activities found in spinach (Spinacia oleracea L.) leaf peroxisomes. In contrast to spinach, however, only little, if any, particulate malate dehydrogenase activity could be attributed to isolated peroxisomes of the three CAM plants.     

Biosynthesis of Leaf Alcohol: Kinetic Study of Lipoxygenase Activation Process Caused by Hydroperoxylinoleic Acid

Quaternary structure of ribulose-1, 5-bisphosphate (RuP₂) carboxylase from the autotrophically grown cells of blue-green alga, Anabaena cylindrica, was studied. Sedimentation coefficient (s_{20, w}) of the enzyme was determined to be 18.3 S by the sucrose density gradient centrifugation. The molecular weight was estimated to be 5.0 × 10⁵ by the Sepharose 4B gel filtration technique. The purification of the enzyme from the algal cells was undertaken by means of sucrose density gradient centrifugation and DEAE-Sephadex A–50 ion-exchange column chromatography, and the structural make-up of the enzyme containing two subunits, A (M. W., 5.2 × 10⁴) and B (M. W., 1.2 × 10⁴) was established by the Na-dodecylsulfate polyacrylamide gel electrophoresis experiment. Structural similarity of the algal RuP₂carboxylase with the spinach enzyme was further demonstrated by the Ouchterlony double immunodiffusion experiment.     

Stabilization of chitin synthetase and purification of chitosomes from several mycelial Mucorales

Stability of chitin synthetase in cell-free extracts from mycelial fungi was markedly improved by the presence of sucrose in the homogenization media. Breakage of mycelium in sucrose-containing buffer yielded enzyme preparations from which chitosomal chitin synthetase could be purified by a procedure involving ammonium sulfate precipitation, gel filtration and centrifugation in sucrose density gradients. Purified chitosomes catalyzed the synthesis of chitin microfibrils in vitro upon incubation with substrate and activators. Chitosomal chitin synthetase from the filamentous form of M. rouxii was similar to the enzyme from yeast cells, except for the poorer stability and diminished sensitivity to GlcNAc activation of the former.     

Microbody of n-alkane-grown yeast

Microbodies appearing abundantly in n-alkane-grown cells of Candida tropicalis pK 233 were isolated by means of sucrose density gradient centrifugation. Electron microscopical observation showed that the microbodies isolated were intact. Localization of catalase and d-amino acid oxidase in the isolated microbodies was confirmed. Isocitrate lyase, malate synthase and NADP-linked isocitrate dehydrogenase were also located in the microbody, but malate dehydrogenase, citrate synthase, aconitase and NAD-linked isocitrate dehydrogenase were not. Neither cytochrome P-450 nor NADPH-cytochrome c reductase, the components involved in the n-alkane hydroxylation system of the yeast, were detected in the microbody fraction.     

Intracellular localization of glycolate dehydrogenase in a blue-green alga

Glycolate dehydrogenase activity was detected in cell-free extracts of Oscillatoria sp. prepared by osmotic lysis of spheroplasts in 0.05 m potassium phosphate buffer, pH 7.5, containing 0.3 m mannitol. Most of the enzyme activity was found in a particulate fraction and localized in the photosynthetic lamellae after centrifugation in a discontinuous sucrose density gradient. Enzyme activity was detected in this fraction both in the presence and absence of the artificial electron acceptor 2,6-dichlorophenolindophenol (DPIP) and a low rate of O(2) uptake was detected in this lamellar fraction. Activity was lost from the lamellar fraction by repeated washing or by treatment with 0.005% Triton X-100 and the solubilized enzyme activity was DPIP-dependent. The data indicate that both glycolate dehydrogenase and its natural electron acceptor are bound to the photosynthetic lamellae in vivo. In contrast, catalase activity was found in the soluble cytoplasmic fraction.     

Localization of L-asparaginase inEscherichia coli

The localization ofl-asparaginase (l-asparagine amidohydrolase, EC 3.5.1.1) EC-2 isoenzyme was studied inEscherichia coli ATCC 9637 grown under conditions of moderate aeration. The enzyme was determined in cell fractions obtained by fraction centrifugation of lysed spheroplasts. When the synthesis of the enzyme was induced byl-asparagine, its amount in the cytoplasmic fraction at the beginning of the induction exceeded as much as five times that in uninduced cells, attaining up to 20% of the total activity. In the course of growth of the culture this activity decreased gradually to zero. The membrane fraction of induced cells contained considerable amount of EC-2l-asparaginase which, at the beginning of the induction, reached up to 6% ot the total enzymic activity; in membrane fraction of control cells the activity was close to zero. The results indicate a relationship of cell structures to thel-asparagine-induced synthesis of the enzyme.     

Isolation of peroxisomes from the dog kidney cortex.

The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.     

Occurrence and biosynthesis of catalase at different stages of seed maturation

It was to be shown whether during the biogenesis of microbodies some of their components were already present in the cell prior to the organelle's assembly. To this end, the occurrence and properties of catalase in soluble and particular fractions of ripening cucumber seeds were examined. Homogenates of seeds from ripening fruits were fractionated by isopycnic density gradient centrifugation, and thus catalase was found in three different fractions: as a soluble enzyme in the gradient supernatant, as a membrane fraction at density d=1.18 kg l^-1, and in association with microbodies. In the early steps of seed formation, catalase was detected at density d=1.18 kg l^-1 and in the gradient supernatant. At a later stage of seed maturation, however, catalase was primarily associated with microbodies which exhibited an equilibrium density of d=1.23 kg l^-1. M _r as well as subunit M _r of catalase were determined, and their close immunological relationship to leaf peroxisomal catalase and glyoxysomal catalase was demonstrated. Biosynthesis of catalase at different stages of seed maturation was investigated by in vivo labeling with l-[³⁵S]methionine, l-[¹⁴C]leucine and -[³H]aminolaevulinic acid. Electrophoretic analysis of de novo synthesized catalase subunits revealed the occurrence of a heavy form (M _r 57,500) in the soluble fraction; this form was preferentially labeled. A light form, M _r 53,500, was detected in microbodies and also in the soluble fraction. The findings lend support to the hypothesis that the rate of catalase synthesis is highest in an early stage of seed formation, when globulins have already been formed, but before de novo synthesis of malate synthase has commenced. Prior to microbody assembling, a cytoplasmic pool of catalase was labeled.Abbreviations EDTA Na₂-ethylenediaminotetraacetate - Hepes 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - M _r molecular weight     

Glycollate metabolism of wheat and rice leaves during senescence and under the influence of growth regulators

The glycollate metabolism of wheat (Triticum vulgare Vill. cv. Sonalika) and rice (Oryza sativa L. ev. Jaya) leaves was studied during senescence by estimating the endogenous levels of glycollate and hydrogen peroxide (H₂O₂) and the activities of glycollate oxidase and catalase. In comparison with light incubation the incubation of excised leaves in the dark caused a decline in the glycollate content and in the activities of glycollate oxidase and catalase, and an increase in the H₂O₂ content, more marked in the leaves of rice than in the leaves of wheat. Glycollate oxidase activity gradually decreased with incubation time, and glycollate metabolism decreased during senescence. The glycollate oxidase in particular and glycollate metabolism of rice were more sensitive to incubation time than those of wheat. Kinetin increased the glycollate oxidase activity and glycollate metabolism during senescence, while ethrel (2-chloroethylpho-sphonic acid) and ABA (abscisic acid) reduced these activities in both plant species.     

Ribonucleic Acid-Dependent Ribonucleic Acid Polymerase in the Immune Response

Ribonucleic acid (RNA)-dependent RNA polymerase activity was demonstrated in the microsomal and ribosomal fraction from the spleen cells of immunized mice. The enzyme activity was solubilized by Triton X-100 from the fraction and partially purified by Biogel A 1.5 M column chromatography. The RNA-dependent RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RNA preparation (iRNA) as template made from the spleens of immunized mice but very low activity was found with an nRNA preparation made from the spleens of normal mice. Incorporation of ³H-UTP markedly decreased in the presence of RNase but not in the presence of DNase. DNA preparations made from the spleens of immunized mice were inactive as template for this enzyme. The iRNA preparation was fractionated by sucrose density gradient centrifugation. A fraction corresponding to 12–13 S was most active as a template. It was followed by a fraction corresponding to 6–7 S. Sucrose gradient analysis of the ³H-UTP-labeled product was attempted. Some properties of this enzyme are described.     

Hydrodynamic parameters and isolation of mitochondria, microperoxisomes and microsomes of rat heart

1. Analytical differential centrifugation of rat heart homogenates revealed a single population of mitochondria and microperoxisomes. Using cytochorme c oxidase, malate dehydrogenase and amine oxidase as mitochondrial marker enzymes, the s̄-value of mitochondria was estimated to s̄ = 10326 ± 406 S (average for the three marker enzymes). The −s-value of microperoxisomes was found to be −s = 1381 ± 40 S using catalase as the marker enzyme. The −s-value for the two orgenelles did not change significantly when the isoosmotic sucrose medium was substituted by an isoosmotic mannitol medium. 2. Analytical differential centrifugation revealed a polydispercity of the microsomal fraction using glucose-6-phosphatase and NADPH-cytochrome c reductase as the marker enzymes. The s̄-values were found to be −s_H1 = 1569 ± 412 S (NADPH-cytochrome c reductase), (glucose-6-phosphatase) and (NADPH-cytochrome c reductase and glucose-6-phosphatase). The recovery of marker enzymes in the isolated subcellular fractions was in the range of 84–94%. 3. When the mitochondrial and microperoxisomal fractions were subjected to isopycnic gradient centrifugation, using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.25 M sucrose medium, buoyant densities of 1.10 g/cm³ (main fraction of mitochondria) and 1.06 g/cm³ (main fraction of microperixosomes) were obtained. The density gradient centrifugation separated microperoxisomes from contaminating lysosomes of high specific activity in acid phosphatase. A value 1.04 g/cm³ was foung for the density of the microsomal fraction. 4. Based on the estimated -values, an optimal procedure is described for the isolattion of mitochondrial and microperoxisomal fractions from rat heart muscle.     

Isolation of L8 and L8S8 forms of ribulose bisphosphate carboxylase/oxygenase from Chromatium vinosum

The enzyme ribulose bisphosphate carboxylase/oxygenase has been purified from Chromatium vinosum. When an extract is subjected to centrifugation at 35,000xg in the presence of polyethylene glycol (PEG)-6000 and the supernatant is treated with 50 mM Mg²⁺ and the precipitate is then fractionated by vertical centrifugation into a reoriented sucrose gradient followed by chromatography on diethylaminoethyl (DEAE)-Sephadex A50, the resultant enzyme contains large (L) and small (S) subunits. Alternatively, centrifugation of extracts at 175,000xg in the presence of PEG-6000 followed by fractionation with Mg²⁺, density gradient centrifugation, and chromatography on DEAE-Sephadex A50 yields an enzyme free of small subunits. The two forms have comparable carboxylase and oxygenase activities and have compositions and molecular weights corresponding to L₈ and L₈S₈ enzymes. The amino acid compositions of L and S subunits are reported. The L₈S₈ enzyme from spinach cannot be similarly dissociated by centrifugation at 175,000xg in the presence of PEG-6000.Abbreviations DEAE diethylaminoethyl - EDTA ethylenediamine-tetraacetate - MOPS 3-(N-morpholino)propanesulfonic acid - PEG polyethylene glycol - RuBisCO d-ribulose 1,5-bisphosphate caboxylase/oxygenase - RnBP d-ribulose 1,5-bisphosphate - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Dedicated to Professor G. Drews on occasion of his 60th birthday     

LOCALIZATION OF ENZYMES WITHIN MICROBODIES

Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm³ which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50–60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [¹⁴C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21–1.22 g/cm³, whereas the original glyoxysomes appeared at density 1.24 g/cm³. Electron microscopy showed that the fraction at 1.21–1.22 g/cm³ was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance.     

Partial purification and characterization of mRNAs encoding glycollate oxidase and catalase

Polyadenylated mRNA was prepared from etiolated and greening leaves of Lens culinaris and cotyledons of Cucumis sativus during the transition from etiolated to photoautotrophic stage. These mRNA preparations were used to identify, by translation in vitro, the precursor forms of glycollate oxidase and catalase, both enzymes being markers of microbodies. The level (per fresh weight) of translatable RNA coding for glycollate oxidase was found to increase ten fold during the first 3 d of illumination of etiolated leaves. For catalase mRNA activity, this increase was less pronounced. Characterizing the products of in-vitro translation directed by the mRNA prepared, we observed a 43-kDa species of glycollate oxidase and a 56-kDa species of apo-catalase. Limited proteolysis of the in-vitro-formed proteins and comparison with the respective mature enzymes present in vivo revealed differences between the cucumber and the lens protein but not between the monomeric precursor and the subunit of mature glycollate oxidase from Lens culinaris. Messenger RNA coding for glycollate oxidase was highly purified by electrophoresis on low-melting-point agarose in the presence of methylmercuric hydroxide. The size of the mRNA was determined to be 1.47 kb. By this procedure, the mRNA for glycollate oxidase in the subfraction could be enriched in such a way that the activity, assayed by translation in a reticulocyte lysate, amounted to 30% of the total translation activity.Abbreviations PAGE polyacrylamide gel electrophoresis - poly(A)⁺ RNA polyadenylated RNA - SDS sodium dodecyl sulfate     

Membranous localization and properties of ATPase of rat liver lysosomes

Summary Lysosomes isolated from rat liver were found to have ATPase activity (EC No. 3.6.1.3). Subfractionation of the lysosomes revealed a membranous localization of ATPase activity. The enzyme has half maximal activity at 0.2mm ATP and is inhibited by high concentrations of ATP. The apparentK _m for divalent metal is 0.2mm, and either ca²⁺ or Mg²⁺ give maximal activity.The ATPase activity has latency when lysosomes are isolated from rats treated with Triton WR-1339. This latency may be due to the presence of internalized sucrose because the activity ofL fraction lysosomes is much less latent and Triton WR-1339 itself is not inhibitory. The latency of glucosamindase, a marker enzyme for lysosomes, contrasts with the low latency of the ATPase and points to an ATPase with an exposed active site in intact lysosomes.     

Isolation of protein bodies on sucrose gradients

Summary Storage protein bodies from sunflower cotyledons during early stages of seed germination were isolated on sucrose density gradients by isopycnic centrifugation. The density of this organelle on the gradients ranged between 1.26 and 1.36 g cm^-3. A proteinase with a pH optimum of 5.2 was associated with this organelle, and is probably responsible for degradation of storage protein. A NADH-dependent cytochrome-c reductase, a membrane marker enzyme with a pH optimum of 8.4, was also present in this organelle fraction.Abbreviations LPA for l-lysine-p-nitroanilide - LPAase for the peptidase which hydrolyzes this peptide This work was supported in part by the National Science Foundation Grant GB-17543, and published as Journal Article No. 5736 of the Michigan Agricultural Experiment Station.Supported by a Deutsche Forschungsgemeinschaft Fellowship.     

Plant leaf alanine: 2-oxoglutarate aminotransferase. Peroxisomal localization and identity with glutamate:glyoxylate aminotransferase.

The distribution of alanine:2-oxoglutarate aminotransferase (EC 2.6.1.2) in spinach (Spinacia oleracea) leaf homogenates was examined by centrifugation in a sucrose density gradient. About 55% of the total homogenate activity was localized in the peroxisomes and the remainder in the soluble fraction. The peroxisomes contained a single form of alanine:2-oxoglutarate aminotransferase, and the soluble fraction contained two forms of the enzyme. Both the peroxisomal enzyme and the soluble predominant form (about 90% of the total soluble activity) were co-purified with glutamate:glyoxylate aminotransferase to homogeneity; it had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration [Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. The evidence indicates that alanine:2-oxoglutarate aminotransferase and glutamate:glyoxylate aminotransferase activities are associated with the same protein. The peroxisomal and soluble enzyme preparations had nearly identical properties, suggesting that the soluble predominant alanine aminotransferase activity is from broken peroxisomes and about 96% of the total homogenate activity is located in peroxisomes.