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H Kindl

Publications and source records attributed to H Kindl.

At least 55 records · Page 3Linked to original sources
PubMed1988

Gene response upon illumination in forming mRNA encoding peroxisomal glycollate oxidase.

Glycollate oxidase is a constituent of leaf peroxisomes. Its biosynthesis is, like the biosynthesis of many chloroplastic proteins, controlled by light, via phytochrome. The level of mRNA coding for glycollate oxidase was determined at different stages of greening of etiolated plant cells. The appearance of glycollate oxidase mRNA in the cytoplasm was measured by hybridization with cDNA containing part of the coding sequence for glycollate oxidase. cDNA was prepared from enriched mRNA, inserted into the Pst I site of pBR 322, and cloned in Escherichia coli DH-1. By differential colony hybridization and hybrid selection, a clone containing a 670 bp sequence complementary to mRNA encoding glycollate oxidase was selected and identified. Northern blot hybridization was used to investigate mRNA levels induced by light. It was found that continuous light affected the formation of glycollate oxidase mRNA. When a large population of microbodies was present in the cells being induced, the immediate mRNA increase was very pronounced, and was detectable as little as 20 min after the beginning of the light treatment. In contrast, a lag period in the mRNA increase was observed when the induction was performed with etiolated leaves which are characterized by the occurrence of a rather small population of microbodies. For comparison, we measured the time-course of formation of mRNA coding for a light-induced chloroplastic protein, i.e., a protein of the light-harvesting complex. The time-courses of levels of the two mRNAs indicate that the program of gene expression differs between the two particular proteins destined either for chloroplasts or for peroxisomes. The formation of glycollate oxidase mRNA could also be stimulated by a short pulse of light, a treatment of 15 s being a sufficient trigger.

Alcohol Oxidoreductases↗
PubMed1986

Plant acyl-CoA oxidase. Purification, characterization, and monomeric apoprotein.

Purified glyoxysomes from cotyledons of germinating cucumber seedlings were used as a source to separate matrix enzymes of the organelle by hydrophobic chromatography. Glyoxysomal acyl-CoA oxidase eluted from the column like hydrophobic proteins and exhibited an Mr of 150,000. An oxidase with identical properties could be prepared in large quantities by a purification procedure starting with crude extracts from cotyledons of 4-day-old etiolated seedlings. The purification procedure included chromatography on phenyl-Sepharose and hydroxylapatite and molecular sieving. 1500-fold purification led to an enzyme of apparent homogeneity characterized by a specific activity of 27 units/mg of protein. Plant acyl-CoA oxidase is a homodimer with a subunit of Mr 72,000. Monospecific antibodies raised in rabbits were used to reveal dissimilarity to the fungal oxidase. The plant enzyme also differed markedly in molecular structure and amino acid composition from the liver peroxisomal enzyme. Glyoxysomal acyl-CoA oxidase acts selectively on fatty acyl-CoAs with 16 or 18 C atoms, cis-9-unsaturated esters with a C16 or C18 acyl moiety being converted with higher rates than saturated or polyunsaturated fatty acyl-CoAs. Besides the enzymatically active organellar form of acyl-CoA oxidase, the monomeric apoprotein was detected when short-term labeling of cotyledons in vivo was performed. The apoprotein (immunoprecipitable by antibodies raised against the glyoxysomal enzyme) did not differ in size from the subunit of the glyoxysomal dimeric enzyme.

Acyl Coenzyme A↗
PubMed1985

Isolation of proteins assembled in lipid body membranes during fat mobilization in cucumber cotyledons.

Lipid bodies from fat-mobilizing cotyledons of cucumber and other Cucurbitaceae were investigated. Proteins and glycoproteins were analyzed by electrophoresis and then used to characterize the lipid body membrane at different stages of cell development. Contaminations by other membranes or organelles were ruled out by comparing the main constituents from the endoplasmic reticulum, cytosol, glyoxysomes and protein bodies with the pattern of the lipid body membrane, considering both the prevalent peptides and the dominating glycoproteins. Among the proteins of lipid body membranes in ripening and germinating cotyledons, a 90-kDa peptide was found as unique marker of lipid bodies at the stage of fat mobilization. The 90-kDa protein was purified, and antibodies against it were raised in rabbits. By means of immunoprecipitation and electrophoretic analysis it was demonstrated that the synthesis of the 90-kDa form located in lipid bodies shows a transient increase and subsequent decline, with maximal values being observed at day 3 of germination. At this stage, the rate of de novo synthesis was compared considering lipid body proteins and other organellar proteins. The 90-kDa protein appeared as the lipid body constituent that is synthesized and assembled in the organelle by far at the highest rates.

Electrophoresis, Polyacrylamide Gel↗
PubMed1984

Purification and properties of a stilbene synthase from induced cell suspension cultures of peanut.

Stilbene synthase ( resveratrol -forming) converts one molecule of rho- coumaroyl -CoA and three molecules of malonyl-CoA into 3,4',5- trihydroxystilbene . Following selective induction of stilbene synthesis in cell suspension cultures of peanut (Arachis hypogaea), the enzyme was extracted and purified to apparent homogeneity by chromatography on DEAE-cellulose and hydroxylapatite. The enzyme was found to be a dimer of estimated Mr = 90,000 exhibiting under denaturing conditions a subunit Mr of approximately 45,000. The isoelectric point was determined with pI = 4.8. The enzyme's high selectivity towards rho- coumaroyl -CoA (Km = 2 microM) as substrate qualified it as resveratrol -forming stilbene synthase. Structurally related CoA esters, e.g. dihydro-rho- coumaroyl -CoA and cinnamoyl-CoA, were converted less than 1/10 as efficiently as rho- coumaroyl -CoA. Malonyl-CoA (Km = 10 microM) could not be substituted by acetyl-CoA. The purified enzyme was free of chalcone synthase activity. Antibodies raised against stilbene synthase were shown to be monospecific and not to cross-react with chalcone synthase.

Acyltransferases↗
PubMed1984

Stilbene Synthase and Chalcone Synthase : Two Different Constitutive Enzymes in Cultured Cells of Picea excelsa.

Cultured cells of Picea excelsa capable of forming stilbenes and flavanoids have been established. Unlike needles of intact plants containing piceatannol (3,3',4',5-tetrahydroxystilbene) and stilbene glycosides the cultured cells converted phenylalanine and p-coumaric acid primarily into resveratrol monomethyl ether (3,4'-dihydroxy-5-methoxystilbene) and naringenin. Partially purified enzyme preparations were assayed for chalcone synthase as well as for stilbene synthase activity converting malonyl-CoA plus p-coumaroyl-CoA into 3,4',5-trihydroxystilbene (resveratrol).Although stilbene synthase and chalcone synthase use the same substrates and exhibit similar molecular properties, i.e. molecular weight and subunit molecular weight, they are two different proteins. This difference was demonstrated by gel electrophoresis and by means of monospecific antibodies.

Journal Article↗
PubMed1983

9,10-Dihydrophenanthrenes as phytoalexins of Orchidaceae. Biosynthetic studies in vitro and in vivo proving the route from L-phenylalanine to dihydro-m-coumaric acid, dihydrostilbene and dihydrophenanthrenes.

Hydroxy derivatives of 9,10-dihydrophenanthrenes, orchinol and hircinol, were isolated from bulbs of Orchidaceae which had been induced to accumulate phytoalexins. Incorporation of radioactive precursors, L-phenylalanine and various hydroxycinnamic acids, has been investigated by feeding experiments in vivo. m-Coumaric acid and dihydro-m-coumaric acid were found to be efficiently incorporated into the dihydrophenanthrene derivatives. Dihydro-m-coumaric acid was not only converted into the dihydrophenanthrenes but was also formed from L-phenylalanine in the same tissue; it was thus proved to be an intermediate. The role of dihydro-m-coumaric acid was substantiated by studies in vitro. An active stilbene synthase was detected in enzyme preparations from induced orchid bulbs and assayed with different CoA esters. The enzyme, characterized on the basis of its substrate specificity, selectively converted dihydro-m-coumaroyl-CoA plus malonyl-CoA into 3,3',5-trihydroxybibenzyl. The role of 3,3',5-trihydroxybibenzyl as physiological intermediate was further corroborated by investigations with intact plants. Both its formation from phenylpropanoids and its conversion into orchinol was demonstrated. The data provided evidence for a biosynthetic sequence originating from L-phenylalanine and leading to 9,10-dihydrophenanthrenes via m-coumaric acid, dihydro-m-coumaric acid, and 3,3',5-trihydroxybibenzyl.

Acyltransferases↗
PubMed1983

Malate synthase: aggregation, deaggregation, and binding of phospholipids.

Octameric malate synthase is located in the glyoxysomes of cucumber cotyledons. The enzyme is predominantly confined to the organelle's membrane and can be solubilized with Mg2+. Separation of cell structures in a zonal rotor afforded, besides glyoxysomes, two other zones with malate synthase activity, viz., in the gradient supernatant and in the range of the endoplasmic reticulum (ER). Malate synthases of these three fractions were purified to apparent homogeneity and classified according to their molecular weight. Differences in subunit molecular weight, however, could not be detected when malate synthases from the three fractions were compared. Mature malate synthase, as well as malate synthase prepared from fractions sedimenting similarly to the ER, exhibited the following behavior with respect to aggregation and deaggregation: at low salt concentrations and in the absence of Mg2+, the enzyme shifted to aggregated forms (approx 100 S); with 2 mM Mg2+, malate synthase deaggregated and occurred predominantly as an octamer (19 S). By changing buffer conditions, mature forms of malate synthase could be interconverted repeatedly between octameric and aggregated forms, whereas a monomeric form (5 S), prepared from soluble fractions assigned to the cytosol, did not oligomerize. The amphipathic properties of malate synthase were demonstrated by the enzyme's capacity for binding phospholipids.

Macromolecular Substances↗
PubMed1983

Oligomerization of malate synthase during glyoxysome biosynthesis.

The octameric malate synthase, found in glyoxysomes of plants, is synthesized as monomeric precursor in the cytoplasm. The precursor form does not possess a different subunit molecular weight than the mature organellar enzyme, but differs from the organellar protein by not oligomerizing and aggregating. This was shown by synthesis in a cell-free reticulocyte lysate system programmed with cucumber poly A+-mRNA followed by immunoprecipitation of the radiolabeled translation products. The precursor form of malate synthase was also detected in vivo in the cytosol of pulse-labeled cucumber cotyledons after immunoprecipitation of the radiolabeled polypeptide. At low salt concentrations, mature malate synthase can be converted into aggregated forms. However, the precursor form obtained either by in vitro translation or by extraction from the cytosol after short pulses of radioactive methionine, could neither be oligomerized into the octameric form nor aggregated into the 100-S form. Processing of malate synthase, assumed to be a requisite for oligomerization, took place rapidly in the glyoxysomes, but proceeded only slowly in the cytosol. This was demonstrated both by the uptake of in vitro-translated malate synthase into glyoxysomes, and by analysis of newly synthesized malate synthase detectable in glyoxysomes in vivo. In both cases the octamer was by far the predominant form.

Cell Compartmentation↗
PubMed1982

Membranes of protein bodies. I. Isolation from cotyledons of germinating cucumber seeds.

Protein bodies were prepared from cotyledons of germinating seeds of cucumber (Cucumis sativus) in different ways: the organelles either obtained from protoplasts by lysis or from cotyledons by mechanical disintegration were separated on sucrose-density gradients. In addition, a non-aqueous procedure was employed to isolate protein bodies. Marker proteins indicative of membranes of other organelles were carefully assayed. By this means contaminations in the purified protein-body fractions could be ruled out. Isolated protein bodies were separated into crystalloids, matrix, and membranes. The membranes were purified and characterized according to their equilibrium density (rho = 1.20 kg/l) on sucrose gradients by flotation or sedimentation. Protein-body membranes labelled in the phospholipid moiety were prepared and analyzed after application of [methyl-14C] choline or [32P] phosphate in vivo.

Amino Acids↗
PubMed1982

Membranes of protein bodies. II. Detection and partial characterization of membrane glycoproteins.

The synthesis of organelle proteins was studied in cotyledons of Cucumis sativus. Protein constituents of protein-body membranes were shown to be synthesized and assembled, at a stage characterized by mobilization of the storage globulin. Besides L-[35S]methionine various labelled hexoses were incorporated into protein bodies of cucumber cotyledons. While D-[U-14C] glucose functioned as precursor of a broad spectrum of glycoproteins, D-[6-3H]glucosamine was selectively incorporated into four glycoproteins of the protein-body membrane. Labelled galactose and mannose, respectively, were preferentially transferred into another set of membrane glycoproteins. The four glycoproteins revealed by labelling with glucosamine were solubilized and purified by chromatography on concanavalin-A-Sepharose.

Autoradiography↗
PubMed1980

A bifunctional enzyme from glyoxysomes. Purification of a protein possessing enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities.

1. Enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase copurified when extracts from cotyledons of 5-day-old cucumber seedlings were fractionated by acetone precipitation, ion-exchange chromatography on CM-cellulose and affinity chromatography on blue-dextran-Sepharose. The protein was purified 600-fold with 16% recovery. 2. Bifunctionality of the protein was substantiated by exactly coinciding activity profiles upon chromatography on hydroxyapatite, CM-cellulose, or blue-dextran-Sepharose respectively. Analysis of the purified protein using isoelectric focusing as well as native electrophoresis confirmed the presence of a bifunctional protein. A monospecific antiserum could be raised against the homogeneous protein. 3. Further characterization of the protein revealed that the two enzyme activities are contained in a single peptide chain of Mr 75000. An extremely alkaline isoelectric point of pH 9.8 was determined. 4. The bifunctional protein was localized in glyoxysomes which were shown to be the exclusive site of beta-oxidation at this stage of germination. At least 10% of the enzyme were attributable to the organelle's membrane.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1980

Purification of glyoxysomal acetyl-CoA acyltransferase.

Acetyl-CoA acyltransferase was extracted from cotyledons of germinating cucumber seeds and purified to apparent heomogeneity. The purification based primarily on cation exchange chromatography and separation on an affinity gel. The enzyme is a dimer consisting of two subunits of molecular weight 45 000. Acetyl-CoA acyltransferase was localized in glyoxysomes, the matrix was found to be the site of thiolase function within the organelle.

Acetyl-CoA C-Acyltransferase↗
PubMed1980

19S cytosolic malate synthase. A small pool characterized by rapid turnover.

A pool of 19S malate synthase was detected in the cytosol. This pool was separated from the microsomal and glyoxysomal malate synthase when cotyledons of germinating seed of Cucumis sativus were fractionated. An early stage of seed germination was selected for our investigations, when 100S "microsomal" malate synthase was also present. 1) When L-[35S]methionine was applied in vivo to label cellular proteins the small pool of 19S malate synthase was found to contain the highest specific activity compared to microsomal or glyoxysomal malate synthase. 2) The kinetics of specific radioactivity in malate synthase during a pulse chase-labelling experiment established that 19S malate synthase was a precursor of microsomal malate synthase. 3) By means of anti-malate synthase antibodies, 100S malate synthase could be recovered on protein A-Sepharose and thus separated from the endoplasmic reticulum. The data suggest that both microsomal and glyoxysomal malate synthase are synthesized in the cytosol rather than at the endoplasmic reticulum according to the signal hypothesis.

Cytosol↗
PubMed1980

Occurrence and biosynthesis of glyoxysomal enzymes in ripening cucumber seeds.

Glyoxysomal enzymes, being necessary during seed germination, are already synthesized at the stage of seed maturation. Two stages of embryogenesis of cucumber seeds (Cucumis sativus) were investigated. One was characterized by the presence of microbodies showing catalase and enoyl-CoA hydratase activities. Microbodies at a later stage contained, in addition, malate synthase and isocitrate lyase. The biosynthesis of three microbody components was followed in a pulse chase-labelling experiment which demonstrated that the biosynthesis of cytosolic species of malate synthase, isocitrate lyase and enoyl-CoA hydratase preceded the appearance of these proteins in microbodies.

Catalase↗